“Regulación epi-genética durante la embriogénesis cigótica en

UNIVERSIDAD DE OVIEDO
Departamento de Biología de Organismos y Sistemas
Programa de Doctorado: “Biología aplicada a la sostenibilidad de recursos naturales
(Mención de calidad)”
“Regulación epi-genética durante la embriogénesis cigótica en
castaño europeo (Castanea sativa Miller)”
“Epi-genetic regulation throughout zygotic embryogenesis in
European chestnut (Castanea sativa Miller)”
TESIS DOCTORAL
Marcos Viejo Somoano
Oviedo, 2015
RESUMEN DEL CONTENIDO DE TESIS DOCTORAL
1.- Título de la Tesis
Español/Otro Idioma:
Regulación epi-genética durante la
embriogénesis cigótica en castaño europeo
(Castanea sativa Miller)
Inglés:
Epi-genetic regulation throughout zygotic
embryogenesis of European chestnut
(Castanea sativa Miller)
2.- Autor
Nombre:
DNI/Pasaporte/NIE:
Marcos Viejo Somoano
Programa de Doctorado: Biología aplicada a la sostenibilidad de recursos naturales (Mención
de calidad)
Órgano responsable: Universidad de Oviedo
FOR-MAT-VOA-010-BIS
RESUMEN (en español)
El castaño europeo (Castanea sativa Miller) es una especie multipropósito distribuida
principalmente en la cuenca mediterránea y extensas áreas en Francia. Posee un gran
valor debido a la calidad de su madera y frutos. Existe un elevado número de cultivares
de interés obtenidos mediante procesos de mejora tradicional, pero la clonación
mediante propagación vegetativa de individuos adultos representa un cuello de botella
debido a su carácter recalcitrante. El castaño suele producir castañas monoembriónicas
como consecuencia del desarrollo de un único primordio seminal, dominante, de entre
todos los incluidos en un ovario dado; aquellos que no entran en el programa
embriogénico son llamados acompañantes y abortan en paralelo al desarrollo del
primordio dominante. Cuando dos o más primordios se desarrollan, se obtienen semillas
poliembriónicas lo cual disminuye su valor comercial.
En base a la problemática expuesta, el objetivo de esta tesis es profundizar en el
conocimiento sobre los posibles mecanismos que afecten al establecimiento de un
primordio dominante que da lugar al embrión maduro y la muerte de los primordios
acompañantes mediante el estudio de variables fisiológicas y (epi)genéticas, así como
analizar posibles interacciones a lo largo de la reproducción sexual y determinar los
mejores explantos que se generan desde la floración hasta la obtención de embrión
maduro para la inducción de embriogénesis somática.
Los resultados muestran que niveles específicos de hormonas, marcas epigenéticas y
expresión génica a lo largo del desarrollo son necesarios para generar la semilla madura.
Así, el destino de los primordios seminales en el ovario se fija durante la polinización
en función de la procedencia del polen: la autopolinización marca los primordios para
abortar mientras que la polinización cruzada da paso al desarrollo del embrión cigótico
y la muerte de los primordios seminales acompañantes. Esta bifurcación en el destino de
los primordios se relaciona con dinámicas específicas en el contenido en ABA y JA. Por
otro lado, la fecundación induce una desmetilación transitoria además de ratios
específicos de CKs y AIA, hormonas que tienen un papel fundamental en la
determinación de la polaridad del embrión. El desarrollo embriogénico posterior así
como el aborto de los primorios acompañantes se asocia con una hipermetilación del
ADN hasta alcanzar la madurez o abortar. Además, el proceso de muerte de los
primordios acompañantes coincide con aumentos en los valores de H3K9me3 y
H3K27me3, marcas epigenéticas asociadas con silenciamiento génico. La maduración
del embrión implica cambios morfológicos que se reflejan en las diferentes dinámicas y
distribuciones de marcas epigenéticas y hormonas. Un fuerte incremento de H4ac marca
el comienzo de la maduración junto con importantes cambios en el ratio ABA:GA4, que
varía durante la maduración hasta alcanzar el final del desarrollo. La expresión
diferencial de genes parece apoyar parte de la cuantificación de hormonas y marcas
epigenéticas estudiadas como ocurre por ejemplo con los niveles de ABA respecto a su
conocido papel en la represión de la expresión de genes que codifican para histones
desacetilasas. Los niveles específicos de las variables estudiadas en un estadio de
desarrollo dado están, además, relacionados con la observación de una ventana de
desarrollo en relación a la inducción de embriogénesis somática que tiene lugar desde
estadios post-fecundación con desarrollo embriogénico hasta el embrión maduro.
Desde la polinización hasta el establecimiento del embrión maduro, puede concluirse
que la transición entre estadios de desarrollo y el destino de los primordios seminales
están controlados mediante una serie de interacciones que integran estímulos externos e
internos. Este control se ejecuta a través de la acción orquestada de diferentes actores
fisiológicos y (epi)genéticos para generar una respuesta global que culmina con el
establecimiento del embrión maduro.
RESUMEN (en Inglés)
European chestnut (Castanea sativa Miller) is a multipurpose species distributed within
the Mediterranean area and extensive areas in France. It possesses paramount value due
to the quality of its wood and nuts. Many cultivars highly appreciated have been
obtained by traditional breeding in the past by crosses between individuals regarding
production of nuts in terms of quality and yield, but cloning due to the recalcitrant
character of adult individuals represents a bottleneck for vegetative reproduction.
Chestnut usually produces monoembrionic nuts due to the development of a unique
ovule, dominant, from all the ovules contained in a given ovary; those which do not
enter in the embryogenic program are called companion ovules and abort in parallel
with the development of the dominant ovule. On the contrary, polyembryonic seeds are
generated when two or more ovules develop a zygotic embryo, which diminishes their
commercial value.
On the basis of the expounded knowledge, the aim of this thesis is to get insight the
understanding of the possible mechanisms affecting the establishment of a dominant
ovule that gives rise to the mature embryo and the associated cell death of companion
ovules by the study of physiological and (epi)genetic variables and analyze their
possible interactions throughout chestnut sexual reproduction as well as evaluate the
best explants produced during the consecutive stages of development for the induction
of somatic embryogenesis from flowering to the establishment of the mature embryo.
Our results show that specific levels of selected hormones, epigenetic marks and gene
expression are necessary throughout in order to achieve the mature seed. The destiny of
the ovules within an ovary has been discovered to be fixed in the early step of
pollination given that the provenance of the pollen tags the ovules for abortion if
autopollination takes place whereas cross-pollination triggers the normal zygotic
embryo development and companion ovules death. This bifurcation in ovules’ fate
seems to be related with specific dynamics in ABA and JA contents. Fertilization of the
ovule induces a transient demethylation status and specific ratios of CKs and IAA,
known for their vital role in the determination of the polarity of the embryo while
posterior embryo development and also the abortion of companion ovules concurs with
hypermethylation of the DNA in the tissues until reaching maturing and death. In
addition, companion ovules degeneration process coincides with increases of the
epigenetic marks associated with genetic silencing status of the chromatin H3K9me3
and H3K27me3. Maturation of the embryo implies morphological changes at the tissue
level that are reflected in different dynamics and distribution of epigenetic marks and
hormones. A strong increase of H4ac marks the beginning of maturation together with
dramatic changes in the ratio ABA:GA4 that varies towards the end of development. In
addition, differential gene expression is likely to support the quantification of hormones
and epigenetic marks studied such as the relation between ABA levels respect to its
repressive role in the expression of histone deacetylases. The specific levels of the
studied variables at a given developmental stage has been found to be related with the
observation of a developmental window leading to somatic embryogenesis induction
from post-fertilization embryogenic stages until the mature embryo.
From the first stimulus consistent on pollination to the establishment of the mature
embryo, it can be concluded that the transition between developmental stages and the
destiny of the ovules are controlled in a cross-talk fashion by the integration of external
and internal cues. Such control is exerted through the orchestrated action of different
physiolocial and (epi)genetic actors that interact in order to generate a global, major
response reflected in the establishment of the mature embryo.
SR. DIRECTOR DE DEPARTAMENTO DE BIOLOGÍA DE ORGANISMOS Y SISTEMAS/
SR. PRESIDENTE DE LA COMISIÓN ACADÉMICA DEL PROGRAMA DE DOCTORADO EN BIOLOGÍA APLICADA
A LA SOSTENIBILIDAD DE RECURSOS NATURALES (MENCIÓN DE CALIDAD)
EstaTesishasidorealizadaen:
UniversidaddeOviedo
DepartamentodeBiologıádeOrganismosySistemas
AreadeFisiologıáVegetal
InstitutoUniversitariodeBiotecnologíade
Asturias
CentrodeInvestigaciónMilleniumSeedBank
(KewGarden,UK)
Financiació npersonalydelainvestigació n:
Becapredoctoralparalaformació neninvestigació n
ydocenciadelprincipadodeAsturias
ProyectoAGL2007-62907/FOR
Có digoepigené ticoyregulació ndesubproteomas
duranteelenvejecimiento-revigorizació ndeespecies
agroforestales.
ProyectoMEC.CIT-010000-2007-5
Obtenció ndematerialesforestalesdereproducció n
dealcornoquedealtacalidadyproductividadde
corcho.
ProyectoAGL2010-22351-C03-01
Factores(epi)-gené ticosenprocesosdedesarrollo,
reprogramació nyadaptació nacambioclimá ticoen
especiesforestales.
ProyectoAGL2011-27904
Regulació n(epi)-genó micadeldesarrolloenespecies
agroforestales.Implicació nenproductividad,
clonació n
yrespuestaaestré sabió tico.
Agradecimientos
Habéis sido muchos los que me habéis acompañado en este camino y cada uno
ha aportado algo valioso. Estos agradecimientos son para vosotros.
María Jesús, gracias por darme la oportunidad y creer en mí. Gracias por tu
punto de vista, siempre válido. Han sido muchos años trabajando juntos en los que he
aprendido mucho de ti y contigo. Gracias por hacer siempre lo que estuvo en tu mano
para que las cosas me fueran lo mejor posible.
Peter, I have no words to describe how much important your support has been
during this time. This thesis wouldn´t have been possible without you. You´re a great
scientist and I´ve enjoyed a lot working with you. Thanks for transmitting your
enthusiasm every day and for believing so much in what you do. You have no idea how
much I´ve learned working by your side. You´ve given me much more than I could ever
give in return.
Rodrigo, gracias por introducirme en el mundo del castaño. Tengo muy
presentes los primeros días en que trabajé contigo. Te marchaste de vuelta a Chile
demasiado pronto pero siempre has estado presente. Gracias por transmitir tus energías
por el trabajo bien hecho. Gracias por seguir colaborando conmigo aun en la distancia.
Espero y deseo que sigamos haciéndolo.
Luis, entré de tu mano en el laboratorio. Siempre recordaré cómo recoger, pesar
y homogeneizar las muestras. Gracias por haber sido un maestro en tantas ocasiones.
Gracias por tus ideas y por compartir tu criterio del que tanto he aprendido. Colaborar
contigo siempre ha sido fructífero y seguro que nos queda mucho por disfrutar
trabajando juntos.
Mónica, gracias por abrirme las puertas del mundo del confocal y las inmunos.
Con lo difícil que me parecía al principio. Gracias por ayudarme siempre que me hizo
falta. Me alegro mucho de haber trabajado contigo.
Estrella, gracias por ser como eres. Hemos trabajado mucho juntos y compartido
muchas comidas y cafés. Siempre has estado ahí para ayudarme. Gracias por tu energía
infinita y por tus consejos.
Rebe, gracias por tu forma de pensar y por compartirla. Eres una gran
trabajadora y has sido ejemplo para mí en muchas ocasiones aunque no lo supieras.
Gracias por compartir tantos buenos momentos dentro y fuera del labo.
Marta, si tu tesis era tanto tuya como mía, aquí tienes otra para compartir a
medias ;-) Me faltaría espacio para agradecerte todo lo que has supuesto en este tiempo.
Gracias por haber estado siempre ahí, incondicionalmente, por tender siempre la mano.
Gracias por reñirme y por ponerte de mi parte. Gracias y mil veces gracias.
Helen, yes única. No cambies nunca. No sé de dónde sacas tanta energía, pero
gracias por compartirla. Gracias por tu forma de ser. Gracias por empujarme siempre y
no dejarme caer.
Víctor. Gracias por ser mi estadístico de confianza. He disfrutado mucho
aprendiendo de ti. Gracias por tu honestidad infinita en el trabajo. Gracias por ser tan
generoso sin pedir nada a cambio y por compartir tan buenos momentos dentro y fuera
del labo.
Mauro, gracias por tu empatía y por tu forma de ver el mundo. Nos queda
mucho por discutir todavía. He aprendido mucho de ti y siempre me haces reflexionar.
Gracias por tus gestos, tus bromas y tu sinceridad.
Sara zoo, gracias por estar siempre ahí. Gracias por las discusiones en las que
siempre aprendo algo y por intentar entenderme siempre. Me alegro de haberte
conocido y de haber compartido tanto contigo.
Sara Kew, gracias por tus análisis y por compartir tu tiempo conmigo. Gracias
por preocuparte y por ponerte de mi parte. Sé que lo conseguirás, así que sigue dándole
duro que estaré ahí para lo que necesites.
Chus, Dani, Mónica. Gracias por los buenos momentos dentro y fuera del
trabajo. Ya no os queda nada. Al final todo sale. Ánimo y mucha suerte.
Gracias a todos los profesores del laboratorio porque siempre me habéis
ayudado. Gracias a Pilar y Enrique por su trabajo y su buen ánimo que siempre alegra
el día.
A mis amigos. No os podéis hacer a la idea de todo lo que habéis significado en
esta etapa. Gracias por dejarme formar parte de vuestras vidas. Gracias por dejarme ser
yo cuando estoy con vosotros. Me habéis dado la gasolina que necesitaba para llegar
hasta aquí. Gracias por las fiestas infinitas, por las risas sin sentido por Skype, por los
planes a lo loco, por los conflictos estúpidos con los que tanto nos reímos después.
Gracias, porque con vosotros el tiempo vuela y las preocupaciones desaparecen. Gracias
por compartir mis risas y mis lágrimas. Gracias por decir tanto con tan poco. No os
puedo querer más. Sé que el tiempo y la distancia no nos van a separar y me siento
tremendamente orgulloso de teneros. Una y mil veces, GRACIAS.
A mi familia. Gracias, básicamente, por aguantarme y quererme tal y como soy.
Gracias por todo lo que me habéis dado siempre que nunca os podré devolver. Gracias
por ser diariamente un ejemplo de superación. Gracias por apoyarme siempre y dejarme
escoger mi camino. Gracias a vosotros he llegado hasta aquí.
Gracias, también, a aquellos que ya no están y de los que tanto he aprendido.
Gracias por poner la semilla de lo que soy hoy.
ABBREVIATIONS
2,4-D
5-mdC
ABA
AU
AUR3
BA
bp
BRs
BSA
cDNA
CKs
CMT
DABCO
DAPI
DMRM
DRM
ELISA
EMBD
EMB
ERFA1
ERFs
GAs
GCN5L
HAT
HDA19
HDA6
HDAC
HDM
HPCE
HPLC
HTM
HUB2
IAA
iRNA
JA
KYP
MET
MSAP
MSL
NML
OVA3
PBS
PCA
2,4-dichlorophenoxyacetic acid
5-methyl deoxy cytidine
abscisic acid
arbitrary units
AURORA3
6-benzylaminopurine
base pairs
brassinosteroids
bovine serum albumin
complementary DNA
cytokinins
chromomethyltransferase
1,4-Diazabicyclo[2.2.2]octane
4`-6-diamidino-2-phenylindole
dynamic multiple multireaction monitoring
domains rearranged methyltransferase
enzyme-linked ImmunoSorbent assay
EMBRYO DEFECTIVE 1345
embryo defective genes
ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR A1
ETHYLENE RESPONSIVE TRANSCRIPTION FACTORS
gibberellins
GENERAL CONTROL NON-REPRESSED PROTEIN5- LIKE GENE
histone acetyl transferase
HISTONE DEACETYLASE19
HISTONE DEACETYLASE6
histone deacetylase
histone demethylase
high-performance capillary electrophoresis
high-performance liquid chromatography
histone methyl transferase
HISTONE MONO-UBIQUITINATION2
indole acetic acid
interfering RNA
jasmonic acid
KRYPTONITE
methyltransferase
methylation sensitive amplification polymorphism
methylation susceptible loci
non methylated loci
OVULE ABORTION 3
phosphate buffered saline
principal component analysis
PCoA
PIN
PTMs
RADSAM
RAM
RdDM
SAH
SAM
SAMe
SAMS2
SE
siRNA
TDMRs
UHPLC
principal coordinate analysis
PIN FORMED transporters
post-translational modifications of histones
RADICAL SAM DOMAIN-CONTAINING PROTEIN
root apical meristem
RNA directed de novo methylation
s-adenosyl homocysteine
shoot apical meristem
s-adenosyl methionine
S-ADENOSYL-L-METHIONINE SYNTHETASE2
standard error
small interfering RNA
tissue-specific differentially methylated regions
ultra-high performance liquid chromatography
INDEX
CHAPTER I
General Introduction ............................................................................................................... 1
1.1. THE FORESTS AND THE CHESTNUT.......................................................................... 1
1.2. RELEVANCE OF THE CHESTNUT................................................................................ 2
1.2.1. Sexual reproduction .................................................................................................... 4
1.2.2. Monoembryony and the death of companion ovules ............................................ 6
1.3. ZYGOTIC EMBRYOGENESIS IN FLOWERING PLANTS.......................................... 6
1.4. HORMONES AND SEXUAL REPRODUCTION .......................................................... 7
1.4.1. Hormone cross-talk during development ............................................................. 10
1.5. EPIGENETIC MECHANISMS ....................................................................................... 12
1.5.1. DNA methylation ...................................................................................................... 13
1.5.2. Post-translational modifications of histones (PTMs) ........................................... 15
1.5.3. Chromatin, epigenetics and control of gene expression during plant
development ........................................................................................................................ 16
1.6. APPROACH AND OBJECTIVES ................................................................................... 20
1.7. BIBLIOGRAPHY .............................................................................................................. 22
CHAPTER II
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller........................................................................................................................ 31
2.1 INTRODUCTION ............................................................................................................. 31
2.2. MATERIAL AND METHODS ....................................................................................... 32
2.2.1. Plant material ............................................................................................................. 32
2.2.2. Histological analysis ................................................................................................. 33
2.2.3. Embryogenic competence evaluation .................................................................... 33
2.2.4. Quantification of DNA global methylation with high performance capillary
electrophoresis (HPCE) ...................................................................................................... 34
2.3. RESULTS ........................................................................................................................... 35
2.3.1. Determination of developmental stages ................................................................ 35
2.3.2. Somatic embryogenesis dynamics .......................................................................... 38
2.3.3. Methylation dynamics .............................................................................................. 39
2.4. DISCUSSION .................................................................................................................... 40
2.5. BIBLIOGRAPHY............................................................................................................... 44
CHAPTER III
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa
reproduction ........................................................................................................................ 49
3.1. INTRODUCTION ............................................................................................................. 49
3.2. MATERIAL AND METHODS ........................................................................................ 52
3.2.1. Plant material ............................................................................................................. 52
3.2.2. MSAP........................................................................................................................... 52
3.2.3. Relative quantification of PTMs by ELISA ............................................................ 55
3.2.3.1. Antibodies specificity .................................................................................... 56
3.2.3.2. ELISA protocol................................................................................................ 56
3.2.3.3. Statistical analysis........................................................................................... 57
3.2.4. Immunohistochemical detection of 5-mdC and H4ac .......................................... 57
3.3. RESULTS ............................................................................................................................ 58
3.3.1. MSAP analysis ........................................................................................................... 58
3.3.2. PTMs quantification .................................................................................................. 62
3.3.3. Immunodetection ...................................................................................................... 66
3.4 DISCUSSION...................................................................................................................... 74
3.5. BIBLIOGRAPHY............................................................................................................... 81
CHAPTER IV
Different hormonal profiles characterize sexual reproduction in C. sativa ............... 87
4.1. INTRODUCTION ............................................................................................................. 87
4.2. MATERIAL AND METHODS ........................................................................................ 88
4.2.1. Plant material ............................................................................................................. 88
4.2.2. Global hormone content ........................................................................................... 89
4.2.3. Determination of embryo moisture content .......................................................... 90
4.2.4. Immunohistochemical detection of ABA and IAA ............................................... 90
4.3. RESULTS ............................................................................................................................ 91
4.3.1. Hormones vs developmental stages clustering and principal component
analysis .................................................................................................................................. 91
4.3.2. Hormones global content ......................................................................................... 96
4.3.3. Moisture content ..................................................................................................... 102
4.3.4. Immunodetection .................................................................................................... 103
4.4. DISCUSSION .................................................................................................................. 111
4.5. BIBLIOGRAPHY ............................................................................................................ 118
CHAPTER V
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction ...................................................................................................................... 125
5.1. INTRODUCTION .......................................................................................................... 125
5.2. MATERIAL AND METHODS ..................................................................................... 127
5.2.1. Plant material ........................................................................................................... 127
5.2.2. RNA isolation and complementary DNA (cDNA) synthesis ........................... 127
5.2.3. Selection of genes for real-time PCR .................................................................... 128
5.2.4. Statistical analysis ................................................................................................... 130
5.3. RESULTS ......................................................................................................................... 130
5.3.1. Validation of chestnut housekeeping genes during reproduction .................. 130
5.3.2. Determination of relative expression of the genes of interest .......................... 132
5.3.3 Determination of conserved domains in the analyzed ESTs ............................. 134
5.4. DISCUSSION .................................................................................................................. 142
5.5. BIBLIOGRAPHY ............................................................................................................ 148
CHAPTER VI
General discussion............................................................................................................... 153
6.1. GENERAL DISCUSSION.............................................................................................. 153
6.2 BIBLIOGRAPHY ............................................................................................................. 163
CONCLUSIONS .................................................................................................................. 169
RESUMEN ............................................................................................................................. 173
INTRODUCCIÓN ............................................................................................................. 173
PLANTEAMIENTO Y OBJETIVOS ................................................................................ 177
RESULTADOS Y DISCUSIÓN ........................................................................................ 178
CONCLUSIONES ............................................................................................................. 184
CHAPTER I
General Introduction
Chapter I
1.1. THE FORESTS AND THE CHESTNUT
Forestry ecosystems represent the greatest source of renewable, high added
value goods in terms of biomass in the emerged land. Importance of forests not only
rely on their production of wood for diverse industries and food for animals and human
consumption, but also their relevance in the climate control and the water cycle must be
taken into account (Foley et al., 2005). The original tree-areas covered by forests have
been shifted due to anthropogenic activities such as crop production or pastures
exploitation, leading to the loss of forestry areas and soil degradation (Foley et al., 2005).
Forests in Europe cover 25 % of the land and the economical associated activities
constitute the 1 % of the gross domestic product according to the Forest Europe (The
Ministerial Conference on the Protection of Forests in Europe, www.foresteurope.org).
The exploitation of economically interesting trees for timber and food production
usually involves clonal plant-mass production by vegetative propagation starting with
the selection of elite genotypes, usually adult trees which intrinsically have important
restrictions in their morphogenic competences that depends on the age and are
genotype dependent (Valledor et al., 2007).
Castanea sativa Miller is a perfect example of a sustainable agroforestry species
integrated in the landscape that has been cultivated and expanded in Europe since
thousands of years ago. Its use since ancient times as a source of timber and food has
represented an income of great importance for the Mediterranean people and also
constitutes the basis of deep cultural connotations (Feijó et al., 1999).
Fruit development in chestnut is of paramount importance as the desired traits
associated with embryo development in commercial terms are seed size, the
polyembryony degree and the intrusion of the episperm within the cotyledons
(Bounous et al., 2001; Fig. 1.1). In France, marron refers to large chestnuts without
episperm intrusion and the main criteria for considering a cultivar as marron is that at
least 88 % of the production must be monoembryonic seeds. In Italy, marron is associated
with specific C. sativa cultivars that include characteristics such as the monoembryonic
character of the seed, the shape of the fruit and the hilar scar or no hollows in the
cotyledons. Moreover, the easy separation of the embryo from the episperm make this
kind of chestnuts ideal for increasing the yield during their processing. In addition,
marron cultivars produce between 70-80 seeds per kg (D’Adda et al., 2003). On the other
1
General introduction
hand, marron varieties have stricter culture requirements and are usually andro-sterile,
so combined plantations are needed in order to ensure their cross-pollination. During
fruition, parthenocarpy is a common phenomenon: when pollination and fertilization
are not successful, the three ovaries can develop into embryo-less nuts along with
normal growth of the bur. Nonetheless bibliography regarding this issue is scant
(Beyhan and Serdar, 2008) although parthenocarpy is much extended in natural
conditions.
Fig. 1.1. Mature chestnut (a), polyembrionic nut with two embryos in transversal cut (b) and immature nuts
showing two lateral parthenocarpic nuts surrounding a central one containing an embryo (c).
Zygotic embryogenesis involves the integration of internal and external cues by
the mother plant and the gametophyte supported by it. The achievement of the mature
embryo being part of the seed is reached by an orchestrated interaction of several factors
in which tight physiological and epi-genetic changes take place in order to control the
consecutive developmental phases that arise during this part of plants ontogenesis.
Moreover, the global physiological status of a given tissue at a given time can be used
for inducing in vitro responses through somatic embryogenesis and constitute an
alternative for the classical clonal propagation methods.
1.2. RELEVANCE OF THE CHESTNUT
The genus Castanea, belonging to the family Fagaceae, was originated in the
northern hemisphere and is part of the warm, deciduous forests. There are 8 species
currently accepted with world-wide distribution: in Asia, C. mollisima Blume, C. henryi
(Skan) Rehder & E. H. Wilson, C. seguinii Dode and C. crenata Siebold & Zuccarini. In
North America, C. dentata (Marshall) Borkhausen, C. pumila (Linnaeus) Miller and C.
ozarkensis Ashe. In Europe, the only representative is C. sativa Miller.
2
Chapter I
European chestnut is a robust, vigorous tree that can reach more than 25 m in
height and displays an open crown. The trunk is short and wide in the plantations for
production of chestnuts, and straight and without branching in the plantations for
timber production. It requires deep, rich soils with moderate acidity: pH 4.5 to 6.5.
Annual medium temperature must be in the range 8 to 15 ˚C and rainfall between 400900 mm per year distributed throughout the year. It grows from sea level to 1000 m
altitude, and while the best range of altitude for nuts production is between 200-600 m,
timber production needs an altitude of 500 to 1000 m (Vieitez et al., 1999).
Distribution in Europe encompasses the Mediterranean region and extensive
areas in France (Fig. 1.2). The production and consumption of chestnuts in Europe in
the last few decades declined from 427,000 T in 1961 to 163,200 T in 2010; this contrasts
with the increasing relevance in the global market of several hybrids and cultivars of C.
sativa regarding the quality of their nuts and their resistance to the chestnut blight.
Chile, Argentina, Australia and New Zealand nowadays culture these hybrids and
cultivars according to the ARFLH (Assemblee des Régions Europeennes Frutières,
Légumières et Horticoles; www.arflh.org) and are around 1 % of the worldwide
production each.
Fig. 1.2. Distribution map of C. sativa. [Taken from EUFORGEN (www.euforgen.org)].
3
General introduction
1.2.1. Sexual reproduction
European chestnut is a monoecious species that requires cross pollination which
can be anemophilous and/or entomophilous. Due to the adhesive nature of the chestnut
pollen, anemophilous pollination is usually performed in conditions of low relative
humidity while entomophilous pollination is mainly accomplished in high relative
humidity conditions.
Male inflorescences are presented as catkins and usually vary in their length
among cultivars and thus, in the number of flowers. There are four classes of unisexual,
male catkins (Vieitez et al., 1999) whose morphology influences the amount of pollen
and its exchange among individuals:
1. Astaminates: sterile flowers without stamens and thus, no pollen
production.
2. Brachystaminate: the filament of the stamen length is 1-3 mm and the
anthers do not exceed the perianth. Poor pollen production.
3. Mezostaminate: the filament of the stamen is 3-5 mm in length and the
anthers are longer than the perianth. Poor pollen production.
4. Longistaminate: The filament of the stamen is 5-7 mm in length and the
anthers easily exceed the perianth. Abundant pollen production
Unisexual staminate catkins grow in the basis of the shoot in a spiral
arrangement while bisexual catkins containing both staminate and pistillate flowers
(Fig. 1.3a) are located on the end of the shoot (Feijó et al., 1999). Female flowers are in
groups of 2 to 5, generally 3, situated in the basis of the bisexual catkin and they are
protected by an involucral bract that will develop into a burr (Fig. 1.3b). Inflorescences
are single or in clusters of 2 or 3 (Botta et al., 1995). Hermaphrodite flowers, sterile, can
also be found and have rudimentary pistils.
Depending on the altitude, the meteorological characteristics of the current year
and the cultivar, full bloom ranges from the end of May to mid-July. It is known that
female flowers are receptive after the shed of the male ones in order to avoid
autopollination. Moreover, different cultivars are known to possess different times of
flowering (Klinac et al., 1995). Staminate flowers in the bisexual catkins bloom after
female flowers have been pollinated avoiding autopollination. Ovaries consist of 6 to 8
loci containing two ovules each from which 6 to 9 styles emerge, usually 7; they are
4
Chapter I
cylindrical, rigid, pale-green and slightly brown in the apex (Vieitez et al., 1999). Once
the pollen grain gets to the stigma, it germinates and crosses the stylus reaching the
ovary in about 10 days. Usually, 10 to 16 tubes penetrate the styles but only one seems
to penetrate the ovary and ultimately fecundate one ovule (Valdiviesso et al., 1993).
Pollination has been found to have a paramount importance because the pollen-donor
plant controls the quality of the seed in terms of size, shape and production (Craddock
et al., 1992).
Fig. 1.3. Bisexual catkin showing two female inflorescences at the basis and the immature male inflorescence
(a) and development of the involucral bract into a burr (b).
After the double fertilization, the embryo and the cellular endosperm start
developing. Chestnuts are exalbuminous seeds, which means that the endosperm is
reabsorbed during the developing of the embryo and the cotyledons adopt the nutrient
storage role filling the majority of the seed at maturity, constituting the gross of the
weight of the seed. The chestnut fruit is an achene composed of the cotyledonary
embryo, an episperm formed by the wall of the ovule that dehydrates at mature and
intimately ligates to the embryo, and the hairy pericarp originated from the wall of the
ovary that hardens and turns color from green in the flower to brown in the mature seed
(Camus, 1929). Usually, the styles persist in the mature fruit. Chestnuts are indehiscent
fruits although the endocarp can break in some cases due to an excessive growing of the
5
General introduction
embryo. An associated problem with nuts storage is the recalcitrant character of
chestnut seeds that make their conservation a challenging issue (Bewley and Black,
1994).
1.2.2. Monoembryony and the death of companion ovules
To date the main unresolved question regarding the developmental model for
reproduction in both the Castanea and Quercus genera is: what is controlling
polyembryony? Given the number of ovules within an ovary and the multiple pollen
grain germination and growth through the style, four different causes for ovule abortion
were proposed by Mogensen (1975) in Q. gambelii that has been extended to C. sativa.
These were: (1) a lack of fertilization, (2) zygote or embryo failure, (3) the absence of an
embryo sac and (4) an empty embryo sac. Feijó et al. (1999) claimed that it is not possible
to distinguish whether monoembryony in chestnut is due to a single fertilization or to
the dominance of the first fertilized ovule over the rest. Moreover, older studies on this
matter, reviewed in Mogensen (1975) speculate that one ovule could dominate the rest
by starving them and redirecting all the available nourishment to itself. The position of
the ovule within the ovary has also been the subject of studies; however, no correlation
with dominance could be established.
1.3. ZYGOTIC EMBRYOGENESIS IN FLOWERING PLANTS
Seeds are the dispersal unit of plants for establishing of the new generation that
ensures the dispersion and persistence of the species through time. Moreover, sexual
reproduction forms a source of genetic variation.
The floral organs comprehend the gametophytic generation that grows and is
supported by the sporophytic generation. After proper pollination, the double
fertilization gives rise to several organs containing tissues comprehending
differentiated cell types. According to Goldberg et al. (1994), embryogenesis in higher
plant serves to “(1) specify meristems and the shoot-root plant body pattern, (2)
differentiate the primary plant tissue types, (3) generate a specialized storage organ
essential for seed germination and seedling development and (4) to enable the
sporophyte to lie dormant until conditions are favorable for postembryonic
development”. In support of the above, these authors proposed the events that can be
6
Chapter I
distinguished during embryogenesis (Table 1.1) from fertilization to the onset of
dormancy of the seed at the end of development.
Table 1.1. Major events of angiosperm plant embryogenesis. Adapted from Goldberg et al. (1994).
Posfertilization-pro-embryo
Terminal and basal cell differentiation
Formation of suspensor and embryo proper
Globular-heart stage
Differentiation of major tissue-type primordia
Establishment of radial (tissue-type) axis
Embryo proper becomes bilaterally symmetrical
Visible appearance of shoot-root (apical-basal) axis
Initiation of cotyledon and axis (hypocotyl-radicle) development
Differentiation of meristems
Organ expansion and maturation
Enlargement of cotyledons and axis by cell division and expansion
Formation of lipid and protein bodies
Accumulation of storage proteins and lipids
Vacuolization of cotyledon and axis cells
Cessation of RNA and protein synthesis
Loss of water (dehydration)
Inhibition of precocious germination
Dormancy
In
the transition
between
developmental
stages throughout embryo
development many morphological, cellular and biochemical changes take place which
are regulated in an orchestrated manner. There are multiple actors involved in the
accomplishment of the mature seed that interact as development progresses. The role
of hormones as intermediates in signaling pathways and the epigenetic mechanisms
that partially control seed development through gene differential expression are
introduced below.
1.4. HORMONES AND SEXUAL REPRODUCTION
Plant hormones govern, in association with other players, the ontogeny of the
plant. The development of the transient gametophyte and the consecution of zygotic
embryogenesis leading to the mature seed is under a tight control exerted by both
external and internal stimuli in a complex fashion in which hormones are involved.
Auxins
Auxins were discovered in the 1930s. Of all the representatives, indole acetic acid
(IAA) is the most abundant and physiologically most relevant. Auxins are synthesized
7
General introduction
by different pathways in apical meristems, young leaves and fruits and developing
seeds (Taiz and Zeiger, 2010) and they are ubiquitous in the plant presenting gradients
and differential distribution (Vanneste and Friml, 2009). Auxins can be considered as a
morphogen as they form a stable concentration gradient, act directly in the cell without
intermediates and the response is proportional to the concentration (Friml et al., 2003).
All development of the organs in the plant require controlled fluxes of auxins for their
formation that follow specific steps: accumulation at the organ´s initiation site,
activation of cellular divisions and the reorganization of the auxin transport for
determining the axis of the developing organ, establishing a maximum in the apical
zone (Benková et al., 2003).
During reproduction, local auxin increments suffice for the initiation and
completion of flowers (Reinhardt et al., 2000). Moreover, the role of auxins has been
described in early embryogenesis in the establishment of the polarity of the embryo
through the PIN-FORMED (PIN) transporters that control the gradient in the tissues
(Vanneste and Friml, 2009). Moreover, IAA controls the establishment and maintenance
of the root apical meristem (RAM) and the position of the shoot apical meristem (SAM;
Taiz and Zeiger, 2010). Auxins also have a central role in fruit set (Ruan et al., 2012), and
during fruit formation they create a source-sink favoring the developing tissues
stimulating cellular division and maintaining cellular expansion (Taiz and Zeiger, 2010).
During seed maturation, IAA (Bewley and Black, 1994) diminishes due to its
transformation into conjugated forms. Moreover, auxins have been classically used in
fruit production due to their ability for the induction of parthenocarpic fruits (Gorguet
et al., 2005).
Gibberellins (GAs)
There are more than a hundred GAs described although only some of them are
biologically active while the rest are not bioactive and are precursors or deactivated
forms (Yamaguchi, 2008). They participate in the initiation of flowering and
determination of the sex of the flower, in the growth of pollen tubes (Taiz and Zeiger,
2010), and they are associated with the normal progression of pollen development
(Huang et al., 2003). They are known to participate in the establishment of the embryo
polarity in somatic embryos derived from microspores of Brassica napus (Hays, 2002)
and to be synthesized in ovules and zygotic embryos in the early phases of
8
Chapter I
embryogenesis (Gorguet et al., 2005). In the later embryo development, GAs, and more
specifically gibberellic acid 4 (GA4), plays a substantial and antagonistic role with
abscisic acid (ABA) in the control of embryo maturation and precocious germination
(White et al., 2000). Moreover, GAs are related with the development of parthenocarpic
fruits, either endogenously (Talon et al., 1992) or when exogenously applied (Mesejo et
al., 2013). GAs are intimately ligated with seed germination as their synthesis in the seed
not only triggers the production of hydrolytically enzymes in order to mobilize the
nutrients storage for feeding the seedling (Karssen et al., 1989), but also induce embryo
elongation and weakening of covering layers (Bewley and Black, 1994).
Cytokinins (CKs)
CKs exert their actions in the context of cellular division and are mainly
produced in juvenile tissues and it is known that all the tissues in active division can
synthesize de novo CKs (Kärkönen and Simola, 1999; Emery et al., 2000). Most of the CKs
derive from the nitrogenous base adenine and can be found as ribosides, ribotides or
glycosides (Taiz and Zeiger, 2010). During development, CKs are involved not only in
proliferating processes, but also in the delay of leaf senescence (Zwack and Rashotte,
2013) and have a central role in the maintenance of meristems, especially in the root
meristem where they induce the differentiation of cells from the quiescent center that
produces a reduction of the size of the meristem (Dello Ioio et al., 2008). During
reproduction, changes in the content of CKs in the seed after anthesis were cited by
Crosby et al. (1981) and later studies have demonstrated their participation in the
determination of the cells of the root meristem (Müller and Sheen, 2008) and their ability
for establishing nutrient sinks in Arabidopsis, where a lower number of developing seeds
within a silique generates bigger seeds rather than a higher number of developing seeds
(Riefler et al., 2006).
Abscisic acid
ABA is a ubiquitous hormone in plants with both short and long-term responses
and usually an antagonist of auxins, CKs and GAs. It is associated not only with
responses to biotic and abiotic stress but also has a significative importance during seed
development and maturation in several phases (Taiz and Zeiger, 2010): in the first stages
of embryo development, ABA levels are low, but once cellular divisions cease, a peak is
found concurring with the storage of nutrient reserves and the acquisition of
9
General introduction
dehydration tolerance while levels finally decrease at embryo and seed maturity
(Ingram and Bartels, 1996). These dynamics are also associated with the inhibition of
precocious germination (Rodríguez-Gacio et al., 2009). In addition, there is a shift
between the ABA production in the seed that in the early embryo development is
produced by the maternal tissues and during maturation by the embryo itself
(Finkelstein et al., 2002).
Brassinosteroids (BRs)
This group of phytohormones was described most recently, in the 1970s (Taiz
and Zeiger, 2010). They are mainly found in the reproductive tissues rather than
vegetative organs and pollen contains the highest concentrations along with seeds
(Montoya et al., 2005). Moreover, they have important roles in fruit development and
maturation (Symons et al., 2008). Most of the research on these hormones was performed
in recent years, discovering their relation with cell-wall modifications, transport and
elongation (Zhu et al., 2013). In addition, BRs stimulate the flux of assimilates from the
leaves to the seed by increasing CO2 assimilation and incrementing the uptake of
glucose in the seed in order to synthetize starch (Wu et al., 2008). The pool of BRs
available in a given tissue at a certain time is controlled in a dynamic fashion by the
tissue BRs production; the cells are at the same time producers and receptors allowing
the control of the endogenous BRs level and the tissue responses (Symons et al., 2008).
Jasmonic acid (JA)
JA is an ubiquitous compound involved in many biological processes such as
plant immune responses against pathogens (Halim et al., 2006) and mechanical wound
response (Wasternack, 2007). It can induce senescence, leaf abscission and inhibition of
germination (Cheong and Choi, 2003). The highest levels of JA are found in young
leaves, flowers and fruits (Creelman and Mullet, 1995) although its possible role in
zygotic embryogenesis has not been studied apart from a possible relation between an
increment in developing fruits with the biosynthesis of ethylene leading to fruit
maturation (Creelman and Mullet, 1995).
1.4.1. Hormones cross-talk during development
Plant growth regulators act in an orchestrate manner during plants ontogenesis.
In addition, the development of the gametophyte supported by the sporophyte means
a high level of complexity since tissues from both generations must act in a coordinate
10
Chapter I
fashion for giving rise to the mature seed and ensure the next generation. Hormones
can act in an antagonistic, cooperative or synergistic way, and they can interfere by
several ways in other hormones actions as proposed by Coenen and Lomax in 1997 (Fig.
1.4).
Fig. 1.4. Schematic representation of the action of hormones on each other. The possible actions of hormone
B over hormone A at various levels are: (1) control of abundance of hormone A, (2) modification of
perception of hormone A, (3) inhibition or stimulation of signal transduction processes induced by hormone
A, (4) regulation of transcription, (5) Post-translational modification and 5- interaction at the response level.
(Modified from Coenen and Lomax, 1997).
There is vast information in the literature regarding hormone interaction during
developmental processes demonstrating that their interactions are of capital importance
in the transduction of signals affecting physiological processes under biotic, abiotic
signals and endogenous cues. Auxins and CKs have been described to control the cell
cycle which in last instance influence cell differentiation and totipotency in several
tissues (Coenen and Lomax, 1997) and their balance also determine the prevalence of
one morphogenic response over others. In addition, auxins and CKs also control the size
of meristems through the control of stem cell proliferation (Dello Ioio et al., 2008).
Auxins act in concert with the rest of hormones including BRs (Choudhary et al., 2012)
during stress responses and development, and distribution alterations have been found
in the apical portion of the shoots when endogenous levels of BRs are altered. The role
of ABA, GAs, CKs, ethylene and auxins have been recently described in the
development and differentiation of the root (Takatsuka and Umeda, 2014). Jasmonates,
on their methylated or free form as JA also act synergistically or antagonistically along
with other phytohormones such as auxins or ABA (Cheong and Choi, 2003).
During reproduction, CKs have been found to be involved in the ovary growth
previously to pollination, when auxins and GAs increase their concentration while CKs
decrease (Matsuo et al., 2012) enhancing cell division and expansion leading to fruit
development. After fertilization, the establishment of the polarity of the embryo is
controlled by the balance and differential distribution of CKs, IAA and GAs (Müller and
11
General introduction
Sheen, 2008). In later developmental stages, IAA enhances GAs production and there is
an essential role of GAs and ABA. ABA peaks coincide with the decreases of IAA and
GAs (Taiz and Zeiger, 2010). The repression of GAs by ABA action in order to induce
desiccation, prevention of precocious germination of seeds (viviparism) and in last
instance, dormancy (Seo et al., 2006), is exerted through transcription factors.
Dormancy and germination of the seed has been described to be controlled by a
dynamic balance between synthesis and catabolism of ABA and GAs (Rodríguez-Gacio,
2009). GAs and CKs have a promoting role in germination while ABA associates with
dormancy and the repression of germination (Wang et al., 2011). ABA exerts part of its
control by promoting the expression of a cyclin inhibitor (Finkelstein et al., 2002)
affecting CKs functions in the cell. The role of auxins in dormancy and germination has
been recently studied by Liu et al. (2013) who described that low levels of auxins are
necessary for seed dormancy release and promote germination and vice-versa.
1.5. EPIGENETIC MECHANISMS
Due to the sessile character of plants, during their ontogenesis they are exposed
to several environmental and biotic stimuli which require plasticity in their responses.
In addition, the start of the developmental programs is also triggered by endogenous
cues. Plant organogenesis is based on the division and differentiation of new tissues and
organs arising from the stem cells contained in the meristems (Reyes and Gruissem,
2002). Part of the tight control, compulsory for proper development, is executed through
differential gene expression by changes in the chromatin state (euchromatin or
heterochromatin) that affect the accessibility to the transcription machinery.
Epigenetics is defined as the versatile and dynamic changes in the DNA and
histones that do not modify the DNA sequence but affect gene function and are
potentially heritable (Grant-Downton and Dickinson, 2006). Thus, epigenetic
mechanisms partially control gene expression and development and are key actors in
genomic imprinting, flowering, embryogenesis or senescence (Grant-Downton and
Dickinson, 2005; 2006).
12
Chapter I
1.5.1 DNA methylation
Methylation of the DNA has been widely studied in plants. It consists on the
addition of a methyl group to the 5’ position of the pyrimidine ring of the deoxycytosine
(5-mdC) in the DNA. It is also possible to find methylated adenine in small amounts in
the DNA (Vanyushin, 2006).
DNA methylation is associated with many genome processes such as
transcription, replication, repair of the DNA, gene silencing and the movement of
transposable elements (Valledor et al., 2007). Thus, methylation of the DNA plays a
predominant role in the normal development of plants ontogenesis in response to
internal cues but also as an intermediate in the transduction cascade of external stimuli
leading to gene expression changes (Chinnusamy and Zhu, 2009). Plants have been
found to show a relative tolerance to aberrant patterns in the DNA methylation not
causing lethality in many cases (Zilberman, 2008).
In Arabidopsis it is known that up to 18.9 % of the cytosines are methylated.
Vanyushin and Ashapkin (2011) found that the methylation state and distribution in a
given expressed gene relates with its translation rate as follows:
-
60 % of all genes appear to be completely demethylated, moderately
expressed and associated with transcription factors.
-
5 % are methylated in the promotor, approximately 200 base pairs (bp)
upstream from the transcription start site and with low expression rates,
generally tissue-dependent.
-
33 % are body-methylated genes while the promotor is methylation-free,
showing high expression rates because of their constitutive nature.
In plants, there are 3 classes of enzymes that catalyze the transfer of the methyl
groups to the DNA from S-adenosylmethionine (SAMe), obtaining deoxymethylcytosine and S-adenosylhomocysteine (SAH):
1. Methyltransferases (MET). MET enzymes methylate CpG islands after
the replication of the DNA using as mold the mother hemymethylated
strand (Finnegan and Kovac, 2000). This group has been found to be very
active in meristems (Ronemus et al., 1996).
13
General introduction
2. Chromomethyltransferases (CMT). This group of methyltransferases
were described in Arabidopsis and other species (Henikoff and Comai,
1998) and is exclusive of plants. These enzymes seem to be involved in
the transition from euchromatin to heterochromatin through the
maintaining of methylation in CpNpG sequences and retrotransposons
(Lindroth et al., 2001).
3. Domains rearranged methyltransferase (DRM). DRM are similar to those
Dnmt3 in mammals (Finnegan and Kovac, 2000) and participate in the
asymmetric methylation of the DNA (Cao et al., 2003) imposing new
methylation patterns.
Moreover, RNA directed de novo DNA methylation (RdDM) can be carried out
by small interfering RNA (siRNA) through the recognition and hybridization of
interfering RNA (iRNA) with specific sequences of the DNA (Jones et al., 2001).
There has been evidence of DNA demethylation for many decades, but the
mechanisms controlling the demethylation events have remained elusive until recently.
There are four possible ways, not entirely confirmed and based on multi-step reactions,
for demethylation of deoxycytidines (reviewed by Zhu, 2009):
1. Base excision repaired by 5-mdC DNA glycosylases in which the 5-mdC
is removed and replaced by an unmethylated deoxycytosine.
2. 5-mdC deamination coupled with G/T mismatch repair by DNA
glycosilases.
3. Oxidative demethylation in which the methyl group is eliminated.
4. The hydrolytic removal of the methyl group releasing it as methanol.
In conclusion, the level and pattern of DNA methylation is not only the
consequence of an homeostatic balance between DNA replication, de novo methylation
and maintaining methylation and demethylation (Hsieh, 2000), but also the coordinated
expression and function of the various kinds of DNA methyltransferases, glycosylases
and other chromatin remodeling factors (Zhu, 2009; Fig. 1.5).
14
Chapter I
Desirable DNA
methylation pattern
Demethylation
Specific
de novo
methylation
Promiscuous
de novo
methylation
DNA methylation remodeling
in response to environmental
or developmental cues
Methyltransferases
Demethylases
Final DNA
methylation
pattern
Undesirable DNA
methylation pattern
Fig. 1.5. Role of active DNA demethylation in establishing DNA methylation patterns. The final DNA
methylation patterns are established by the combined action of DNA methyltransferases and demethylases.
(Modified from Zhu, 2009).
1.5.2. Post-translational modifications of histones (PTMs)
The compacting of nuclear DNA into a higher complexity structure is achieved
by its association with histones. The first compacting step, the 11 nm chromatin fiber, is
composed of 146 base pairs of DNA wrapped around an octamer of histones of four
classes grouped into two tetramers each encompassing H2A, H2B, H3 and H4, what is
known as a nucleosome and constitutes the basic unit of compaction in eukaryotes.
Histones comprehend 3 domains that include a globular domain involved in the
histone-histone interaction, an N-terminal tail for histones H3 and H4 and the C- and
N-terminal tails for histones H2A and H2B. Of these domains, the tails of the histones
are susceptible for the addition of different chemical groups that bind to specific amino
acid residues including methylation, phosphorylation, acetylation, ubiquitination,
glycosylation, sumoylation, biotinylation, carbonylation and ADP ribosylation
(Ruthenburg et al., 2007). Of these, methylation and acetylation are the most studied
epigenetic marks. Chromatin status as euchromatin or heterochromatin is essential for
nuclear processes such as DNA replication, transcription, DNA repair and
recombination; PTMs are known to be involved in these processes and acetylation is the
major mark related with the open and closed conformations (Lusser et al., 2001).
Generally, only one type of PTM mark can be found at the same residue giving as a
result that some marks such as methylation of the lysines excludes other modifications
(Rice and Allis, 2001).
The PTMs found at a specific time in a given cell are the result of the action of
several effectors: readers, writers and erasers that act in a concerted manner giving the
cell the needed plasticity required for the initiation of the developmental patterns.
15
General introduction
Histone methylation is exerted by histone methyltransferases (HTMs) and acetylation
by histone acetyltransferases (HATs; Rice and Allis, 2001). On the contrary, though the
existence of histone demethylases (HDMs) and histone deacetylases (HDACs) was
predicted many years ago, they have been only recently characterized. Several examples
of PTMs and the catalytic enzymes directing the modifications are contained in Table
1.2 as reviewed by Berr et al. (2011).
Table 1.2. Writers and erasers of histones PTMs (Modified from Berr et al., 2011).
Type
Histone
acetyltransferases
GNAT
Gene
Full name
Specificity
AtGCN5/HAG1
H3K14, H3K27
MYST
HAM1/HAM2
Histone deacetylases
RPD3/HDA1
General control non-respressible
5
Histone acetyltransferase of the
myst family 1/2
AtHD1/AtHDA19/RPD3A
Histone deacetylase 1
HD2-like
AtHDA6/RPD3B
Histone deacetylase 6
H3K9, H4K5,
H4K8, H4K12,
H4K16
H3K4, H4K5,
H4K12
Histone
ubiquitination
E2
E3
UBC1/UBC2
HUB1/HUB2
Ubiquitin carrier 1/2
Histone monoubiquitination
1/2
H2B
H2B
SDG2/ATXR3
SDG27/ATX1
Set domain group 2
Set domain group 27
H3K4me3
H3K4me2/3
FLD
MEE27/JMJ15
IBM1/JMJ25
Flowering locus D
Maternal effect embryo arrest 27
Increase in bonsai methylation 1
H3K4me
H3K4me1/2/3
H3K9me2
Histone
methyltransferases
SET domain group
Histone
demethylases
LSD1-type
Jumonji (Jmj)
H4K5
Histone PTMs are both associated with short-term responses due to
environmental and internal cues and with long-term responses, in which case they can
be heritable (Sims and Reinberg, 2008). The histone code hypothesis claims that “distinct
histone modifications, on one or more tails, act sequentially or in combination to form a
‘histone code’ that is read by other proteins to bring about distinct downstream events”
(Strahl and Allis, 2000).
1.5.3. Chromatin, epigenetics and control of gene expression during plant development
The development of plants ontogenesis is controlled by a complex interactive set
that integrates stimuli and responds in consequence through different effectors. The
modulation of the responses greatly depends on gene expression, and the accuracy of
16
Chapter I
these responses depends on a tight control mediated by the dynamic regulation of the
chromatin status and the cellular response machinery.
There are 3 ways by which chromatin status can be modified from a structural
point of view according to Pfluger and Wagner (2007; Table 1.3):
1. By the alteration of the tails of the histones (PTMs) which change the
interaction between DNA and nucleosomes generating or blocking
binding sites to the DNA for proteins.
2. ATPases that use the energy from the hydrolysis of the ATP for altering
the position or composition of the nucleosomes.
3. DNA methylation that interferes with binding elements or other proteins
including transcription factors and recruits other proteins
Table 1.3. Types of chromatin modifications that alter gene expression. Modified from Pfluger and Wagner
(2007).
Chromatin
alteration
Histone
modifications
Chromatin
remodelling
DNA
methylation
Type
Acts upon
Mechanism
Ubiquitination
Lysine
Methylation
Lysine, arginine
Distances histones from
DNA
Recruitment of other
chromatin regulators
Acetylation
Lysine
Phosphorylation
Serine, threonine
SWI/SNF
Sliding to new position
Activation and
repression
SWR1
Nucleosome
position and
occupancy
Histone exchange
H2A.Z histone variant
incorporation
Activation
CG and non-CG
Promoter
Repression
CG
Gene (less at 5’
and 3’) ends
Inhibition of transcription
factor binding
May reduce transcription
elongation
Charge neutralization,
recruitment of other
chromatin regulators
Charge neutralization,
recruitment of other
chromatin regulators
Outcome of
transcription
Activation (H2B)
Activation
(H3K4, H3K36)
or repression
(H3K9, H3K27,
H4K20, H4R3)
Activation
Activation
Repression
As seen in Table 1.3 specific marks and effectors are associated with different
outcomes regarding transcription. Moreover, transcription is also associated with the
position at which the PTMs are located within a specific gene (Figure 1.6).
17
General introduction
The effects of the different PTMs regarding physiological processes range from
the control of basal activities within the cell such as the assembly of chromatin
(H3K9me, Soppe et al., 2002) and the control of the cell cycle (H3S10 phosphorylation,
Jenuwein and Allis, 2001) to the responses to external stimuli (dehydration stress, van
Dijk et al., 2010).
Fig.1.6. Distribution of chromatin modifications over genes and their relationship with expression in a
schematic. TSS, transcription start site, 5´ and 3´ UTRs and the coding region are indicated. (Adapted from
Roudier et al., 2009).
In general terms, the acetylation of histones H3 and H4 in promotor regions is
associated with active gene expression (Lusser et al., 2001). These and other PTMs, such
as H3K9ac, H3K9me3 and H3K27ac were found in euchromatic regions in transcription
areas and in a great variety of tissues and organs (Charron et al., 2009). Heterochromatin,
on the contrary, is usually associated with high levels of H3K9me and low levels of
H3K4me (Jasencakova et al., 2003). Our group has developed extensive work regarding
PTMs (histone H4 acetylation) and their association with ontogenetic processes such as
bud dormancy and release in chestnut (Santamaría et al., 2009), the floral bud
development in azalea (Meijón et al., 2009) or needle maturation of pine (Valledor et al.,
2010).
The setting of epigenetic marks is dependent on the action of catalytic enzymes,
and those activities have been found essential in Arabidopsis for zygotic embryogenesis.
The down-regulation of AtHD2A (a HDAC) has as a consequence the abortion of the
18
Chapter I
embryo (Lusser et al., 2001) while its overexpression represses several genes involved in
the development and maturation of the seed (Zhou et al., 2004). Early embryo
development has been also associated with changes in H3K27me (Köhler and
Makarevich, 2006), but maybe the most important role of epigenetics in reproduction
has to do with the control of the imprinting, as embryo development is mainly
controlled by the maternal genome while the paternal genome is silenced through DNA
methylation and H3K27me (reviewed in Vaillant and Paszkowski, 2007). Thus, there is
a delay in the activation of the parental genome due to the necessity of a proper genic
dosage that must be expressed from the maternal genome in the first steps of embryo
development (Lohe and Chaudhury, 2002). Moreover, deficiencies for H3K27me have
been associated with the repression of essential genes for flowering and seed
development, among others (Zhang et al., 2007).
Other HDACs can be differentially regulated by abiotic factors such as cold,
osmotic or saline stresses as well as by ABA, JA or salicylic acid (Chinnusamy and Zhu,
2009), and these same stresses can also induce differential gene expression through the
hypo or hypermethylation of the DNA. In studies on AtHD1 (HDAC) mutants, it was
demonstrated that this enzyme has a role in the development and regulation of gene
expression in response to environmental signals (Tian et al., 2005). These authors found
several reversible and local changes in the acetylation of lysines in H4 associated to
specific loci that do not affect other epigenetic marks.
The relationship between DNA methylation and PTMs has been thoroughly
studied in recent years, finding well-stablished associations. Soppe et al. (2002) found
that H3K9me is essential for heterochromatin compacting and it is known to be directed
by DNA methylation. The same authors described the mechanism by which
heterochromatin is constituted through the cross-talk between MET1, KYP
(KRYPTONITE, a HMT) and DDM1 (a nucleosome remodeler), involving the
deacetylation of H4K16ac prior to the methylation of H3K9 and the DNA. Fischle et al.
(2003) described that the ubiquitination of H2B is a prerequisite for H3K4 and H3K79
methylation and would also be involved in the physical opening of the chromatin
making it accessible for the methyltransferases (Martin and Zhang, 2005). DNA
methylation can be the result of the action of the histone deacetylase 6 (HDA6) that
would act through the siRNA silencing pathway (Loidl, 2004). On the other hand, the
HAT GCN5 interacts with H3S10p, which directs the acetylation of H3K14ac (Lee et al.,
19
General introduction
2010), and also interferes with the siRNA pathway with a general repressive effect in
their production leading to the inhibition of gene silencing (Kim et al., 2009).
These interactions exemplify the great number of PTMs that in last instance
define specific chromatin configurations that affect gene expression during the
developmental pathways in plants. Taking into account that DNA and histones are the
building blocks of the chromatin, and that PTMs and DNA methylation act in concert,
as described above, a top level of control was proposed, known as the epigenetic code,
which would comprehend both kinds of epigenetic modifications (Sims and Reinberg,
2008).
1.6. APPROACH AND OBJECTIVES
Forestry species constitute a source of renewable raw materials, food and
energy. The increasing interest in some species due to their valuable characteristics
make them desirable targets for the sustainable exploitation of forests, which needs of
in depth knowledge of their physiology and reproduction. European chestnut, due to
its twofold interest as timber and nut production tree, has been subject of study for
many years regarding the production of clonal seedlings by asexual propagation
methods although there are important bottlenecks associated with these techniques. The
great importance of chestnuts as fruits justifies the study of the sexual reproduction
since proper zygotic embryogenesis must be accomplished in order to obtain valuable
seeds. The integration of external and internal cues resulting in seed formation needs
the coordination of many actors that play their roles in a complex network. General
signaling and effectors such as hormones, the dynamics and plasticity of epigenetic
mechanisms and the differential gene expression, lead to the imposition of the various
developmental programs carried out during reproduction.
Taking into account the previous work of our research group carried out in other
species regarding physiological events (i.e. phase change, flowering or ageing) and the
insights acquired on epi-genetics associated to these processes and the importance of
proper sexual development, the general objective of this thesis is the characterization of
the zygotic embryogenesis and the death of companion ovules during the development of the
sexual reproduction in C. sativa Miller through the identification and the interaction of
20
Chapter I
epigenetic marks with hormones and differential gene expression. This general objective is
supported by the study of 4 partial goals:
1. The histological characterization of the chestnut sexual embryogenesis
from flowering to mature seed, focusing discrete developmental stages
that encompass several distinct steps such as cross- and autopollination,
embryo development and associated companion ovules death and
embryo maturation.
2. The epigenetic characterization through the quantification of several
epigenetic marks (Global DNA 5-mdC levels, sequence-specific
methylation and PTMs relative quantification) in the developmental
stages previously defined; and the spatial-temporaal distribution by
immunolocalization along development of 5-mdC and H4ac as
representatives of general repressive and activator marks, respectively.
3. The physiological characterization of the distinct developmental stages
defined above by the global quantification of plant growth regulators.
Immunolocalization of ABA and IAA as main actors during
reproduction will be also assayed.
4. The differential gene expression profiles of selected genes related to
epigenetic
regulation
and
embryo
development
throughout
reproduction.
21
General introduction
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28
CHAPTER II
DNA methylation patterns and in vitro
responses during sexual embryogenesis in
C. sativa Miller
Chapter II
2.1. INTRODUCTION
The European chestnut is a monoic, autosterile species with cross-pollination
(McKay, 1942). Due to the twofold economic interest of this species, timber and nuts, it
has been introduced as a crop in South America, Australia, and New Zealand.
Worldwide Nut production is 500,000 T/year (Sauer and Wilhelm, 2005), with the
European chestnut (C. sativa Mill.) and hybrids such as C. crenata  C. sativa being the
major contributors.
The quality of seeds depends on limiting their degree of polyembryony.
Monoembryony is the desirable character in commercial terms, and occurs when only
one ovule from the 16 usually contained in the ovary gives rise to a seed. It involves the
degeneration of companion ovules, which is a common event in angiosperms even
when enough pollen is received (Sutherland, 1987). Obtaining trees with
monoembryonic marron nut production is the main goal of chestnut improvement
programs in which somatic embryogenesis is regarded as an important tool. Enhancing
the occurrence of monoembryonic nuts requires knowledge of the means of sexual
embryogenesis control.
Despite the work carried out on chestnut floral biology and embryo
development in terms of histodifferentiation and the death of ovules after pollination
(Peano et al., 1990; Botta et al., 1995), information about the molecular control of this
process has not been available. The asynchrony of the development of the ovules within
the cluster, and finally the degree of polyembryony, is probably due to differential gene
expression among ovules.
Much of the profile of gene expression in plants is controlled by epigenetic
mechanisms, of which DNA methylation has been widely studied in plants (Valledor et
al., 2007). Methylation of DNA is considered a determining factor in the imposition and
maintenance of the genetic patterns that characterize the ontogenetic developmental
stages of an individual, being essential for correct plant development to such an extent
that variations in DNA methylation profile lead to abnormal development patterns
(Finnegan et al., 1996). Variation in the levels of global DNA methylation throughout
development allows its use as a molecular indicator of processes related to aging,
reinvigoration and maturity, both in angiosperms (Baurens et al., 2004) and
gymnosperms (Fraga et al., 2002a, b). The involvement of methylation mechanisms on
31
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
somatic embryogenesis has been partially demonstrated in Leuterococcus senticosus
(Chakrabarty et al., 2003). Plant production based on somatic embryogenesis constitutes
a valid alternative to clonal mass propagation of superior tree genotypes (Sauer and
Wilhelm, 2005) and to the regeneration of genetically modified specimens of Castanea
(Carraway et al., 1994). Unfortunately, the use of somatic embryogenesis in chestnut is
only effective when induced from ovules and immature embryos (Sauer and Wilhelm,
2005).
In this study we examine the role of epigenetic regulation throughout sexual
embryogenesis and its relationship with its ability to induce somatic embryogenesis.
Using burrs from chestnuts trees with and without cross-pollination, we analyzed
stages from anthesis to mature seed in order to determine changes in global DNA
methylation levels with respect to: (1) whether fertilization induces changes in DNA
methylation, (2) the behaviour of ovules that are not effectively fertilized in terms of
methylation levels and (3) the relationship of DNA methylation levels to somatic
embryogenesis induction from ovules and zygotic embryos.
2.2. MATERIAL AND METHODS
2.2.1. Plant material
Burrs from 15 adult C. sativa trees growing in chestnut stands in Asturias, Spain,
were collected every twelve days from mid-July until mid-October (from anthesis to
seed). This collecting frequency allowed us to establish the developmental pattern of
sexual reproduction for this species in this particular region. Burrs from 6 isolated trees
at different locations were also taken at the same time. Burrs were stored at 4 ºC till their
analysis. Each collection time chosen correlates to a developmental stage.
Ovaries were collected (Fig. 2.1) taking into consideration their location (central
or lateral) within the burrs. Ovules and embryos from developing ovaries were
characterized according to parameters including relative size, shape, length of cluster
of ovules/ovary ratio and colour measured in the first stages; once it was possible to
distinguish the zygotic embryo from the fertilized ovule, size and colour were the
parameters measured.
32
Chapter II
2.2.2. Histological analysis
Tissues extracted from burrs
were fixed in FAA [4:1:1 (v/v) 50 %
ethanol:formalin:glacial acetic acid], for
48 h. Tissues were then dehydrated in 30
min changes of 25 %, 50 %, 70 %, 96 %
ethanol and two changes of 100 %
ethanol. Samples were then introduced
into
toluene
for
an
hour,
then
toluene:paraffin for 45 min at 60ºC and
finally embedded in paraffin overnight
in an oven at 60 ºC. Samples were
sectioned at 10 µm in a microtome and
then, slides were introduced into two 5
min changes of xylol and rehydrated in
a decreasing ethanol gradations (100 %,
96 %, 70 %, 50 %, and 25 %) and distilled
water for 5 min for each step. Samples
then were stained with Toluidine Blue
O (0.1 %
in phosphate buffer –
monosodic and disodic phosphate at
0.05 M- pH 6.8) for 1 min, washed in
distilled water and then in ethanol 100
%. They were visualized under a light
Fig. 2.1. Isolation of clusters, companion and
fertilized ovules from burrs at any stage.
microscope.
2.2.3. Embryogenic competence evaluation
Following macromorphological characterization, the ovaries were sterilized as
previously described (Giovannelli and Giannini, 2000). The cupule was removed under
aseptic conditions and nuts were excised; explants were extracted by dissection. Single
ovules, embryonic axes, and cotyledons were tested for embryogenic competence. One
hundred explants of each kind of tissue taken from each stage were tested.
33
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
Somatic embryogenesis was tested as described by Sauer and Wilhelm (2005),
with modifications. The induction medium used consisted of MS medium (Murashige
and Skoog, 1962) supplemented with 5 µM 2,4-dichlorophenoxyacetic acid and 0.5 µM
6-benzylaminopurine (BA). After one week of culture, the explants were transferred to
fresh induction medium for another two weeks. Finally, the explants were grown for
four weeks in MS basal medium supplemented with 0.88 μM BA. Embryogenic
competence was quantified throughout the previously described developmental stages.
In those phases with the highest embryogenic competence, necrosis and callus
responses were also quantified.
2.2.4. Quantification of DNA global methylation with high performance capillary
electrophoresis (HPCE)
Samples (ovules, embryonic axis and cotyledons) of 40-50 mg fresh weight were
stored at -80ºC until analysis were taken from ovaries. At least 3 biological replicates
per developmental stage and type of sample were done.
After sample homogenization, genomic DNA isolation was performed with a
plant genomic DNA extraction kit (DNeasy Plant Mini, Qiagen) with the addition of 20
l of RNAse A (Qiagen) to assure DNA-free RNA. Once samples had been dried in a
DyNA Vap, the genomic DNA extract was resuspended in 3 l of distilled water.
Enzymatic hydrolysis was conducted as per Fraga et al. (2002c) with some
modifications: DNA samples were denatured at 95 ºC for 4 min to which 0.75 l of 10
mM ZnSO4 and 1.25 l of nuclease P1 (Sigma Aldrich, USA, 200 units ml-1 in 30 mM
C2H3O2Na) were added. Mixtures were incubated overnight at 37 ºC. Additions of Tris
(0.5 M, pH 8.3; 1.25 l) and 0.5 l of alkaline phosphatase [Sigma, USA, 50 units ml-1 in
2.5 M (NH4)2SO4] were made and the solution was incubated for an additional 2 h at 37
ºC. Samples were centrifuged for 20 min at 15,000 g, and the supernatant was stored at
4 ºC.
Samples were analyzed in a capillary electrophoresis system (CIA, Waters
Chromatography, USA) as described by Fraga et al. (2002c) with modifications. An
uncoated fused silica capillary (Waters Chromatography, 600 · 0.075 mm2 I.D. with
effective length 570 mm) and 47,8 mM NaHCO3, 72,8 mM SDS, pH 9.6 buffer were used.
Running conditions were 20 ºC, and an operating voltage of 10 kV was applied for 20
min. Samples were injected hydrostatically at 98 mm upon the cathode for 30 s (Hasbún
34
Chapter II
et al., 2008). Absorbance was measured at 254 nm on the column. Quantification of the
relative methylation of each DNA sample was performed as the percentage of
methyldeoxycytidines (mdC) of total deoxycytidines (dC + mdC).
Statistical analyses were performed using the SigmaStat v2 software package for
Windows. Normal data distribution was verified by means of the Kolmogorov-Smirnov
test. Differences in mean methylation percentages between stages and types of samples
were analyzed with an ANOVA test. Paired means were compared using the HolmSidak test when Normal distribution was found, applying a 5 % of significance for this
test.
2.3. RESULTS
2.3.1. Determination of developmental stages
From anthesis to mature seed formation, seven developmental stages were
defined, based on macromorphological (Fig. 2.2) and histological traits (Fig. 2.3). In
order to characterize the overall process, global methylation levels and embryogenic
responses were also analyzed once the stages were established.
Stage E1. Clusters of ovules generally consist of 16 ± 2 ovules (Fig. 2.2c); they
were homogeneous in size and round (Fig. 2.2d, Fig. 2.3a). The ratio of the length of the
clusters of ovules/length of the ovary was 1:2. Ovules and stigmas were white coloured
and hollow.
Stage E2. The ratio of the length of the clusters of ovules/length of the ovary was
1:3 due to ovarian and ovule growth (Fig. 2.2f). Ovules (Fig. 2.2h) were still
homogeneous in both shape and size (Fig. 2.2g), receptive (Fig. 2.3b) and the embryo
sac was still visible (Fig. 2.3c). They remained white (Fig. 2.2h). Stigmas began to become
necrotic at the apical end (Fig. 2.3d) after pollination.
35
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
Figure 2.2. Determination of developmental stages. Female flower (a), Ovary with style and stigmas (b),
cluster of ovules (c), ovule at pre-pollination stage (d), burr (e), ovary with style and stigmas (f), cluster of
homogeneous ovules (g), ovule at pre-fertilization stage (h), burr (i), ovary with style and stigmas (j), cluster
of heterogeneous ovules from a central ovary (k), cluster of homogeneous ovules from a lateral ovary (l),
fertilized ovule (m), companion ovule at post-fertilization stage (n), ovule at post-fertilization stage (o),
burr (p), ovary with style and stigmas (q), cluster of heterogeneous ovules (r), fertilized ovule (s), ovule
with evident signs of necrosis (t). Bars are 1 cm. in figures (a), (e), (f), (i), (j), (p) and (q); for the remaining
pictures, they are 1 mm. Names in boxes correspond with the names of the tissues analyzed.
Stage E3. Loss of homogeneity among ovules within the same ovary was evident.
As fertilization takes place (Fig. 2.3e) stigmas became necrotic at the apical end and one
ovule becomes dominant, sometimes two or more in the case of polyembryony;
dominant ovules (E3D) increased their size fourfold with respect to the others (E2C and
E2I from isolated trees, Fig. 2.2k). Companion (unfertilized) ovules maintained the same
shape, size, and colour as in stage two (Fig. 2.2n), but they started to collapse (Fig. 2.3f).
36
Chapter II
Asynchrony in development was found depending on the location of the ovary within
the burr (Fig. 2.2k, l). More advanced stages of development were found in central
ovaries compared to lateral ones, which presented ovule clusters that appeared similar
to stage two cases, although their general phenological appearance corresponded to
stage three (Fig. 2.2j). On isolated trees, no difference among ovules (E3I) at this stage
was detected; homogeneity of clusters of ovules remained as in previous stages.
Figure 2.3. Sexual embryogenesis from anthesis to mature seed. Stage E1: cluster of developing ovules (a);
stage E2: mature ovule (b), mature ovule showing embryo sac at micropilar end (c) and stilus filled up with
collapsed tissue after pollination (d); stage E3: ovule showing globular embryo (e), companion ovule at
stage 3 without effective fertilization (f); stage E4: embryonic axis showing the bipolar structure (g) and
companion ovules degenerating (h); stage E6: set up embryonic axis (i); stage E7: mature embryonic axis
showing RAM and cotiledonar ligaments (j). Size bars are 1 mm except for (c) and (e) in which are 100 µm
in length.
Stage E4. The dominant ovule (E4D) continued to grow and became yellow (Fig.
2.2s), showing a clear bipolar embryonic axis (Fig. 2.3g) as observed by microscopic
analysis, while companion ovules became necrotic (Fig. 2.2t, Fig. 2.3h). On isolated trees,
there were no differences and cluster of ovules (E4I) remained stage 2-like.
Stage E5. White embryonic axis (E5A) measured 2.5±0.5 mm in length and
cotyledons were clearly visible (Fig. 2.4a). Ovaries started to turn brown. In the isolated
trees, ovules turned necrotic at this stage, but parthenocarpic phenomena allowed some
ovaries to grow and mature in subsequent stages.
Stage E6. The ovary was occupied by the dominant ovule (Fig. 2.4b). The mean
length of the embryonic axis (E6A) was 3.4±0.4 mm, continued elongating (Fig. 2.3i) and
was yellow, except for the radicle.
37
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
Stage E7. Ovaries achieved the typical colour of mature chestnuts (Fig. 2.4c).
Volumes of the dominant ovule and ovary were slightly increased. The embryonic axis
(E7A) was completely formed (Fig. 2.3j) and yellow, except for the cotyledonary
ligaments, which remained white. Mean length, 4.1±0.2 mm.
Fig. 2.4. Embryo and seed development: development of embryo at stage 5 (a), and 6 (b), mature seed (c).
Size bars are 1 cm in length.
2.3.2. Somatic embryogenesis dynamics
Responses obtained from tissues taken from the seven previous developmental
stages defined are presented in Table 1.
Table 2.1. Percentages of somatic embryogenic responses throughout stages indicated and kind of explant.
Explant
Ovules
Fertilized ovules
Companion ovules
Embryonic axis
Cotyledon tissues
E1
0
E2
0
Developmental stages
E3
E4
E5
67
0
E6
E7
22
15
0
19
71
0
76
20
Ovules from stages E1 and E2, before fertilization, became necrotic when
cultured. An embryogenic response was first induced from stage E3, once fertilization
had taken place. Only dominant ovules were able to develop embryogenic masses while
companion ones did not undergo this response and subsequently necrotized.
The highest embryogenic induction (76 %) was measured for immature axes at
stage E5, which showed heterogeneous and asynchronous embryo differentiation (Fig.
2.5b). Companion ovules that did not undergo necrosis, developed non-embryogenic
calluses (Fig. 2.5a).
38
Chapter II
Fig. 2.5. In vitro callus generated after somatic embryogenesis induction. Non-embryogenic callus with rests
of the ovule indicated by an arrow (a) and embryogenic callus with somatic embryos indicated by arrows
(b).
Ovules taken at the same time from isolated trees in which non-cross pollination
was assured did not respond with embryogenesis inductions in any of the cases
observed.
2.3.3. Methylation dynamics
Specific levels of 5-mdC were quantified during ovule development,
fertilization, throughout sexual embryo development (Fig. 2.6). Ovule development
takes place during stages E1 and E2. During ovule development no significant
differences on DNA methylation levels were found either from cross and non-cross
pollination
trees.
Fig. 2.6. DNA methylation levels from anthesis to mature seed. Arrow indicates fertilization time. Between
points, different letters indicate significant differences between means (p≤0.05). ANOVA test and
subsequent Holm-Sidak test.
39
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
At stage E3, the fertilized ovules became dominant and underwent a 2.5 %
demethylation acquiring a hypomethylated status (9.5 %) when compared with
companion ovules located on the same cluster which maintained a higher methylation
level (11.2 %).
The 5-mdC levels in ovules taking into account position of ovaries (lateral or
central) were quantified in Figure 2.7. No differences between ovules before fertilization
were noted, independent of their location. After fertilization, dominant ovules from
central ovaries are more methylated than the ones from lateral ovaries.
Fig. 2.7. Global methylation levels of corresponding ovules from the first three sexual stages depending on
position of ovaries within the burr. Between spots, different letters indicate significant differences between
means. (p≤0.05). ANOVA test and subsequent Holm-Sidak test.
From stage E4 on embryo differentiation and methylation increased (Fig. 2.6).
Degenerated companion ovules were hypermethylated with respect to developing
tissues and after stage E4 it was not possible to perform the DNA extraction due to the
necrotic status of the tissues. The embryonic axis and cotyledons formed at stage E5
gave values of 15.8 % and 12 % of 5-mdC respectively. In stages E6 and E7 embryonic
axis was hypermethylated reaching 19 % of global methylation in the last stage while
cotyledons maintained 12 % of methylation.
2.4. DISCUSSION
Results showed that sexual embryogenesis in the chestnut occurs with specific
increases and decreases in DNA methylation levels as demonstrated for different
40
Chapter II
developmental processes in animals (Ikegami et al., 2009) and in plants (Santamaría et
al., 2009).
The pollination phase can be located with certainty based on macro and
micromorphological observations and also supported by the histodifferentiation studies
done in C. sativa by Botta et al. (1995) and in C. crenata (Nakamura, 2001).
Prior to fertilization both in cross and non-cross pollinated trees, ovules maintain
size and equal constant methylation levels. In cross-pollinated trees, once fertilization
occurs, at least one ovule per cluster increases in size and experiences demethylation
just before the initiation of zygotic embryo differentiation. Conversely, in non-cross
pollinated trees where fertilization does not occur, the size of ovules and methylation
levels are maintained. This result indicates that demethylation takes place after
fertilization and before entrance into sexual embryo development.
In plants the acquisition of new competences such as reproductive ability seem
to be accompanied with transient demethylation of mature vegetative meristems, as has
been shown for the azalea (Meijón et al., 2008) and validated by the use of flower
inducing photoperiods. Similar results concerning the association of methylation with
vegetative/reproductive competence have also been described for radiata pine
(Valledor et al., 2007; 2009).
In chestnut, changes in genomic methylation levels were also found during the
different stages of bud development (dormancy, burst and growth; Santamaría et al.,
2009) and during phase-change (Hasbún et al., 2007).
In plant and animal models, DNA methylation has become accepted as an
essential epigenetic mechanism for maintenance of cell patterns and one of the most
important alternatives of gene control during the progress of plant development. In pine
it has been shown that DNA methylation plays important roles during ageing and
reinvigoration processes, as well (Fraga et al., 2002a). Micrografting and intensive
pruning, which are among the most effective techniques for the reinvigoration of pines
species, promote progressive demethylation along with the acquisition of in vitro
micropropagation competence Fraga et al., 2002b).
The results presented here indicate that under cross-pollinated conditions only
fertilized ovules experience demethylation, while the companion ovules increase their
41
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
DNA methylation level and this leads to degeneration. Hypermethylation is, with
limited exceptions, associated with gene silencing and is also related to cell death
(Lippman, 2004) and ageing. Publications indicate the existence of a gradual increase of
5-mdC during tree ageing, which is related with inability to respond to any external
stimuli, including in vitro tissue culture (Valledor et al., 2007). Since abortion of proembryos is well-known in this species (Mckay and Crane, 1938), the hypermethylation
of companion ovules appears to be a key event with regard to degeneration, but in noncross pollinated trees the DNA methylation level is maintained and the degeneration
noted because of the absence of effective fertilization.
The effect of fertilization on DNA methylation was validated by studying
asynchrony in flower receptivity. In chestnut, fertilization occurs first in central ovaries
(Bounous et al., 2002). At a time when both central and lateral ovaries have been
fertilized, dominant and companion ovules from central ovaries are hypermethylated
with respect to their homologous from lateral ovaries. Moreover, the dominant ovule,
independent of its location, is hypomethylated as compared to its companion ovules.
Taking into account that companion ovules from lateral ovaries maintain methylation
levels from previous stages, it can be claimed that fertilization may contribute to the
hypomethylation of dominant ovules and the absence of fertilization to the
hypermethylation of companion ovules.
Once the embryo is established, cotyledons and embryonic axis are formed
during seed development. The development of the embryonic axis is an ongoing process
with a strong increase in methylation, reaching the highest level at seed maturity, which
is related to the quiescent status. Similar results were found in Silene latifolia (Zluvova
et al., 2001). Conversely, cotyledons, as a source of energy for seeds, regulate their
metabolic activity and maintain constant methylation levels.
With respect to the induction of somatic embryogenesis, it was shown that only
fertilized ovules that enter the embryogenic program after demethylation are capable of
undergoing somatic embryogenic induction. Before reaching embryo maturity,
embryonic axis and cotyledons also respond to induction, whereas companion ovules
were not capable of inducing somatic embryogenesis in any of the cases studied. The
highest percentage of embryogenic induction was obtained during the initiation of the
sexual embryogenic program, from stage E3 to stage E5, as was also shown by Sauer
42
Chapter II
and Wilhelm (2005) for the same species. Moreover, our results coincide with those
obtained by Şan et al. (2007), who when testing immature cotyledons found similar
embryogenic responses.
The “developmental window” for somatic embryogenesis responses includes
stages from fertilization to embryo maturity, being necessarily a period of transient
decrease of methylation after fertilization for the later development of somatic
embryogenesis responses.
In conclusion, in this study we have demonstrated that fertilization occurs
together with a transient demethylation phase. Further seed development is
accompanied by an increase of DNA methylation. Embryogenic induction of somatic
embryogenesis always takes place after demethylation of dominant ovule.
43
DNA methylation patterns and in vitro responses during sexual embryogenesis in C.
sativa Miller
2.5. BIBLIOGRAPHY
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Bounous G. 2002. Il Castagno: coltura, ambiente ed utilizzazione in Italia e nel
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Carraway DT, Wilde HD, Merkle SA. 1994. Somatic embryogenesis and gene
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Chakrabarty D, Yu KW, Paek KY. 2003. Detection of DNA methylation changes
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Finnegan EJ, Peacock WJ, Dennis ES (1996) Reduced DNA methylation in
Arabidopsis thaliana results in abnormal plant development. Proceedings of the National
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Fraga M, Cañal MJ and Rodríguez R. 2002a. Phase-change related epigenetic and
physiological changes in Pinus radiata D. Don. Planta 215: 672–678.
Fraga M, Cañal MJ and Rodríguez R. 2002b. Genomic DNA methylation–
demethylation during aging and reinvigoration of Pinus radiata. Tree Physiology 22:
813–816.
Fraga M, Uriol E, Diego LB, Berdasco M, Esteller M, Cañal MJ, Rodríguez R. 2002c.
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2’-deoxycytidine in genomic DNA: application to plant, animal and human cancer
tissues. Electrophoresis 23: 1677–1681.
Giovannelli A, Giannini R. 2000. Reinvigoration of mature chestnut (Castanea sativa)
by repeated graftings and micropropagation. Tree Physiology 20: 1243–1248.
Hasbún R, Valledor L, Rodríguez JL, Santamaría E, Ríos D, Sánchez M, Cañal MJ,
Rodríguez R. 2008. HPCE quantification of 5-methyl-20-deoxycytidine in genomic
DNA: methodological optimization for chestnut and other woody species. Plant
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Hasbún R, Valledor L, Santamaría E, Cañal MJ, Rodríguez R. 2007. Dynamics of
DNA methylation in chestnut trees development. Acta Horticulturae 760: 563–566.
Ikegami K, Ohgane J, Tanaka S, Yagi S, Shiota K. 2009. Interplay between DNA
methylation, histone modification and chromatin remodelling in stem cells and during
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Lippman Z, Martienssen R. 2004. The role of RNA interference in heterochromatic
silencing. Nature 431: 364–370.
McKay JW. 1942. Self-sterility in the Chinese chestnut (Castanea mollisima).
Proceedings of the American Society for Horticultural Science 36: 293–298.
McKay JW, Crane HL. 1938. The immediate effect of pollen on the fruit of the
chestnut. Proceedings of the American Society for Horticultural Science 41: 156–160.
Meijón M, Valledor L, Rodríguez JL, Hasbún R, Santamaría E, Feito I, Cañal MJ,
Berdasco M, Fraga MF, Rodríguez R. 2008. Plant epigenetics. In: Epigenetics in biology
and medicine. pp 225–239, London, UK.
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tobacco tissue culture. Physiologia Plantarum 15: 473–497.
Nakamura M. 2001. Pollen tube growth and fertilization in Japanese chestnut
(Castanea crenata Sieb. et Zucc.). Journal of the Japanese Society for Horticultural Science
70: 561–566.
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e di fruttificazione di cultivar europee, orientali ed ibridi del genere Castanea Mill.
Annali della Facolta di Scienze Agrarie della Universita degli Studi di Torino 16: 83–89.
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PE, Cañal MJ, Rodríguez R. 2009. Acetylated H4 histone and genomic DNA methylation
patterns during bud set and bud burst in Castanea sativa. Journal of Plant Physiology
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immature cotyledons of some European Chestnut (Castanea sativa Mill.) cultivars.
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Wilhelm E. 2000. Somatic embryogenesis in oak (Quercus ssp). In vitro Cellular &
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2265–2273.
46
CHAPTER III
Epigenetic scenarios in ovules and
zygotic embryos throughout C. sativa
reproduction
Chapter III
3.1. INTRODUCTION
The persistence of spermatophytes depends on sexual reproduction that involves
the coexistence of a transient gametophytic generation supported by the sporophytic one
(Caudhury and Berger, 2001). The interaction of both structures in the same individual
during the time that reproduction lasts is under the control of the gametophytic haploid
genome and the sporophytic diploid one. In chestnut, cross-pollination is required
(McKay, 1942) and leads to double fertilization which constitutes the starting point of
sexual embryogenesis in, usually, one of the ovules within the ovary and the associated
death of companion ovules giving rise to monoembryonic nuts. Abortion of ovules from
isolated trees under autopollination conditions emphasizes the necessity of proper
pollination for the development of posterior zygotic embryo development (Viejo et al.,
2010). After fertilization, early embryogenesis is mainly controlled by the maternal
genome while the paternal genome is silenced (Vielle-Calzada et al., 2000) and the
posterior embryo development is the result of the coordination between both parental
genomes. In Arabidopsis it has been determined that from the 16,000 genes expressed
from flowering to embryo maturation, 289 (1.8 %) are exclusively expressed during these
developmental stages (Le et al., 2010). Thus, specific genes must act in a coordinated
spatial-temporal way from the very beginning of embryogenesis in order to generate
and develop the tissues that form the embryo. A high degree of plasticity involving
differential gene expression during the imposition and consecution of embryo
development is partially mediated by chromatin reorganization.
Chromatin constitutes the first level of genome packaging in eukaryotes. The
smallest unit of chromatin is the nucleosome, consisting on 147 bp of DNA wrapped
around an octamer of core histones containing two copies of each type (H2A, H2B, H3
and H4). Therefore, chromatin is a dynamic heteropolymer and its ability for compacting
and relaxing is not only necessary for the cell-cycle but also represents part of the
extremely imbricate mechanism for controlling gene expression in eukaryotes
(Margueron et al., 2005). Its actions rely on two possible, opposite configurations,
considered as “open” or “close”, known as euchromatin and heterochromatin that
correlate with activated or repressed transcriptional situations, respectively. Besides,
chromatin has been shown to be a barrier for transcription: when histones are
eliminated, transcription is activated (Lauria and Rossi, 2011) making it more accessible
49
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
for transcription. The variety of players involved in controlling gene expression includes
the epigenetic mechanisms, important for plant gene regulation and development.
Epigenetically controlled gene expression depends on two factors: on the one
hand, the modifications of DNA by cytosine methylation and the tails of the histones by
several kind of chemical modifications (e. g., methylation, acetylation, ubiquitination,
etc…) that do not alter the nucleotide sequence; on the other, the interaction of these
epigenetic marks with the transcriptional machinery and modifiers of the chromatin
giving as a result the modulation of the expression of the involved genes through a
homeostatic balance between writers, readers and erasers of the epigenetic marks (Berr
et al., 2011).
Cytosine methylation is the most studied epigenetic mark of the DNA although
it has been recently described that adenines can also be methylated, which could be
involved in both the control of DNA replication and gene expression (Vanyushin and
Ashapkin, 2011). Cytosine methylation effects on transcription are likely to depend on
differential distribution in the genes: while methylated promotor regions are associated
with inhibition of transcription, methylation in the body of the gene is usually associated
with active transcription depending on the level of methylation (Zilberman et al., 2007).
In general terms, the importance of cytosine methylation during embryogenesis has been
demonstrated in studies conducted with mutants for the methyltransferase MET1 and
the double mutant MET1/CMT3 (Xiao et al., 2006) where alterations in the distribution
of the methylated cytosine patterns led to developmental abnormalities as also noticed
in the parental imprinting after fertilization regarding endosperm development
(Finnegan et al., 2000a). Our previous study on global DNA methylation (Chapter 2;
Viejo et al., 2010) showed association between the global methylation status in a given
developmental stage during embryogenesis with the in vitro morphogenic capabilities
and the determination of the fate of the tissues. Moreover, the DNA methylation status
has also been studied in several species since Reyna-López and coworkers developed
the Methylation Sensitive Amplification Polymorphism (MSAP) analysis for fungi in
1997. This technique has become feasible for evaluating epigenetic variations in a variety
of systems (Hao et al., 2004; Fang and Chao, 2008; Herrera and Bazaga, 2013).
Histones PTMs constitute the other main epigenetic mechanism that is
coordinated along with DNA methylation and chromatin remodeling effectors. Histones
50
Chapter III
divide into 2 domains: a globular one that participates in the interaction with the rest of
histones from the octamer and the tails that protrude from the nucleosome (Loidl, 2004)
where the epigenetic marks mainly are. Histones H3 and H4 have a N-terminus with
residues that are subject to modification, while histones H2A and H2B are known to
have both N- and C-termini with modification sites. PTMs found in plants comprehend
methylation and acetylation of lysines and arginines, phosphorylation of serines and
threonines, and ubiquitination and sumoylation of lysines (Peterson and Laniel, 2004).
From the variety of PTMs that can be found in the histones, H4ac, H3ac and H3K4me3
are associated with euchromatin and gene transcription activation while H3K9me3 and
H3K27me3 are usually found in heterochromatin and correlate with repression of
transcription (reviewed in Pfluger and Wagner, 2007).
DNA methylation and histones PTMs have been described as interdependent
(Mathieu et al., 2005) in that DNA methylation affects the epigenetic marks contained
within the histone tails and vice versa as described for H3me and DNA methylation
(Soppe et al., 2002). DNA methylation and PTMs also act in concert on critical steps
during development as seen for DNA methylation and H3K27me for controlling
genomic imprinting (Vaillant and Paszkowski, 2007). The same way, DNA methylation
and H4ac are likely to play a role in the transition from vegetative to floral bud in Azalea
(Meijón et al., 2010). Similarly, a correlation was described between acetylated and
methylated lysines of H3 and gene expression during Arabidopsis deetiolation (Charron
et al., 2009) and spatial-temporal changes were noticed under the application of different
stresses (drought, Kim et al., 2008 or salt, Chen et al., 2010).
In spite of all this accumulated knowledge, the control of sexual reproduction by
epigenetic marks from a combined spatial-temporal point of view remains unstudied in
forestall plants. The aim of this study is to provide insight into the role of epigenetic
marks in the reproduction of chestnut through the characterization by relative
quantification of some of the repressive and activation epigenetic marks during seed
development, their distribution within the tissues and the determination of DNA
methylation
sequence-specific
changes
by
MSAP
analysis
throughout
the
developmental stages from flowering until embryo maturation.
Moreover, given the importance of the PTMs dynamics during development, in
this work we have developed an ELISA relative quantification procedure based on
51
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
previous studies in animal cell cultures (McKrittik et al., 2004; Dai et al., 2007; Dai et al.,
2011; Dai et al., 2013) that allows the relative quantification of PTMs in plant tissues.
3.2. MATERIAL AND METHODS
3.2.1. Plant material
Burrs were collected in 2011 from mid-July to mid-November from several openpollination trees in chestnut stands in Carreño (Asturias, Spain) basing on the previously
defined developmental stages of the ovules (E1 to E4) and growing embryos (E5 to E7)
(Chapter 2; Viejo et al., 2010) but not taking into account the central or lateral position of
the ovaries within the inflorescence. Burrs from isolated trees (autopollinated) were
collected at the same collecting dates for stages E2 and E3 and used as controls for proper
cross-pollination/fertilization events.
Burrs were immediately dissected extracting ovaries and obtaining ovules from
stages 1 to 4 and embryonic axes from stages 5 to 7.
3.2.2. MSAP
In order to obtain a general view of the DNA methylation, developmental stages
E1, E2, E2I, E3D, E3C, E3I, E5A and E7A were used for the MSAPs analysis.
Four samples per developmental stage and 100 mg per sample were
homogenized in a mortar with liquid nitrogen. Genomic DNA isolation was performed
with the plant genomic DNA extraction kit DNeasy Plant Mini (Qiagen) following
manufacturer´s instructions. All the DNA extractions were diluted to 5 ng/µl and stored
at -20 ˚C until use. The protocol for MSAPs analysis was developed basing on the one
described by Hasbún et al. (2011) for AFLPs.
An aliquot of 5.5 µl of diluted genomic DNA per sample were digested with 5 U
of EcoRI and 4U of HpaII or MspI (New England Biolabs) for 2 h at 37 ˚C in separated
reactions. Additionally, the ligation was performed in the same reactions for 1 h at 16 ˚C
using 1x T4 ligase DNA (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 1 mM
ATP), 50 µM NaCl and 50 ng of bovine serum albumin (BSA) and adding adaptors for
EcoRI, HpaII and MspI (2 pmol/µl, Table 1). After the incubation, the enzymes were
inactivated at 65 ˚C for 20 min and the products were diluted 1:5 in LowTE buffer [1mM
52
Chapter III
Tris-HCl, 0.1 mM Ethylenediaminetetraacetic acid (EDTA) pH 8] and stored at -20 ˚C
until use.
The pre-amplification was carried out in a total volume of 25 µl containing 5 µl
of restricted-ligated DNA, 0.2 µM EcoRI and HpaII/MspI pre-selective primers (Table
1), 0.5U Taq DNA polymerase (Invitrogen), 1x Taq DNA polymerase buffer, 0.1 mM
dNTPs and 1.5 mM MgCl2. PCR conditions were 72 ˚C for 4 min, 94 ˚C for 30 s and 28
cycles at 94 ˚C for 20 s, 56 ˚C for 1 min, 72 ˚C for 2 min and a final extension cycle at 72
˚C for 10 min. After pre-amplification, 15 µl of the PCR products were diluted 1:10 in
LowTE buffer.
The selective amplification was performed in a volume of 12.5 µl. The three EcoRI
+ HpaII/MspI primer combinations used were: E-ACA + HM-ACC, E-ACA + HM-CCG
and E-ACA + HM- TAGC. Each reaction contained 2.5 µl of the diluted pre-amplification
PCR products, 0.1 µM EcoRI and HpaII/MspI selective fluorescent primers (Table 1), 0.5
U Taq DNA polymerase (Invitrogen), 1x Taq polymerase buffer, 0.1 mM dNTPs and 1.5
mM MgCl2. PCR conditions were 94 ˚C for 2 min and 30 cycles at 94 ˚C for 20 s, 66 ˚C for
1 min, 72 ˚C for 2 min and a final extension cycle at 60 ˚C for 30 min.
Table 3.1. Adaptors and primers used in the MSAP analysis.
Name
EcoRI
Adaptors
HpaII/MspI
Preselective primer
Selective primer
EcoRI+0
HpaII/MspI+0
E-ACA + HM-ACC
E-ACA + HM-CCG
E-ACA + HM-TAGC
Sequence (5’- 3’)
CTCGTAGACTGCGTACC
AATTGGTACGCAGTCTAC
GATCATGAGTCCTGCT
CGAGCAGGACTCATGA
GACTGCGTACCAATTC
ATCATGAGTCCTGCTCGG
ATCATGAGTCCTGCTCGGACC
ATCATGAGTCCTGCTCGGCCG
ATCATGAGTCCTGCTCGGTAGC
Selective amplifications were separated by capilar electrophoresis in an
automated sequencer ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems) and
data obtained were analyzed with GeneMapper 5 software (Applied Biosystems). In
order to analyze the methylation status in the samples, MSAP polymorphic bands were
scored as 1 (present) or 0 (absent) and used to generate a binary matrix as proposed by
González et al. (2007; Fig. 3.1).
53
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
Fig. 3.1. Methylation and demethylation events carried out by HpaII/MspI endonuclease digestion in the
sequence CCGG and the band pattern obtained codified as 0 and 1 values. Taken from González et al. (2007).
HpaII and MspI are isoschizomers that recognize and digest CCGG sites but
present differential sensitivity to the methylation state (Fig. 3.2): while HpaII is
insensitive to full methylation of cytosines in both DNA strands, it cleavages the
hemimethylated target if only one strand is methylated and MspI only digests targets
with internal methylated cytosines. Thus, comparing amplification fragments from
EcoRI + HpaII and EcoRI + MspI allows to detect changes in the methylation status
between samples.
Fig. 3.2. Cleavage specificity of the isoschizomers HpaII and MspI for the sequence CCGG. Taken from
Pérez-Figeroa (2013).
A second analysis was performed using R Statistical Environment (R core team,
2012) core functions plus the package msap (Pérez-Figueroa, 2013) with the scoring
54
Chapter III
information obtained from González et al. (2007). The generated binary matrix was used
in subsequent steps following msap package instructions for obtaining the denominated
“non methylated loci”, (NML), and “methylation susceptible loci”, (MSL). Finally, a
principal coordinate analysis (PCoA) was performed using the package ade4 (Dray and
Dufour, 2007).
3.2.3. Relative quantification of PTMs by ELISA
Developmental stages used for ELISA were E1, E2, E2I, E3D, E3C, E3I, E4D, E4C,
E5A, E6A and E7A.
Nuclei purification was performed based on Haring et al. protocol (2007) with
modifications. 300 to 500 mg of material was grinded in liquid nitrogen to a fine powder
and used immediately. The powder was transferred to a 12 ml polypropylene tube and
8 ml of ice-cold freshly prepared extraction buffer A (0.44 M sucrose, 10 mM Tris-HCl
pH 8.0, 5 mM Beta-Mercaptoethanol and 1mM PMSF) were slowly added and gently
mixed at 4 ˚C for 30 min in an overhead shaker. The resulting solution was filtered
through 3 layers of Miracloth (20-50 µm pore size) into a new tube and the filtrate was
centrifuged at 3,000 g for 15 min at 4 ˚C. Supernatant was carefully removed and the
pellet was smoothly resuspended in 5 ml of ice-cold freshly prepared extraction buffer
B (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2, 1 % Triton X-100, 5 mM BetaMercaptoethanol and 0.15 M PMSF). After 10 min of gently mixing at 4 ˚C in an overhead
shaker, the solution was centrifuged at 3,000 g for 10 min at 4 ˚C. The supernatant was
removed, resuspended in 5 ml of ice-cold freshly prepared extraction buffer B, mixed at
4 ˚C in an overhead shaker and centrifuged at 3,000 g for 10 min at 4 ˚C two times more.
After removing the supernatant, the pellet was smoothly resuspended in 8 ml of ice-cold
freshly prepared extraction buffer C (0.25 M sucrose, 10 mM Tris-HCl pH 8.0, 10 mM
MgCl2, 5 mM beta-mercaptoethanol and 0.15 M PMSF). The resulting solution was
centrifuged at 3,000 g for 10 min at 4 ˚C. After removing the supernatant the pellet
containing the nuclei was stored at -80 ˚C overnight or the acid isolation of nuclear
proteins was continued.
Acid extraction of nuclear proteins was performed according to Shechter et al.
(2007) with modifications. The pellet obtained in the nuclei purification was
resuspended in 400 µl of H2SO4 0.4 N and transferred to a 2 ml microcentrifuge tube
55
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
placed in ice. The resulting solution was vigorously vortexed and then mixed at high
speed in an overhead shaker at 4 ˚C for 20 min after which the solution was sonicated
with 2 pulses of 15 s at 100 % amplitude in a 200 W ultrasonic processor. Tubes were
kept in ice water and cooled at least for 30 s between pulses and then centrifuged at
16,000 g for 10 min at 4 ˚C. Supernatant was transferred to a new tube and 140 µl of a
saturated solution of trichloroacetic acid was added. Tubes were incubated on ice for 30
min and centrifuged at 16,000 g for 15 min at 4 ˚C. Supernatant was removed and the
pellet was washed with cold acetone (-20 ˚C) and vortexed until the pellet was released
from the tube. After centrifuging at 16,000 g for 10 min at 4 ˚C, the supernatant was
removed and the pellet washed with cold acetone and centrifuged as described above
for 2 times more. After the last supernatant removal, tubes were left open at room
temperature for drying. The dry pellet was resuspended in 50 µl of urea 8 M at room
temperature and stored at -20 ˚C until use.
Quantification of the extracted acid proteins was performed following Bradford´s
method.
3.2.3.1. Antibodies specificity
Previously to the performance of the ELISA, the specificity of the antibodies was
assessed by Western blot following the protocol described by Valledor et al. (2010).
Specificity was checked by observing a single band after developing the Western blot.
Antibodies for histone H4 (Antibodies Online, ref. ABIN1819578), H4ac (Upstate, ref. 06866), histone H3 (Antibodies online, ref. ABIN619545), histone H3ac (Millipore, ref. 06599), histone H3K4me3 (Millipore, ref. 04-745), histone H3K9me3 (Upstate, ref. 07-523)
and histone H3K27me3 (Millipore, ref. 07-449) were used.
3.2.3.2. ELISA protocol
Protein extracts were diluted to 0.1 µg/µl and 0.8 µg of protein was used per
reaction to remain within the linear response range of the assay. Four biological
replicates and 2 technical replicates were used per developmental stage assayed. Protein
adsorption in 96-well plates consisted on the addition of 8 µL of diluted protein to each
well plate plus 42 µL of 1x phosphate buffered saline (PBS). Three negative controls
consisting of 50 µL of 1x PBS were also considered. After sealing the plate with
Parafilm®, it was incubated overnight at 4 ˚C. The plate was briskly shaken in order to
remove the adsorption mixture and it was washed 4 times with 200 µL/well of 0.5 %
56
Chapter III
Tween® 20 in PBS at room temperature in a total time of 10 min, removing the washing
solution between washes. Wells were blocked by adding 200 uL of 5 % BSA, 0.05 %
Tween-20 in PBS and incubating 1h at room temperature. After removing the blocking
solution as previously described, 50 µL/well of the primary antibody diluted 1:1000 in
5 % BSA, 0.05 % Tween® 20 in PBS was added and incubated for 1h at room temperature.
Plate was washed 4 times as indicated above and 50 µL/well of secondary antibody
(anti-rabbit IgG coupled to Alkaline Phosphatase) was added, diluted 1:5000 in 5 % BSA,
0.05 % Tween-20 in PBS. After 1h incubation at room temperature, the plate was
developed by adding 200 µL/well of fresh p-nitrophenylphosphate solution and
incubated 45 min at 37 ˚C. Absorbances were measured at 405 nm.
3.2.3.3. Statistical analysis
Four biological and 2 analytical replicates from every developmental stage were
used. Raw absorbance values were checked to be in the linear response range of the
assays. PTMs values were normalized for nucleosome loading in identical sampledistributed ELISA plates with antibodies for H3 and H4 corresponding with the PTMs
assayed. Values were represented as arbitrary units and expressed relative to the
developmental stage E1.
Data was analyzed using the statistical program SPSS (SPSS Inc., USA) for
normality and variance homogeneity (Kolmogorov-Smirnov and Levene tests,
respectively). Differences between stages were contrasted by one-way analysis of
variance (ANOVA) and post hoc Duncan test. The level of significance was 0.05.
3.2.4. Immunohistochemical detection of 5-mdC and H4ac
Tissues from developmental stages E1 to E7 were used except for samples from
developmental stages E3I and E4C due to their advanced degradation status that did not
allowed proper sectioning. At least three samples per developmental stage were studied.
Immunohistochemical detection was performed according to Pérez et al. (2015) with
modifications: samples were fixed in 4 % paraformaldehyde (w/v) overnight at 4 ˚C
under vacuum. Samples were then sectioned at 50 µm thickness using a cryomicrotome
Leica CM1510-S (Leica Instruments) and stored at -20˚C until used. Sections were
dehydrated in an ascending series of ethanol (25 %, 50 %, 75 %, and 100 %; 5 min each)
and subsequently rehydrated (100 %, 75 %, 50 %, 25 %; 5 min each). Slides were
permeabilized by incubating 45 min in 2 % cellulase in PBS (w/v) followed by 30 min in
57
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
0.1 % Tween 20 in PBS (v/v) and subsequent DNA denaturalization in 2 N HCl for 30
min. After the blocking reaction [10 % BSA in PBS (w/v) for 10 min] sections were
incubated with mouse antibody anti-5-mdC (Millipore, ref. MABE146) or rabbit
antibody anti-H4ac (Upstate, ref. 06-866) which bonds to every lysine residue acetylated
in the histone tail. Antibodies were diluted 1/50 in 1 % BSA in PBS (w/v) and applied
to the slides for 1 h. Alexa Fluor 488-labelled anti-mouse polyclonal antibody
(Invitrogen, ref. A-11001) was used as secondary antibody against anti-5-mdC antibody
and Alexa Fluor 488-labelled anti-rabbit polyclonal antibody (Molecular Probes, ref. A11008) for the H4ac detection. Secondary antibodies were diluted 1/25 and applied for
1 h in dark. In order to stain the nuclei, slides were counterstained with a DAPI (4`-6diamidino-2-phenylindole, Fluka) solution [1 μg/ml, 1 % Triton in PBS (v/v)] for 15 min
in darkness and three rinses with distillate water (5 min each) were performed. Sections
were fixed to the slides with a Mowiol® solution (Valnes and Brandtzaeg, 1985) without
polyvinyl alcohol or 1,4-Diazabicyclo[2.2.2]octane (DABCO). Negative controls showed
no fluorescence signal (data not shown). Fluorescence was visualized using a confocal
microscope (Leica TCS-SP2-AOBS) connected to a workstation and the images were
processed with Leica Software (LCS 2.5).
3.3. RESULTS
3.3.1. MSAP analysis
MSAP summary results (Tables 3.2 and 3.3) showed that more than half of the
fragments generated by this technique corresponded with monomorphic loci in the
transitions between developmental stages studied.
From the changing loci that were analyzed, differences between the percentages
of methylation and demethylation events when comparing developmental stages varied
from 0.2 to 4.6 % which make them not conclusive at this level. Nevertheless, differences
in the percentages of changes manifested that cross-pollination (Table 3.2, E1-E2) and
fertilization
(Table
3.2,
E2-E3D)
concurred
with
a
mean
of
12.4
%
of
methylation/demethylation events and no specific loci were found with the
combination of primers analyzed. The posterior embryo growth (Table 3.2, E3D-E5A)
and maturation (Table 2, E5A-E7A) stages, on the contrary, presented a mean of 41.0 %
of changes and a percentage of 21.6 and 22.8 % of specific loci, respectively. Besides,
58
Chapter III
transition from E3D to E5A was dominated by specific methylation events (39.3 %) while
maturation (E5A to E7A) showed predominance of specific demethylation loci (84.5 %).
Percentages of epigenetic changes in autopollination (Table 3.3, E2-E2I) showed
14.5 % for methylation and 16.3 % for demethylation which appeared slightly higher
than their homologues for cross-pollination (Table 3.2, E1-E2; 12.9 and 12.7 %,
respectively) and presented a low rate of specific loci for methylation (0.91 %) and
demethylation (5.1 %) contrasting with cross-pollination. Moreover, both kinds of
pollination shared methylation and demethylation loci in 50 % and 30 % of the loci
obtained (data not shown). The transition of ovules from pre-abortive stages (E2I and
E2; Table 3.3) to abortive ovules (E3I, E3C; Table 3.3) took place with a range of the
percentages of changes from 9.1 to 16.5 %. Of these methylation and demethylation
events, less than 1/3 of both epigenetic changes took place on the same loci (data not
shown) for both abortive pathways while the rest were shared with other developmental
transitions. Exclusive loci from E2I to E3I regarding methylation and demethylation
were 22.0 and 13.2 %, respectively. Specific loci for E2 to E3C transition presented lower
values: 5.4 % for exclusive methylations and 8.9 % for demethylations.
59
Table. 3.2. Summary of the MSL obtained by the informatics analysis performed in R for the embryogenic development. Developmental transitions between stages with the
different events characterized are shown. Percentages of methylation and demethylation events are given in reference to the global number of changing loci. Changing loci
percentage is in reference to the total loci. The specific loci events for methylation and demethylation are given in reference to the total of changing specific loci.
Specific
loci
Global loci
E1-E2
60
ACAACC
ACACCG
ACATGCA
Methylation
18
10
24
Demethylation
12
22
17
Changing loci
152
127
122
HpaII specific
24
24
18
MspI specific
25
9
Monomorfic
176
Others
E2-E3D
ACAACC
ACACCG
ACATGCA
11
13
17
23
20
11
160
121
115
66
18
13
25
16
50
29
10
207
188
571
168
73
62
47
182
Total loci
328
334
310
Methylation
0
0
Demethylation
0
Changing loci
0
E3D-E5A
ACAACC
ACACCG
ACATGCA
71
45
39
44
36
58
137
102
126
56
10
7
15
13
52
11
13
213
195
576
191
79
65
49
193
972
328
334
310
0
0
0
0
0
0
0
0
0
0
0
0
Total
52
(12.9 %)
51
(12.7 %)
401
(41.2 %)
E5A-E7A
ACAACC
ACACCG
ACATGCA
40
48
58
57
47
37
105
114
123
32
2
6
7
15
10
34
5
13
13
31
232
184
607
223
220
187
630
1
1
4
6
1
0
8
9
972
328
334
310
972
328
334
310
972
0
0
30
14
17
7
0
11
0
0
0
3
3
12
19
22
19
0
0
0
33
17
29
26
22
30
Total
41
(10.3 %)
54
(13.6 %)
396
(37.5 %)
Total
155
(42.4 %)
138
(37.8 %)
365
(37.5 %)
61
(39.5 %)
18
(13.0 %)
79
(21.6 %)
Total
146
(42 %)
141
(41.2 %)
342
(35.1 %)
18
(28.57 %)
60
(84.5 %)
78
(22.8 %)
Table. 3.3. Summary of the MSL obtained by the informatics analysis performed in R for the abortive pathways. Developmental transitions between stages with the different
events characterized are shown. Percentages of methylation and demethylation events are given in reference to the global number of changing loci. Changing loci percentage is
in reference to the total loci. The specific loci events for methylation and demethylation are given in reference to the total of changing specific loci.
Specific
loci
Global loci
E1-E2I
ACAACC
ACACCG
ACATGCA
Methylation
22
13
28
Demethylation
21
32
18
Changing loci
160
143
131
HpaII specific
18
10
15
MspI specific
25
15
Monomorfic
168
Others
E2I-E3I
E2-E3C
ACAACC
ACACCG
ACATGCA
Total
ACAACC
ACACCG
ACATGCA
19
44
14
77 (16.5%)
9
19
9
35
7
26
68 (14.6 %)
18
13
25
167
174
124
465
(47.8 %)
163
124
117
43
16
37
9
62
30
13
14
57
18
58
16
12
17
45
18
14
19
51
191
179
538
161
160
186
507
165
210
193
568
74
73
52
199
81
74
58
213
88
65
50
203
Total loci
328
334
310
972
328
334
310
972
328
334
310
972
Methylation
1
2
1
4
(5.1 %)
0
18
1
2
0
0
Demethylation
0
0
0
0
3
3
3
0
1
4
Changing loci
1
2
1
4
(0.9 %)
3
21
4
2
1
4
Total
63
(14.5 %)
71
(16.3 %)
434
(44.6 %)
19
(22.0 %)
9
(13.2 %)
28
(6.0 %)
Total
37
(9.1 %)
56
(13.8 %)
404
(41.5 %)
2
(5.4 %)
5
(8.9 %)
7
(1.7 %)
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
Principal Component Analysis (PCoA) was performed for the MSL in the
genome using the 8 development stages and the 3 combinations of primers (Fig. 3.3).
Fig. 3.3. PCoA analysis for the selected developmental stages analyzed by MSAP. Coordinate 1 comprises
17.8 % of the variability and coordinate 2, 15.1 %. In green, developmental stages from the normal zygotic
embryo development and in red, developmental stages of the abortive pathway. Ellipses show replicates
dispersion within developmental stages.
The two first coordinates explained 32.9 % of the total variance. Overall,
fertilization (E3D) had none or slight negative correlation with both coordinates, staying
in the center of the plot while the rest of the developmental stages showed more extreme
displacement. Coordinate 1 correlated inversely with the progression of development as
earlier stages grouped to the top right of the plot. Post-fertilization stages, on the
contrary, correlated negatively and stage E7 clearly separated in the first component
from the rest of the stages. It is worth highlighting the proximity between both kinds of
pollination on this coordinate while coordinate 2 appeared to explain pollination type.
3.3.2. PTMs quantification
The verification of the antibodies specificity (Fig. 3.4) showed a single band for
all the antibodies assayed in the Western blot.
62
Chapter III
Fig. 3.4. Bands after Western blot with the different antibodies. Marker (a), H3 (b), H3ac (c), H3K4me3 (d),
H3K9me3 (e), H3K27me3 (f), marker (g), H4 (h) and H4ac (i).
All the epigenetic marks quantified (Table 3.4) showed significant differences
through development. The strongest variation among developmental stages was found
for H4ac [17.41 arbitrary units (AU)] while the smallest range was found in H3K27me3
(8.71 AU).
Table 3.4. Relative quantification of PTMs selected for histones H4 and H3 through development. Means in
AU and standard error (SE) are shown. Differences between stages were contrasted by one-way analysis of
variance (ANOVA) and post hoc Duncan test (p≤0.05).
Developmental stage
Mark
H4ac
Statistics
E1
E2
E2I
E3D
E3I
E3C
E4D
E4C
E5A
E6A
E7A
Mean
3.58
de
4.51
cde
3.96
cde
3.33
e
3.74
ed
0.56
f
5.08
cd
5.37
c
14.48
a
8.45
b
5.33
c
SE
0.38
0.18
0.23
0.25
0.68
0.21
0.29
0.42
0.88
0.59
0.65
15.22
a
11.42
b
7.65
c
5.03
e
5.29
de
7.76
c
4.33
e
2.35
f
12.11
b
12.41
b
6.87
cd
0.35
0.33
0.14
0.23
0.24
1.19
0.08
0.13
1.21
0.60
0.73
23.39
a
18.44
b
11.47
de
7.64
f
10.18
e
15.80
c
5.98
f
4.15
g
12.66
d
16.16
c
11.47
de
SE
0.75
0.69
0.54
0.19
0.49
0.57
0.20
0.37
0.34
0.67
1.10
Mean
5.93
b
5.69
bc
3.75
de
3.53
e
2.87
e
13.86
a
2.51
e
4.03
cde
3.03
e
6.03
b
5.35
bcd
SE
0.19
0.33
0.24
0.36
0.22
2.60
0.07
0.25
0.10
0.20
0.74
Mean
9.57
b
6.76
c
5.12
d
3.55
ef
5.11
d
11.97
a
3.26
f
4.79
de
5.39
d
7.70
c
6.83
c
SE
0.74
0.29
0.14
0.10
0.43
0.12
0.17
0.35
0.38
0.32
0.93
Mean
H3ac
SE
H3K4me3
H3K9me3
H3K27me3
Mean
Dynamics of the epigenetic marks showed a similar pattern (Fig. 3.5) during the
normal development from flowering (E1) to mature embryo (E7A). All the marks of
63
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
histone H3 (Fig. 3.5a, b, c, d) showed significant decreases in their relative amount after
both kinds of pollination while H4ac (Fig. 3.5e) did significantly change. Moreover,
autopollination was associated with stronger decreases than cross-pollination in stage
E2 for H3ac, H3K4me3 and H3K27me3, while only a decrease in H3K9me3 was
observed after auto-pollination without a decrease after cross-pollination (Fig. 3.4, Table
3.4). Ovules from cross-pollination continued the mentioned decrease towards
fertilization (E3D) for all four H3 marks while H4ac remained unchanged in both E3D
and E3I. On the other hand, autopollination was associated with stable values at E3I for
all the histone modifications except for H3ac that presented a significant decrease (Fig.
3.5, Table 3.4). The starting point of ovule abortion in cross-pollinated trees (E3C ovules)
presented important differences between marks when compared with stage E2:
H3K9me3 reached 2.5 times the level found in E2 while H3K27me3 nearly doubled. On
a contrasting fashion, significant reductions in the relative amount of PTMs were found
for H3K4me3 and H3ac, while the decrease for H4ac was more than 90 % for H4ac in
E3C ovules (Fig. 3.5, Table 3.4). The progression of the abortion pathway at stage E4
(E4C ovules) concurred with a reversion in the relative amounts of PTMs for H3K9me3,
H3K27me3 and H4ac to similar values found at stage E2 while in H3ac and H3K4me3
the decreases continued reaching the lowest levels in development. After the initiation
of embryogenesis in E3D ovules, same pattern was found during embryo development
for all the H3 marks quantified. Thus, ovules at E3D and E4D presented transient low
values followed by significant increments at E5A once the embryo was clearly
differentiated into axis and cotyledons. Final developmental stages concurred with a
peak at E6A and decreasing (H3ac, H3K4me3) or maintained (H3K9me3, H3K27me3)
values at E7A for the H3 epigenetic marks studied. H4ac displayed a different pattern
than the H3 marks. Although H4ac did not show a significant transient decrease in E3D
ovules, it increased in E4D and sharply peaked in E5A, returning to values similar to
E4D in the mature embryo at E7A.
64
Chapter III
Fig. 3.5. Relative abundance of histone H3 PTMs and H4ac and dynamics throughout development. Data
are normalized for E1 in each panel.
65
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
3.3.3. Immunodetection
Immunolocalization of the epigenetic marks 5-mdC and H4ac displayed tissuespecific distribution throughout the developmental stages analyzed. From ovaries at
anthesis (E1) to mature embryo (E7), there is a divergence in the pathways taken by the
ovules depending on the type of pollination. After cross-pollination (E2), ovules enter
the embryogenic pathway (E3D) that will end up with the mature embryo at E7 and
there is an associated abortion of companion ovules (E3C). Ovules under
autopollination conditions (E2I) also died in posterior developmental stages.
Pollination
In the ovules at anthesis (E1), a methylation signal was only found in the walls
of the ovary (Fig. 3.6b, c) while histone H4ac was ubiquitous in both ovules and ovary
tissues (Fig. 3.7b, c). The histological study showed that pollination was associated with
an increment in the ovule size upon both types of pollination (Fig. 3.6d, g; Fig. 3.7d, g).
Immunolocalization after cross-pollination showed contrasting patterns for 5-mdC and
H4ac: methylation signal remained in the ovarian tissues and appeared in the ovule
(Fig. 3.6e, f) while H4ac signal was strongly reduced in the embryo sac (Fig. 3.7e, f).
Autopollination (E2I) showed high intensity for H4ac in the ovules’ outer integument
(Fig. 3.7h, i) and homogeneous signal in the inner integuments and embryo sac, which
constituted an intermediate status between the ubiquitous signal in E1 and the loss of
signal in the central part of the ovule in E2. The methylation signal in ovules after
autopollination (E2I; Fig. 3.6h, i) displayed higher signal intensities in the inner
integuments and embryo sac, more present in the basal part of the ovule than in the
apical part of compared with ovules after cross-pollination.
Fertilization
Early embryogenesis after fertilization at stage E3D (Fig. 3.8a-f; Fig. 3.9a-f)
showed dissimilar patterns for methylation and H4ac. Methylation signal was hardly
found in the growing embryo (Fig. 3.8b, c, e, f), contrasting with the clear signal found
in the inner integuments and the rests of the endosperm. H4ac localization, on the
contrary, displayed high intensity in the growing embryo (Fig. 3.9b, c, e, f) and also in
the rest of tissues of the ovule with similar concentration in the outer and inner
integuments. The increment in size of the dominant ovule (Fig. 3.8a) contrasted with the
lack in growth of the companion ovules (E3C; Fig. 3.8g), which only showed
66
Chapter III
methylation signal in the inner integuments and embryo sac (Fig. 3.8h, i). Histone H4ac
was only found in the inner integuments but was absent in the embryo sac (Fig. 3.9h, i).
Embryo expansion
Ovules at stage E4D showed a dramatic increase in size compared with previous
E3D stage. The growing embryo within the ovule already had differentiated axis with
provascular bundles and cotyledons (Fig. 3.10a; Fig. 3.11a). Methylation signal at this
stage was vague and mainly located in the root meristem and the final zone of the
growing cotyledons (Fig. 3.10b, c). H4ac, on the contrary, was ubiquitous and more
present in the external cell layers of the embryo (Fig. 3.11b, c).
Embryo maturation
In the posterior stages of embryo expansion and maturation (E5A, E6A and E7A)
the axis experienced an increase in size and length (Fig. 3.10d, g; Fig. 3.11d, g) until
reaching its final shape (Fig. 3.10j; Fig. 3.11j). Methylation signal displayed a dynamic
pattern: at stage E5A (Fig. 3.10e, f), 5-mdC mainly accumulated in the root cap, the RAM
and the provascular bundles next to it. On the contrary, the signal was very low in the
half part of the embryo closer to the SAM, which was strongly labelled. At stage E6A
(Fig. 3.10h, i) methylation signal became ubiquitous with high intensity in the
provascular tissues, the root cap, epidermis and the SAM where 5-mdC spread to the
peripheral zone. At this point, RAM showed much less methylation intensity than at
E5A and there was a diffuse presence all over the hypocotyl. The distribution pattern
changed in E7A where 5-mdC became abundant in the peripheral zone of SAM and
remained low in the RAM with a strong signal gradient towards the SAM (Fig. 3.10k, l).
The same gradient was also found in the subepidermical layers of cells. Remarkably, an
opposite gradient was found in the provascular bundles where the signal also
disappeared from the cells surrounding that tissue. The root cap was the only tissue that
maintained methylated in a stable fashion from E5A to E7A.
Histone H4ac distribution was ubiquitous from stages E5A to E7A except for its
absence in the root cap (Fig. 3.11e, f, h, i, k, l). Provascular bundles, SAM and RAM,
presented high signal as well as the epidermis at stages E5A and E6A while mature axis
(E7A) was found to lose the signal in the provascular tissues, remaining high in cells
bordering the provascular bundles, and in the subapical zone of the SAM. Moreover,
67
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
H4ac was high in the lower hypocotyl and low in the entire RAM; as well as low in the
upper hypocotyl except for the provascular bundle border.
Fig. 3.6. Immunodetection of 5-mdC using confocal microscopy throughout development in longitudinal
sections. Differential interference contrast (DIC) of an ovule and surrounding ovary tissues previous to
pollination (E1) (a), 5-mdC signal at E1 (b), merged of DAPI (in blue) and 5-mdC (in green) signals at E1
(c), DIC of ovules after cross-pollination at E2 (d), 5-mdC signal at E2 (e), merged image of DAPI (in blue)
and 5-mdC (in green) signals (f), DIC of an ovule without cross-pollination (E2I) (g), 5-mdC signal at E2I
(h), merged of DAPI (in blue) and 5-mdC (in green) signals at E2I (i). Size bars are 0.2 cm. OW, ovarian wall;
OI, outer integument; II, inner integument; EM, embryo sac.
68
Chapter III
Fig. 3.7. Immunodetection of H4ac using confocal microscopy throughout development in longitudinal
sections. DIC of an ovule and surrounding ovary tissues previous to pollination (E1) (a), H4ac signal at
E1 (b), merged of DAPI (in blue) and H4ac (in green) signals at E1 (c), DIC of ovules after crosspollination at E2 (d), H4ac signal at E2 (e), merged image of DAPI (in blue) and H4ac (in green) signals
(f), DIC of an ovule without cross-pollination (E2I) (g), H4ac signal at E2I (h), merged of DAPI (in blue)
and H4ac (in green) signals at E2I (i). Size bars are 0.2 cm.
69
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
Fig. 3.8. Immunodetection of 5-mdC using confocal microscopy throughout development in
longitudinal sections. DIC of the dominant ovule at stage 3 (E3D) after fertilization containing a
developing embryo inside (a), 5-mdC signal at E3D (b), merged of DAPI (in blue) and 5-mdC (in green)
signals for E3D (c), DIC of the developing globular embryo (E3D) (d), 5-mdC signal in the developing
embryo (e), merged of DAPI (in blue) and 5-mdC (in green) signals of the embryo (E3D) (f), DIC of a
companion ovule at stage 3 showing embryo sac degradation (E3C) (g), 5-mdC signal in E3C (h), merged
of DAPI (in blue) and 5-mdC (in green) in the companion ovule without fertilization at stage 3 (E3C) (i).
Size bars are 0.2 cm except for (d) in which size bar is 50 µm. EB, developing embryo; OI, outer
integument; EN, rests of the endosperm; II, inner integument.
70
Chapter III
Fig. 3.9. Immunodetection of H4ac using confocal microscopy throughout development in longitudinal
sections. DIC of the dominant ovule at stage 3 (E3D) after fertilization containing a developing embryo
inside (a), H4ac signal at E3D (b), merged of DAPI (in blue) and H4ac (in green) signals for E3D (c), DIC
of the developing globular embryo (E3D) (d), H4ac signal in the developing embryo (e), merged of DAPI
(in blue) and H4ac (in green) signals of the embryo (E3D) (f), DIC of a companion ovule at stage 3
showing embryo sac degradation (E3C) (g), H4ac signal in E3C (h), merged of DAPI (in blue) and H4ac
(in green) in the companion ovule without fertilization at stage 3 (E3C) (i). Size bars are 0.2 cm except
for (d) in which size bar is 50 µm.
71
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
Fig. 3.10. Immunodetection of 5-mdC using confocal microscopy throughout development in
longitudinal sections. DIC of cotyledonary embryo at E4D (a), 5-mdC signal in E4D (b), merged of DAPI
(in blue) and 5-mdC (in green) signals for E4D (c), DIC of embryonic axis at E5A (d), 5-mdC signal in
E5A (e), merged of DAPI (in blue) and 5-mdC (in green) signals for E5A (f), DIC of embryonic axis at
stage E6A (g), 5-mdC signal in E6A(h), merged of DAPI (in blue) and 5-mdC (in green) signals for E6A
(i), DIC of mature embryonic axis at stage E7A (j), 5-mdC signal in E7A (k), merged of DAPI (in blue)
and 5-mdC (in green) signals for E7A (l). Size bars are 0.5 cm. EA, embryonic axis; COT, cotyledon; PB,
provascular bundles; RC, root cap; HY, hypocotyl.
72
Chapter III
Fig. 3.11. Immunodetection of H4ac using confocal microscopy throughout development in longitudinal
sections. DIC of cotyledonary embryo at E4D (a), H4ac signal in E4D (b), merged of DAPI (in blue) and
H4ac (in green) signals for E4D (c), DIC of embryonic axis at E5A (d), H4ac signal in E5A (e), merged of
DAPI (in blue) and H4ac (in green) signals for E5A (f), DIC of embryonic axis at stage E6A (g), H4ac
signal in E6A (h), merged of DAPI (in blue) and H4ac (in green) signals for E6A (i), DIC of mature
embryonic axis at stage E7A (j), H4ac signal in E7A (k), merged of DAPI (in blue) and H4ac (in green)
signals for E7A (l). Size bars are 0.5 cm
73
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
3.4. DISCUSSION
The control of chestnut reproduction has remained elusive for years. In spite of
studies of its floral biology and reproduction (Botta et al., 1995; Feijó et al., 1999), focused
approaches regarding epigenetics have only been carried out in our previous work
analyzing global methylation levels (Chapter 2; Viejo et al., 2010) for this species. The
present work associates the achievement of the sexual reproduction in chestnut with
epigenetic changes in a spatial-temporal fashion throughout the developmental stages
analyzed.
Validation of epigenetic techniques during chestnut reproduction
The heterogeneity of tissues and developmental pathways during chestnut
reproduction make MSAP a good technique to apply in this species in order to assess
the differentiation of developmental stages. This variety derived from the normal
progression of reproduction, along with the fact that DNA methylation in plants takes
place in specific sequences (Finnegan et al., 2000b), led to the identification of 643 MSL
of which 47 % were polymorphic. Although methylation in plants has been described
as the integral part of the regulatory networks in plants governing development
(Finnegan
et
al.,
2000a),
surprisingly,
no
differences
in
the
number
of
methylation/demethylation events were found within a given developmental
transition. This fact contrasted with our previous study (Chapter 2; Viejo et al., 2010)
where significant differences in global methylation levels appeared during
development. Although some results in plants (Meijón et al., 2010) and animals (Yang et
al., 2011) established associations between the changes in MSAPs and global
methylation levels, there are two limitations that affect the way MSAPs can be
interpreted: (1) the isoschizomers HpaII and MspI do not recognize all the possible
cytosine-methylation islands and (2) the combination of primers in the preselective and
selective PCR only cover the genome in a partial fashion. Due to these limitations, MSAP
technique only provides a partial scan of the genome, making it not suitable for global
quantifications of DNA methylation. In spite of the above, MSAP have proven their
informative character during chestnut reproduction in terms of the number of changes
between developmental transitions and the loci specific changes as discussed below.
The relative quantification of the PTMs assayed by ELISA was successful taking
into account the variety of tissues used. The use of a modified protocol base in the one
74
Chapter III
developed by Shechter et al. (2007) has proven its reliability and reproducibility in a
wide range of tissues from flowering to the mature seed in chestnut. The posterior
Western blot and ELISA analysis have determined that the yield and quality of the
protein isolations is sufficiently high for performing these techniques. In addition, this
has been the first time that a relative quantification of PTMs has been achieved in a
forestal plant species. Thus, we have develop a new, simple and cheap methodology,
not technically demanding for the relative quantification of histone epigenetic marks in
a non-model species. Moreover, the presence of specific patterns in the relative
quantification of PTMs regarding the normal progression of embryogenesis and the
abortion of ovules strongly supports the study in Arabidopsis mutants by AlvarezVenegas and Avramova (2005).
Finally, the immunolocalization of 5-mdC and H4ac in the tissues during
reproduction brought to light the importance of global, tissue-specific dynamics from
flowering to the mature embryo in a specific fashion not only for each antibody but also
between epigenetic marks. This complementary distribution of the epigenetics marks in
the tissues is in strong support of the global methylation levels (Chapter 2; Viejo et al.,
2010) and the quantification and dynamics of the histones PTMs quantified in this
chapter.
Pollination
The first stimulus in angiosperm sexual reproduction is pollination. In the case
of chestnut, cross-pollination is compulsory to enter the embryogenic pathway (McKay,
1942). However, both cross- and autopollination did not show predominance of
methylation or demethylation events in the MSAP analysis, associated with a low
percentage of global changes compared to the global number of changing loci. The fact
that the ovule is not mature even after pollination (Feijó et al., 1999) seems not to have
an evident effect at MSAP level. On the contrary, the appearance of some exclusive loci
changing in autopollination at E2I and latter in E3I supports the growing body of
evidence that indicates an early establishment of ovules´ destiny depending on the kind
of pollination and also supports the immunolocalization results as differential
distribution was found when comparing cross and autopollination. Moreover, the
variation in the exclusive loci involved in the transitions from E1 to E2I and E2I to E3I
75
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
possibly control the entrance into the abortive pathway in E2I and E3I taking into
account the position of these stages in the PCoA analysis.
DNA methylation has been usually associated with the repression of
transcription (Tariq and Paszkowski, 2004) in contrast with H4ac (Meijón et al., 2010).
Pollination in chestnut does not seem to follow this claim as H4ac signal has been found
to display a similar distribution in ovules as 5-mdC. Ovules at E1 showed
hyperacetylation of H4 (as shown in figure 6B), result that is consistent with the general
association between H4ac, and also H3ac, with active euchromatic regions in plants
(Hollender and Liu, 2008). After cross-pollination, H4ac disappeared from the embryo
sac, a tissue that showed H4ac signal under autopollination conditions, the latter case
resembling E1 distribution, and could be associated both with the absence of a valid
pollination signal and the entrance in the death pathway. H4 hyperacetylation has been
recently reported to be involved in the induction of cell death in animals (Jeong, 2014),
although this fact has not yet been observed in plants. Furthermore, the relative content
of H4ac showed an increment in cross-pollination in accordance with the active growth
of the ovules, as discussed above and in contrast with the absence of change in
autopollination which is congruous with the idea of an early determination of ovule fate
triggered by the different type of pollination. The rest of analyzed marks showed
decreases, contrasting with H4ac dynamics and independently of their attributed active
(H3ac, H3K4me3) or repressive (H3K9me3, H3K27me3) role and the type of pollination.
Moreover, the decline in H3 marks was stronger for autopollination than crosspollination indicating a differential response. This is the first report for quantification of
histones PTMs in pollination.
Fertilization and ovule abortion
In chestnut, fertilization not only triggers the establishment of the polarity of the
embryo and the generation of the endosperm, but also constitutes the entrance of the
companion ovules (E3C) into the abortive pathway. After the maternal and paternal
genomes are combined in the double fertilization, the tight control of the genomic
imprinting is exerted by epigenetic mechanisms (Köhler and Makarevich, 2006;
reviewed in Vaillant and Paszkowski, 2007) including DNA methylation and changes
in H3K27 methylation. Moreover, the epigenetic reprograming after fertilization
76
Chapter III
through DNA and histones methylation is fundamental for controlling the parental
contribution by the maternal genome (Autran et al., 2011).
Our previous study (Chapter 2; Viejo et al., 2010) associated a decrease in the
levels of global methylation with this reprogramming in chestnut although this
dynamic was not found to have repercussion in the MSAP analysis, neither in the global
number of methylation events, nor in specific methylated/demethylated loci. This
result might be due to the differential contribution of the maternal tissues (ovule´s
integuments) which dominates at this stage and could be masking the small amount of
tissue corresponding to the early embryo as noticed by the histological study.
Immunolocalization of epigenetic marks, on the contrary, showed hardly 5-mdC
signal in the early embryo in strong contrast with the highly acetylated histone H4 at
this stage, which would be in support of the reprogramming processes taking place at
the tissue level in which the repressive mark of methylation was underrepresented and
the activation H4ac was strongly present. On the contrary, H4ac relative quantification
did not show changes after fertilization and the rest of the epigenetic marks quantified
from histone H3 displayed reductions after pollination regardless of their association
with active or repressive chromatin status. This shared pattern seems to characterize the
normal variation in terms of global chromatin epigenetic content rather than being
associated with specific developmental changes.
The transition of ovules after cross-pollination to companion ovules (E3C) not
only differentiates from the dominant ones (E3D) in the appearance of MSAP specific
loci, but also in the dynamics regarding relative quantification of epigenetic marks.
Thus, activation marks such as H4ac underwent a transient decrease at E3C and also
reduced its presence in the ovule, specifically to the inner integument, which is in
contrast with 5-mdC distribution that also covered the embryo sac and maintained
similar distribution as abortive ovules E2I. The rest of activation marks from histone H3,
H3ac and H3K4me3, significantly decreased their content in companion ovules E3C in
contrast with repressive marks H3K9me3 and H3K27me3 that transiently increased
indicating a direct relationship between the entrance in the abortive pathway of E3C
ovules and the repression of the expression of genes tissue-dependent as already
described for H3K27me3 (Zhang et al., 2007). H3K9me3, on the contrary, has not been
77
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
found to be tissue-specific (Charron et al., 2009) although its dynamics in chestnut not
only resembled H3K27me3 but also is a clear indicative of ovule abortion in E3C ovules.
The relative quantification of epigenetic marks and their combination clearly set
up an epigenetic scenario that marks the abortion of companion ovules as supported by
the transient increase found in E3C for H3K9me3 and H3K27me3 that reached former
values in E4C. It is remarkable that the two kinds of ovule abortion (E3I vs. E3C) studied
showed clear differences in the PCoA analysis regarding methylation changes but also
in the dynamics of the PTMs quantification, suggesting differential mechanisms for the
death pathway and also a possible role of the dominant ovule in the exertion of the
companion ovules´ fate.
Embryo expansion
At E4D, the embryo showed a clearly differentiated axis and cotyledons that will
develop until its final form at E7 stage. This transition stage between the dominant ovule
as E3D and the beginning of embryo axis maturation at E5A was characterized by a faint
signal of 5-mdC, with more presence in the internal part of cotyledons and root cap and
possibly associated with the active developmental status as H4ac was ubiquitous and
also showed an increment in the relative quantification in contrast with the rest of
activation marks studied such as H3ac and H3K4me3 and repressive (H3K9me3 and
H3K27me3) that maintained similar levels at E3D. This differential display between
histones H4 and H3 seems to be indicating that the embryogenic character of the zygotic
embryo at this point is related with low amounts of the H3 epigenetic marks studied
while H4 opposes these dynamics.
Embryo maturation
Final stages of embryo growth and maturation are closely related with high
amounts of changes in the methylation status according with the MSAP analysis. From
E3D to E5A and E5A to E7A, cell elongation and deposition of storage compounds
prevails in parallel with cell differentiation does to ensure the proper distribution of
cellular types within the embryo (Santos-Mendoza et al., 2008). On this scenario, and in
contrast with the rest of developmental stages, it can be claimed that embryo
development and maturation concurs with an increasing number of changes in DNA
methylation. Moreover, maturation also represented the highest rate of specific
methylation-changing loci, supporting the variety of cellular types and processes going
78
Chapter III
on such as dehydration, storage compounds accumulation or the entering in quiescence.
The variability during this final stages made noteworthy that the specific methylation
loci predominated in E3D to E5A while there was an inversion on this correlation in the
maturation from E5A to E7A. Overall, these results were consistent with Zhang et al.
(2011) and the description of TDMRs (Tissue-specific Differentially Methylated
Regions) in sorghum during development. These methylation differences were also
reflected in the PCoA analysis as embryogenesis development negatively correlates
with coordinate 1. Changes in DNA methylation also correlated with specific dynamics
in the histones epigenetic marks content. H4ac appeared to mark the beginning of
maturation since it peaked in E5A, an earlier stage than the peak of the rest of the marks
from histone H3. This differential dynamics which would be pointing to a collaborating
role of histones H4 and H3. Besides, for the first time in a plant species PTMs have been
quantified during the zygotic embryogenesis.
The immunolocalization from stages E5A to E7A confirmed the extraordinary
variability and plasticity that plants possess by the use of signal gradients and specific
spatial-temporal patterns. Thus, taking into account a permissive role, the peak of H4ac
at E5A can be considered an earlier step to tissue differentiation. This idea was reflected
in the dynamics of the acetylated H4 distribution in the tissues during maturation and
would explain the general loss of signal towards E7A and the disappearance from the
provascular bundles as well as the progressive loss of signal in the meristems which is
in accordance with a previous study in the apical meristem of Hordeum vulgare
(Braszewdka-Zaleska et al., 2013) regarding tissue differentiation dynamics in seedlings.
5-mdC localization, on the contrary, seemed to follow the opposite pattern, spreading
from the meristems to all other tissues in the mature embryo (E7A) when it disappeared
from the RAM and the surrounding provascular bundles but also from the central zone
of the SAM. Thus, maturation can be claimed to have a direct relationship with specific
changes in the amount and disposition of epigenetic marks and that their dynamics are
necessary for the maturation of the zygotic embryo.
In conclusion, specific epigenetic marks dynamics have been associated with the
normal development of sexual embryogenesis in chestnut. MSAP specific loci changes
and variations in the histones PTMs could be behind the differential status of chromatin
and gene expression associated with the variety of cell types and functions found in this
study.
In addition, the differential localization of some repressive (5-mdC) and
79
Epigenetic scenarios in ovules and zygotic embryos throughout C. sativa reproduction
activation (H4ac) marks in the tissues are associated with the great variety of
developmental scenarios found in chestnut reproduction. From flowering to the mature
embryo, the combination of the epigenetics marks studied can be specifically addressed
to distinct and complementary developmental pathways needed for the normal
development of reproduction such as zygotic embryo development and ovule abortion.
80
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Different hormonal profiles characterize
sexual reproduction in C. sativa
Chapter IV
4.1. INTRODUCTION
European chestnut is a highly appreciated species in the Mediterranean region
in terms of food and timber production, as well as landscape preservation. Seed quality
requirements mainly relies on the monoembryonic character of the nuts (Peano et al.,
1990) and it is the aim of breeding programs. Nevertheless, the understanding of proper
embryo development focusing on the establishment of mono or polyembryony through
the production of dominant ovules and companion ones remains unstudied.
Zygotic embryogenesis in plants is a complex process that starts with the double
fertilization and finishes with the mature embryo. In chestnut it has been studied in
histological terms (Botta et al., 1995) and at the epigenomic level by quantification of
global DNA methylation (Chapter 2; Viejo et al., 2010), associating DNA methylation
status with several hits during reproduction (pollination, fertilization, embryo set,
abortion/death of ovules, growing and maturing of the embryo). Nevertheless, there
are no studies of this species regarding physiological characterization, including
moisture content during embryo development which determines maturity or the spatial
and temporal role of hormones which are known to play decisive roles during
reproduction processes (Bewley and Black, 1994). Moreover, there is an evident lack of
literature regarding the role of hormones in ovule death.
Plant hormones play important roles during ontogenesis through external
stimuli and endogenous signals. Factors as concentration, gradient, ratio between two
or more hormones and localization in a tissue can determine the developmental
pathway for a specific cell or group of cells at any development stage. Auxins were the
first studied group and IAA is the main representative. It is ubiquitous in the plant and
plays an important role in early zygotic embryogenesis establishing the axial pattern
(Harada et al., 2010) and later in the determination of the shoot and radical apical
meristems in the embryo axis during tissue differentiation (Vanneste and Friml, 2009).
Hormones can show inverse relationships, as happens with CKs and auxins during the
formation of the axial pattern during embryo development (Müller and Sheen, 2008). In
late embryo development, CKs ribosides are arrested in the seed while controlling
embryo growth through the ratio to auxins (Morris, 1997). ABA also has a well-known
action during embryo development showing low levels in early embryogenesis,
reaching a maximum while the embryo is growing, and decreasing during maturation
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Different hormonal profiles characterize sexual reproduction in C. sativa
and the acquisition of dehydration tolerance (Finklestein et al., 2002). Participation of
GAs in embryo development depends on their ratio to ABA and is linked with embryo
growth and maturation; they also play a role in the growth of the pollen tube (Swain
and Singh, 2005). JA is well known for its role in plant defense, but there are few data
supporting its possible action during reproduction except for a suggested role during
ABA decrease at maturity of the embryo (Hays et al., 1999). BRs are a recent group of
molecules accepted as plant regulators and although they participate in signaling along
with the rest of hormones (mainly GAs) in a wide range of processes such as flowering
and embryo development, their roles are not clear so far (Zhu et al., 2013).
The aim of this study was to determine the hormonal and physiological status
through reproduction development focusing on defined developmental stages and the
mailstones that determine proper embryo development: pollination, fertilization and
early embryo development versus the death of companion ovules and embryo growing
and maturation. A global hormone characterization was performed for IAA, ABA, GAs,
CKs, JA and BRs. Given the substantial role of IAA and ABA during embryogenesis and
that hormone quantification of whole organs do not always offer the right insight into
their roles, localization of these hormones was performed by immunohistochemistry in
order to shed light on the hormonal control during chestnut reproduction at defined
developmental stages. Moreover, moisture content during maturation of the embryo
was also quantified.
4.2. MATERIAL AND METHODS
4.2.1. Plant material
Burrs were collected from mid-July to mid-November of 2012 from several openpollination trees in chestnut stands in Carreño (Asturias, Spain) and classified in
accordance with the previously defined developmental stages: ovules (E1 to E4) and
growing embryo (E5 to E7; Chapter 2; Viejo et al., 2010) not taking into account the
location of the ovary within the burr. Infructescences from isolated trees were collected
at the same collecting dates for stages E2 and E3 (2I and 3I, respectively) and used as
controls for proper cross-pollination/fertilization events. Burrs were immediately
dissected to extract ovaries and obtain ovules from stages E1 to E4 and embryonic axes
and cotyledons [proximal (PC) and distal portions (DC)] from stages E5 to E7.
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Chapter IV
4.2.2. Global hormone content
Tissues from stages E1 to E7 were immediately frozen in liquid nitrogen and
then stored at -80 ºC until analysis. Analyses of 3 biological and 3 analytical replicates
were performed for each sample. Global content of 15 hormones (ABA; IAA; JA; zeatin
riboside, ZR; dihydrozeatin, DHZ; dihydrozeatin riboside, DHZR; BA; isopentenyl
adenine, iP; isopentenyl adenosine, iPA; GAs GA3, GA4 and GA7; epibrasinolide, 24EB;
homobrasinolide, HBI; castasterone, BK) were quantified according to Pan et al. (2010)
with modifications: 60-100 mg of lyophilized tissue were ground into a fine powder
with a mortar and pestle and 500 μl of 2-propanol:H2O:concentrated HCl (2:1:0.002,
v/v/v) with internal standards (30-60 ng) were added, followed by agitation for 30 min
at 4 °C. CH2Cl2 (1 mL) was added followed by another 30 min of agitation at 4 °C. Two
phases formed with the plant debris in the interphase. The lower phase was collected,
concentrated in 2 ml glass vials with nitrogen flow and stored at -20 °C until analysis.
Samples were re-suspended in 150 μl of 100 % methanol and filtered through a
0.2 μm regenerated cellulose filter (Agilent Technologies). All the compounds were
separated and quantified by an ultra-high performance liquid chromatography
(UHPLC) in a 6460 Triple Quad LC/MS (Agilent Technologies) using the protocol
described by Novak et al (2008) for CKs and performed for the plant growth regulators
analyzed. A chromatographic separation was made using a reverse phase column
(Zorbax SB-C18 2.1 x 50 mm column). The column was held at 40 ºC and the mobile
phase used in the chromatography consisted of (A) 99.9 % MeOH: 0.1 % COOH and (B)
ammonium formate (15 mM, pH 4). A linear gradient of MeOH from 10 % to 50 % and
then reaching 100 % in 7 and 2 minutes respectively was used for elution. Plant growth
regulators were quantified by dynamic multiple multireaction monitoring (DMRM) of
their [M+H]+ and the appropriate product ions, using optimized cone voltages and
collision energies for diagnosis of each hormone analyzed. All the solvents used were
high-performance liquid chromatography (HPCL) grade.
Data collected was transformed into µmol hormone/g dry weight for each
developmental stage and mean values were statistically analyzed with Kruskal-Wallis
(significance level 0.05) in R Statistical Environment (R core team, 2012) core functions
plus the package agricolae (de Mendiburu, 2014). Moreover, packages cluster (Maechler
et al., 2013) and gplots (Warnes et al., 2013) were used for clustering hormones mean
values (Ward´s hierarchical method) and represented in a heat map. Principal
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Different hormonal profiles characterize sexual reproduction in C. sativa
component analysis of the data was also performed with package FactoMineR (Lê S et
al., 2008).
4.2.3. Determination of embryo moisture content
At each developmental stage fresh and dry weight were recorded and
determined gravimetrically before and after drying embryos at 103 ºC for 17 h. Moisture
content was expressed as a percentage on a fresh weight basis and calculated as: (Fresh
weight – Dry weight) / Fresh weight x 100. Percentages of moisture content were
transformed using arcsin prior to data analysis.
Data was analyzed using the statistical program SPSS (SPSS Inc., USA) for
normality and variance homogeneity (Kolmogorov-Smirnov and Levene tests,
respectively). Differences between stages were contrasted by one-way analysis of
variance (ANOVA) and post hoc Duncan test. The level of significance was 0.05.
4.2.4. Immunohistochemical detection of ABA and IAA
Tissues from all developmental stages were used except for cotyledons, which
due to their histological homogeneity in later stages did not reflect any specific
distribution of the hormones. Isolated ovules from stage 3 (E3I) and companion ovules
from stage 4 (E4C) were also excluded from the immunohistochemical detection due to
the impossibility of maintaining integrity when sectioning. At least three samples per
developmental stage were studied and negative controls were performed, consisting of
the substitution of the primary specific antibody for the hormone with a 1 % BSA
solution. Immunolocalization was performed according to Pérez et al. (2015), with
modifications: samples were fixed with 3 % (w/v) paraformaldehyde in 4 % (w/v) 1ethyl-3-(3-dimethylaminopropyl) carbodiimide (©Sigma-Aldrich Co.) containing 0.1 %
(v/v) Triton® X-100 overnight at 4 ºC under vacuum. Samples were then sectioned at
50 µm using a cryomicrotome Leica CM1510-S (Leica Instruments). Sections were
dehydrated in an ascending series of ethanol (25, 50, 75 and 100%; 5 min each) and
subsequently rehydrated (100 %, 75 %, 50 %, 25%; 5 min each). Slides were
permeabilized by incubating them 30 min in 0.1 % Tween 20 in PBS and unspecific
unions were blocked by a reaction with 20 % bovine serum albumin in PBS for 30 min.
Sections were then incubated with rabbit antibody anti-ABA or anti-IAA (Agrisera, ref.
AS09 446) diluted 1/100 in 1 % BSA at 4 ºC overnight. Alexa Fluor 488-labelled antirabbit polyclonal antibody (Invitrogen, ref. A1108) diluted 1/25 in 1 % BSA was used as
90
Chapter IV
the secondary antibody. The slides were counterstained with a DAPI (Fluka) solution (1
µg/ml, 1 % Tritón in PBS) for 15 min in darkness and washed 3 times (5 min each) with
distillated water. Sections were fixed to the slides with a Mowiol® solution (Valnes and
Brandtzaeg, 1985) without polyvinyl alcohol or DABCO. Fluorescence was visualized
using a confocal microscope (Leica TCS-SP2-AOBS) connected to a workstation and the
images were processed with Leica Software (LCS 2.5). Negative controls showed no
fluorescence signal (data not shown).
4.3. RESULTS
4.3.1. Hormones vs developmental stages clustering and principal component analysis
Temporal variations in global content of hormones regarding developmental
stages from flowering to mature embryo showed clear differences (Fig. 4.1). The heat
map and clustering offered a general view in which three main groups could be
identified
divided
by
developmental
stage.
Fig. 4.1. Dendrograms obtained by agglomerative hierarchical clustering for developmental stages and
hormones global levels. The heat map indicates response values close (clear tones) and distant (darker
tones) from the samples’ average. Experimental replicates were used to obtain dendrograms (n = 3).
Firstly, the cluster at the bottom of the figure showed medium values for
hormone concentrations, and mainly corresponded with anthesis (E1) and controls for
zygotic embryo development (E2I, E3I and E3C). A second cluster was associated with
91
Different hormonal profiles characterize sexual reproduction in C. sativa
medium to high hormone concentrations in actively growing stages such as early
embryogenesis (E3D) or with late embryo development characterized by active cellular
divisions and elongation (E5A, E6A).
It also included E4C that forms an advanced stage of abortion. The third cluster,
localized at the top, showed low to medium hormone values and mostly contained
cotyledons of developmental stages associated with nutrient storage during maturation,
as well as E4D and E7A.
Hierarchical clustering of hormones in the studied developmental stages
revealed the relation of ABA and GA4 in a strongly separated group from the rest of
regulators which could be divided in four clusters: one was represented by JA, HBI and
24EB. A second group contained all the CKs along with the rest of GAs analyzed while
IAA and BK were separated in a third and fourth groups, respectively.
Three PCAs were performed in order to supplement the general impression
provided by the clustering and heat map. A first PCA included all the developmental
stages and tissues comprised from E1 to E7 (Fig. 4.2).
Fig. 4.2. Principal component analysis factor maps for Developmental stages E1 to E7. Developmental stages
distribution (a) and hormones distribution (b). Arrows represent contribution intensity and direction of
contribution.
The first component for the whole development analysis explained 40.18 % of the
variability while the second one was 13.8 % decreasing to 11.18 % in the third and 9.30
% in the fourth. The first component mainly associated negatively with samples
regarding maturation (E5-E7; Fig. 4.2a) while hormones (Fig. 4.2b) grouped in the
92
Chapter IV
positive part of the axis and was mainly represented by GA3, GA7 and some CKs (Table
4.1).
Table 4.1. Principal component analysis components’ summary for developmental stages E1 to E7. List of
the hormones contributing to each principal component. Hormone, contribution intensity as correlation for
each principal component and probability value (p value) are shown.
Hormone
BA
iP
GA7
IPA
GA3
DHZR
DHZ
BK
HBI
ZR
EB
Component 1
Correlation
0.94
0.94
0.93
0.92
0.80
0.79
0.69
0.45
0.19
0.19
0.16
p value
0
0
0
0
0
0
0
0
3.27e-09
1.47e-02
4.74e-02
Hormone
EB
HBI
ZR
JA
DHZ
GA3
ABA
GA4
Component 2
Correlation
0.72
0.69
0.64
0.41
0.18
-0.30
-0.31
-0.37
p value
0
0
0
1.09e-07
1.90e-02
1.24e-04
7.16e-05
1.68e-06
The second component was associated with early developmental stages from E1
to E4 in association with JA, BRs and CKs. The distribution of developmental stages and
correlation of hormones in the global PCA associating with early embryogenesis and
maturation justified its subdivision into two other PCAs, one from flowering to early
embryo development (E1 to E4D developmental stages; Fig. 4.3) and another one
regarding embryo maturation (E5 to E7 developmental stages; Fig. 4.4).
In the PCA from flowering to early embryo development (Fig. 4.3a) the first
component accumulated 35.5% of the total variability and was associated in the positive
part with the death of companion ovules (E4C) and cross-pollination (E2) while later
stages regarding embryo development (E3D and E4D) moved towards the negative part
of this component changing in their hormonal association from CKs and GA3 and GA7
to GA4 and IAA (Fig. 4.3b, Table 4.2).
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Different hormonal profiles characterize sexual reproduction in C. sativa
Fig. 4.3. Principal component analysis factor maps for Developmental stages E1 to E4. Developmental stages
distribution (a) and hormones distribution (b). Arrows represent contribution intensity and direction of
contribution.
The second component that explains less than half the variability of the first one
(16.5 %) is linked to developmental stages that have not yet entered the embryogenic
program from E1 to E3 (Fig. 4.3a) and BRs, ABA and JA (Fig. 4.3b, Table 4.2). The third
and fourth components explained 15.39 % and 9.15 % of the variability, respectively.
Table 4.2. Principal component analysis components’ summary for developmental stages E1 to E4. List of
the hormones contributing to each principal component. Hormone, contribution intensity as correlation for
each principal component and probability value (P value) are shown.
Hormone
BA
GA7
iPA
iP
GA3
BK
DHZ
DHZR
IAA
GA4
Component 1
Correlation
0.94
0.94
0.88
0.81
0.72
0.61
0.58
0.58
-0.35
-0.49
P value
0
0
0
0
7.02e-13
8.41e-09
4.97e-08
5.87e-08
2.31e-03
1.02e-05
Hormone
JA
HBI
EB
BK
ABA
GA3
GA4
IAA
Component 2
Correlation
0.70
0.58
0.53
0.50
0.44
-0.31
-0.56
-0.65
P value
7.05e-12
6.07e-08
1.57e-06
7.64e-06
1.01e-04
6.42e-03
1.99e-07
6.36e-10
In contrast, in the embryo maturation PCA (Fig. 4.4) the first component
accumulated the 68.98 % of variability and 9.36 %, 5.49 % and 4.42 % for the second,
third and fourth, respectively.
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Chapter IV
Fig. 4.4. Principal component analysis factor maps for Developmental stages E5 to E7. Developmental stages
distribution (a) and hormones distribution (b). Arrows represent contribution intensity and direction of
contribution.
Most of the developmental stages were in the negative side of the first
component (Fig. 4.4a) but hormones correlating with this component mostly exerted its
influence in the positive part linked to E5A, E6A and E5PC (Fig. 4.4b, Table 4.3). The
second component explained less than 10 % of variability being E6A the only sample
that clearly separated from the rest and was mainly associated with ABA (Fig. 4.4b).
Table 4.3. Principal component analysis components’ summary for developmental stages E5 to E7. List of
the hormones contributing to each principal component. Hormone, contribution intensity as correlation for
each principal component and probability value (p value) are shown.
Hormone
DHZR
BA
iPA
iP
GA7
IAA
GA3
JA
HBI
EB
DHZ
ZR
BK
GA4
ABA
Component 1
Correlation
0.96
0.95
0.95
0.95
0.93
0.90
0.88
0.82
0.81
0.80
0.79
0.73
0.69
0.60
0.44
p value
0
0
0
0
0
0
0
0
0
0
0
1.040e-14
5.33e-13
1.66e-09
2.84e-05
Hormone
ABA
BK
DHZ
IAA
GA3
GA7
iPA
iP
Component 2
Correlation
0.82
0.42
0.37
0.26
-0.21
-0.23
-0.23
-0.23
p value
0
7.38e-05
5.64e-04
1.80e-02
4.87e-02
3.80e-02
3.45e-02
3.14e-02
95
Different hormonal profiles characterize sexual reproduction in C. sativa
4.3.2. Hormones global content
Hormones global concentrations during the analyzed developmental stages
showed specific variations (Table 4.4). IAA concentrations fluctuated from 0.64 up to
more than 8.47 µmol/g dry weight (Fig. 4.5a). Pollination between stages 1 and 2 went
along with a small increment and after fertilization IAA reached a maximum at stage
E4D but maintain low levels in embryonic axes and cotyledons in subsequent stages.
Maturity of the embryo in E7 coincided with the lowest level of IAA of the axes while
cotyledonary tissues maintained low levels during development. In the absence of
pollination (E2I), IAA levels remained similar to those in E1 with a further decrease in
E3I; which was in strong contrast with the fertilized stage E3D. Companion ovules at
both stages 3 and 4 also contained low levels of IAA.
Strong variations were found in ABA concentrations (Fig. 4.4b). From 1 to 28.59
µmol /g dry weight, this hormone showed the strongest variations in concentration. It
showed a similar response to pollination as IAA. After fertilization there was a dramatic
increase in E5A in the global levels peaking in the E6A and decreasing again to low
levels at E7A. Cotyledonary tissues maintained lower levels than the axes. The absence
of pollination concurred with a near 8-fold increase in ABA content in E2I, which was
transient, declining in E3I. Meanwhile companion ovules showed higher levels of ABA
at E3C but there was a decrease at stage E4C.
JA (Fig. 4.4c) varies from 0.93 to 20.62 µmol/g dry weight and showed different
global dynamics than ABA or IAA, showing a peak in E1; hormone content
progressively decreased until E4D. A transient increase was found in the growing axis
of E5 ovules. Proximal and distal cotyledonary tissues showed contrasting patterns in
E5PC/E5DC and E6PC/DC and maintained similar and higher levels than the axis at
stage E7A. The lack of pollination in E2I resulted in the highest values of hormone that
decreased in E3I although the level was still higher than in the dominant ovule at E3D.
In contrast, no differences were found in the companion ovules but their levels
were higher than their dominant equivalents.
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Chapter IV
Table 4.4. Hormones concentrations in the different developmental stages analyzed. Cotyledonary tissues
maintained lower levels than the axes. The absence of pollination concurred with a near 8-fold increase in
ABA content in E2I, which was transient, declining in E3I. Meanwhile companion ovules showed higher
levels of ABA at E3C but there was a decrease at stage E4C.
Developmental
Hormone (µmol hormone/g dry weight)
stage
ABA
IAA
JA
ZR
DHZ
DHZR
BA
iP
E1
1.53±0,12,h,i
1.34±40,59e
11.98±0,35a,b
11.92±1,26a
1.03±0,12e
0.76±0,04f,g
1.18±0,07d,e
0.76±0,004e
E2
1.81±0,07g
1.76±0,08b
3.1±0,18c,d
1.68±0,45b
1.47±0,06a,b
1.3±0,05a,b
1.88±0,11a
1.4±0,05a
E3D
1.52±0,11h,i
5.23±0,19a
1.44±0,03g
15.68±0,72a
2.2±0,14a
1.5±0,02a
1.43±0,005b
1.08±0,03b
E4D
2.25±0,02f
8.48±0,8a
1.16±0,04h
0.69±0,03e
0.75±0,02f,g
0.66±0,02h
1.06±0,03e
0.81±0,01d
E5A
11.83±0,10b
1.45±0,05c,d
2.63±0,1d
1.24±0,1b,c
1.31±0,05b,c
1.16±0,05b,c
1.87±0,07a
1.44±0,02a
E5PC
4.41±0,53d,e
1.19±0,09e
1.78±0,1e,f
1.1±0,11b,c
1.18±0,1d,e
1.01±0,09d,e
1.89±0,07a
1.35±0,05a
E5DC
5.64±0,24c
0.65±0,01h
0.93±0,04i
0.54±0,02g
0.58±0,01h
0.52±0,01i
0.83±0,02f
0.59±0,01e,f
E6A
28.59±1,14a
1.45±0,09c,d
1.53±0,09g
0.97±0,08c,d
1.1±0,06c,d
E6PC
4.47±0,36c,d
0.73±0,06g,h
0.95±0,1i
0.55±0,07f,g
0.63±0,06g,h
0.45±0,01i
0.71±0,01f
0.5±0,01f
E6DC
9.83±0,23b
0.82±0,01f,g,h
1.71±0,05f
0.69±0,01e,f
0.78±0,01f,g
0.7±0,01g,h
1.1±0,02e
0.8±0,01d
0.79±0,02f
0.98±0,06c,d 1.39±0,007b,c
0.71±0,01g,h 1.16±0,0002d
1±0,005b,c
E7A
3.36±0,05e
0.99±0,01e
1.06±0,02h,i
0.68±0,02e,f
E7PC
1.23±0,02j
0.85±0,01f,g
1.46±0,04g
0.69±0,01e,f
0.79±0,004f,g 0.7±0,004g,h
1.11±0,007e
0.83±0,01d
E7DC
1.00±0,07j
0.86±0,02f,g
1.44±0,04i
0.69±0,01e,f
0.79±0,01f,g
0.7±0,01g,h
1.12±0,01e
0.8±0,009d
E2I
2.59±2,6b
1.2±0,08d,e
20.62±0,04a
0.8±0,03d
0.96±0,03d,e
0.86±0,03d,e
1.28±0,003c
0.92±0,002c
0.71±0,07d
0.8±0,004d
E3I
12.21±0,14i
0.86±0,07f
3.65±0,2b,c
16.27±0,72a
0.97±0,08d,e
1.73±0,09a
1.36±0,06b,c
E3C
2.87±0,2f
1.09±0,03e
2.01±0,1e
0.8±0,04d
0.93±0,05d,e
0.78±0,01e,f
1.26±0,01c
0.9±0,01c
E4C
1.56±0,05h
1.61±0,03b,c
2.26±0,21e
1.28±0,08b,c
1.32±0,02b,c
1.17±0,01b,c
1.88±0,02a
1.34±0,01a
iPA
GA3
GA4
GA7
24EB
HBI
BK
E1
0.7±0,01e
0.43±0,01f
1.12±0,06e,f
0.43±0,01e
4.19±0,25a
1.03±0,04a
0.51±0,03d,e,f
E2
1.39±0,02a
0.71±0,02a,b
0.7±0,02h
0.72±0,03a
0.94±0,01b
E3D
0.91±0,005b
0.6±0,01b,c
0.72±0,02h
0.58±0,01b
0.79±0,02c,d
0.61±0,004a,b 0.57±0,03c,d
0.37±0,01e,f
0.45±0,009f
E4D
0.69±0,02e
0.52±0,02d,e
7.76±0,15a
0.41±0,01e
0.5±0,008h
0.28±0,01h
0.33±0,002h
E5A
1.29±0,02a
0.84±0,02a
4.91±0,11b
0.73±0,03a
1.05±0,03a,b
0.45±0,01c,d
1.04±0,02a
E5PC
1.21±0,04a
0.84±0,04a
3.07±0,13c
0.72±0,02a
0.69±0,02d,e
0.44±0,03d,e
0.47±0,03f
E5DC
0.53±0,01f
0.44±0,01f
1.82±0,07d,e
0.32±0,007f
0.34±0,008j
0.22±0,004j
0.27±0,01i
E6A
0.9±0,002b,c
0.62±0,02b
3.05±0,17c
0.36±0,009f
0.97±0,04a
0.28±0,02h,i
0.54±0,003b,c 0.62±0,003e,f
E6PC
0.46±0,01f
0.38±0,04f
3.19±0,12c
0.27±0,006f
0.29±0,01j
0.18±0,006f
E6DC
0.71±0,01d,e
0.54±0,01c,d
2.56±0,07d
0.44±0,006e
0.66±0,008e,f
0.27±0,003i
E7A
0.73±0,01d
0.46±0,008e,f
0.68±0,02h
0.44±0,007e
0.5±0,01h
0.37±0,01g
0.56±0,004c,d
,e
0.28±0,003h,i 0.37±0,006g
0.31±0,01g
E7PC
0.71±0,004d,e
0.46±0,008f
0.6±0,02i
0.43±0,002e
0.47±0,01i
E7DC
0.71±0,008d,e
0.44±0,01f
0.56±c0,01i
0.44±0,005e
0.48±0,01h,i
0.28±0,005h,i
0.33±0,01h
E2I
0.82±0,001c
0.53±0,01c,d
0.69±0,02ih
0,5±0,001c,d
0.54±0,002g
0.38±0,01f
0.63±0,01b,c
0.38±0,01g
E3I
0.87±0,04b,c
0.52±0,03d.e
0.87±0,03g
0,47±0,0004c
0.55±0,01g
0.37±0,01f
E3C
0.86±0,02b,c
0.53±0,02d
0.84±0,04g
0,55±0,02b,c
0.61±0,02f
0.37±0,007f
0.5±0,02e,f
E4C
1.23±0,009a
0.74±0,01a
1.1±0,05f
0,73±0,009a
0.83±0,006c
0.5±0,005b,c
0.69±0,01a,b
CKs (Fig. 4.6) levels varied from less than 0.44 to more than 16.26 µmol/g dry
weight with strong differences among the analyzed members of this hormone group.
The hormones iP, iPA [MEP (methylerythritol phosphate) pathway] and BA (Fig. 4.6a,
b, c, respectively) presented similar dynamics and also with DHZR (Fig. 4.6d) and DHZ
[Fig. 4.6E; MVA (mevalonate) pathway]. ZR (Fig. 4.6f) was the exception, where stages
E1, E3D and E3I were 10-fold times higher than DHZR and DHZ. The three of them
showed similar patterns including an increase after pollination (E2) and fertilization
97
Different hormonal profiles characterize sexual reproduction in C. sativa
(E3D) where ZR, DHZR and DHZ peak and then a transient decrease was found in the
forming embryo at stage E4D that recovered at E5A and E5PC; it gradually decreased
towards to the final maturation stage (E7).
Fig. 4.5. Global hormone content throughout development. IAA (a), ABA (b) and JA (c). Different letters
indicate significant differences among means. (p ≤ 0.05. Kruskal-Wallis test). Dark grey bars correspond
with proper embryogenic development from bloom to mature embryo axis while white bars are for
cotyledonary tissues. Soft grey bars correspond with controls for development where samples from isolated
trees are in diagonal lines and companion ovules with net. Error bars represent SE.
98
Chapter IV
Fig. 4.6. Citokinins global content throughout development. iP (a), iPA (b), BA (c), ZR (d) DHZR (e) and
DHZ (f). Different letters indicate significant differences among means (p≤ 0.05, Kruskal-Wallis test). Dark
grey bars correspond with proper embryogenic development from bloom to mature embryo axis while
white bars are for cotyledonary tissues. Soft grey bars correspond with controls for development where
samples from isolated trees are in diagonal lines and companion ovules with net. Error bars represent SE.
99
Different hormonal profiles characterize sexual reproduction in C. sativa
Increasing content of DHZR and DHZ could be found at stage E4C when ovules
have entered the abortion pathway, as well as in E3I for DHZR and ZR. iP, iPA and BA
presented three peaks, first after pollination (E2), also in the companion ovules at stage
4 (E4C) and in E5 in both axis (E5A) and proximal parts of cotyledons (E5PC). Isolated
samples E2I showed lower values respect to pollinated (E2) ovules while E3I had not
differences with fertilized ovules (E3D). Companion ovules maintained similar values
to dominant ovules (E3D) and performed an important increase at stage E4 (E4C). A
similar pattern was found for all the CKs in cotyledonary tissues, with double the
amount of hormone at stage E5PC than E5DC; an inversion of this relationship at stage
E6; and very similar levels in the mature tissue (E7PC and E7DC), similar to the axis
(E7A).
Global levels of the 3 GAs analyzed showed very similar patterns for GA3 (Fig.
4.7a) and GA7 (Fig. 4.7b) and they presented four peaks in the same developmental
stages (E2, E5A, E5PC, and E4C) as for iP, iPA and BA. The concentration range for GA3
and GA7 showed relative small differences varying between 0.28 and 0.84 µmol
hormone/g dry weight while GA4 (Fig. 4.7c) ranges from 0.56 to 8.76 µmol hormone/g
dry weight followed a different pattern during pollination and fertilization although
similar dynamics could be found in later stages. Controls of development showed
medium values for isolated ovules (E2I, E3I) and companion ovules at stage E3 (E3C) in
GA3 and GA7 while companion ovules at stage E4 (E4C) increased their content in both
hormones to the highest values found in the samples analyzed. In contrast, GA4 controls
maintained low values in these developmental stages similar to those found in stages
previous to embryo differentiation at stage E4. Proximal cotyledonary tissues in GA 3
and GA7 contained similar levels as E5A while the distal portion (E5DC) contained half
the amount of hormone. This pattern was inverted at stage E6 with less difference
between the cotyledonary parts. Maturity of the seed concurred with near-identical
levels of cotyledonary parts, similar to axes. In contrast, GA4 content in the cotyledons
in E5 and E6 was higher in the proximal portion of the cotyledon (E5PC, E6PC) than in
the distal parts at maturity (E7PC, E7DC) and slightly lower than the axis (E7A).
100
Chapter IV
Fig. 4.7. GAs and BRs global content throughout development. GA3 (a), GA4 (b), GA7 (c), 24EB (d), BK (e)
and HBI (f). Different letters indicate significant differences among means (p ≤ 0.05. Kruskal-Wallis test).
Dark grey bars correspond with proper embryogenic development from bloom to mature embryo axis
while white bars are for cotyledonary tissues. Soft grey bars correspond with controls for development
where samples from isolated trees are in diagonal lines and companion ovules with net. Error bars represent
SE.
Active BRs 24EB (Fig. 4.7d) and HBI (Fig. 4.7e) showed a similar pattern during
development. 24EB was 4 times more abundant than HBI in anthesis (E1) reaching 4.19
µmol hormone/g dry weight although the rest of developmental stages and controls
101
Different hormonal profiles characterize sexual reproduction in C. sativa
levels contained similar values to HBI. The precursor BK (Fig. 4.7f) showed maximum
values during axis development at stages E5 and E6 when the embryo is expanding.
High values could be found as well in isolated ovules at stage E2 (E2I) and in companion
ovules at stage E4 (E4C). In the three studied BRs similar dynamics could be observed
for cotyledonary tissues which resembled the dynamics found in CKs, GA3 and GA7.
4.3.3. Moisture content
During the final stages of embryo expansion and maturation the embryonic axis
doubled in fresh weight (Fig. 4.8), along with an increase in size (Figs. 4.13 and 4.14)
while the moisture content decreased from 87.37 % to 66.11 %. The proximal parts of the
cotyledons at developmental stage E6 (E6PC) showed a transient increase in moisture
content, dropping to 45.84 % at stage E7 (E7PC).
Fig. 4.8. Fresh and dry weight in complete axis and moisture content in axis, proximal and distal cotyledons
during stages 5, 6 and 7. Means and standard errors are represented. Different letters indicate significant
differences (p ≤ 0.05, ANOVA and post hoc Duncan tests).
102
Chapter IV
The distal part of the cotyledons showed continuing loss of moisture reaching
values similar to the proximal part of the cotyledons at stage E7 (48.48 %). Differences
in moisture content between axis and cotyledons were slightly higher at stage E5
(around 30 %) than at mature embryo at stage E7, where cotyledons were approximately
20 % more dehydrated than axis is. Maturation was also associated with the increase of
axis fresh weight from 1.17 mg to 7.73 mg.
4.3.4. Immunodetection
Immunolocalization of IAA and ABA presented specific distribution, depending
on the developmental stage. From anthesis (E1) to mature embryo (E7) ovules can enter
the embryogenic pathway giving rise to embryos that consist of axis and cotyledons, or
die when cross pollination is prevented (developmental stages E2I, E3I) or because of a
lack of fertilization (developmental stages E3C, E4C).
Anthesis and pollination
IAA immunodetection prior to fertilization (Fig. 4.9) showed signal in the tissues
of the ovarian wall and septum but not in the ovule at anthesis (E1; Fig. 4.13b,c). After
cross-pollination IAA signal was distributed in the entire ovules although stronger
intensity was found in the outer integuments (Fig. 4.9e, f). When cross-pollination is not
accomplished (E2I; Fig. 4.9h, i), IAA signal spread into the nucellus. ABA signaling
showed a similar distribution as IAA at stage E1 (Fig. 4.10b, c), without any signal in the
ovule and also after cross-pollination (Fig. 4.10e, f), where it was only found in the outer
integument of the ovule while the inner integument and nucellus were free from the
hormone. The absence of cross-pollination (E2I) was characterized by a dramatic
increase of signal in the whole ovule (Fig. 4.10h, i) that was less pronounced in the
nucellus.
Fertilization
Embryogenesis initiated after fertilization in E3D (Fig. 4.11a-f) concurred with a
very high signal of IAA in the outer integument and to a lesser extent in the embryo and
surrounding endosperm (Fig. 4.11B, c, d, f). Companion ovules (E3C) showed signs of
degradation (Fig. 4.11g) with ubiquitous IAA signal in the whole ovule (Fig. 4.11h, i)
and less intensity in the apical zone. At stage E3D ABA was hardly found in the ovule
but some signal appeared in the endosperm (Fig. 4.12b, c). In the detailed embryo ABA
103
Different hormonal profiles characterize sexual reproduction in C. sativa
had a limited presence (Fig. 4.12e, f). Companion ovules showed signs of tissue
degradation (Fig. 4.12g) and intense ABA signal in the nucellus and inner integument
but a much lower presence in the outer integument of the ovule (Fig. 4.12h, i).
Embryo expansion and maturation
During embryo expansion and maturation (stages E4 to E7; Fig. 4.13a-l) several
changes take place not only in terms of size and shape but also in the determination and
differentiation of new tissues with specific functions such as meristems, vascular tissues
or protective structures (root cap). Growing of the axis was noticeable due to the
increase in size and length (Fig. 4.13a, d, g, j). IAA signal varied significantly in its
distribution throughout development although it was ubiquitous (Fig. 4.13b, c, e, f, h, i,
k, l). Developing embryos at stage 4 (E4D) showed intense IAA signal in the adaxial
zone of cotyledons as well as in the endosperm while hormone was hardly present in
the forming axis (Fig. 4.13b, c). The growing axis at stage 5 (E5A) was characterized by
maintaining a strong signal in the meristems and a gradient could be found from the
RAM to the subapical zone of the SAM (Fig. 4.13e, f); the cortex showed a remarkably
high signal. The opposite situation was found at stage 6 (E6A; Fig. 4.13h, i) where IAA
signal was concentrated in the vascular tissues next to the RAM but most of the signal
disappeared in the rest of the tissues. Signal in the vascular bundles expanded at stage
7A through the vascular bundles and its presence was high in the root cap and the
elongation zone while both meristems displayed a low signal (Fig. 4.13k and l).
ABA signal at stage E4D (Fig. 4.14b, c) was strongly distributed in the adaxial
part of the cotyledons and to a lesser extent in the axis. ABA in E5A was ubiquitous with
less presence in the vascular tissues (Fig. 4.14e, f). In the final steps of maturation the
signal showed a gradient (Fig. 4.14h, i, k, l) opposite to the one found in IAA with higher
intensity next to SAM but no hormone is found in the RAM or vascular bundles. The
mature axis (E7A) presented the opposite gradient than E6A for ABA signal (Fig. 4.14k,
l) except for the maintained signal in SAM. The distribution of ABA in the vascular
bundles and the disappearance of signal in the cortex was also noticeable as well as its
presence in the root cap which did not occur in stages E5A and E6A. This distribution
was similar to that of IAA.
104
Chapter IV
Fig. 4.9. Immunodetection of IAA using confocal microscopy throughout development in longitudinal
sections. DIC of an ovule and surrounding ovary tissues previous to pollination (E1) (a), IAA signal at E1
(b), merged of DAPI (in blue) and IAA (in green) signals at E1 (c), DIC of ovules after cross-pollination at
E2 (d), IAA signal at E2 (e), merged image of DAPI (in blue) and IAA (in green) signals (f), DIC of an ovule
without cross-pollination (E2I) (g), IAA signal at E2I (h), merged of DAPI (in blue) and IAA (in green)
signals at E2I (i). Size bars are 0.2 cm.
105
Different hormonal profiles characterize sexual reproduction in C. sativa
Fig. 4.10. Immunodetection of ABA using confocal microscopy throughout development in longitudinal
sections. DIC of an ovule and surrounding ovary tissues previous to pollination (E1) (a), ABA signal at E1
(b), merged of DAPI (in blue) and ABA (in green) signals at E1 (c), DIC of ovules after cross-pollination at
E2 (d), ABA signal at E2 (e), merged image of DAPI (in blue) and ABA (in green) signals (f), DIC of an ovule
without cross-pollination (E2I) (g), ABA signal at E2I (h), merged of DAPI (in blue) and ABA (in green)
signals at E2I (i). Size bars are 0.2 cm.
106
Chapter IV
Fig. 4.11. Immunodetection of IAA using confocal microscopy throughout development in longitudinal
sections. DIC of the dominant ovule at stage 3 (E3D) after fertilization containing a developing embryo
inside (a), IAA signal at E3D (b), merged of DAPI (in blue) and IAA (in green) signals for E3D (c), DIC of
the developing globular embryo (E3D) (d), IAA signal in the developing embryo (e), merged of DAPI (in
blue) and IAA (in green) signals of the embryo (E3D) (f), DIC of a companion ovule at stage 3 showing
embryo sac degradation (E3C) (g), IAA signal in E3C (h), merged of DAPI (in blue) and IAA (in green) in
the companion ovule without fertilization at stage 3 (E3C) (i). Size bars are 0.2 cm except for (d) in which
size bar is 50 µm.
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Different hormonal profiles characterize sexual reproduction in C. sativa
Fig. 4.12. Immunodetection of ABA using confocal microscopy throughout development in longitudinal
sections. DIC of the dominant ovule at stage 3 (E3D) after fertilization containing a developing embryo
inside (a), ABA signal at E3D (b), merged of DAPI (in blue) and ABA (in green) signals for E3D (c), DIC of
the developing globular embryo (E3D) (d), ABA signal in the developing embryo (e), merged of DAPI (in
blue) and ABA (in green) signals of the embryo (E3D) (f), DIC of a companion ovule at stage 3 showing
embryo sac degradation (E3C) (g), ABA signal in E3C (h), merged of DAPI (in blue) and ABA (in green) in
the companion ovule without fertilization at stage 3 (E3C) (i). Size bars are 0.2 cm except for (d) in which
size bar is 50 µm.
108
Chapter IV
Fig. 4.13. Immunodetection of IAA using confocal microscopy throughout development in longitudinal
sections. DIC of cotyledonary embryo at E4D (a), IAA signal in E4D (b), merged of DAPI (in blue) and IAA
(in green) signals for E4D (c), DIC of embryonic axis at E5A (d), IAA signal in E5A (e), merged of DAPI (in
blue) and IAA (in green) signals for E5A (f), DIC of embryonic axis at stage E6A (g), IAA signal in E6A (h),
merged of DAPI (in blue) and IAA (in green) signals for E6A (i), DIC of mature embryonic axis at stage E7A
(j), IAA signal in E7A (k), merged of DAPI (in blue) and IAA (in green) signals for E7A (l). Size bars are 0.5
cm.
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Different hormonal profiles characterize sexual reproduction in C. sativa
Fig. 4.14. Immunodetection of ABA using confocal microscopy throughout development in longitudinal
sections. DIC of cotyledonary embryo at E4D (a), ABA signal in E4D, (c) merged of DAPI (in blue) and ABA
(in green) signals for E4D (b), DIC of embryonic axis at E5A (d), ABA signal in E5A (e), merged of DAPI (in
blue) and ABA (in green) signals for E5A (f), DIC of embryonic axis at stage E6A (g), ABA signal in E6A
(h), merged of DAPI (in blue) and ABA (in green) signals for E6A (i), DIC of mature embryonic axis at stage
E7A (j), ABA signal in E7A (k), merged of DAPI (in blue) and ABA (in green) signals for E7A (l). Size bars
are 0.5 cm.
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Chapter IV
4.4. DISCUSSION
An overview on hormonal control during sexual reproduction
The combined analysis of hormones levels from flowering to mature embryo in
the clustering and PCA showed four developmental scenarios based on the
physiological status of the samples triggered by consecutive milestones in chestnut
reproduction: pollination, fertilization, early embryo development associated with
differential ovule abortion and maturation of the embryo. Clustering separation of
developmental stages into three groups clearly emphasized the importance of crosspollination as the main effector leading to sexual embryo development. Active embryo
development concurred with the highest values in hormone levels indicating active
roles while maturation and non-embryonic stages concurred with medium or low levels
due to a decrease in or low level of metabolism. The performed PCAs supported, in
addition, the tight control exerted by the studied hormones in the coordination of the
sequential developmental stages by modulating the sense and strength of their actions.
Thus, PCA analysis for the first 4 stages of development clearly demonstrated the
heterogeneity of the physiological processes going on as variability explained by
components 1 and 2 is hardly over 50 % and coincided with association of specific
hormones for non-embryogenic related stages (E1, E2I, E3C and E3I). The initial
reproductive development of ovules (E2 and E3D) along with E4D was governed by a
different set of hormones, mainly CKs and GAs GA3 and GA7; in addition, E4D was
characterized distinctly by its positive correlation with GA4 and IAA. The proximity of
E2 and E4C in the PCA revealed the plasticity in the way that the hormones act in the
physiology of the plant, showing the action of both CKs and GAs in the ovules’ response
to pollination (E2) previous to the entrance in the embryogenic pathway and the death
of ovules at stage E4C. In contrast to early embryo development, all hormones acted in
a similar way during maturation, as the PCA shows for component 1 while much less
variation was found in component two, emphasizing the strongest direction of
development in order to achieve maturity.
Pollination
Pollination concurred with several hormonal changes in terms of global content
and it has an evident effect on the ovules. (Fig. 4.15), representing the relative
contribution of every hormone in the developmental stages analyzed, showed that
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Different hormonal profiles characterize sexual reproduction in C. sativa
cross-pollination (E2) was associated with decreases in JA, ZR, 24EB and increases in
the rest of CKs, IAA, ABA and JA.
Fig. 4.15. Hormonal load throughout developmental stages based on concentrations quantified.
The dramatic increase of ABA and JA representing more than 70 % of the
hormonal content in autopollination (E2I) suggests a differential mechanism in chestnut
regarding pollination that determines not only the destiny of those ovules leading to
fertilization in E2 and abortion in E2I but also the pollen recognition at the stigma in the
ovules. Localization of ABA in the ovules showed how upon cross-pollination the
hormone spread from the outer integument in E1 to the inner in E2. Similar results were
obtained for immature ovules from Arabidopsis and cucumber prior to pollination (Peng
et al., 2004); in contrast, autopollination was characterized by an ubiquitous signal in the
ovule which suggests cross-talk between stigma and ovules after pollination, as well as
support the growing body of evidence pointing to an early determination of ovule fate.
Moreover, the increase of IAA as described by Hayata et al. (2002) for muskmelon
coincided with our results at the ovule level after cross pollination where the increase
of CKs global levels except for ZR was also found. Besides, this IAA increment is in
accordance with its role for ovule development (Sundberg and Østergaard, 2009) that
continues after pollination (Feijó et al., 1999). Pollination of any kind also had a clear
effect on IAA distribution within the ovules; while cross-pollination concurred with the
appearance of signal mainly in the integuments of the ovule, IAA is ubiquitous after
112
Chapter IV
self-pollination. This result was the opposite of that found in Olea europaea (Solfanelli et
al., 2006). Nevertheless, in Petunia the only stimulus of pollination was found to be
sufficient for the development of parthenocarpy, and the reduction of CKs in the global
load of hormones in E2I was in accordance with the sink effect of the stigma for CKs
(Kovaleva and Zakharova, 2004). CKs in E3I ovules constituted more than 70% of the
global hormone load at that stage, coinciding with the known role of CKs along with
other plant regulators in parthenocarpy (reviewed in Sundberg and Østergaard, 2009)
which is known to occur in chestnut under autopollination conditions and act in
collaboration with IAA and GA3 as recently proposed (Ding et al., 2013). Individual
increments in the global hormone level found for GA3 and GA7 resembled CKs
dynamics, which coincided with their known role during pollination (reviewed by
Pharis and King, 1985). GAs along with BRs (except for BK) were found in small
quantities during pollination which was reflected in their contribution to the global
hormone load (Fig. 4.15), but PCA analysis showed important correlation of both
hormone groups with pollination events suggesting a putative role in the process.
Fertilization and ovule abortion
Subsequent to fertilization (developmental stage 3) the main unresolved
question in chestnut reproduction arises: which is the mechanism by which usually only
one fertilized ovule develops into the monoembryonic seed while the rest of the ovules
abort within the containment of the ovary? This question was contemplated by
Mogensen in 1975 for Quercus and is in extension applied to Castanea as noted by Feijó
et al. (1999). Mogensen (1975) concluded that there could be an (up to date)
undetermined mechanism by which the first fertilized ovule would induce the death of
its companions. Fertilization marks the beginning of zygotic embryogenesis and the
dominant ovule (E3D) was characterized by an increase in IAA that together with CKs,
the latter constituting almost 70 % of the global hormone content in the ovules,
determines the polarity of the embryo as described for several species by Bennici and
Cionini (1979) and Steves and Sussex (1989). The dominance of CKs at this stage
coincided with the observations by Lulsdorf (2013) in two Cicer species and was also in
accordance with the capacity of CKs of creating source-sink relationships (Riefler et al.,
2006), essential for embryo development. The examination of the individual behaviour
of CKs after fertilization showed how the highest increase corresponded with ZR (MVA
pathway) as described by Lulsdorf (2013) for CKs in general, or remained invariable
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Different hormonal profiles characterize sexual reproduction in C. sativa
(DHZR and DHZ) but decreased in the MEP CKs (iPA and iP) and also BA, which in
the case of iPA and iP could be associated with their role as substrate for MAV CKs.
Similarly to dominant ovules (E3D), ovules from isolated trees (E3I) displayed peaks in
ZR and DHZR that could be a reflection of the active growth of the ovary regarding
parthenogenesis; these results are in accordance with Ding et al. (2013) where the crosstalk is described between CKs and other hormones in the tomato parthenogenesis.
Early embryogenesis at E3D was associated with a small decrease in ABA global
levels which was reflected in the low abundance and the signal distribution of the
dominant ovule. In contrast, the growth promoting effect of IAA is evident since it was
found in the developing ovule and in every tissue of the embryo. This pattern regarding
IAA and ABA is in accordance with Obroucheva (2014) under cross-pollination
conditions. However, in ovules that do not enter the embryogenic pathway (E3C), the
same distribution for IAA and ABA was found although histological signs of tissue
degeneration appeared. This could be explained by the cut-off of assimilates from the
plant, maintaining the same distribution as in the previous developmental stage (E2).
Moreover, increases of JA and ABA were found in companion ovules (E3C) and
maintained in the posterior developmental stage (E4C), which can be associated with
the beginning of cell death. This phenomenon has been associated with increments of
JA in cells (Overmyer et al., 2003), and would also be explained by the fact that JA is a
subproduct of the lipase and lipoxygenase activity in cell membranes mediated by the
alteration of ion channels associated with cell damage (Creelman and Mullet, 1995).
Content analysis of hormones did not seem to provide sufficient information to
elucidate whether the dominant ovule is governing the death of its companions after
being fertilized, although it contributed to the growing body of evidence proposed by
Mogensen in 1975. On the one hand, clustering separated E3C and E3I into one
subgroup in the PCA which indicated their similarity, distinct from the rest of the
developmental stages; on the other hand, there was a clear differential hormonal load
when comparing stages E3C and E3I probing the involvement of fertilization in the
abortion response. The equimolar relationship of the hormones appeared to be involved
in the fate of ovaries at this developmental stage.
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Chapter IV
Embryo expansion
Embryo expansion started in stage E4D. A growing axis and cotyledons could
be clearly identified in the histology and IAA showed a strong presence in the adaxial
part of cotyledons as well as in the endosperm which is in consonance with Vanneste
and Firml (2009), who described the production of this hormone in the endosperm of
Arabidopsis even when most of it is reabsorbed in later development. Proliferation of
organs at this stage concurred with an inversion in the percentages of IAA and CKs (Fig.
4.15), which is in accordance with the capacity of IAA to induce cytokinin biosynthesis
as shown in the individual hormones levels graphs in the later stage E5A; this result is
in accordance with the IAA:CKs ratio that govern organ proliferation (Su et al., 2011).
IAA has also been associated with the promotion of GA1, GA3, GA4 and GA7 synthesis
(Slater et al., 2000); however, we only found a concurring peak for GA4 at E4D. This
result would point to differential roles of GA3/GA7 vs GA4. Indeed, auxins have been
associated with the conversion of GA9/GA20 to the active forms GA4/GA1 (Hedden et
al., 2000), which is in support of our observations.
Embryo maturation
GA4 was intimately associated with ABA during embryo development and seed
filling in a determinant dynamic balance (Alabadi et al., 2009; Liu et al., 2010). The
beginning of embryo maturation in E5A entailed an inversion of the ratio GA4:ABA (Fig.
4.15) due to the increase of ABA global levels, which is in accordance with Seo et al (2006)
who described the negative regulation of GAs synthesis by ABA. The promoting
germination effect of GA4 in the immature embryo would be blocked by ABA as claimed
by Rodríguez-Gacio (2009) and for axes from stages 5 to 7 (E5A, E6A,E7A) ABA
predominated over GA4 reinforcing its key role in embryo maturation in accordance
with Nambara and Marion-Poll (2003). ABA showed the strongest change in global
levels during development at E6Axis when a drop in moisture content was observed;
this would be in accordance with the positive regulation of storage compounds
(Finkelstein et al., 2002). The final decrease in ABA content found in both axis and
cotyledons also resembled the Arabidopsis model for zygotic embryos. Globally, the
maturation dynamics for ABA is in accordance with Pérez et al. (2015) for the Fagaceae
Q. suber.
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Different hormonal profiles characterize sexual reproduction in C. sativa
ABA global content during the last stages of embryo development was reflected
in its tissue distribution and contrasted with IAA, suggesting dynamic roles during the
process. ABA disappeared from the axis in E6 (E6A) including the signal in the vascular
tissues. Taking into account that the ABA antibody recognizes not only the free form of
ABA but also its conjugate form as an ester (Weiler, 1980), this pattern could be due to
the hormone transport through the axis and also its presence at embryo maturity in E7
(E7A) could be related with water transport related with dehydration of the embryo
(Sauter and Hartung, 2000). Given that the maintenance of stem cells in RAM is ABAdependent (Zhang et al., 2010) and that the root meristem governs the next step of
development of the embryo (germination), these balances in the signal distribution
suggest tissue-specific control of RAM functions during maturation. ABA was
differentially distributed in the root cap between the root cap proper and the root cap
initials. Schraut et al. (2004) described the root cap as a tissue with anion trap properties
for ABA, which would affect the availability of hormone for the RAM. Our results
support this idea showing a strong presence of ABA in the root cap initials at E5A with
RAM ABA signaling and its disappearance in later stages as RAM shows no ABA while
the mature embryo displays ABA signal only in the root cap. The dynamism of signal
gradients during maturation along with the gradual disappearance and migration to
the leaf primordia of ABA in SAM emphasized that transient distributions of hormones
seem to be intrinsic during normal embryo development. A similar pattern for signal
distribution was found for IAA in SAM, which along with the gradual loss of IAA in
the RAM during maturation resulting in its absence in E7A supports the idea of the key
role played by IAA in the establishment and determination of meristems (Dinneny and
Benfey, 2008). Besides, during E5 and E6, the axis showed a decreasing signal gradient
from RAM to SAM that is lost in maturity at stage E7. These results are in accordance
with the widely accepted idea of IAA acting as a morphogen through the regulation of
its own distribution along the target tissues (Berleth, 2001).
The last steps of embryo development until maturity requires the translocation
of assimilates from the photosynthetic organs of the plant for seed filling as shown by
the augmentation of dry weight of the embryos from E5 to E7. Clustering and PCA
analysis showed strong convergence of all hormones during maturation. CKs are
known to be involved not only in the establishment of source-sink relationships during
plant development but also in the strength of this relationship as reviewed by Roitsch
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Chapter IV
and Ehneβ (2000). Moreover, studies in cytokinin receptors (Riefler et al., 2006)
demonstrated their importance in seed size, which can be behind the relative high
amount of CKs in the cotyledons during maturation. CKs share dynamics with BRs and
also GAs whose medium values could be partially associated with the accumulation of
fatty acids in the seed (Chen et al., 2012), suggesting coordinated action during embryo
maturation. PCA analysis strongly supported this explanation as CKs and GAs are the
hormones that correlate the strongest with late embryo development. Moreover, the
proximal parts of cotyledons seemed to contribute to the partial dehydration of the
embryo as noted in the transitory increase of their fresh weight at E6PC along with the
decrease of the axis fresh weight at that stage (E6A), indicating the translocation of water
from the embryonic axis as it increases its dry weight towards maturity.
Summarizing, accomplishment of the mature embryo within the seed as unit of
dispersion encompasses several crucial steps, beginning with pollination and finishing
with the full-grown seed as a quiescent independent organism, on which the successful
establishment of the next generation depends. The external stimuli and the endogenous
control of the sexual reproduction are controlled through dynamic changes in the
hormone content, effectively influenced by both the mother plant and the embryo in a
coordinated and complex fashion. Plant hormones, although discovered many decades
ago, still reveal novel aspects of plant sexual reproduction. In this work we described
not only the spatial-temporal variations in endogenous hormone levels during zygotic
embryogenesis and ovules death, which depending on the pollination donor concur
with differential hormonal responses with the same result; but also tried to shed light
on the cross-talk among them, as shown by the dynamic changes in hormones ratios
such as GA4:ABA or IAA:CKs, which in last instance stand for a reflection of the
physiological status of the different tissues through zygotic embryo development until
reaching maturity. Moreover, most of the hormones followed very close dynamics
obeying to their global levels range as observed with GAs, CKs and BRs, indicating that
basal levels must be maintained during embryo development.
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Different hormonal profiles characterize sexual reproduction in C. sativa
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CHAPTER V
Gene expression dynamics linked to
physiological milestones in C. sativa
sexual reproduction
Chapter V
5.1. INTRODUCTION
Sexual reproduction involves several crucial steps from anthesis to mature seed.
In the perennial chestnut tree flowering, pollination, fertilization, seed growth and
filling and the establishment of the mature embryo must be performed in a manner that
ensures the viability of the next generation within a specific time of year. Among the
factors associated with reproduction, individuals’ interactions during the pollination
window are critical for the initiation of sexual embryo development. Thus, cross
pollination is necessary in order to start the embryogenic pathway and is concomitant
with the death of companion ovules, which constitutes a common way to avoid
polyembryony that would otherwise reduce the quality of the seed. The control of
reproduction is ultimately controlled at the genetic level, in which gene expression must
be orchestrated in a strict fashion responding to the complexity of the mother plantembryo interactions and the ongoing processes taking place.
Of all the mechanisms involved in the regulation of gene expression, epigenetic
marks are known for their key role during ontogenesis as previous studies reported
(reviewed in Viejo et al., 2012) for several developmental processes such as phase
change, flowering or embryo development in several species. The combination of
reversible epigenetic modifications in a given genome, in a specific spatial-temporal
spot as a consequence of environmental and endogenous conditions, constitutes the
epigenome. It is composed by DNA cytosine methylation and PTMs (Chinussamy and
Zhu, 2009), the latter including a great variety of modifications in aminoacids residues
such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation and
ADP ribosylation (Yu et al., 2011).
In previous works on chestnut, we associated different dynamics for global
levels of DNA methylation (Viejo et al., 2010), histone H4 acetylation and other PTMs
with defined developmental stages during reproduction as well as the histological
distribution of 5-mdC and H4ac from anthesis to mature embryo (see Chapters 2 and 3).
Even if global levels of epigenetic marks are analyzed and their localization in the
tissues during reproduction reveal the connection between epigenetic marks and
physiology, there is an evident lack of knowledge of this species regarding differential
gene expression associated with the epigenetic control of the processes in progress at a
specific time of development. Some embryo-related housekeeping genes have been
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reproduction
found to be specifically expressed during embryogenesis and are compulsory for its
success such as the embryo defective genes (EMB genes; Tebbji et al., 2010) and also
aminoacyl-tRNA synthetases (Berg et al., 2005). After fertilization, the phase of early
embryogenesis starts with the establishment of the embryo within the ovary. This
developmental stage is concomitant with high rates of cell division which must be under
strict control in order to define the shape and size of the embryo. Among the genes
involved in cell division, Aurora genes are well characterized in both animals and plants
(Hofmann, 2011) and are responsible for the phosphorylation of the 10th serine residue
of the tail of the histone H3 (H3S10) in a fluctuating fashion during the cell cycle.
Besides, these proteins collaborate with other players involved in epigenetic gene
regulation such as the HAT GCN5 which is known to acetylate several residues in H3
and H4 associated with specific gene expression during flowering and embryo
development (Servet et al., 2010). Another HAT with a domain resembling the GCN5
domain that is involved in many biological processes is the subunit ELP2 of the
Elongator complex, a multiprotein complex that interacts with RNA polymerase II, in
which the subunit ELP3 also participates and constitutes the catalytic unit (Wang et al.,
2013). Deacetylation by HDACs and acetylation by HATs take place in an antagonistic
manner in order to maintain the required epigenetic marks through chromatin during
development (Berr et al., 2011). Of the 18 HDACs described in Arabidopsis (Luo et al.,
2012), HDA6 and HDA19 have been associated with a wide variety of processes such as
abiotic stress response (Perrella et al., 2013), pathogen response (Zhou et al., 2005), or
repression of embryonic properties after germination (Tanaka et al., 2008), among
others. HDA6 in Arabidopsis is necessary for jasmonate response, senescence and
flowering (Wu et al., 2008) and is also involved in the transposable element silencing
(Liu et al., 2012) by direct interaction with methyl transferase 1 (MET1).
DNA methylation by methyl transferases can depend on SAMS which generates
SAMe, a universal donor of methyl groups, and methylation is known to govern specific
developmental stages during plants life (Li et al., 2011). On the other hand, ethylene,
which has a role during plant reproduction, is synthesized by SAMS (Yang and
Hoffman, 1984). Methyl groups are also involved in histones PTMs, and depending on
the combination of histones epigenetic marks, transcription activation or repression
takes place (Zhang et al., 2007). Ubiquitination of histones, specifically histone H2B, is
126
Chapter V
associated to gene regulation itself and through complexes with methylated histones
(Gu et al., 2009).
Despite the vast knowledge accumulated in Arabidopsis regarding the control of
gene expression and its relation with epigenetic marks, references regarding differential
gene expression in chestnut is scant. Santamaría et al. (2011) published an interesting
work in chestnut using as experimental system the dormant and non-dormant buds and
their association with epigenetics-related genes. In order to contribute to a better
understanding of chestnut reproduction concerning epigenetic control and ovules fate,
the aim of this work is to study the expression profile of genes associated with epigenetic
control throughout chestnut reproduction.
5.2. MATERIAL AND METHODS
5.2.1. Plant material
Burrs were collected in 2013 from mid-July to mid-November from several openpollination trees in chestnut stands in Carreño (Asturias, Spain) based on the previously
described developmental stages of the ovules (E1 to E4) and growing embryos (E5 to
E7; Chapter 2; Viejo et al., 2010) whithout taking into account the location of the ovaries
within the burr. Burrs from isolated trees (autopollinated) were collected at the same
collecting dates for stages E2 and E3 and used as controls for proper crosspollination/fertilization events.
Burrs were immediately dissected extracting ovaries and obtaining ovules from
stages E1 to E4 and embryonic axes from stages E5 to E7. Material was frozen in liquid
nitrogen and stored at -80 °C.
5.2.2. RNA isolation and complementary DNA (cDNA) synthesis
Total RNA was isolated from 100 mg of frozen tissues on different
developmental stages following Santamaría et al. (2010) using a concentration of 1.4
mg/ml of PVP from the described in that work. RNA concentration and quality was
estimated using a Picodrop Microliter UV/Vis Spectrophotometer (Picodrop, United
Kingdom) and by horizontal electrophoresis in agarose gels (1.5 % and 0.5 µg/ml
ethidium bromide), using lambda DNA (New England Biolabs) as marker. The 28S/18S
127
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
ratio was also determined with the KODAK 1D Image Analysis software (KODAK) to
verify RNA quality.
Total RNA was purified using the RNeasy Plant Mini Kit (Qiagen, Crawley, UK)
and 1 µg of RNA was reverse transcribed using the RevertAidTM First Strand cDNA
Synthesis Kit (Thermo Scientific) according to the supplier protocol and using 1 µl of
oligo (dT)18 primer. Samples were incubated at 25 °C for 10 min, 60 min at 37 °C, 60
min at 42 °C and a final step of 10 min at 70 °C in an Applied Biosystems 2720 Thermal
Cycler (Applied Biosystems).
5.2.3. Selection of genes for real-time PCR
Housekeeping genes during chestnut reproduction
To select a pair of constitutive genes as endogenous control for mRNA
quantification, eight genes were tested (Table 1). The selection of expressed sequence
tags (ESTs) of the genes was made based on their presumed constitutive expression and
taking into account previous work in chestnut (Santamaría et al., 2010) and in other
Fagaceae species (Quercus suber; Marum et al., 2012; Soler et al., 2008).
The evaluation of their housekeeping character was performed with the geNorm
software (Vandesompele et al., 2002) to select the two most stable genes. Subsequently,
the reference genes for normalizing data were also selected based on the parameters of
highest stability across samples (lower standard deviations) and highest abundance
(lower Ct) according to Soler et al. (2008).
Table 5.1. List of primers designed from the ESTs included in Santamaría et al. (2010) used for the evaluation
of endogenous controls. PL, product length.
Gene
ATUB1
ATUB4
ATUB5
ATUB6
ATUB7
BTUB5
GAPDH
ACTIN
Genbank
accession nº
HO847568
HO847484
HO847557
HO847390
HO847578
HO847563
HO847579
Universal
Forward primer (5’ 3’)
Reverse primer (5’3’)
CTCCAACTGTGGTTCCTGGT
ATTGAGCGACCCACCTACAC
GGCATTTGTGCACTGGTATG
TTGAGGGGATGGGTAGACAG
CGAGTAGCACCGAGACATCA
GCCCCGATAACTTCGTTTTT
GTGACAGCAGGTCGAGCATA
TCCATCATGAAGTGCGATGT
CCCTCTTCGCCTACATCAAA
TTCTGGGGTATGGAACCAAG
AGCAGACTCAGCACCAACCT
CTGTTGGTGGAGGAACAGGT
CTGTCTACCCATCCCCTCAA
GCCTCCTTACGAACAACGTC
TCAACCACACGGGAACTGTA
AACCTCCGATCCAGACACTG
PL
(bp)
132
139
114
110
104
115
115
188
Selection of genes for real-time PCR
Genes of interest for real-time PCR were selected based on their involvement in
epigenetic
128
mechanisms
[AURORA3
(CsAUR3),
ETHYLENE-RESPONSIVE
Chapter V
TRANSCRIPTION FACTOR A1 (CsERFA1), GENERAL CONTROL NON-REPRESSED
PROTEIN5-
LIKE
GENE
(CsGCN5L),
HISTONE
MONO-UBIQUITINATION2
(CsHUB2), RADICAL SAM DOMAIN-CONTAINING PROTEIN (CsRADSAM), SADENOSYL-L-METHIONINE SYNTHETASE2 (CsSAMS2), HISTONE DEACETYLASE6
(CsHDA6) and HISTONE DEACETYLASE19 (CsHDA19)] and their relation with
embryogenesis [EMBRYO DEFECTIVE 1345 (CsEMBD) and OVULE ABORTION 3
(CsOVA3)].
All the primers were designed based on the ESTs from Santamaría et al. (2010)
with Primer 3 web 4.0.0 (Rozen and Skaletsky, 1999; Table 5.2) except for CsHDA6 and
CsHDA19 that were obtained from previous work in Q. suber (Pérez et al., 2015)
Table 5.2. List of primers used for the Real Time-PCR of the genes of interest. PL, product length.
Gene
AUR3
EMBD
ERFA1
GCN5L
HUB2
OVA3
RADSAM
SAMS2
HDA6
HDA19
Genbank
accession nº
HO847372
HO847205
HO847234
HO847125
HO847187
HO847407
HO847524
HO847411
JZ719311
JZ719310
Forward primer (5’ 3’)
Reverse primer (5’3’)
ATTCCGAACGCATTTTCTTG
GGTTTGGGCACTTTCTTTCA
ACCACGCATCCTAAAAGCAG
CGCATCTTTCTTCGCTCTCT
TGCCTGCTTTGTCTTCACAC
TTGGACCTTGCGAACAAGTA
GGTTAGTGCAAGGCGAACAT
CTGGAAAGATTCCCGACAAG
CCTCGCCGACGTTAAACCTCTTG
CACCATGGAGATGGTGTGG
CTCATGACAATACGCCACG
CTCCAAGGAGCATAGCCATC
CCTCAAGCAGCCTAGAACCA
TCATCGTCTCCATCCTCCTC
ACGCAGAAGCAGAGGCATAC
AGCAGCAGCAGGAGTTTCTC
GCTTGGAACCTCAGCTATCG
TCCCTTCCGAAGTGTCCATA
CGGCCAACCCCTCCGACAT
TCGCGTATGTCACCTGTACC
PL
(bp)
143
126
194
147
155
149
149
148
129
107
Validation of genes putative identity
Putative identity of the ESTs analyzed was performed in two ways: ESTs were
blasted with a nucleotide BLAST (basic local alignment search tool- nucleotide;
BLASTn) confirming their identity according with Santamaría et al. (2011) for Castanea
genes and Quercus genes from Pérez et al. (2015; data not shown). On the other hand,
after designing the primers and prior to performing real-time RT-PCR, putative identity
of the PCR products from the chestnut genes was assessed by sequencing and then
alignment (ClustalW2 software) with the ESTs previously described (Santamaría et al.,
2011), obtaining 100 % of query identity (data not shown). Likewise, PCR products from
Quercus primers QsHDA6 and QsHDA19 were sequenced and then aligned, obtaining
100 % of query identity (data not shown). All the PCR products were sequenced on an
ABI 3700 automated sequencer (Perkin-Elmer, Foster City, CA, USA) and inconsistences
in the sequences corrected with Geneious 8.4.2 software prior to their analysis.
129
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
Real-time PCR conditions
The cycling conditions comprised an initial 20 s polymerase activation at 95 °C
followed by 50 cycles at 95 °C for 1 s and 60 °C for 20 s; this was followed by a final
incubation of 15 s at 95 °C, 15 s at 60 °C and 15 s at 95°C. Each PCR was performed in
duplicate for each biological replicate and three biological replicates were used for each
stage. PCR efficiency was tested using a standard curve for each gene with LinReg PCR
11.0 software (Ruijter et al. 2009). Analysis of dissociation curves was performed to check
gene-specific amplification with the SDS 2.3 software (Applied Biosystems) and agarose
gel electrophoresis (1 % and 0.5 µg/ml ethidium bromide) of the PCR products was
used to verify amplicon size.
Relative expression analysis
For each gene of interest, relative expression values were calculated and
expressed as fold-change using the ΔΔCt method (Livak and Schmittgen, 2001),
normalized for the selected housekeeping genes using the mean of their Ct values and
expressed relative to developmental stage E1.
5.2.4. Statistical analysis
Changes in gene expression among developmental stages were analyzed by
Kruskal-Wallis test (significance level 0.05) in R Statistical Environment (R core team,
2012) core functions plus the package agricolae (de Mendiburu, 2014).
5.3. RESULTS
5.3.1. Validation of chestnut housekeeping genes during reproduction
All of the potential housekeeping genes were successfully amplified and their
stability values were analyzed using GeNorm algorithm (Vandesompele et al., 2002).
Their expression stability value showed that ACTIN and GAPDH were the two most
stable genes (Fig. 5.1) while BETA TUBULIN1 was the least stable.
130
Chapter V
Fig. 5.1. Stability values of candidate reference genes calculated using GeNorm software and cDNA samples
from all the developmental stages.
The constitutive expression of the reference genes studied was also validated by
a box and whisker plot (Fig. 5.2). ACTIN showed the lowest standard deviation
associated with high stability among the developmental stages evaluated and the lowest
Ct value corresponding with high transcript abundance. Among the batch of genes
examined, GAPDH displayed low standard deviation, although higher than ACTIN,
and a Ct value close to the median of all the reference genes analyzed.
Taking into account the GeNorm and the box and whisker plot analysis, ACTIN
and GAPDH were selected as reference genes for the normalization of the genes of
interest.
131
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
Fig. 5.2. Box and whisker plot of the variation of candidate reference gene expression. Each box indicates
the 25/75 percentiles and the horizontal line inside each box indicates the median. Whiskers represent the
maximum and minimum values and outliers are indicated by dots.
5.3.2. Determination of relative expression of the genes of interest
All the analyzed genes showed significant differences between developmental
stages (Fig. 5.3). CsAUR3 expression decreased during fertilization and maturation of
the embryo with a transient increase in E6Axis. Abortion of ovules from autopollinated
trees showed a decrease after pollination and in the later stage E3I. Companion ovules
(E3C) presented lower expression levels than dominant ovules after fertilization (E3D).
Cross-pollination had an effect on CsEMBD as expression peaked in E2 while a decrease
was found in autopollination (E2I) and maintained in the posterior E3I stage.
Development of the embryo towards maturation went along with a gradual decrease in
expression up to E7 where the axis presented the lowest expression level. Companion
ovules showed a medium expression value, lower than the dominant ovule. CsERFA1
also showed differential expression depending on the kind of pollination accomplished:
while cross pollination did not change its expression, autopollination increased its value
and decreased in the next stage E3I. Embryo development from E3D to E7 concurred
with a decrease up to the beginning of maturation at stage E5 and increased two-fold in
E6 and three-fold in E7 with respect to E5.
132
Chapter V
Fig. 5.3. Relative gene expression of CsAUR3, CsEMBD, CsERFA1, CsGCN5L, CsHUB2, CsOVA3,
CsRADSAM, CsSAMS2, CsHDA6 and CsHDA19 during the 10 developmental stages studied assessed by
real time-PCR. Expression is relative to ACTIN and GAPDH and normalized for stage E1. Identical letters
above the bars indicate no significant differences between stages. (p ≤ 0.05. Kruskal-Wallis test).
133
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
The highest expression level was found in the companion ovules (E3C)
contrasting with the maintenance of low levels in the developing embryo. CsGCN5L
expression dynamics were characterized by a transient decrease upon fertilization and
a second peak in expression later during embryo development, decreasing to a
minimum at maturity (E7). CsHUB2 expression was associated with increases after
pollination in both pollination types and maintained level in E3I. Embryo development
concurred with no variation in the expression levels including companion ovules. Very
similar dynamics were found for CsOVA3 with similar expression values. Slight but
significant variation in expression was found for CsRADSAM. CsSAMS2 expression
profile showed a transient increase after cross-pollination in E2 ovules but not in E2I.
Embryonic development develops with similar values after fertilization (E3 stages on)
with higher values in companion ovules (E3C) than in dominant ovules (E3D) and
maturity of the embryo associates with a transient increase in E6A previous to embryo
maturation at E7. On the other hand, the development of normal reproduction was
characterized by the increase after fertilization in E3D and a transient decrease during
embryo expansion at E4D and then the recovery of expression levels after fertilization
until the end of development. Autopollination (E2I), in contrast with cross-pollination,
concurred with an increase in the expression level that is maintained in E3I. The
expression pattern for CsHDA6 and CsHDA19 was shared for both genes presenting a
gradual decrease in expression as embryogenesis progressed, reaching the lowest value
in maturity at stage E7. Companion ovules (E3C) showed lower levels than their
dominant homologue (E3D) while autopollination (E2I) concurred with a transient
decrease in both CsHDA6 and CsHDA19, returning to high values in the abortive ovules
E3I.
5.3.3. Determination of conserved domains in the analyzed ESTs
The ESTs from the genes of interest were subject to BLASTx and conserved
domains were found in every single EST analyzed. In the Figure 5.4a it is shown that
the EST from CsAUR3 contains a conserved catalytic domain with presence in AuroraB kinases among others and that the EST CsEMB (Fig. 5.4b) contains a WD40 domain
associated with signal transduction in eukaryotes. Figure 5.5a shows that CsRFEA1
contains a DNA binding AP2 domain that interacts with transcription factors involved
in the ethylene responsiveness while CsGCN5L (Fig. 5.5b) contains a GCN5L1 domain.
CsHUB2 gene (Fig. 5.6) possesses two domains within the EST analyzed, a TATA
134
Chapter V
element modulatory factor and also a GCN5L1 domain. CsOVA3 (Fig. 5.7a) contains a
glutamyl-tRNA synthetase conserved domain and the EST for CsRADSAM an
Elongator protein 3 domain (Fig. 5.7b). Finally, from the C. sativa ESTs studied,
CsSAMS2 (Fig. 5.8) has been found to possess an S-adenosylmethionine synthetase
domain.
CsHDA6 and CsHDA19, shown homology with the conserved domains Class I
histone deacetylases (Fig. 5.9a) and histone deacetylase 1 domain (Fig. 5.9b),
respectively.
135
a
Domain
Castanea s. HO847372
Pp XP_007220840.1
Pm XP_008232701.1
Md XP_008354754.1
Pxb XP_009379282.1
Tc XP_007052016.1
Eg EYU32016.1
STKc_Aurora-B_like, cd14117, 7.78e-46
VEHQLRREIEIQSHLRHPNILRLYNYFHDRKRIYLILEYAPRGELYKELQKHGRFDEQRTATFMEELADALHYCHEKKVIHRDIKPENLLMGYKGELKIADFGWS
KIQHQLKREMEIQTSLRHPNILRLYGWFHDSERIFLILEYAHGGELYGELRKRGFLSENKAATYIMSLAQALAYCHEKHVIHGDIKPENLLLDHEGRLKIADFGWS-----------------------KIQHQLRREMEIQTSLRHPNILRLYGWFHDDERIFLILEYAHGGELYGLLRKTNYLSEEQAATYILSLTQALAYCHEKNVIHRDIKPENLLLDHEGRLKIADFGWSVQSRSKRQTMCGTLDYLAPEMVEN
KIQHQLRREMEIQTGLRHPNILRLYGWFHDDERIFLILEYAHGGELYGLLRKTNYLSEKQAATYILSLTQALAYCHEKNVIHRDIKPENLLLDHEGRLKIADFGWSVQSRSKRQTMCGTLDYLAPEMVEN
KIQHQLRREMEIQTSLRHPNILRLYGWFHDDDRIFLILEYAHGGELYGLLRKTTYLSEKQAATYILSLTEALAYCHEKHVIHRDIKPENLLLDHEGRLKIADFGWSVQSRSKRHTMCGTLDYLAPEMVEN
KIQHQLRREMEIQTSLRHPNILRLYGWFHDDDRIFLILEYAHGGELYGLLRKTTYLSEKQAATYILSLTEALAYCHEKHVIHRDIKPENLLLDHEGRLKIADFGWSVQSRSKRHTMCGTLDYLAPEMVEN
RIHHQLRREMEIQTSLRHPNILRLYGWFHDSERIFLILEYAFGGELYKELRKNGHLSEKQAATYIASLTKALAYCHEKHVIHRDIKPENLLLDHEGRLKIADFGWSVQSTSKRRTMCGTLDYLAPEMVEN
RLHHQLRREMEIQTGLRHPNVLRLYGWFHDDERIFLILEYAHGGELYKELRKLGNLSERKAATYIASLTQALAYCHEKHVIHRDIKPENLLLDHEGRLKIADFGWSVQSRSKRHTMCGTLDYLAPEMVEN
:::***:*******.*****:*********.:*********.***** ***
***.:*******::********:*** ***********************
100
188
188
190
190
190
194
b
Domain
Castanea s. HO847205
Pp XP_007202301.1
Pm XP_008241233.1
Nn XP_010271653.1
Vv XP_002282694.1
Eg XP_010036689.1
Mn XP_010101568.1
Domain
Castanea s. HO847205
Pp XP_007202301.1
Pm XP_008241233.1
Nn XP_010271653.1
Vv XP_002282694.1
Eg XP_010036689.1
Mn XP_010101568.1
cd00200, WD40,
7.92e-09
LTGHTGEVNSVAFSPDGEKLLSSSSDGTIKLWDLSTG----KCLGTLR----GHENGVNSVAFSPDGYLLASGSEDGTIRVWDLRTG-----------ECVQTL
--------------------------LQGHTQDVNMVMWHPTLDVLFSCSYDNTVKVWADDDD--DWQCVQTLGEPNNGHSSTVWALSFNKTGDKMVTCSDDLTPKIWEMDGTSMQSADGYAPWRHLCTI
LATCGRDKTVWIWEVQPGNEFDCVAVLQGHTQDVKMVQWHPSRNLIFSCSYDNTVKIWADEGDDDDWACVQTLGETNNGHSSTVWALSFNDGGDKMVTCSDDLTLKIWGTDNEKMQSTDDFVPWRHLCTL
LATCGRDKTVWIWEVQPGNEFDCVAVLQGHTQDVKMVQWHPSRNLIFSCSYDNTVKIWADEGDDDDWACVQTLGETNNGHSSTVWALSFNDGGDKMVTCSDDLTLKIWGTDNEKMQSTDDFVPWRHLCTL
LATCSRDKSVWIWEVQPGNEFECVAVLQGHTQDVKMVKWHPFMDVLFSCSYDNTVKVWAEDGDTDDWHCVQTLGEPNNGHTSTVWALSFNSTGDKMVTCSDDLTLKIWETDSRIPQETDGYMPWRHLSTL
LATCSRDKSVWIWEVQPGNEFECVSVLQGHTQDVKMVQWHPIMDVLFSCSYDNTVKIWAEDGDSDDWHCVQTLGESNNGHTSTVWALSFNPEGDKMVTCSDDLTVKIWDTDSITMQAGEGYAPWKHLCTL
LATCGRDRSVWIWEVLPGNEFECASVLQGHTQDVKMVQWHPTMDILFSCSYDNTIKIWAEDGD-DDWHCVQTLSEANGGHTSTVWALSFNTAGDKMVTCSDDLTIKVWETDSAKMMSGDGYVPWSHVCTL
LATCGRDKTVWIWEVLPGNEFECAAVLQGHTQDVKMVQWHPTVDVLFSCSYDNSIKIWADEGDDDDFVCVQTLDEPSNGHTSTVWALSFNSSGDKMVTCSDDLTLKIWGTDLERMHSGDGYAPWRHLCTI
********:** *** :::*******::*:**::.* *: *****.*...**:********* ************ *:* *
:.: ** *:.*:
SGHTN-SVTSLAWSPD
TGYHDRTIFSVHWSSEGIIASGAADDAIRFFVED--KDGLVDGPSYKLLLKEEKCTWHGYKFQCMEPWGKTTIDFC---------------SGYHDRTIFSVHWSRDNIIASGAADDTIRFFVENDDKDGLVDGPSYKLLLKKEKAHDMDINSVQWSPGEDRILASAADDGTIKIWELTSAGSGYHDRTIFSVHWSRDNIIASGAADDTIRFFVENDDKDGLVDGPSYKLLLKKEKAHDMDINSVQWSPGEDRILASAADDGTIKIWALTSAGTGYHDRTIFSVHWSSEGVIASGAADDAIRLFIEN--KDGLVDGPSYKMLLKKEKAHDMDVNSVQWSPKEQRLLASASDDGTIKIWEMVPSSSGYHDRTIFSAHWSREGIIATGAADDAIRFFVES--KDGLVDGPLYKLMLKKEQAHDMDINSVQWSSGENRLLASASDDGTIKIWELASITSGYHDRTIFSVHWSREGIIASGAADDAIRFFVES--KDGSVDGPSYKMILRNEKAHDMDVNSVRWSHG-----VSLS----LSI-------AGYHDRTIFSVHWSREGIIASGAADDAIRFFVEDNEKDGLVDGPKYKLLLKEEKAHEMDVNSVQWSPGEKRLLASASDDGTIKIWELASVPY
:*********.*** :.:**:*****:**:*:*.*** **** **::*::*:.
. :
.
102
260
260
264
255
260
261
176
351
351
353
344
347
353
Fig. 5.4. Translated amino acid alignments of C. sativa CsAUR3 (a), CsEMB (b) with truncated orthologous sequences from 6 different species. Genbank accessions are provided
with species names. ClustalW2 was applied to perform the alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Below the alingment, “*” indicates identical residues, “:”
indicates conserved substitutions and “.” Indicates semi-conserved substitutions. Coincident residues between the conserved domain and the sequences are highlighted in red.
Conserved domain name and its similarity with C. Sativa sequence is provided in the first line. Pp, Prunus persica; Pm, Prunus mume; Md, Malus domestica; P×b, Pyrus ×
bretschneideri; Th, Theobroma cacao; Eg, Erythranthe gutatta; Nn, Nelumbo nucifera; Vv, Vitis vinifera; Mn, Morus notabilis.
a
Domain
Castanea s. HO847307
Tc XP_007025492.1
Vp ADC94860.1
Cs XP_006467696.1
Ej AFG26327.1
Pxb XP_009352827.1
Pp XP_007212119.1
cd00018, AP2, 6.68e-15
YRGVRQRPWGKWVAEIRDPS-GGRRIWLGTFDTAEEAARAYDRAALKLRGSSAVLNFP
P--QRHQIN-TSSSLEPARNIGKKHYRGVRRRPWGKYAAEIRDSARHGARIWLGTFTTAEEAALAYDKAAFRMRGAKALLNFPAEVVAASSVQRFNPNSSMVSSTKHD------------SESSGS--SR
P--PRNQSS-ATNMLEPSRTIAKKHYRGVRRRPWGKYAAEIRDSTRNGARVWLGTFVTAEEAALAYDRAAFRMRGAKALLNFPAEVVAASSVQRLRPNLSSTSSEKTNPDSGSSCSTLSISESESS--TT
PPIKHQTTP-TSNALEPARTIAKKHYRGVRRRPWGKYAAEIRDSAKHGARTWLGTFETAEEAALAYDRAAFRMRGSKALLNFPAEVVAASSQRTPKPNLSSENLKRDASNSSNSSAASSIRCSASS--AS
P--QRSEVH-TSSMLEPKRTISKKHYRGVRRRPWGKFAAEIRDSARHGQRIWLGTFETAEEAALAYDRAAFRMRGTKAMLNFPAEIVAASSPPTSSVHRFRPSFSIPCSLNSKNSTTTSDSSGSSGMLSV
P--QRHHIHRQPNRLEPTKNIGKKHYRGVRRRPWGKYAAEIRDSARQGARVWLGTFNTAEEAALAYDRAAFRMRGTKAMLNFPAEIVAASSPPTSSVHRFRPSFSIPCSLNSKNSTTTSDSSGSSGMLSV
P--QRHHIHRQPNRLEPTKNVGKKHYRGVRRRPWGKYAAEIRDSARQGARVWLGTFNTAEEAALAYDRAAFRMRGTKAMLNFPAEIVAASSPPTSSVHRFRPSFSIPCSLNSKSSITTSDSSGSSSMPSV
P--QRHQINHQPNGLEPSKNIGKKHYRGVRRRPWGKYAAEIRDSARHGARVWLGTFSTAEEAALAYDRAAFRMRGTKALLNFPPDVVAASSSPSSSIHRVRPSFSVPCSSNSNS--TTSDSSGSSSLLSI
*
:
.. *** :.:.**************:*******:::* * ***** **********:**
113
160
158
142
180
180
163
b
Domain
Castanea s. HO847125
Mn XP_010087167.1
Md XP_008355074.1
Jc KDP45729.1
Rc XP_002517726.1
Vv XP_002270028.1
Cc XP_006446618.1
Domain
Castanea s. HO847125
Mn XP_010087167.1
Md XP_008355074.1
Jc KDP45729.1
Rc XP_002517726.1
Vv XP_002270028.1
Cc XP_006446618.1
GCN5L1, pfam06320, 2.16e-29
LLKEHQAKQAELREVQERLRREAIASANALTDALVDTVNAGVAQAYANQKRLEAEAKALQATSAAFAKQTEQWLTLIENFNTALKEIGD
---PPQLPLASGRVPSSSPSSSFSFDAQHADPGGLEAALLQIMHDHHHTSFRLRDQAERAKKDAIQNAARVSDLLVDAVNSGVQESFINEKRIEREIRALAVTIARFMKQTNQWLTPTHAINTAVKEIGD
MYSPTPLPVAR-ARVTP---------SSDAEPGGLESSLLQLVHDHHQSSLRLRELTEKKKKEAIRNAARVSDLLVEAVNGGVQEFFVNEKCIELEIRALAATITRFMKQTDQWLAATHAINTAVKEIGD
MHSQTSLPLAHGAVASPSTFS----FSSEPEPGGVEEALLQLVQDHHHVSLRLRDATEKATKDAIKKAARAADLMVEAVNGGVQEAFVNEKRIEYEIRALAATIARFSKQTDQSLSVTHSMNTAIKEIGD
-MYPPQLPLARTRVVSPQDVD-----KSQAEPGGLEASLLQLMQDHHNTSLRLRDRTEKAKKDAIRNAVRVSDLLMDAVNGGVQESFINEKRIELEIRALAVTISRFMRQTDQWLAATHAINTAIKEIGD
-MSSPQLPLARPRVASPWEID-----KPQPEPGSLEGSLLQLIQDHHQTSLRLRENTEKAKKDAIRKAAKVSGLLMDAVNGGVQESFINEKRIEFEIRALAATVSRFMRQTDHWLTATHAINTAIKEIGD
-MFTQPLPVARARVLSPAEIE-----RPNADPSGLEASLLQLIQDHHQTSLKLRDETEKAKNDAIRTAMRVSDLLVDTVNGGVQEAFINEKRIELESRALTATVIRFAKQTHQWLAASHAINTAIKEIGD
MSSPPQFPPASTRVQATAETEKAQADTTAAAAGGLEASLLQLIQNHQHSSLKLREQTERAKRYSVRHAERVSDLLTDALNGGVQESYVIEKRIELEIRTLAATIAKFMKQTDQWLATSHAINTAVKEIGD
:* *
:.
. ...:* :***::::*:: *::**: :*: .. ::: * :.:.*: :::*.**** :: ** ** * *:*:.*: :* :**.: *: :*::***:*****
VENWARSIENDMKTIASALEEAYEA
FENWMKTMEFDCKSITAAIQNIHQA-PFCKLCSMFFVK-SVI-Y-LLHSNETCCSTSIFWCL-WRCPSSNSPPPTIKVSRKKMY
FENWMKTMEFDCKSVVAAIHNIHQE----------------------------------------------------------FENWMKIMEFDCKSITAAIHNIHQA----------------------------------------------------------FENWMKTMEFDCKSISTAIRNIHQ-----------------------------------------------------------FENWLKTMEFDCKSINAAIRTIHQ-----------------------------------------------------------FENWMKTMDFDCRSINAAIRNIHQP----------------------------------------------------------FENWMKTMDLDCKSINAAIRNIYQD----------------------------------------------------------****:* *::**:*: :**:.*:*
127
120
136
124
124
124
130
206
145
151
148
148
149
155
Fig. 5.5. Translated amino acid alignments of C. sativa CsERFA1 (a) and CsGCN5L (b) with truncated orthologous sequences from 6 different species. Genbank accessions are
provided with species names. ClustalW2 was applied to perform the alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Below the alingment, “*” indicates identical
residues, “:” indicates conserved substitutions and “.” Indicates semi-conserved substitutions. Coincident residues between the conserved domain and the sequences are
highlighted in red. Conserved domain name and its similarity with C. Sativa sequence is provided in the first line. Th, Theobroma cacao; Vp, Vitis pseudoreticulata; Cs, Citrus sinensis;
Ej, Eriobotrya japonica; P×b, Pyrus × bretschneideri; Pp, Prunus persica; Mn, Morus notabilis; Md, Malus domestica; Jc, Jatropha curcas; Rc, Ricinus communis; Vv, Vitis vinifera; Cc, Citrus
clementina.
Domain
Castanea s. HO847187
Ga KHG11716.1
Gr KJB23336.1
Tc XP_007014753.1
Vv XP_010651350.1
Jc KDP44344.1
Cc XP_006445836.1
Domain
Castanea s. HO847187
Ga KHG11716.1
Gr KJB23336.1
Tc XP_007014753.1
Vv XP_010651350.1
Jc KDP44344.1
Cc XP_006445836.1
Domain
Castanea s. HO847187
Ga KHG11716.1
Gr KJB23336.1
Tc XP_007014753.1
Vv XP_010651350.1
Jc KDP44344.1
Cc XP_006445836.1
TMF_TATA_bd, pfam12325, 7.78e-04
RRL--------------------------------------------------------KKMSLKLRWKKLCRMQGERILN----------------QSFV-WLRLCLKKWE---------WKLS-SDGRRQ---------------YSKQLKDLQNELKDDKFIQSSRLYTLLNDQLQHWNAEMEQYKALIDALQTDRFLVMRREKELNMKAETADAVRNTINNADSRIEELELQLQKCIIERNDLEIKMEEAIQDAGRNDIKAEIRVMASALSKE
YSKQLKDLQNELKDDKFIQSSRMYTLLNDQLQHWNAEMEQYKALTDSLQTDRFLVMRREKELNMKAETADAVRNTINNADSRVEELELQLQKCIIERNDLEIKMEEAIQDAGRNDIKAEIRVMASALSKE
YSEQQQDLQNELKDEKFVQSSRLYTLLSDQLQHWNAEVEQYKALTDALQTDRFLVMRREKELNLKAESADAARNIIDNADSRIEELELQLQKCIIERNDLEIKMEEAIQDAGRNDIKAEFRVMASALSKE
LSKQLQDLQNELKDDKYVYSSRPYTLLNDQLQHWNAEAERYKLLTDSLQADRAQVVRREKELNAKSELADAARSVIEN-DSKIEELELQLQKCLIEKNDLEVKMKEALQDSGRKDIKAEFHVMASALSKE
LLKELEDIKDELKDDKHVQSSRLYNLVNDQLQHCNAEAERYKALTSSLQADRSLVVRREKEVNVKIESADAARSTIDTAESRIEELELQLKNCVIEKNDLEIKMEEAIQDSGRKDVKAEFRVMAAALSKE
LSKQLENLQNELNDDKYVHSSRLYNLVNDQLQHWNVEVERYKALTDSLLIDRSLVLRREKEINVRAESADAARNTVDDSESRIERLEVQLQKSIIEKNDLGLKMEEAIQDSGRKDIKAEFRVMASALSKE
* :
* .:
:: *: :*
:* : *.: *::
: : .*. *:
GCN5L1, pfam06320, 3.54e-17
--------------EGELASLKDELARLEAERDEARQEIVKLTEENEE----LKELKKEIEELEKELEDLETTLELLGEKSERRADVVDLKE
VAQAYANQKRLEAEAKALQATSAAFAKQ
--------------PHDALSLSEEAQSLKAQLDRKSNEMQSLSDKCAEQMMEIKSLKELIDKLQKEKLELQIFLDLYGQESHDNRDLVDIKESERRAHSQAEVLRNALDEHSLELRVKAANEAEAACQQR
MGMMEAQLNRWKETAHEAISLHEEAQALKALLSDKTNLQKHLAEECAEQIVEIKSLNDMIEKMQKEKLELQIFLDMYGQEGYDNRDVMEIRESENRAHSQAEILKNALDEHSLELRVKAANEAEAACQER
MGMMEAQLNRWKETAHEAISLHEEAQALKALLSDKTNLQKRLAEECAEQIAEIKSLNDMIEKLQKEKLELQIFLDMYGQEGYDNRDVMEIRESKNRAHSQAEILKNALDEHSLELRVKAANEAEAACQER
MGMMEAQLNRWKETAHEAISLREEAQTLKDVLSDKTNQGKRLAEECAEQIVEIKSLKGLIEKLQKEKLELQIFLDMYGQEGYDNRDVMEIREAENRAHSQAEVLKNALDEHSLELRVKAANEAEAACQER
MGMMESQLNRWKETAHEALSLREQVQSLKALLNKKTNEQKCLADKCEEQMVEIKSLKALIEKLQKGKLELQIFVDMHGQESYDNRDLMEIKESEHKAHMQAEVLRNALDEHSLELRVKAANEAEAACQQR
MGMMEAQLNRWKQTAHEALSLREKSESLRASLTEKTNEQKCLTRKCAEQISEIKSLKTLIEKLQKEKLELQIILDMYGQEGYDSRDMLEIKESERKARLQAEVLRSALDEHGLELRVKAANEAEAACQQR
MGMMEAQLNRWKETADEALSLREKAVSLKVSLSAKTNEQKRLTDKCVEQMAEIKSLKALIEKLQKDKLESQIMLDMYGQEGHDPRDLMEIKESERRAHSQAEVLKNALDEHSLELRVKAANEAEAACQQR
..:*:** *: :*: * *:* : *: :* **: *****: :*:*:** *** **::*::***.:* **:::*:*::.:*: ***:*:.*****.****************:*
T----EQWLTLIENFNTALKEIGDVENWARSIENDMKTIASALE---EAYEA
LSAAEAEIADLRVKLDASERDVLELTEAIRNKDAEAEAYIAEIETIGQAYEDMQTQNQHLLQQVTERDDYNLKLVSESVKTKQAQNALLLEKQALEKQLQQINASIESLKMRISHSEEQMEPCLTEVIKC
LSVAEVEIADLRAKLDASERDVLELTEAIKSKDRESETYISEIETIGQAYEDMQTQNQHLLQQMTERDDYNIKLVSESVKTKQAHSFLLSEKQALARQLKQVNSSIESVKMRIGQSEEQIKVCLTDAVKF
LSVAEVEIADLRAKLDASERDVLELTEAIKSKDRESETYISEIETIGQAYEDMQTQNQHLLQQMTERDDYNIKLVSESVKTKQAHSFLLSEKQALARQLKQVNSSIESVKMRIGQSEEQIKVCLTDAVKF
LSVAEAEIAELRAKLDASERDVLELKEAIKSKDLESEAYISEIETIGQAYEDMQTQNQHLLQQMTERDDYNIKLVSESVKTKQAQSFFLTEKQTLARQLEQVNSSIKSVKMRIAHSEEQMKVCLTEAIKS
LSAAEAEIADLRAKLDASERDVLELKEAIRIKDVEAEAYISEIETIGQAYEDMQTQNQHLLQQVTERDDYNIKLVSESVKTKQMQSFLLSEKQALAKQLQQVNNALESLKMRIAQSEEQMKVCLAEALKY
LSAAEAEIAELRMKLDTSERDVWELTEAIKSKDREAEAYISEIETIGQAYEDMQTQNQHLLQQVAEREDYNIKLVSESVKTKQAQSSLLSEKQALTKQLQQVNASVEYVKMRIAQSEEQMKVCLTEAIRY
LSAAEAEIIELVAKLDASERDVMELEEAMKSKDREAEAYIAEMETIGQAFEDMQTQNQHLLQQVAERDDLNIKLVSESVKTKQVQSFLLSEKQALARQLQQINALVESAKLRILHAEEQMKACLTEALRY
**.**.** :* ***:***** ** **:: ** *:*:**:*:******:*************::**:* *:*********** :. :* ***:* :**:*:* :: *:** ::***:: **::.::
26
458
458
202
251
460
474
162
594
594
338
387
596
610
292
724
724
468
517
726
740
Fig. 5.6. Translated amino acid alignments of C. sativa CsHUB2 with truncated orthologous sequences from 6 different species. Genbank accessions are provided with species
names. ClustalW2 was applied to perform the alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Below the alignment, “*” indicates identical residues, “:” indicates
conserved substitutions and “.” Indicates semi-conserved substitutions. Coincident residues between the conserved domain and the sequences are highlighted in red or blue
in the case of the second conserved domain. Conserved domain name and its similarity with C. Sativa sequence is provided in the first line. Pp, Prunus persica; Pm, Prunus
mume ; Nn, Nelumbo nucifera; Vv, Vitis vinifera; Eg, Eucalyptus grandis; Mn, Morus notabilis; Md, Malus domestica; Jc, Jatropha curcas; Rc, Ricinus comumunis; Vv, Vitis vinifera;
Cc, Citrus clementina.
a
Domain
Castanea s. HO847407
Gr KJB12357.1
Mn XP_010090722.1
Cs KDO68365.1
Cc XP_006422417.1
Vv CAN72214.1
Pt XP_002313571.2
Domain
Castanea s. HO847407
Gr KJB12357.1
Mn XP_010090722.1
Cs KDO68365.1
Cc XP_006422417.1
Vv CAN72214.1
Pt XP_002313571.2
GluRS_core, Cd00808, 3.09e-40
VRTRFAPSPTGFLHIGGARTALFNYLFARKHGGKFILRIEDTDQERSVE
HRITELNLGLRLRLRTMATTLIGTTPSWMRISEVAP---PPSIFRRSCLFFHKRFGSSSSSSRSFSISAASAEDKAEV---VRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRVEDTDLERSTK
----------------MAALVAGTP--WMRIRVIP--EFAPPFIFR----------RHFR--RNFSVRASIDSDAP-----VRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRIEDTDLERSTR
-----------------MATIVGAP--WTRIRFYP--EVAPPFLRRSPLFYRSKRIQDFRRIRTFSVSAKNSGEERN----VRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRIEDTDLERSTK
----------------MASIVAATP--WSRIRTITKLELAPPIFLQSS--------VYYCKRRRFSVAASLSTNTNKVDGQVRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRIEDTDLERSTK
----------------MASIVAATP--WSRIRTITKLELAPPIFLQSS--------VYYCKRRRFSVAASLSTNTNKVDGQVRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRIEDTDLERSTK
-----------------MASLVGSP--WMKIRVIP--EVAPPILRRSS--------SLFR--RSFSVSCSXEAPPKKLEGEVRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRIEDTDLERSTK
---------------MANSIIAGTP--WMRIRVIP--EISFPILRSSSSLYNHKVSFLFPTRRRFSVSAIASTEKEQ----VRVRFAPSPTGNLHVGGARTALFNYLFARSKGGKFVLRIEDTDLERSTK
: : .:. * :*
.
. .::
* **: .
**************************************:*********:
EAEEAILEALKWLGIDWDEGPDVGGPYGPYRQSQR
QSEDALLRDLSWLGLHWDEGPGVGGDYGSYRQSERNSL-------------------------------ESEEAVLRDLAWLGLDWDEGPGVGGDYGPYRQSERNAMYKQYAEKLLESGHVYRCFCSNEELEKMKEIAE
ESEEAMLQDLSWLGLDWDEGPGVGGDYGPYRQSERNSMYKHYAEKLLESGHVYRCFCSNEELEKMKEIAK
ESEEAVLQDLSWLGLDWDEGPGVGGDYGPYRQSERNSLYKQYADKLLESGHVYRCFCSNEELEKMKEIAK
ESEEAVLQDLSWLGLDWDEGPGVGGDYGPYRQSERNSLYKQYADKLLESGHVYRCFCSNEELEKMKEIAK
QSEEALLQDLSWLGLHWDEGPGVGGDYGPYRQSERNSLYKQHAEKLLESGHVYQCFCSNEELEKMKEIAK
ESEEAVLRDLSWLGLDWDEGPGVGGDYGPYRQSERNSLYKQHAEKLVESGHVYRCFCSNEELEQMKEIAK
:**:*:*:**:****.************.*******::
124
93
105
104
104
99
107
162
163
175
174
174
169
177
b
Domain
Castanea s. HO847524
Pxb XP_009362442.1
Mn XP_010104491.1
Pt XP_006376236
Pe XP_011001084.1
Rc XP_002521816.1
Vv CBI18159.3
Domain
Castanea s. HO847524
Pxb XP_009362442.1
Mn XP_010104491.1
Pt XP_006376236
Pe XP_011001084.1
Rc XP_002521816.1
Vv CBI18159.3
Elp3, smart00729, 6.00e-19
VQSGDDEVLKAINRGHTVEDVLEAVELLREAGP-IKVSTDLIVGLPGETEEDFEETLKLLKELGPDRVSIFPLSPRPGTPLAKMYKRLKPPTKEERAEL
--------SFLHVPVQSGSDTVLNAMNREYTVSEFRTVVDTLTELVPGMQIATDIIYGFPGETDEDFAQTVSLVKEYKFPQVHISQFYPRPGTPAARMKKVPSNVVKKRSRELTSIFEAFTPYNGMEGRL
VLRHPCVYSFLHVPVQSGSDAVLTAMNREYTVSEFKTVVDTLTELVPGMQIATDIICGFPGETDEDFTQTLSLIKEYKFSQVHISQFYPRPGTPAARMKKVPSTLVKKRSRELTSAFEAFAPYVGMEGRV
VLHHPCVYSFLHVPVQSGSDAVLTAMNREYTVSEFRTVVDTLTELVPGMQIATDIICGFPGETDEDFAQTIGLINKYKFPQVHISQFYPRPGTPAAKMKKVPSTIVKKRSRELTSVFEAFTPYNGMEGRV
VLRHPCVYSFLHVPVQSGSDAILTAMNREYTVNEFRTVVDTLTELVPGMQIATDIICGFPGETDKDFSQTVNLIKAYKFAQVHISQFYPRPGTPAARMKKVPSNIVKQRSRELTSVFEAFTPYNGMEGRV
VLRHPCVYSFLHVPVQSGSDAILTAMNREYTVNEFRTVVDTLIELVPGMQIATDIICGFPGETDKDFSQTVNLIKAYKFAQVHISQFYPRPGTPAARMKKVPSNIVKQRSRELTTVFEAFTPYNGMEGRV
VLRHPCVYSFLHVPVQSGSDNVLNAMNREYTVSNFRTVVDTLTELVPGMQIATDIICGFPGETDDDFAQTVSLINEYKLPQVHISQFYPRPGTPAARMKKVPSNIVKKRSRELTAVFEAFTPYNGMEGRV
VLRHPCVYSFLHVPVQSGSDAILSAMNREYTVTEFRTVVDTLTELVPGMQIATDIICGFPGETDEEFAQTVSLIQEYRFPQVHISQFYPRPGTPAARMKKVPSAVVKKRSRELTSIFEAFTPYNGMEGRV
************ :*.********.:*:****** ************* *******.:*:**:.*:: *::.****************:****** :**:******: ****:** *****:
ERIWITEIATDGIHLVGHTKGYVQVLVIAPESMLGTSAIVKITSVGRWSVFGEVIETINHINDESSLRNNKPSQAKCSPCTNPIESCAC------------------------------ERIWITDIATDGIHLVGHTKGYVQVLVAAPESMLGTSAIAKITSVGRWSVFGEVIETIPHINDRTASTNETRSQEKCFPGANNCETCACSTEPETCACGPESCGGQATPGECAVTRNDVL
ERIWITDIATDGVHLVGHTKGYVQVLVVAPESMLGTSAIVKITSVGRWSVFGEVIETIQDVNYKKTSSTRSSSENKCSPCSDPCNACASSRVQETCACGPEGCGG-TTLEESAVSTNAIP
ERIWITDIAADGIHLVGHTKAYVQVLIVAQESMLGTSAIVKITSVGRWSVFGEVIETLNQINQKSKSVEKMLSEEKCSPCSDPCDSCACSGESEPCACGPESCGGQSTIEQSDVLQNEVL
ERIWITDIATDGIHLVGHTKAYVQVLIVAQESMLGTSAIVKITSVGRWSVFGEVIETLNQTNQKSKSVEKMLSEEKCSPCSDPCDSCACSGESEPCACGPESCGGQSTSEQSDVLQNDVL
ERIWITEIATDGIHLVGHTKGYVQVLVIAPETMLGTSAIVKITSVGRWSVFGEVIQTLNQTNRGVASAEKMPSGGKYSPCSDPCETCACSKEPESCACGPESCGGQNPLEESAIAQNDML
ERIWISEIATDGIHLVGHTKGYMQVLVVAPRSLMGTSAIVKITSVGRWSVFGELIETLNQVNDNISLNEEKFSLGKCSPCSVPGEICACSREAEPCACEPQSCEGKISMEEGSVSRKDML
*****::**:**:*******.*:***: * .:::*****.*************:*:*: . *
. * * * :
: **.
122
350
421
412
425
430
350
211
470
540
532
545
550
470
Fig. 5.7. Translated amino acid alignments of C. sativa CsOVA3 (a) and CsRADSAM (b) with truncated orthologous sequences from 6 different species. Genbank accessions
are provided with species names. ClustalW2 was applied to perform the alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Below the alignment, “*” indicates
identical residues, “:” indicates conserved substitutions and “.” Indicates semi-conserved substitutions. Coincident residues between the conserved domain and the sequences
are highlighted in red. Conserved domain name and its similarity with C. Sativa sequence is provided in the first line. Gr, Gossypium raimondii; Mn, Morus notabilis; Cs, Citrus
sinensis; Cc, Citrus clementina; Vv, Vitis vinifera; Pp, Populus trichocarpa; P×b, Pyrus × bretschneideri; Pe, Populus euphratica; Rc, Ricinus comumunis; Vv, Vitis vinifera.
Domain
Castanea s. HO847411
Gr KJB24465.1
Cm XP_008447451.1
Pxb XP_009367157.1
Md XP_008343205.1
Fxa AFI38954.1
Fv XP_004288342
Domain
Castanea s. HO847411
Gr KJB24465.1
Cm XP_008447451.1
Pxb XP_009367157.1
Md XP_008343205.1
Fxa AFI38954.1
Fv XP_004288342
S-AdoMet_synt_C, pfam02773, 5.13e-51
IGGPQGDAGLTGRKIIVDTYGGWGAHGGGAFSGKDPTKVDRSAAYAARWVAKSLVAAGLARRCLVQVSYAIGVAEPLSIMVDTYGTSKKSEEELLEIVRKNFDLRPGVIVKMLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVANGLARRAIAQVSYAIGVPEPLSVFVDTYGTGKIPDKEILKIVKENFDFRPGMITINLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVANGLARRCIVQVSYAIGVPEPLSVFVDSYGTGKIPDKEILQIVKENFDFRPGMITINLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVAAGLARRAIVQVSYAIGVPEPLSVFVDTYGTGKIPDKEILKIVKENFDFRPGMITINLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVANGLARRAIVQVSYAIGVPEPLSVFVDTYGTGKIPDKEILKIVKETFDFRPGMITINLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVANGLARRAIVQVSYAIGVPEPLSVFVDTYGTGKIPDKEILKIVKETFDFRPGMITINLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYVVRQAAKSIVANGLARRAIVQVSYAIGVPEPLSVFVETYGTGKIPDKEILKIVKENFDFRPGMITINLD
LDEKTIFHLNPSGRFVIGGPHGDAGLTGRKIIIDTYGGWGAHGGGAFSGKDPTKVDRSGAYIVRQAAKSIVANGLARRALVQVSYAIGVPEPLSVFVETYGTGKIPDKEILKIVKENFDFRPGMITINLD
*************************************************************:********** *****.:.****************::********
LKKP---IYQQTAAYGHFGRDD--FPWE
LEGGGNGRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS-IVLSYPLLFNACFNCYFDE-FACLLAAII-NYSSCSNPLPKISSSIISFLFIFYFKYIFHFYVIMLQVS-CNEKLMRILSKLWH
LKRGGNGRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS------------------------------------------------------------------------------------LKRGGNSRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS------------------------------------------------------------------------------------LKRGGGGRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS------------------------------------------------------------------------------------LKRGGGGRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS------------------------------------------------------------------------------------LKRGGNKRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS------------------------------------------------------------------------------------LKRGGNKRFLKTAAYGHFGRDDPDFTWEVVKPLKWEKPQS------------------------------------------------------------------------------------****:****.**************: **. *********************************
130
353
353
353
353
353
353
251
393
393
393
393
393
393
Fig. 5.8. Translated amino acid alignments of C. sativa CsSAMS2 with truncated orthologous sequences from 6 different species. Genbank accessions are provided with species
names. ClustalW2 was applied to perform the alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Below the alignment, “*” indicates identical residues, “:” indicates
conserved substitutions and “.” Indicates semi-conserved substitutions. Coincident residues between the conserved domain and the sequences are highlighted in red.
Conserved domain name and its similarity with C. Sativa sequence is provided in the first line. Gr, Gossypium raimondii; Cm, Cucumis melo; P×b, Pyrus × bretschneideri; Md,
Malus domestica; F×a, Fragaria × ananassa; Fv, Fragaria vesca.
a
Domain
Quercus s. JZ719311
Cs XP_004138094.1
Gm XP_003525556.1
Cc XP_006439900.1
Cs XP_006476865.1
Tc XP_007036338.1
Nn XP_010248621.1
Domain
Quercus s. JZ719311
Cs XP_004138094.1
Gm XP_003525556.1
Cc XP_006439900.1
Cs XP_006476865.1
Tc XP_007036338.1
Nn XP_010248621.1
Cd09991, HDAC_classI, 6.92e-88
VGNYYYGQGHPMKPHRIRMTHSLILSYGLYKKMEIYRPRPATAEELTKFHSDDYIDFLRSVSPDNMK--EFKKQLERFNVGEDCPVFDGLY
---------------------------------------IGKYYYGQGHPMKPHRIRMAHNLIVHYSLHRRMEINRPFPANPSDIRRFHSDDYVEFLASVTPETLSDHSFSRHLKRFNVGEDCPVFDGLF
--MSDD-------IHGGASLPS-GPDGRKRRVTYFYEPTIGDYYYGQGHPMKPHRIRMAHNLIVHYGLHRRMEINRPYPAGPEDIRRFHSDDYVDFLASVSPETLSDHAFSRHLKRFNVGEDCPVFDGLF
MGMEEESSNNSSIIEGGASLPSTGSDAKKRRVTYFYEPTIGDYYYGQGHPMKPHRIRMAHNLIVHYSLHRRMEINRPFPASPADIRRFHSDDYVDFLSSVSPETLADSAFSRHLKRFNVGEDCPVFDGLF
--MEEP--------TEGASLVS-GPDGKKRRVSYFYEPTIGDYYYGQGHPMKPHRIRMAHNLIVHYGLHRRMEVNRPFPAGPSDIRRFHTDEYVEFLASVSPESSGDPSFSRHLKRFNVGEDCPVFDGLF
--MEEP--------TEGASLVS-GPDGKKRRVSYFYEPTIGDYYYGQGHPMKPHRIRMAHNLIVHYGLHRRMEVNRPFPAGPSDIRRFHTDEYVEFLASVSPESSGDPSFSRHLKRFNVGEDCPVFDGLF
--MEDS--------AGGASLPS-GPDAKKRRVTYFYEPTIGDYYYGQGHPMKPHRIRMAHNLIVHYSLHRRMEINRPFPAGPADIRRFHTDEYVDFLNSVSPESISDPTYSRHLKRFNVGEDCPVFDGLF
--MEDA--------SGSASLPG-GPDAKKRRVCYFYEPSIGDYYYGQGHPMKPHRIRMAHNLIVHYSLHRRMEVNRPYPAGPDDIRRFHSDDYVEFLASVTPETLHDHTHSRHLKRFNVGEDCPVFDGLF
**.************************.******:***:**.* ******:*:**:** **:**: * :.********************
91
120
130
119
119
119
119
EYCQLYAGGSIAAAVKLNRGQADIAINWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGD
GFCQSSAGGSIGAAVKLNRGDADIALNWAGGLHHAKKSEASGFCYVNDIVLGILELLKVHRRVLYVDIDVHHGD-------------------------------------------------------GFCQASAGGSIGAAVKLNRGDADIAINWAGGLHHAKKSEASGFCYVNDIVLGILELLKYHKRVLYIDIDVHHGDGVEEAFYTTDRVMTVSFHKFGDFFPGTGHIKDVGVGTGKNYALNVPLNDGMDDDSF
PFCQASAGGSLGAAVKLNRADADIAINWAGGLHHAKKSEASGFCYVNDIVLGILELLKVHRRVLYVDIDVHHGDGVEEAFYTTDRVMTVSFHKFGDFFPGTGHVKDIGVGSGKNYAVNVPLNDGMDDESF
GFCQASAGGSIGAAVKLNRGDADIAVNWAGGLHHAKKSEASGFCYVNDIVLGILELLKVHRRVLYVDIDVHHGDGVEEAFYTTDRVMTVSFHKFGDFFPGTGHIRDVGAGQGKYYALNVPLNDGLDDESF
GFCQASAGGSIGAAVKLNRGDADIAVNWAGGLHHAKKSEASGFCYVNDIVLGILELLKVHRRVLYVDIDVHHGDGVEEAFYTTDRVMTVSFHKFGDFFPGTGHIKDVGAGQGKFYALNVPLNDGLDDESF
GFCQASAGGSIGAAVKLNRGDADIAINWAGGLHHAKKSEASGFCYVNDIVLGILELLKVHRRVLYVDIDVHHGDGVEEAFYTTDRVMTVSFHKFGDFFPGTGHIRDVGVGNGKHYALNVPLNDGMDDESF
PFCQASAGGSIGAAVKLNRGDADIALNWAGGLHHAKKSEASGFCYVNDIVLGILELLKVHRRVLYVDIDIHHGDGVEEAFFTTDRVMTVSFHKFGDYFPGTGHLKDIGAGPGKYYAMNVPLNDGMDDESF
***:*****:********.*****:******************************** *:****:***:****
165
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249
249
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249
b
Domain
Quercus s. JZ719311
Rc XP_002531796.1
Vv XP_002283371.1
Gm XP_009358923.1
Pxb XP_009358923.1
Md XP_008348955.1
Fv XP_004290100.1
Domain
Quercus s. JZ719311
Rc XP_002531796.1
Vv XP_002283371.1
Gm XP_009358923.1
Pxb XP_009358923.1
Md XP_008348955.1
Fv XP_004290100.1
Cd10010, HDAC1, 1.06e-111
AGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEAFYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYY
-------------------------------------------AGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGYGKGKYY
RFNVGEDCPVFDGLYSFCQTYAGGSVGGAVKLNHGLCDIAINWAGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGYGKGKYY
RFNVGEDCPVFDGLYSFCQTYAGGSVGGAVKLNHGLCDIAVNWAGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGFGKGKYY
RFNVGEDCPVFDGLYSFCQTYAGGSVGGAVKLNHDQCDIAVNWAGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDVRDIGYGKGKYY
RVNVGEGRPVFDGLYSFCQTYAGGSVGGAVKLNHGICDISINWAGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGYGKGKYY
RFNVGEDCPVFDGLYSFCQTYAGGSVGGAVKLNHGICDISINWAGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGYGKGKYY
RFNVGEDCPVFDGLYSFCQTYAGGSVGGAVKLNHGICDISINWAGGLHHAKKCEASGFCYVNDIVLAILELLKQHERVLYVDIDIHHGDGVEEAFYTTDRVMTVSFHKFGDYFPGTGDIRDIGYGKGKYY
***************************************************************************:****:******
AVNYPLRDGIDDESYEAIFKPVMSKVMEMFQPSAVVLQCGADSLSGDRLGCFNLTIKGHAKCVEFVKSFNLPMLMLGGGGYTIRNVARCW
SLNVPLDDGIDDESYHFLFKPIIGKVMEIFRPGAVILQCGADSLSGDRLGCFNLSIKGHAECVRFMRSFNVPLLLLGGGGYTIRNVARCW-----------------------------SLNVPLDDGIDDESYHFLFKPIIGKVMEVFKPGAVVLQCGADSLSGDRLGCFNLSIKGHAECVKFMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGMDVDDKMPQHEYYEYFGPDYT
SLNVPLDDGIDDESYHFLFKPIIGKVMEVFRPGAVVLQCGADSLSGDRLGCFNLSIKGHAECVRYMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGIEVDDKMPQHEYYEYFGPDYT
SLNVPLDDGIDDESYHFLFKPIIGKVMEVFRPGAVVLQCGADSLSGDRLGCFNLSIRGHAECVKYMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGIEVDDKMPQHEYYEYFGPDYT
SLNVPLDDGIDDESYHYLFKPIIGKVMEIFKPGAVVLQCGADSLSGDRLGCFNLSIKGHAECVRYMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGAEIEDKMPQHEYYEYFGPDYT
SLNVPLDDGIDDESYHYLFKPIIGKVMEIFKPGAVVLQCGADSLSGDRLGCFNLSIKGHAECVRYMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGAEIEDKMPQHEYYEYFGPDYT
SLNVPLDDGIDDESYHYLFKPLIGKVMEIFRPGAVVLQCGADSLSGDRLGCFNLSIKGHAECVKYMRSFNVPLLLLGGGGYTIRNVARCWCYETGVALGAEIEDKMPQHEYYEYFGPDYT
****************:****:******:*:****:********************:******::*************************
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350
350
Fig. 5.9. Translated amino acid alignments of QsHDA6 (a) and QsHDA19 (b) with truncated orthologous sequences from 6 different species. GeneBank accessions are
provided with species names. ClustalW2 was applied to perform the alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Below the alignment, “*” indicates
identical residues, “:” indicates conserved substitutions and “.” Indicates semi-conserved substitutions. Coincident residues between the conserved domain and the
sequences are highlighted in red. Conserved domain name and its similarity with Quercus suber sequence is provided in the first line. Rc, Ricinus communis; Vv, Vitis
vinifera; Gm, Glycine max; Cc, Citrus clementina; Cs, Citrus sinensis; Th, Theobroma cacao; Nn, Nelumbo nucifera; P×b, Pyrus × bretschneideri; Md, Malus domestica; Fv,
Fragaria vesca.
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
5.4. DISCUSSION
During sexual reproduction in chestnut, several developmental pathways play
a role in the ovules, giving rise to mature embryos and dead ovules in mature nuts after
cross-pollination or to dead ovules contained in parthenocarpic nuts in autopollinated
trees. The high complexity of zygotic embryogenesis and the number of factors involved
in its correct development complicated the choice of genes for analysis. Thus, the set of
genes studied here are an attempt to shed some light on the variability of ovule and
tissues fates associated with embryo specific genes and epigenetic marks. Our results
showed differential gene expression for all the genes studied during development
depending on the type of pollination.
CsEMBD seemed to be strongly associated with the beginning of the zygotic
embryogenesis as shown by the differential level of expression found after cross
pollination in contrast with autopollination. It seems that the peak triggered by crosspollination led to fertilization. These results concur with Solanum chacoense (Tebbji et al.,
2010), in which loss of function mutants for EMB 1345 abort before reaching the globular
embryo stage (defined as E3D in chestnut), supporting the essential role for CsEMBD in
chestnut for the proper development of zygotic embryogenesis. Besides, in that work
specific expression levels of the so called EMB genes were associated with zygotic
developmental stages. Our results showed that CsEMBD expression dropped during
embryo late development and maturity, which contrasts with the maintenance of
medium levels of expression in ovules after autopollination (E2I, E3I) and in companion
ovules (E3C), pointing to a certain expression level required for proper embryogenesis
while autopollination was associated with basal levels of expression at the beginning of
ovule abortion. Moreover, both AtEMB1345 and CsEMBD code for a protein similar to
CIA1 that has not only been well characterized for its role in the CIA pathway involved
in the basic cell physiology (Balk and Pilon, 2011) through its participation in the Fe-S
cluster assembly, but it has also been recently demonstrated to play an essential role in
the zygote development with the rest of proteins encompassed in the CIA pathway
(Buzas et al., 2014).
CsOVA3 is also encompassed in the EMBD genes and encodes for a glutamatetRNA synthetase. Aminoacyl-tRNA synthetases are vital for normal embryo
development, functioning in translation within the basal cell activities; loss of function
142
Chapter V
mutants in Arabidopsis led to ovule abortion or early embryo lethality (Berg et al., 2005).
We have found similar expression levels during development with the exceptions of
pollination that triggered an increase, probably associated with the maturation of the
ovule before being receptive as shown for the high expression values in E2; and ovules
from isolated trees E2I and E3I, although the last ones have already entered the death
pathway. Normal embryo development was associated with similar expression levels
until maturity, probably due to the essential function of these enzymes.
Epigenetic marks are largely known for playing important roles during the
ontogenesis of plants and our group pioneered the study of methylation and histone H4
acetylation in C. sativa (Santamaría et al., 2009). Although a first approximation linking
global methylation levels and zygotic embryogenesis was already published (Viejo et
al., 2010), the results presented in this work manifest the plasticity regarding relative
gene expression necessary for the zygotic embryogenesis-associated processes taking
place from chestnut flowering to maturation of the embryo.
During early embryo development a strict division pattern must be followed in
order to establish the radial pattern and the determination of the future organs
(meristems and cotyledons). Aurora genes encode for cyclin-dependent kinases and
have been widely studied in animals and plants as they have an essential role in the
formative cell divisions that give rise to the body of the embryo (Van Damme et al.,
2011). Presumably, higher expression of these genes is found in actively dividing tissues
(Demidov et al., 2005) as we observed during ovule development at stage E1 and after
cross-pollination at E2 for CsAUR3. In contrast, there was a gradual decrease in the
expression of this gene when autopollination takes place at stages E2I and E3I pointing
to early mechanisms of pollen recognition and ovules fate determination, although
these ovules did not show differences in size respect to stage E2. Lower values found in
companion ovules (E3C) with respect to E3D could also be associated with their destiny
as they abort. The expression dynamics of this gene is in accordance with results
obtained by Demidov et al. (2005) during embryogenesis given that CsAUR3 expression
level declined with maturation and the highest values were maintained during early
embryo development at E3D and embryo expansion at E4D. AtAURORA3 is known to
phosphorylate serines 10 and 28 in histone 3 (Kawabe et al., 2005), a modification that is
encompassed in the histones PTMs. Moreover, phosphorylation of H3S10 has been
143
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
shown to facilitate H3K14 acetylation by GCN5 in Sacharomyces cerevisiae (Lo et al., 2000)
and GCN5 in Arabidopsis is known to acetylate the same lysine residue H3K14
(Benhamed et al., 2006). This makes it likely that cross-talk between these two proteins
could be partially behind the control of the developmental stages studied, coinciding its
expression dynamics in cross pollination (E2) and early embryogenesis (E3D) when
embryo polarity is established resembling results in Arabidopsis by Long et al. (2006).
CsGCN5L presented high values during embryo development and maturation
(except for stage E7), which is in consonance with previous works in Arabidopsis where
this gene is related with the maintenance of the root meristems (Servet et al., 2010) and
transit amplifying cells proliferation (Kornet and Scheres, 2009). It has been described
to be highly expressed during embryogenesis along with other acetyl-histone related
actors such as HDA19 (Long et al., 2006). AtHDA19 is a histone deacetylase
constitutively expressed during the plants life cycle (Zhou et al., 2005) and Perella et al.
(2013) predicted a high degree of redundancy as HDACs are part of a gene family. Yu
et al. (2011) described the controlling role of HDACs during transitions between
developmental stages (Yu et al., 2011). Besides, Chen and Wu (2010) claimed redundant
roles for HDA19 and HDA6 in seed germination and stress responses. These studies
could be in support of the similar dynamics found for CsHDA19 and CsHDA6 relative
expression during chestnut reproduction. Moreover, HDA19 is vital for embryogenesis
as double mutants for HDA19 and HSL1 are lethal and AtHDA19 is also highly
expressed in early embryogenesis (Zhou et al., 2013) which is consistent with our results.
Abortive ovules (E3C) presented half the expression level compared with dominant
ovules (E3D) for both genes, which supports that determination of ovules fate in
chestnut zygotic embryogenesis is partially controlled by epigenetic mechanisms. A
different pattern was found in ovules from isolated trees, where increasing expression
of CsHDA6 and CsHDA19 was associated with the entrance and establishment of the
death pathway in ovules at E3I.
HDA6 and HDA19 in Arabidopsis are known for repressing embryonic
characteristics after germination (Tanaka et al., 2008); similarly, an increase in expression
of these genes in chestnut, when ovules enter the abortion pathway after
autopollination, could act as an autoincompatibility mechanism. Thus, high expression
levels of CsHDA6 and CsHDA19 in the developmental stages studied seem to be
144
Chapter V
associated with opposite developmental processes: those regarding zygotic
embryogenesis (E1; E2 and E3D) but also death of ovules in E3I. Wu et al. (2008)
proposed the active role of HDA6 in senescence, which could be partially behind its
expression profile in abortive ovules. The acquisition of the zygotic embryo
development from stages E4 to E7 was associated with a gradual decrease of both HDAs
expression, reaching minimum values in the mature embryo; this pattern is in
agreement with the absence of AtHDA6 transcripts in mature seeds described by Wu et
al. (2008) while AtHDA19 has been found to be ubiquitous in the plant (Zhou et al., 2005).
Moreover, Zhou et al. (2013) described the repression of embryogenic characteristics
during seed development which is in consonance with the low expression of the
CsHDA6 and CsHDA19 during the last stages of embryo development.
Epigenetic marks of histones, such as deacetylation managed by HDAs, has been
associated with other actors involved in vital functions for the plant, i.e. stress response
and senescence (Zhou et al., 2005) mediated by JA and ethylene. In maize it has been
described that differential expression levels of the machinery for synthesis and response
to ethylene along with differential sensitivity to the hormone in a given tissue controls
the destiny of these tissues as is the case with the developing embryo and the endosperm
(Gallie and Young, 2004). Ethylene responses are mediated by ETHYLENE
RESPONSIVE TRANSCRIPTION FACTORS (ERFs) that interact with the DNA in order
to regulate the expression of stress responsive genes (Fujimoto et al., 2000). The
expression of CsERFA1 showed a peak in companion ovules (E3C) in contrast with the
levels found in early embryogenesis, which seems to lead to differential responses
mediated by ERFs after fertilization. This high level of expression might be related with
stress and senescence as described by Chen et al. (2002). The development of the zygotic
embryo, in contrast, was associated with low levels of CsERFA1 presenting the lowest
value at the beginning of the maturation phase (E5) and slightly increasing towards the
mature embryo, which can be associated with the described role of ethylene in ripening
fruits (Alexander and Grierson, 2002).
Ethylene is a product of the biosynthesis route in which SAMS participates by
catalyzing the transformation of methionine into SAMe that eventually produces
ethylene (Yang and Hoffman, 1984). This relationship between ethylene and SAMe
could explain the peak in the expression of CsSAMS2 in companion ovules (E3C) after
145
Gene expression dynamics linked to physiological milestones in C. sativa sexual
reproduction
fertilization. Moreover, SAMe is a universal methyl donor involved in the methylation
of DNA and histones (Li et al., 2011). Cross-pollination in chestnut concurred with an
increment in CsSAMS expression while after autopollination (E2I) there was no change
in expression and it decreased in successive stages (E3I). This fashion is in accordance
with Gómez-Gómez and Carrasco (1998) who found that pollination/fertilization
leading to proper zygotic embryogenesis induces an increment of SAMS1 mRNA
synthesis. Fertilization associated with a decrease in CsSAMS2 expression and low
levels are maintained until maturity with exception of the transient spike in E6. This
expression pattern, in association with the different phases the embryo went through
during embryo expansion from developmental stage E4 to mature embryo at stage E7,
could be due to the variety of processes assigned to different SAMS enzymes (Lindroth
et al., 2001). Other proteins involved in the metabolism of SAMe are members of the
RADICAL SAM family, which generate radicals and catalyze several reactions
including unusual methylations (Sofía et al., 2001). CsRADSAM shows homology with
the elongator complex subunit 3 (ELP3) that is known to have a role in the
demethylation of the parental genome in mouse (Okada et al., 2010). However, up to
date it has not been possible to establish its direct involvement in DNA methylation in
somatic cells and also if this function was conserved in plants (Wang et al., 2013). ELP3
is also known for possessing a HAT domain in the carboxyl end suggesting that both
the HAT activity and the ability for cleaving SAMe must be functional or
mechanistically associated (Chinenov, 2002). Besides, the HAT catalytic domain is
similar to GCN5 and can acetylate H3 in vitro (Wittschieben et al., 1999). Thus, two
different but possibly complementary activities (DNA methylation and HAT) can be
assigned to RADSAM. CsRADSAM dynamics during the developmental stages studied
showed differential expression associated with the kind of pollination, increasing in
autopollination and maintained in abortive ovules in E3I. Cross-pollination did not
show changes in expression but fertilization did (E3D), and there was also an increase
in companion ovules (E3C) with lower levels than the dominant ovule, which suggests
a role of this gene in the determination of ovules´ fate. If we consider its action as HAT,
its dynamics during embryo development from stage E4 to E7 is similar to GCN5 in the
extent that high levels of expression were found until the end of development (not for
E7 in GCN5). This pattern is in contrast with the HDACs HDA6 and HDA19 also
146
Chapter V
studied, which suggests that transcription of CsRADSAM is more likely to be involved
in histone acetylation rather than in methylation.
Histone post-translational modifications also rely on the activity of HUB2 that
catalyzes, within a complex, the ubiquitination of the histone H2B which is associated
with transcription and translation elongation. Moreover, H2B monoubiquitination is a
pre-requisite for the methylation of lysine residues 9 and 79 of histone H3 (Weake and
Workman, 2008). CsHUB2 expression profile showed an increase after pollination but
the rest of development did not show differences between normal embryo development
derived from cross-pollination and autopollination. The finding of two catalytic
domains, a GCN5L1 and a TATA, the last related with gene transcription, confers to
CsHUB2 possible activities beyond its participation in H2B ubiquitination that could be
complementary. This fact make this protein to encompass an elevated degree of
complexity and make not possible to offer a plausible explanation far from that CsHUB2
expression profile might be due to its participation in several processes.
In summary, from the analysis of gene expression dynamics we can conclude
that different patterns are followed in association with specific processes such as crosspollination (E2) vs. autopollination (E2I) or the contrast between the dominant ovule
(E3D) and the companion ovules (E3C). The destiny of ovules seems to be pre-fixed
depending on the kind of pollination accomplished, giving rise to developing embryos
and companion ovules that abort, as do ovules from isolated trees. Specifically, the
dichotomy zygotic embryogenesis/ovule death is supported by differential expression
of genes that, in the case of histone acetylation, perfectly reflects the antagonistic action
of HATs and HDACs in CsGCN5 expression versus CsHDA6 and CsHDA19.
Autopollination associated with autoincompatibility, on the other hand, also showed
different expression dynamics of those genes at the posterior embryo expansion stage
(E4D) and maturation (E5 to E7). On the contrary, CsHUB2, CsRADSAM and CsOVA3
cannot be associated with embryo development and maturation.
147
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reproduction
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General discussion
Chapter VI
6.1. GENERAL DISCUSSION
Zygotic embryogenesis: a general overview
A successful sexual embryogenesis is crucial to the new generation in terms of
dispersion, individuals’ multiplication and increase in genetic variability. For the
accomplishment of these goals, successful embryo development is necessary. In the case
of C. sativa, as a fruit tree, the quality of the nuts is intimately connected with their
economic value. The set of the nut in the beginning of the reproduction, involving the
dichotomy embryogenesis vs ovule death that in last instance determines the
polyembryony character of the seed, has not been studied from a physiological and epigenetic perspective as well as the rest of the zygotic development until reaching
maturity.
From anthesis to mature embryo, we have defined 7 stages of development that
encompass the variety of pathways followed by a specific ovule from the very first
stimulus, pollination, that determines the fate of the ovules containing in that given
pollinated flower. Thus, autopollination has been found to be enough for triggering
seed development but the lack of fertilization in this case produces parthenocarpic fruits
with aborted ovules inside; on the contrary, cross-pollination possesses the capacity of
inducing zygotic embryo development in posterior developmental stages. Once the
cross-pollination pathway is entered, specific spatial-temporal processes occur as
fertilization takes place, dividing the 16 ovules within an ovary in two groups: the
dominant ovule(s) that gives rise to the seed, and the companion ovules that abort. The
setting of the zygotic embryo in the dominant ovule is in parallel with the abortion of
the rest of the ovules that will remain atrophied accompanying the consecutive
morphogenic changes of the dominant one throughout development until the seed
reaches maturity.
The great amount of complex physiological and morphological changes that
ovules undergo, once their destiny is established, can only be unravelled by splitting
their study into small parcels of knowledge that together, and taking into account their
interactions, conform a global-view drawing of the exciting area of study that chestnut
reproduction provides.
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General discussion
The study of part of the actors that possess a role and exert their coordinated
actions in plant development such as plant regulators, epigenetic marks and differential
gene expression, have shown characteristic spatial-temporal dynamics through
development and depending on the pathway followed by the ovules within a given
ovary under both types of pollination.
Taking into account the 7 developmental stages defined from anthesis to mature
seed, this general discussion has been divided into 4 main developmental hits:
pollination, fertilization and ovule abortion, embryo expansion and embryo maturation.
Pollination: a small thing makes the difference
Pollination constitutes the first stimuli during reproduction in chestnut and the
quality of this input (cross- or autopollination) leads to embryo development and
companion ovules’ abortion in the case of cross-pollination and to the death of the
ovules in the case of autopollination. The pollen autoincompatibility in the genus
Castanea was already described by McKay (1942).
Several hormonal changes take place during pollination depending on the type,
resulting in a dramatic increase of JA in autopollination while in cross-pollination
ovules showed a decrease of this hormone, indicating an early signal transduction once
the pollen gets to the stigma. Moreover, spatial distribution results were in agreement
with Peng et al. (2006) as ABA, under cross-pollination conditions, spread from the outer
integument in E1 ovules to the inner in E2. Autopollination, on the contrary, concurred
with ubiquitous signal of ABA in the ovule. On the other hand, IAA with its classically
attributable growth-promoting role, presented an increase in cross-pollination which is
a prerequisite for ovule maturation (Sundberg and Østergaard, 2009) and increases of
CKs (except for ZR) were also found in support of ovules’ growth. Again, a differential
response was also found in spatial terms as cross-pollination associated with IAA which
located in the integuments while it is ubiquitous in autopollination. Parthenocarpy, as
a common phenomenon in C. sativa although not studied so far, was found to take place
in autopollinated trees, which coincided with the increases of IAA and GA3 in the ovules
that would act in collaboration with CKs as recently described in tomato by Ding et al.
(2013) in the progress of this response.
Pollination, greatly affecting the hormonal load of ovules, has not been found to
have repercussion in terms of global DNA methylation levels that did not show any
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changes after pollen arrival. The distribution of 5-mdC in the tissue, on the contrary, is
differentially affected by both kinds of pollination as the signal spreads more intensely
in the embryo sac and the internal integuments in E2I ovules, while E2 ovules showed
only increments in the outer integuments. This differential distribution could be related
to the imposition of the divergent developmental programs, as the MSAP technique for
finding specific methylation status of the genome found specific changes in E2I ovules,
which differentiated from cross-pollinated ones in that they did not show any change.
In addition, this differential response depending on the type of pollination was
intensified in E3I ovules with an increase in the number of specific loci found. These
differences in the specific loci found might be behind the entrance into the abortive
pathway of E2I ovules. Differential gene expression of CsSAMS2 was also found
depending on the type of pollination. Our results coincide with Gómez-Gómez and
Carrasco (1998) who described a substantial increment in SAMS1 mRNA synthesis
when pollination and fertilization lead to proper zygotic embryogenesis. On the
contrary, the maintaining of CsSAMS2 expression levels in E2I and posterior decrease
in E3I support that study given the absence of zygotic embryogenesis in these ovules.
Other epigenetic marks concerning histones, such as H4ac, showed
hyperacetylation in E1 ovules while the signal is differentially distributed in E2 and E2I.
Surprisingly, E2I ovules showed hyperacetylation of H4, which could be associated
with cell death as reported in animals (Jeong, 2011). It is noteworthy the finding of
similar patterns among all the H3 marks analyzed independent of their active or
repressive character which points to differential roles of the histones H3 and H4 during
pollination. Gene expression of HDAs seems to function in an imbricate fashion in
coordination with hormones as shown by E2I ovules with an ABA peak, while in the
next developmental stage E3I ovules experienced an increment in CsHDA6 and
CsHDA19 mRNAs production. This is supported by previous studies that demonstrated
an association between ABA and HDACs expression (Pfluger and Wagner, 2007; Zhou
et al., 2005); moreover, these dynamics would indicate a medium term response.
Acetylation would also be behind the maturation of the ovules prior to fertilization
involving cell elongation and division, the latter partially controlled by the
phosphorylation of H3S10 (Kawabe et al., 2005) through AURORA genes, which
constitutes a pre-requisite for the acetylation of H3K14 by GCN5 (Lo et al., 2000). The
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General discussion
cross-talk between these enzymes could be reflected in the same expression pattern
associated with pollination in chestnut orthologues for GCN5 and AURORA genes.
The results obtained in this thesis along with the related bibliography, feed a
growing body of evidence which let us to conclude that pollen arrival not only triggers
a transduction signalling reflected in a variety of changes in the ovule, but also tags the
ovules by determining their dichotomous fate: embryogenesis and associated
companion ovules’ death in cross-pollination vs ovules’ death in autopollination.
Fertilization and ovule abortion: the sole survivor
Fertilization after cross-pollination not only marks the starting point for zygotic
embryo development but is also accompanied by the death of the companion ovules.
This bifurcation in the ovules´ fate has a vital significance since it constitutes the basis
of monoembryony. While the dominance of a single ovule has been described macroand micromorphologically in the European chestnut (Botta et al., 1995; Viejo et al., 2010,
Chapter 2), and recently in the Chinese chestnut (C. mollissima) by Zou et al. (2014), an
as yet unresolved question arose phrased by Mogensen (1975) regarding the genus
Quercus, another member of the Fagaceae, which also applies to chestnut: what is the
mechanism by which usually only one ovule develops and the rest abort within a given
ovary? Our results indicated several changes in hormones, epigenetic marks and
differential gene expression that shed light on the control of this developmental pattern.
The imposition of a new developmental program in the dominant ovule (E3D)
was characterized by an increase in the size of the ovule. Both increments in IAA and
CKs were characterized at this developmental stage; these hormones are important
regulators for the establishment of the polarity of the embryo and, moreover, CKs are
known for their capacity of creating source-sink relationships (Riefler et al., 2006),
fundamental for embryo growth and expansion. On the contrary, hormones such as
ABA that do not promote growth showed low abundance in its global level as well as a
scant distribution in the tissues while IAA was found in every tissue of the ovule
including the developing embryo. The epigenetics of fertilization has proven that the
entrance in the embryogenic pathway has a clear transient demethylating effect in the
dominant ovule (Chapter 2; Viejo et al., 2010) and such a transient decrease similarly
occurs in other plant tissues with the start of a new developmental program (azalea,
Meijón et al., 2010; pinus, Valledor et al., 2010); moreover, this transient decrease was
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Chapter VI
reflected in the decrease of CsSAMS expression. These changes, on the contrary, have
not been related with specific methylation events as found with the MSAP analysis,
which could be due to the contribution to the analytical sample volume by the ovules’
tissues coming from the mother plant that constitute the gross of the sample compared
with the developing embryo.
The diffuse signal for 5-mdC localization in E3D ovules is in agreement with the
general association between hypomethylation and the activation of gene expression and
the euchromatin status (Valledor et al., 2007). On the contrary, histone H4ac was present
in every tissue of the E3D ovules as expected, although statistical differences were not
found when quantifying its relative level. The embryonic character of this
developmental stage concurred with the same dynamics for the rest of PTMs studied,
independently of their repressive or activator function, which would be associated with
the normal progression of the development or with a masking effect as postulated for
the MSAP analysis. Gene expression related to epigenetic marks showed that the
expression dynamics of CsHDA19 is consistent with previous studies in embryogenesis
(Zhou et al., 2013). The same high expression values were found for CsHDA6, and since
this gene belongs to the same family as CsHDA19 and functional redundancy was
suggested (Perrella et al., 2013), this may explain the similarity in expression pattern for
the developmental stages.
What is clear is that E3D ovules are in a given developmental state associated
with the hormonal load and DNA epigenetic scenario that make them susceptible to
morphogenic in vitro responses, as these ovules are the first explants that positively
responded to somatic embryogenesis induction (Chapter 2; Viejo et al., 2010). Thus, a
physiological window is opened allowing this morphogenic response, involving the
redetermination of the tissues and cells fate.
In strong contrast with the somatic embryogenesis abilities of the E3D ovules,
companion ovules E3C not only did not show somatic embryogenesis responses under
the same induction conditions, but increases in genes expression associated with
responses to senescence such as CsERFA or CsSAMS were found. CsSAMS expression
pattern corresponds with the hypermethylation found in companion ovules that enter
the abortive pathway; the expression peak for CsERFA1, as responsive gene for
ethylene, might be related with the death of E3C ovules as the expression of ERFs have
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General discussion
been associated with senescence (Chen et al., 2002). Moreover, ethylene is a product of
SAM activity (Yang and Hoffman, 1984) which would also be in accordance with the
high expression of CsSAMS in companion ovules E3C. Given the association between
hypermethylation with gene silencing and cell death (Lippman and Martienssen, 2004),
and comparing this fact with the absence of change in the methylation levels of E3I
ovules, it can be concluded that hypermethylation of companion ovules after
fertilization and the beginning of the zygotic embryogenesis in E3D must be the
consequence of some mechanism involving the supremacy of one of the ovules over the
rest. On the other hand, post-translational methylation of histones transiently increased
in E3C ovules as happened for H3K9me3 and H3K27me3, the latter mark involved in
the silencing of tissue-specific genes (Zhang et al., 2007). The differential dynamics of
PTMs in E3C compared with E3I ovules that decrease in their PTMs content, along with
the differences found in the MSAP specific loci associated to these developmental
stages, supports the theory that points to differential epigenetic regulation of the growth
arrest by which the ovules that do not generate a zygotic embryo abort.
Embryo expansion: filling the void
E4D ovules contained the growing embryo which showed a bipolar embryo axis
with initial meristems and expansion cotyledons. The differentiation of tissues
concurred with important changes at several levels such as the inversion in the IAA:CKs
ratio that is known to govern organ proliferation (Su et al., 2011). This result is also
supported by the immunolocalization of IAA which is ubiquitous in the embryo with
predominance in the cotyledons. Similarly, ABA is also ubiquitous in the embryo but is
more concentrated in the embryonary axis underlining the different action of these
hormones during this developmental stage. Moreover, the specific values of IAA and
ABA found between the embryogenic stage E4D and its homologous E3C and E4C
clearly confirm the involvement of these phytohormones in the establishment of the
differential fate of the ovules. At the gene expression level, it is known that EMB genes
such as AtEMBD and other related to the CIA pathway affect ABA and auxins synthesis
(Balk and Schaedler, 2014) which could also explain the differences in the gene
expression levels for CsEMBD and CsOVA3 between E4D and companion ovules (E4C)
in the abortive pathway. From the genes that play a role during zygotic embryogenesis,
AtGCN5 is known for being involved in the differentiation of the root tissues (Servet et
al., 2010) though dispensable and it is also fundamental for the meristem activity and
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Chapter VI
stem cell maintenance (Kornet and Scheres, 2009); these functions could explain the
increase of CsGNC5 in the ovules at E4D which in addition to its actions in multiprotein
complexes regarding histone acetylation is in accordance with the decrease of CsHDA19
expression at this stage in order to maintain an acetylation balance involving chromatin.
Nevertheless, H4ac but not H3ac showed an increase in E4D ovules corresponding with
its ubiquitous localization in the embryo and in contrast with the methylation signal
that remains faint as happened in E3D ovules that experimented an increase of 5-mdC
in E4D ovules as long as the differentiation of the tissues became evident (Botta et al.,
1995).
Embryo maturation: rearing the next generation
The final developmental stages leading to the mature embryo are characterized
by a dramatic increase in size of the cotyledons and the elongation and differentiation
of the tissues of the embryonic axis until the seed reaches its final form and size. During
these steps, several changes take place at the physiological level.
One of the most studied paradigms in embryo maturing is the interrelation
between GA4 and ABA, which act in a dynamic balance (Alabadi et al., 2009; Liu et al.,
2010). Our results showed a changing ratio between these hormones during normal
embryo development as in the final stages of development (E5A, E6A, E7A) the ratio
GA4:ABA was inverted favouring ABA due to the increase of this hormone and
reflecting the negative regulation of GAs by ABA according to Seo et al. (2006). The
increase in ABA at stage E5 is supposed to counteract the promoting effect on
germination of GA4 as described by Rodríguez-Gacio (2009). Seed filling and the
acquisition of the dehydration tolerance have been described to depend on the
hormonal balance and was associated in chestnut with a drop in the moisture content
of the embryo in E6A according with Finkelstein et al. (2002). Finally, ABA dynamics
during maturation of the zygotic embryo of C. sativa resembled that recently described
in Q. suber in somatic embryos (Pérez et al., 2015).
In contrast with ABA increase, IAA content in the axes diminished at stage 5 and
maintained low until the end of development, which has been described as a
consequence of its transformation into conjugated forms of the hormone (Bewley and
Black, 1994). Maturity of the embryo also concurred with the actions of CKs, involved
in the translocation of assimilates to the embryo and the strength of the source-sink
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General discussion
relationships (Roitsch and Ehneβ, 2000) and partially determine the size of the seed
(Riefler et al., 2006), which is reflected in the relative high level of CKs in the cotyledons
during chestnut embryo maturation. It is noteworthy that the dynamics are shared
between CKs, BRs and GAs pointing to coordinated actions during embryo maturation.
Gene expression during embryo maturation has been found to be partially
controlled by ABA as proposed by Chinnusamy et al. (2008) who described a downregulation exerted over HDACs. This reference coincides with the progressive decrease
of CsHDA6 and CsHDA19 from stages 4 until 7 and this dynamics is also supported by
the low expression of AtHDA6 in mature seeds (Wu et al., 2008) although AtHDA19 has
been described to be ubiquitous in the plant (Zhou et al., 2005).
The importance of epigenetics became evident looking at the dynamics of
quantified marks and their variety. Maturation taking place from stages E5A to E7A has
been reported to concur with a progressive increase in DNA global methylation levels
that ends with its highest value at E7A (Chapter 2; Viejo et al., 2010), in accordance with
Zluvova et al. (2001), while cotyledons maintain a lower level of methylation once they
are differentiated at stage E4. Taking into account that axis maturation concurs with a
series of cell differentiations giving rise to complex tissues such as the meristems, and
given the progressive increase in the total number of methylation changes and changes
in specific loci from E3D to E7A quantified by MSAP, it can be claimed that maturation
of the embryonic axis is associated with specific changes in DNA methylation.
Cotyledons, on the other hand, maintained a lower and constant level of methylation
from stages E5 to E7, possibly explained by their active metabolic status until the end of
development which is likely related with assimilates translocation as demonstrated by
CKs levels in the cotyledons previously discussed. Moreover, specific patterns in
histones PTMs were found, and specifically, acetylation of H4 is likely to play a role at
the beginning of maturation in E5A when it peaked while the rest of PTMs from histone
H3 followed a delay in the same dynamics. Contrary to changes in DNA methylation,
the mature axis (E7A) was associated with low abundance of PTMs no matter their
activation or repressive character.
The overall scenario encompassing hormones, epigenetic marks and associated
differential gene expression was reflected in the ability of axes and cotyledons to
produce somatic embryos as embryogenic capacities decreased, along with the increase
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Chapter VI
in global methylation levels for the axes, while cotyledons maintained a stable
percentage of somatic embryogenesis induction corresponding with low methylation
levels. Similar results in somatic embryogenesis induction in chestnut have been
reported by Sauer and Wilhelm (2005) and Şan et al. (2007).
It is noteworthy that, in a global context of maturation, several of the quantified
parameters showed transient changes at stage E6, coinciding with the reduction of
somatic embryogenesis ability of cotyledons and a decrease in moisture content as well
as in GA3, GA7 and BRs in the proximal parts of cotyledons while these compounds
increased in the distal parts of cotyledons. At the same time, the abundance decreased
in the embryonic axes for H3ac, H3K4me, and for CsAURORA and CsSAMS expression.
These changes are indicative for the preparation of the embryo to reach its quiescent
status at maturity. In addition, the spatial-temporal distribution of hormones (ABA,
IAA) and epigenetic marks (H4ac, 5-mdC) in the axes also showed dynamic balances
towards maturity. These observations point to an active role of the quantified
parameters and their orchestrated actions influencing the highly complex pathway of
embryo maturation.
Major transient changes in the immunolocalization during maturation were
found in the embryonic axes. The decline of ABA in E6A could be related with the
ongoing dehydration taking place in the embryo according with (Sauter and Hartung,
2000) and also suggests an active role in the control of the meristem activity supporting
its known role in the maintenance of stem cells in the RAM (Zhang et al., 2010). In the
same way, the progressive loss of IAA signal in RAM suggests an active role in the
establishment and determination of meristems as described by Dinneny and Benfey
(2008). Moreover, the widely accepted idea of IAA acting as a morphogen through the
regulation of its own distribution along the target tissues (Berleth, 2001) was supported
by the decreasing signal gradient from RAM to SAM in stages E5 and E6 that is lost at
maturity. The distribution of the epigenetic mark H4ac, corresponded with the peak of
the relative levels of this modification at E5A and the later loss of signal in the tissues in
E7A including the meristems, as was described for Hordeum vulgare regarding tissue
differentiation dynamics in seedlings (Braszewdka-Zaleska et al., 2013). The permissive
role in terms of development and chromatin configuration that has been attributed to
H4ac contrasted with the silencing role of DNA methylation and its reflection in the
distribution of 5-mdC in the axis which followed the opposite pattern, spreading from
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General discussion
the meristems to all other tissues in the mature embryo (E7A), disappearing from the
RAM and the surrounding provascular bundles as well as from the central zone of the
SAM.
In conclusion, maturation is characterized by specific spatial-temporal changes
in the intensity and distribution of hormones and epigenetic marks needed for
accomplishing the maturation of the embryo. Moreover, spatial distribution in the
tissues of the actors involved in zygotic embryogenesis have provided substantial
knowledge supporting and complementing the view offered by the variety of analysis
carried out throughout development.
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CONCLUSIONS
Conclusions
CONCLUSIONS
1. Chestnut sexual reproduction is associated with specific DNA methylation
dynamics in cross-pollinated trees. Fertilization exerts a transient demethylation
in the dominant ovule triggering the embryonic developmental program while
hypermethylation was associated with death of the companion ovules and
maturation of the embryo.
2. The absence of change in the methylation level of ovules from autopollinated
trees suggests different mechanisms involving DNA methylation in the abortion
of ovules depending on the type of pollination.
3. There is a “developmental window” during chestnut zygotic embryogenesis
captured by the embryonic character of the explants and their global
methylation levels.
4. Epigenetic post-traductional modifications in histones H3 and H4 from
flowering (E1) to the mature embryo (E7) can be specifically linked to distinct
and complementary developmental pathways such as zygotic embryo
development and ovule abortion.
5. Spatial-temporal distribution of H4ac and 5-mdC in the analyzed stages of
development points to differential roles of these epigenetic marks in the control
of tissue differentiation after fertilization and the establishment of meristems
during embryo maturation.
6. The increase in sequence-specific methylation changes detected by MSAP
during zygotic embryo maturation, as well as the finding of exclusive loci that
differentially characterize the death routes entered by companion and
autopollinated ovules, point to underlying and distinct epigenetic mechanisms
controlling these processes.
7. Distinctive hormonal concentrations defining specific hormonal loads during
the progression of zygotic embryogenesis are necessary for the imposition of the
different developmental patterns and tissues encompassed in chestnut
reproduction.
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Conclusions
8. Early pollen recognition and associated early bifurcation of ovules’ fate
depending on the kind of pollination is suggested by the dramatic increase of JA
and ABA after autopollination, leading to death, and the absence of change
leading to zygotic embryogenesis in cross-pollination.
9. The endogenous levels of GA4 and ABA during embryo development and
maturation, along with embryo moisture decrease towards quiescence of the
embryo, support the well-known cross-talk between these growth regulators
and their relevance in C. sativa reproduction.
10. Differential distribution in form of gradients and concentrations of ABA and
IAA signals throughout the developing tissues confirm the differential role of
these hormones during the progression of zygotic embryogenesis with special
attention to the meristems during maturation of the embryo and the abortion of
ovules.
11. An early tagging of the ovules depending on the type of pollination is reflected
at the molecular level since 8 from the 10 genes studied showed distinctive
expression patterns at that stage.
12. Differential gene expression of epigenetic-related genes has been found
necessary during the progression of zygotic embryo development associating
higher expression levels with metabolically active stages of development and a
further progressive decrease in expression towards embryo maturation.
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RESUMEN
Resumen
INTRODUCCIÓN
Los ecosistemas forestales suponen una fuente de materias primas de alto valor
en términos de biomasa. El 25 % de la superficie europea está cubierta por bosques. La
explotación de especies forestales se basa en la selección de genotipos élite que
normalmente son individuos adultos cuyas capacidades morfogénicas están mermadas,
lo cual limita enormemente las opciones para su reproducción asexual.
Castanea sativa es un ejemplo perfecto de especie agroforestal integrada en el
paisaje. Ha sido cultivada y expandida en Europa durante miles de años gracias al alto
valor de su madera y frutos siendo sustento económico para los pueblos mediterráneos
además de poseer profundas connotaciones culturales.
La correcta fructificación del castaño es de vital importancia, ya que las
características requeridas en términos comerciales son el tamaño de la semilla, el grado
de poliembrionía y la intrusión del episperma en los cotiledones. Existen varias
clasificaciones que recogen diferentes criterios para la clasificación de los cultivares
entre los que poseen alto valor (marrones) y los normales.
La consecución de la embriogénesis cigótica necesita de la integración de
estímulos externos e internos a través de la planta madre y el gametófito que ésta
sustenta. La formación de la semilla madura se alcanza mediante la interacción
orquestada de factores fisiológicos y (epi)genéticos. Además, el estado fisiológico global
de un tejido en un tiempo dado del desarrollo puede determinar la capacidad de generar
respuestas morfogénicas in vitro como la embriogénesis somática, que constituye una
alternativa a los métodos clásicos de propagación clonal.
Las hormonas poseen importantes funciones durante la ontogénesis. Factores
como su concentración, gradiente o ratio y la localización en un tejido, determinan el
programa de desarrollo para una célula o grupos de células. Las auxinas fueron el
primer grupo de reguladores vegetales estudiados, siendo el ácido indol acético (AIA)
el representante más importante. Es ubicuo en la planta y juega un papel fundamental
durante la embriogénesis en el establecimiento de los patrones axiales del eje
embrionario así como en la determinación de los meristemos durante la diferenciación
tisular. Las hormonas pueden mostrar relaciones inversas como ocurre con las
citoquininas (CKs), que actúan junto al AIA en la formación del eje del embrión y más
adelante en su crecimiento. El ácido abscísico (ABA) es un regulador fundamental de la
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embriogénesis; valores bajos se asocian a la embriogénesis temprana mientras que los
valores máximos se alcanzan antes de la entrada en el proceso de maduración y la
adquisición de la tolerancia a la deshidratación. Las giberelinas (GAs) actúan en
conjunto con el ABA durante la maduración y el crecimiento del embrión. Además, se
relacionan con el crecimiento del tubo polínico. El ácido jasmónico (JA), aunque
conocido por sus acciones en la defensa de las plantas, no se ha relacionado con el
desarrollo embrionario más allá de una posible acción relacionada con la disminución
de ABA en la maduración. Los brasinosteroides (BRs) son el último grupo de
reguladores del crecimiento descritos, y aunque se conoce su participación en un amplio
número de procesos junto con otras hormonas (mayormente GAs), sus papeles no han
sido esclarecidos hasta el momento.
Durante la reproducción sexual, el control de la expresión génica se ejerce
parcialmente por mecanismos epigenéticos tales como la reorganización de la
cromatina. La importancia de la embriogénesis cigótica es clara teniendo en cuenta que
hay 289 genes de expresión exclusiva en esta etapa del desarrollo de entre los 16.000
genes expresados durante la embriogénesis. La formación de tejidos y diferenciaciones
sucesivas necesitan de una plasticidad morfogénica que se consigue a través de la
expresión diferencial de genes durante la imposición y consecución del desarrollo
embrionario.
La cromatina constituye el primer nivel en el empaquetamiento del genoma de
organismos eucariotas. La unidad más pequeña de la cromatina es el nucleosoma,
consistente en 147 pares de bases (pb) de ADN enrolladas alrededor de un octámero de
histonas que contiene 2 copias de cada uno de los tipos (H2A, H2B, H3 y H4). De este
modo, la cromatina es un heteropolímero dinámico formado por nucleótidos y
proteínas. Su importancia para la condensación del ADN durante la replicación celular
no resta relevancia a su papel en el control de la expresión génica. La cromatina posee
dos configuraciones, abierta (eucromatina) y cerrada (heterocromatina) que se
relacionan con estados de activación o represión de la transcripción.
El control epigenético de la expresión génica recae en dos actores principales que
no modifican la información contenida en el ADN: por un lado, la metilación de las
cisotinas del ADN y la modificación química de las colas de las histonas por procesos
de metilación, acetilación, ubiquitinización, etc…, y por otro, la interacción de estas
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marcas epigenéticas con la maquinaria de transcripción y modificadores de la
cromatina. Debe existir un balance para mantener la homeostasis ejercido por escritores,
lectores y eliminadores de las marcas epigenéticas que en último término regulan la
expresión génica asociada a un determinado proceso de desarrollo.
La metilación del ADN es la marca epigenética más estudiada. Sus efectos a nivel
de expresión génica dependen de su distribución en los genes: las regiones promotoras
metiladas se asocian con inhibición de la transcripción mientras que la metilación en el
cuerpo del embrión afectan a la expresión del gen dependiendo de la densidad de
citosinas metiladas. La importancia de la metilación del ADN en la embriogénesis ha
sido demostrada mediante el uso de mutantes para las metiltransferasas MET1 Y CMT3.
Alteraciones de los patrones de metilación conllevan anormalidades en el desarrollo.
Además, niveles específicos de metilación se relacionan con la imposición de
determinados patrones de desarrollo durante la embriogénesis cigótica al igual que se
ha encontrado que afectan a las capacidades morfogénicas in vitro cuando se utilizan
embriones cigóticos como explantos iniciales. Aparte de cuantificaciones de metilación
globales mediante electroforesis capilar de alta resolución (HPCE), el uso de la técnica
de MSAP (methylation sensitive amplification polymorphism) se ha utilizado en la
última década para evaluar variaciones epigenéticas en varios organismos.
Las modificaciones postraduccionales de histonas (PTMs) constituyen el otro
gran mecanismo epigenético que se coordina con la metilación del ADN y los factores
remodeladores de la cromatina. De los dos dominios que tienen las histonas, las colas
que sobresalen del núcleo es donde se encuentran las marcas epigenéticas. Las
modificaciones de histonas son muy variadas: los residuos de lisinas y argininas pueden
estar metilados y acetilados, las serinas y treoninas, fosforiladas, y las lisinas pueden
además estar ubiquitinadas o sumoiladas. De entre estas modificaciones, H4ac, H3ac y
H3Kme3 son ejemplos de marcas asociadas a la eucromatina y la activación de la
transcripción mientras que H3K9me3 y H3K27me3 se localizan normalmente en
regiones heterocromáticas y se relacionan con la represión de la transcripción.
La metilación del AND y las PTMs han sido descritas como interdependientes
de modo que la primera afecta al contenido de las segundas en las colas de las histonas
y viceversa como ocurre con la metilación de la histona H3. Estas relaciones se
materializan en momentos clave del desarrollo como por ejemplo el control de la
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impronta genómica mediante la interacción entre la metilación del ADN y la metilación
de H3K27. También se han descrito diferentes interacciones que implican cambios
espacio-temporales durante la aplicación de estreses.
A pesar del conocimiento acumulado, el control de la reproducción sexual en
especies forestales desde una visión que combine las variaciones en el espacio-tiempo
permanece sin estudiar.
Durante la ontogenia de las plantas, la combinación de marcas epigenéticas
reversibles en un genoma dado, en un tejido y momentos dados, como consecuencia de
señales externas e internas, constituye el epigenoma. Si bien las marcas epigenéticas y
su localización en los tejidos durante la reproducción pueden revelar ciertas conexiones
con la respuesta fisiológica, la expresión diferencial de genes específicos durante la
reproducción sexual en castaño no ha sido abordada desde este punto de vista. Existen
múltiples genes cuya expresión es obligatoria para el correcto desarrollo de la
embriogénesis tales como el grupo de los genes EMB. El gran número de divisiones
celulares que tienen lugar en las primeras etapas de la embriogénesis definen la forma
y tamaño del embrión. Los genes aurora están implicados en división celular y son
responsables de la fosforilación de H3K10 de forma fluctuante durante el ciclo celular.
Además, estas proteínas colaboran con otros actores implicados en la regulación
epigenética de genes como las histona acetiltransferasa (HAT) GCN5 que acetila varios
residuos en las histonas H3 y H4 asociada con expresión de genes específicos durante
la floración y el desarrollo embrionario. La desacetilación mediante la acción de histonas
desacetilasas (HDACs) tiene lugar de manera antagonista junto con las HATs de modo
que mantienen un balance homeostático de las marcas epigenéticas en la cromatina a lo
largo del desarrollo. De las 18 HDACs descritas en arabidopsis, HDA6 y HDA19 se han
asociado con una amplia variedad de procesos tales como respuestas al estrés abiótico,
respuesta a patógenos o represión de las propiedades embriogénicas tras la
germinación, entre otras. La metilación de las citosinas del ADN depende de la
donación de un grupo metilo por parte de la S-adenosil metionina. Otras marcas
epigenéticas como la ubiquitinización de la histona H2B se asocia con la regulación
génica y también actúa en colaboración con la metilación de histonas.
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Resumen
PLANTEAMIENTO Y OBJETIVOS
Las especies forestales constituyen una fuente renovable de materias primas,
alimento y energía. El creciente interés por algunas especies debido a determinadas
cualidades como ocurre con el castaño gracias al alto valor de su madera y frutos en el
mercado, las hace objeto de explotación sostenible. Para ello, un conocimiento profundo
de su fisiología y reproducción es necesario.
La producción de planta clonal, en el caso del castaño, ha sido abordada desde
hace décadas mediante técnicas tradicionales que se ven limitadas debido al carácter
recalcitrante de los individuos adultos élite donde se muestran las características, tanto
madereras como frutales, de interés. La calidad de las castañas, teniendo en cuenta que
la mayor parte de la semilla está formada por los cotiledones, depende del correcto
desarrollo del embrión cigótico. Dicho desarrollo suele ocurrir de forma diferencial en
uno de los primordios seminales mientras que el resto, acompañantes, degeneran. En
algunos casos, varios de los primordios seminales se desarrollan, dando lugar a frutos
poliembriónicos que restan valor a la semilla. Los mecanismos de control de este
fenómeno permanecen sin estudiar aun cuando de ellos depende la calidad de la
semilla. Por ello, la importancia de la castaña como producto de alto valor justifica el
estudio de la reproducción sexual de esta especie.
El desarrollo del embrión cigótico tiene lugar a través de la integración, de forma
coordinada, de estímulos externos e internos en una compleja red de señalización.
Actores tales como las hormonas, los mecanismos epigenéticos y la expresión diferencial
de genes son la base para la imposición de la variedad de programas de desarrollo que
tienen lugar durante la formación de la semilla.
Teniendo en cuenta el trabajo previo de nuestro grupo de investigación en
sistemas experimentales relacionados con eventos fisiológicos clave en la ontogénesis
de las plantas (cambio de fase, floración o envejecimiento), el conocimiento acumulado
en la (epi)genética de estos procesos y la importancia de un desarrollo embriogénico
conforme en castaño, el objetivo general de esta tesis es la caracterización de la
embriogénesis cigótica y la muerte de los primordios acompañantes enmarcadas en el
desarrollo de la reproducción sexual a través de la identificación e interacción de marcas
epigenéticas con las hormonas y la expresión génica diferencial. Este objetivo principal
se basa en cuatro objetivos parciales:
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1. La caracterización histológica de la embriogénesis sexual en castaño desde la
floración hasta la semilla madura teniendo en cuenta hitos en el desarrollo como
son la autopolinización, la polinización cruzada, el desarrollo embrionario junto
con la muerte asociada de primordios acompañantes así como la maduración del
embrión.
2. La caracterización epigenética a través de la cuantificación de marcas
epigenéticas (5-mdC , H4ac, H3ac, H3K4me3, H3K9me3 y H3K27me) en los
estadios de desarrollo anteriormente descritos; además, la distribución espacial
de 5-mdC y H4ac como representantes de marcas epigenéticas represoras y
activadoras de la expresión génica.
3. La caracterización hormonal de los estadios de desarrollo ya definidos mediante
la cuantificación global de hormonas y la inmunolocalización de ABA y AIA
como actores fundamentales durante la reproducción.
4. El estudio de la expresión génica diferencial de genes relacionados con
regulación epigenética y desarrollo embrionario a lo largo de la reproducción
mediante PCR cuantitativa en tiempo real.
RESULTADOS Y DISCUSIÓN
Las cuantificación de marcas epigenéticas, hormonas y expresión génica durante
los estadios de desarrollo estudiados, así como su distribución espacio-temporal, han
demostrado estar asociadas al establecimiento y consecución de los diferentes patrones
de desarrollo.
Desde la floración (estadio E1) hasta el embrión maduro (E7), los primordios
seminales reciben una serie de estímulos que se ven reflejados en su estado fisiológico.
Dichos estímulos comienzan con la polinización, que determina el destino de los
primordios. Así, la autopolinización es suficiente para generar frutos partenocárpicos
que contienen primordios abortados en su interior; por el contrario, la polinización
cruzada induce el desarrollo del embrión cigótico en las siguientes fases del desarrollo.
De este modo, tras la polinización y posterior fecundación, los primordios se dividen en
dos grupos: uno constituido por el, normalmente único, primordio dominante, y otro
por los primordios acompañantes que degeneran. Estos primordios permanecen
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atrofiados acompañando al dominante durante todo el desarrollo embrionario hasta la
maduración.
Polinización
La polinización (autopolinización o polinización cruzada), determina la entrada
en el programa embriogénico o la degeneración de los primordios. La autopolinización
produce un fuerte incremento en los niveles de JA mientras que la polinización cruzada
tiene el efecto contrario en, mostrando los primordios una disminución en su contenido;
este hecho pone de manifiesto la existencia de una señalización temprana entre el
estigma y los primordios que determinará su destino. Por otro lado, la distribución de
ABA, hormona usualmente asociada a procesos de estrés, se encontró ubicuamente en
los primordios seminales procedentes de autopolinización (E2I) mientras que aquellos
de polinización cruzada (E2) mostraron una distribución cambiante desde los
integumentos externos a los internos tras la polinización en E1. Otros reguladores del
crecimiento mostraron incrementos en polinización cruzada como el AIA o las CKs
excepto por el RZ, lo cual podría estar relacionado con el estado inmaduro de los
primordios en el momento de la polinización. Por otro lado, incrementos en AIA, CKs
y GAs podrían asociarse en condiciones de autopolinización con la partenocarpia de los
ovarios, que crecen hasta el final del desarrollo.
Otros cambios a nivel epigenético mostraron diferencias en su distribución en
los primordios como ocurre con la 5-mdC, que se expande por todo el embrión en
primordios E2I mientras que en la polinización cruzada sólo aparece en la zona externa
de los integumentos. Cambios de metilación específicos en el genoma detectados por
MSAP detectaron loci específicos tras la autopolinización, apoyando a una
diferenciación temprana en el destino de los primordios comentada anteriormente.
Además, esta respuesta diferencial se incrementa en el tiempo, pues el número de loci
exclusivos en primordios E2I fue de 4, mientras que la transición a primordios E3I se
produjo con 28 cambios. La acetilación de la histona H4 mostró una distribución espacial
diferencial, estando de forma ubicua en los primordios E2I, lo cual podría estar
relacionado con una activación de genes relacionados con la muerte celular como se ha
descrito en animales. El control de la acetilación de histonas mediante HDACs parece
estar relacionado con hormonas como el ABA, que tras la autopolinización mostró un
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pico en E2I, tras lo cual, en primordios E3I, se tradujo en un incremento de los niveles
de expresión de CsHDA6 y CsHDA19 en concordancia con estudios previos.
Fecundación y degeneración de primordios seminales
La fecundación tiene como resultado la entrada en el programa embriogénico
del primordio dominante (E3D) así como la muerte asociada de los primordios
acompañantes (E3C). Esta bifurcación en el destino de los primordios constituye la base
de la monoembrionía. Este paradigma durante la reproducción es bien conocido y ha
dado lugar a una cuestión, aún sin resolver, que podría enunciarse como ¿cuál es el
mecanismo mediante el cual, normalmente, sólo un primordio se desarrolla mientras
que el resto degeneran? Nuestros resultados indicaron cambios en los niveles
endógenos de hormonas, marcas epigenéticas y expresión génica diferencial que han
arrojado luz sobre el control de estos patrones de desarrollo.
La imposición del programa de embriogénesis cigótica en el primordio
dominante se acompañó de importantes incrementos en AIA y CKs, hormonas
conocidas por su importante papel en el establecimiento de la polaridad del embrión.
Además, las CKs están implicadas en el establecimiento de relaciones fuente-sumidero.
La distribución ubicua de AIA en el embrión y tejidos del primordio contrastó con la
práctica ausencia de ABA, que además se encontró en baja concentración en la
cuantificación global.
Quizá uno de los cambios más interesantes tras la fecundación en los primordios
E3D fue la desmetilación transitoria del ADN, que coincidió con un descenso en la
expresión de CsSAMS2, aunque no se encontraron cambios exclusivos en loci mediante
MSAP. Por otro lado, la hipometilación de E3D también se vio reflejada en la
distribución de 5-mdC, que apenas mostraron señal. Este estado hipometilado
concuerda con otros procesos fisiológicos en los que también se produce una
desmetilación global antes de la entrada en programas de desarrollo específicos, y
podría estar relacionada con la activación génica y un estado eucromático de la
cromatina. La cuantificación de H4ac, por el contrario, no mostró diferencias respecto a
primordios E2, lo que hubiera sido esperable dada la asociación general entre acetilación
de histonas y estados permisivos de la cromatina para la transcripción génica.
El estado fisiológico global de los dos tipos de primordios tras la fecundación
tuvo un claro efecto cuando se probó su capacidad para generar respuestas
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morfogénicas in vitro. De este modo, primordios E3D son los primeros con capacidad
para generar embriones somáticos. Por el contrario, primordios E3C mostraron una nula
respuesta embriogénica junto con incrementos en la expresión de genes asociados a
estrés como CsERFA1 o a la metilación del ADN (CsSAMS2) en línea con la
hipermetilación cuantificada en los mismos. Al comparar la hipermetilación de los
primordios E3C con el mantenimiento de los niveles de 5-mdC en primordios E3I,
parece clara la existencia de algún mecanismo dirigido, o al menos asociado, a la
supremacía de un primordio dominante (E3D). Además, las PTMs asociadas con
silenciamiento génico cuantificadas (H3K9me3 y H3K27me3) mostraron un fuerte
incremento transitorio en los primordios acompañantes (E3C), lo cual apoya los
resultados obtenidos para los niveles de metilación. Estos resultados muestran que
existe una vía diferencial asociada a la degeneración y muerte de los primordios según
éstos procedan de un ovario con un primordio dominante o de un ovario que ha sido
autopolinizado.
Expansión del embrión
El primordio E4D contiene un embrión polarizado en el que se distingue el eje
embrionario y los cotiledones en expansión. En este estadio se produjo una inversión en
el ratio AIA:CKs como consecuencia del desarrollo de los órganos. La presencia
mayoritaria de AIA en los cotiledones contrastó con la de ABA, más presente en el eje
embrionario, apuntando a los diferentes papeles que juegan estas hormonas durante el
desarrollo embrionario.
Los genes embryo defective se sabe que están relacionados con funciones clave
durante el desarrollo embrionario como por ejemplo la síntesis de ABA y auxinas, lo
cual podría estar tras las diferencias en la expresión de los genes CsEMBD y CsOVA3
entre primordios E4D y E3C, más elevado en los primeros. Por otro lado, genes
implicados en la acetilación de histonas como CsGCN5, vieron modificados sus valores
de expresión, aumentando en este caso en asociación con su conocido papel en la
diferenciación de tejidos de la raíz y también en la actividad meristemática y el
mantenimiento de las células madre. CsHDA19, por el contrario, disminuyó su
expresión. Estos resultados concuerdan con el aumento de H4ac, que además mostró
una disposición ubicua en el embrión mientras que apenas se obserbó señal para 5-mdC.
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Maduración del embrión
Los estadios finales de desarrollo en los que el embrión madura se caracterizan
por un fuerte incremento en el tamaño de los cotiledones y la elongación y
diferenciación de los tejidos del eje embrionario hasta que la semilla adquiere su forma
y tamaño finales.
Uno de los paradigmas en la maduración de las semillas es la relación entre GA4
y ABA. Nuestros resultados mostraron un balance dinámico entre estas hormonas. Así,
el ratio GA4:ABA se invirtió favoreciendo al ABA lo cual estaría relacionado con el efecto
inhibitorio de esta última sobre la biosíntesis de GAs. El aumento de ABA en E5,
además, estaría relacionado con la inhibición de la germinación precoz impulsada por
GA4. El pico de ABA también tendría relación con la tolerancia a la desecación del
embrión.
El AIA, en contraposición al ABA, disminuye en el estadio E5 y permanece en
valores bajos hasta el final de la maduración debido a su paso a formas conjugadas no
activas. Las CKs, por el contrario, mantienen niveles relativamente altos, sobre todo en
los cotiledones, atribuibles a su papel en el establecimiento de relaciones fuentesumidero además de influir en el tamaño de la semilla. Patrones similares en la
concentración de CKs, BRs y GAs durante estos últimos estadios de desarrollo apuntan
a su acción coordinada durante la maduración del embrión. La relación entre hormonas
y expresión génica parecen estar relacionadas teniendo en cuenta el descenso progresivo
en la expresión de CsHDA6 y CsHDA19 durante la maduración coincidiendo con el pico
de ABA en E5, lo cual apoya resultados previos en los que se describe la inhibición en
la expresión de HDACs por esta hormona.
Por otro lado, la importancia de los mecanismos epigenéticos ha mostrado ser
de vital importancia durante esta etapa del desarrollo. La maduración concurrió con un
aumento progresivo de los niveles de metilación en los ejes embrionarios (E5A a E7A)
mientras
que
los
cotiledones
mantuvieron
niveles
de
metilación
estables,
probablemente debidos a su metabolismo activo asociado a la acumulación de reservas.
Además, el incremento en metilación global en los ejes se correspondió con aumentos
en los eventos de metilación y desmetilación cuantificados mediante MSAP, lo cual
apoya el establecimiento y maduración de la variedad de tejidos que están presentes en
el embrión maduro. Por otro lado, las PTMs mostraron la misma dinámica en sus
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valores sin importar su carácter represivo o activador, incrementándose en E5A para
luego disminuir hasta valores mínimos al final del desarrollo (E7A).
Ha sido curioso encontrar grandes cambios transitorios en el estadio E6A en
varios de los parámetros estudiados. Por un lado, hubo una reducción en la capacidad
embriogénica de los cotiledones junto con descensos en los valores de GA3, GA7 y BRs.
Por el otro, incrementos en las partes distales de los cotiledones para GA3, GA7, ABA y
BRs, así como en los ejes embrionarios para H3ac, H3K4me, CsAURORA y CsSAMS2, y,
finalmente en el contenido en agua en la porción distal de los cotiledones (E6PC).
Además, la distribución espacio temporal de hormonas (ABA y AIA) y marcas
epigenéticas (H4ac y 5-mdC) en los ejes mostraron patrones dinámicos hasta alcanzar
la maduración. Estas observaciones apuntan a un papel activo de los parámetros
cuantificados y su acción orquestada durante la maduración embrionaria.
La desaparición de ABA en el estadio E6A podría estar relacionada con la
deshidratación previa a la maduración en el embrión tal como apuntan estudios
anteriores, y además esta desaparición del ABA sugiere un papel activo en el control de
la actividad meristemática como también ha sido descrito. Del mismo modo, la pérdida
progresiva de AIA
en el meristemo radical apunta a su importancia en el
establecimiento y determinación de los meristemos. Además, la ampliamente aceptada
idea del AIA actuando como un morfogén a través de la regulación de su propia
distribución en los tejidos apoya el gradiente decreciente desde el meristemo radical al
del tallo entre los estadios E5A y E6A para desaparecer finalmente en E7A. Por otro
lado, la acetilación de H4 se pierde con el transcurso de la maduración como ya se ha
observado, y además su distribución sigue una dinámica diferente a la de la metilación,
en contraste con sus funciones complementarias. De este modo, la acetilación de H4
siguen un patrón contrario en el que 5-mdC en el eje se expande desde los meristemos
hacia el resto de tejidos desapareciendo del meristemo radical y los haces provasculares
cercanos así como de la zona central del meristemo del tallo.
Estos resultados muestran que la maduración está caracterizada por cambios espaciotemporales específicos en la intensidad y distribución de hormonas y marcas
epigenéticas, necesarios para alcanzar la madurez embrionaria.
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CONCLUSIONES
1. La reproducción sexual en castaño se asocia con dinámicas de metilación del
ADN específicas cuando ocurre bajo condiciones de polinización cruzada. La
fecundación provoca una demetilación transitoria en el primordio dominante
que desencadena el programa embriogénico mientras que la hipermetilación se
asoció con la muerte de los primordios acompañantes y la maduración del
embrión.
2. La ausencia de cambios en los niveles de metilación de primordios procedentes
de árboles autopolinizados sugiere la implicación de diferentes mecanismos
relacionados con la metilación del ADN en el aborto de primordios dependiendo
del tipo de polinización.
3. Existe una “ventana del desarrollo” durante la embriogénesis cigótica en castaño
que depende del carácter embriogénico de los explantos y sus niveles globales
de metilación.
4. Las modificaciones epigenéticas postraduccionales de las histonas H3 y H4
desde la floración (E1) hasta el embrión maduro (E7) se pueden asociar a vías de
desarrollo diferentes y complementarias como la embriogénesis cigótica y el
aborto de primordios acompañantes.
5. La distribución espacio-temporal de H4ac y 5-mdC en los estadios de desarrollo
analizados apunta a diferentes roles de estas marcas epigenéticas en el control
de la diferenciación tisular tras la fecundación así como en el establecimiento de
los meristemos durante la maduración del embrión.
6. El incremento de cambios de metilación secuencia-específicos detectados
mediante MSAP durante la maduración del embrión cigótico, así como el
hallazgo de loci exclusivos que caracterizan diferencialmente las rutas de muerte
de primordios acompañantes y primordios autopolinizados, apuntan a
diferentes mecanismos epigenéticos subyacentes de control durante estos
procesos.
7. Concentraciones distintivas de hormonas definen cargas hormonales específicas
durante la progresión de la embriogénesis cigótica y son necesarias para la
imposición de los diferentes tejidos y patrones de desarrollo durante la
reproducción del castaño.
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8. El reconocimiento temprano del polen y la bifurcación temprana del destino de
los óvulos en función del tipo de polinización se sugieren debido al importante
aumento de JA y ABA tras la autopolinización, los cuales conducen a la muerte,
mientras que la ausencia de cambios conducen a la embriogénesis cigótica
cuando hay polinización cruzada.
9. Los niveles endógenos de GA4 y ABA durante el desarrollo del embrión y la
maduración junto con la disminución del contenido en agua hasta alcanzar la
quiescencia apoyan la bien conocida relación entre estos dos reguladores del
desarrollo y ponen de manifiesto su relevancia en la reproducción de C. sativa.
10. La distribución diferencial en forma de gradientes y concentraciones de ABA e
IAA a través de los tejidos en desarrollo confirman el rol diferencial de estas
hormonas durante la progresión de la embriogénesis cigótica con especial
mención a los meristemos durante la maduración del embrión y el aborto de los
óvulos.
11. Un marcaje temprano de los óvulos dependiendo del tipo de polinización se
refleja a nivel molecular puesto que 8 de los 10 genes estudiados muestran
patrones de expresión distintivos en esos estadios del desarrollo.
12. La expresión génica diferencial de genes relacionados con procesos epigenéticos
es necesaria durante la progresión del desarrollo del embrión cigótico, asociando
niveles altos de expresión con estadios de desarrollo metabólicamente activos
mientras que la maduración se asocia con un disminución en la expresión.
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