Xylem cells cooperate in the control of lignification and cell death

Xylem cells cooperate in the control of
lignification and cell death during plant
vascular development.
Sacha Escamez
Umeå Plant Science Centre
Fysiologisk Botanik
Umeå 2016
“[…] longitudinal capillary sap-vessels, through which rooted plants
draw nourishment to every part from the earth.”
Vegetable Staticks: Or, An Account of some Statical Experiments on the Sap in VEGETABLES, etc.
London: W. and J. Innys and T. Woodward, 1727
Stephen Hales (1677-1761), “The Newton of plant physiology”
Sacha Escamez pursued his higher education at the University of Caen in
France. There, Sacha received a master’s degree in plant physiology for his
work on sulphur nutrition in canola plants at the French Institute for
Agronomical Research (UMR-INRA-950 Plant Ecophysiology, Agronomy and N, C, S
Nutrition, University of Caen, France).
Sacha has then been recruited as a PhD
student at the Umeå Plant Science Centre, in order to conduct research on the
development of the vascular tissue which conducts water in plants. This
doctoral thesis presents the findings of his research, discusses its implications
and tells about possible applications.
Umeå Plant Science Centre
Fysiologisk Botanik
Umeå universitet, 901 87 Umeå
www.umu.se / www.upsc.se
ISBN 978-91-7601-400-4
Xylem cells cooperate in the control
of lignification and cell death during
plant vascular development.
Sacha Escamez
Umeå Plant Science Centre
Fysiologisk Botanik
Umeå 2016
Responsible publisher under swedish law: the Dean of the Medical Faculty
This work is protected by the Swedish Copyright Legislation (Act 1960:729)
ISBN: 978-91-7601-400-4
Ev. info om Omslag/sättning/omslagsbild:
Elektronisk version tillgänglig på http://umu.diva-portal.org/
Tryck/Printed by: KBC Service Centre, Umeå Universitet
UMEÅ, SVERIGE/SWEDEN 2016
“The acid test of empirical scientific content in an argument is
to see what happens when you try to unpack it by stating its
opposite and ask for an empirical test between the two. If no
such test exists, then we are dealing either with sociological,
polemical viewpoints, which can differ according to the stance
of the speaker, or we are dealing with metaphor, and of
course, we could be dealing with both since metaphor is a
favourite recourse of polemicists.”
Denis Noble
In: The Music of Life: Biology Beyond the Genome, Oxford University
Press , Oxford, UK (p. 14)
Papers
Published papers and manuscripts presented
in the printed version of this thesis
Paper I (published):
Pesquet, E., Zhang, B., Gorzsás, A., Puhakainen, T., Serk, H., Escamez, S.,
Barbier, O., Gerber, L., Courtois-Moreau, C., Alatalo, E., Paulin, L.,
Kangasjärvi, J., Sundberg, B., Goffner, D., and Tuominen, H. (2013). Noncell-autonomous postmortem lignification of tracheary elements in Zinnia
elegans. The Plant Cell 25, 1314-1328.
Paper II (published):
Escamez, S., André, D., Zhang, B., Bollhöner, B., Pesquet, E., and
Tuominen, H. (2016). METACASPASE9 modulates autophagy to confine cell
death to the target cells during Arabidopsis vascular xylem differentiation.
Biology Open. doi: 10.1242/bio.015529. [Epub ahead of print].
Paper III (manuscript):
Zhang, B., Miskolczi, P.C., Escamez, S., Turumtay, H., Vanholme, R.,
Hedenström, M., Cao, Y., Ma, L., Bhalerao, R.P., Boerjan, W., and
Tuominen, H. (manuscript). PIRIN2 controls chromatin modification of
lignin biosynthetic genes and modulates the non-cell autonomous xylem
lignification in Arabidopsis thaliana.
Paper IV (manuscript):
Escamez, S., Zhang, B., Oikawa, A., Sztojka, B., Sathitsuksanoh, N., Eudes,
A., Scheller, H.V., and Tuominen, H. (manuscript). The bHLH62
transcription factor is involved in the PIRIN2-dependent regulation of
lignification in the xylem of Arabidopsis thaliana.
Other contributions from the author of
this PhD thesis
(This list of contributions only includes work related to the thesis)
Review article:
Escamez, S., and Tuominen, H. (2014). Programmes of cell death and
autolysis in tracheary elements: when a suicidal cell arranges its own corpse
removal. Journal of Experimental Botany 65, 1313-1321.
Book chapter:
Ménard, D., Escamez, S., Tuominen, H., and Pesquet, E. (2015). Life
beyond death: The formation of xylem sap conduits. In Plant Programmed
Cell Death (Springer), pp. 55-76.
Patent (filed in August 2014 but not published yet):
Boerjan, W., Vanholme, R.M.I., Turumtay, H., Tuominen, H., Escamez, S.
and Zhang, B. (patent). Modification of the plant PIRIN genes to alter lignin
properties and to reduce lignocellulosic biomass recalcitrance.
Innehåll/Table of Contents
Innehåll/Table of Contents
i
Simplified summaries in different languages
iii
Enkel sammanfattning (Svenska /Swedish)
iii
Simplified summary (English)
iv
Abstrakt (Svenska)
xvii
Abstract (English)
xviii
Preface
xix
Abbreviations
xx
I. Introduction
1
I.1. General context, scope and relevance of the research
1
I.2. Xylem differentiation in higher plants
2
I.2.1. Xylem cell types
2
I.2.1.a. Vascular stem cells
2
I.2.1.b. Xylem sap-conducting cells: tracheary elements
4
I.2.1.c. Cells specialised in mechanical support
4
I.2.1.d. Cells specialised in metabolic support
5
I.2.2. Specification of xylem cell fate
5
I.2.2.a. Molecular factors involved in xylem specification
5
I.2.2.b Hormonal induction of TE differentiation in experimental systems
6
I.2.3. The four modules of the xylem differentiation programmes
8
I.2.3.a. The differentiation programmes of TEs and fibres consist of four
modules
8
I.2.3.b. The molecular master switches controlling the xylem differentiation
programmes
10
I.2.3.c. Polysaccharidic Secondary Cell Wall deposition
12
I.2.3.d. Lignification
14
I.2.3.e. Programmed Cell Death
19
I.2.3.f. Programmed autolysis
25
I.3. Aims of the research
29
II. Results and discussion
30
II.1. New regulations of xylem lignification
30
II.1.1. Identification of new regulators of non-cell autonomous TE lignification 30
II.1.1.a. Screening for candidate genes potentially regulating lignification 30
II.1.1.b. Confirmation of candidate genes as regulators of lignification
31
II.1.1.c. Identifying genes regulating non-cell autonomous lignification
33
II.1.2. Transcriptional regulation of non-cell autonomous lignification
34
II.1.2.a. Transcriptional regulation of lignin biosynthetic genes at the
chromatin level
34
II.1.2.b. Transcriptional regulation of lignin biosynthesis by protein
complexes
37
i
II.1.3. Conclusions and perspectives on new regulations of xylem lignification 41
II.2. New regulations of xylem cell death
42
II.2.1. Restriction of cell death to the target cell type
42
II.2.1.a. Dying TEs implement safeguards to protect the surrounding cells 42
II.2.1.b. The spatial restriction of cell death requires tight regulation of
autophagy in TEs
44
II.2.2. Conclusions and perspectives on new regulations of xylem cell death
47
Acknowledgements
49
References
53
ii
Simplified summaries in different
languages
Umeå Plant science centre är ett center för experimentell växtbiologi.
Centret består av forskargrupper från både Umeå universitet och Sveriges
lantbruksuniveritet (SLU) och har forskare från över 40 olika länder. Detta
internationella klimat som då bildas bidrar till att skapa ett mångkulturellt
samarbete vilket gynnar forskningen på flera sätt. För att spegla en av dessa
positiva mångkulturella effekter på forskningen har jag bett mina kollegor
från olika delar av välden att översätta denna förenklade sammanfattningen
av min forskning på deras respektives modersmål.
Enkel sammanfattning (Svenska
/Swedish)
Växter är levande organismer som använder solenergi för att
omvandla koldioxid och vatten till socker. Därför har landlevande
växter utvecklat ett vaskulärt system som kallas xylem för att
transportera vatten från jorden upp till deras olika växtdelar. I träd
bildar detta xylem det som i dagligt tal kallas ved, vilket gör att detta
xylem är den huvudsakliga delen i den biomassa som blir till material
i pappersmassa och bioenergi production. Xylemets betydelse ur både
det ekologiska och biologiska perpektivet så väl som utvecklingen av
dess användningsområde, har drivit forskningen till det yttersta för
att förstå hur denna vävnad bildas. Forskningen som presenteras i
den här avhandlingen visar studien av bildandet av xylemceller som
sköter vattentransporten. Dessa vattentransporterande celler bygger
en tjock vägg runt dem som förstärks av en rigid polymer som kallas
lignin som gör att de kan hålla emot för det höga tryck som bildas av
vattenflödet vid transporten. Min forskning visar upptäckten att
inkorporeringen av lignin polymeren i denna tjocka cellvägg behöver
hjälp av närstående xylemceller, vilket visar att det faktiskt finns ett
samarbete mellan olika xylemceller. Dessutom måste dessa
vattentransporterande xylemceller organisera deras egen död så att
deras väggar lämnas kvar och bildar ett ihåligt rör som vattnet kan
strömma igenom. När de gör det använder de riskabla molekylära
“verktyg” för sin självförstörelse. Jag var dessutom involverad i
upptäckten som visar att under denna självförstörelse genomför dessa
vattentransporterande celler också åtgärder för att skydda de
omgivande cellerna, vilket återigen visar på ett cellulärt samarbete
under xylembildandet.
iii
The Umeå Plant Science Centre is a dynamic institute for research on
plant biology. This institute regroups both Umeå University and the
Swedish University of Agricultural Sciences (SLU) and employs
around two-hundred persons from over forty different countries. This
very international environment gives a great opportunity for sharing
different cultures, which in the end fosters research. To reflect this
cultural wealth and its positive effect on research, I have asked
colleagues from different parts of the world to translate the simplified
summary of my research into their mother tong.
Simplified summary (English)
Plants are living organisms which use solar energy to turn carbon
dioxide and water into sugar. Hence, the plants that live on land
surfaces have evolved a vascular tissue called xylem to draw water in
the soil and to transport it within their body. In trees, the xylem tissue
forms what is commonly called the wood, which means that the xylem
represents an important source of biomass for materials, pulp and
paper and biofuel production. The biological, ecological and
economical importance of xylem have driven research endeavours to
understand how this tissue is formed. The work presented in this
thesis has studied the formation of the xylem cells that conduct the
water. These water conducting cells build a thick wall around them
which gets reinforced by a rigid polymer called lignin such that they
can sustain the pressure from the water flow. I participated in
discovering that the deposition of the lignin polymer requires the help
from neighbouring cells, which demonstrates the existence of
cooperation between different xylem cells. In addition, the waterconducting xylem cells must organize their own death so as to leave
their walls empty, which forms hollow “tubes” in which the water can
flow better. When they do so, the water conducting xylem cells use
dangerous molecular “tools” to self-destruct. I was involved in
discovering that during their self-destruction, these water conducting
cells also implement measures to protect the surrounding cells, which
constitutes another example of cellular cooperation during xylem
formation.
iv
Le Centre pour la Science des Plantes d’Umeå (Umeå Plant Science Centre,
UPSC) est un institut dynamique de recherche biologique sur les plantes. Cet
institut est un regroupement auquel participent l’Université d’Umeå (Umeå
University) et l’Université Suédoise pour les Sciences Agriculturales (SLU).
L’UPSC emploie environ deux-cents personnes venant de plus de quarante
pays. Cet environnement résolument international représente une
formidable opportunité de partage culturel, ce qui favorise grandement la
recherche scientifique. Pour refléter cette richesse culturelle et son effet
positif sur la recherche, j’ai demandé à nombre de mes collègues des quatre
coins du monde de traduire le résumé simplifié de mes travaux dans leurs
langues maternelles.
Résumé simplifié (Français / French)
Les plantes sont des organismes qui utilisent l’énergie solaire pour
fabriquer du sucre à partir de CO2 et d’eau. De ce fait, les plantes qui
peuplent les milieux terrestres ont évolué de manière à acquérir un
tissu vasculaire appelé le xylème, qui transporte l’eau tirée du sol au
sein du corps des plants. Chez les arbres, le xylème constitue le bois,
ce qui signifie que le xylème représente une importante source de
biomasse pour la production de matériaux, de patte à papier et de
biocarburants. L’importance biologique, écologique et économique du
xylème ont conduit à d’intenses efforts de recherche pour comprendre
la formation de ce tissu. Le travail présenté dans cette thèse constitue
une étude sur la formation des cellules qui conduisent l’eau au sein du
xylème. Ces cellules conductrices d’eau construisent une paroi
cellulaire épaisse renforcée par un polymère rigide appelé la lignine,
permettant de supporter la pression exercée par le flux d’eau. J’ai
participé à découvrir que la déposition de la lignine nécessite
l’intervention des cellules avoisinantes, ce qui démontre l’existence
d’une coopération entre les cellules du xylème. De plus, les cellules
conductrices d’eau du xylème organisent leur propre mort de manière
à ne laisser que leurs parois, qui une fois vides forment ainsi des
« tubes » dans lesquels l’eau circule plus efficacement. Pour que cela
se produise, les cellules conductrices d’eau utilisent des « outils »
moléculaires dangereux afin de s’autodétruire. J’ai participé à
découvrir que pendant leur autodestruction, les cellules conductrices
de l’eau mettent également en place des mesures de protection pour
épargner les cellules voisines. Ces mécanismes protecteurs
constituent un autre exemple de coopération cellulaire lors de la
formation du xylème.
v
Vereinfachte Zusammenfassung
(Deutsch /German)
Pflanzen sind lebende Organismen, die Sonnenenergie verwenden,
um Kohlenstoffdioxid und Wasser in Zucker umzuwandeln.
Landpflanzen haben dafür ein Gefäßsystem namens Xylem
entwickelt, welches das Wasser aus der Erde herauszieht und durch
den Pflanzenkörper transportiert. In Bäumen bildet das Xylem jenes
Gewebe, welches gemeinhin als Holz bezeichnet wird. Dies bedeutet,
das Xylem eine wichtige Quelle für Biomasse ist, die für
Baumaterialien, Zellstoff-, Papier- und Biobrennstoffproduktion
verwendet werden kann. Nicht nur die ökologische und biologische
Bedeutung
des
Xylems,
sondern
auch
die
vielfältigen
Verwendungsmöglichkeiten, haben Wissenschaftler dazu veranlasst,
die Entstehung dieses Gewebes zu erforschen. Die in dieser
Doktorarbeit präsentierten Studien befassen sich mit der Entstehung
jener Xylem Zellen, die das Wasser transportieren. Diese Zellen
bilden eine dicke Zellwand, die durch ein steifes Lignin Polymer
verstärkt wird, sodass sie dem Wasserdruck standhalten kann. Ich
habe zu der Entdeckung, dass die Einlagerung von Lignin die Hilfe
von benachbarten Zellen benötigt, beigetragen. Diese Entdeckung
demonstriert, dass verschiedene Xylem Zelltypen zusammen
arbeiten. Weiterhin müssen die wassertransportierenden Zellen ihren
eigenen Tod organisieren, sodass nur ihre Wände zurück bleiben,
welche zusammen hohle „Röhren“ bilden, in denen das Wasser
fließen kann. In diesem Prozess benutzen diese Zellen gefährliche
molekulare „Werkzeuge“ um sich selbst zu zerstören. Ich war daran
beteiligt herauszufinden, dass die sterbenden Zellen Maßnahmen
ergreifen, um ihre benachbarten Zellen zu schützen. Dies ist ein
weiteres Beispiel für die zellulare Zusammenarbeit während der
Xylem Bildung.
vi
Umeå Plant Science Center jest dynamicznie rozwijającym się instytutem
skupiającym naukowców z dziedziny biologii roślin. Instytut składa sie z
dwóch jednostek: Umea University oraz Swedish University of Agricultural
Sciences (SLU). Zatrudnionych jest tutaj ponad dwieście osób pochodzących
z ponad czterdziestu krajów. Stanowi to bardzo międzynarodowe środowisko
i daje ogromną możliwość wymiany kulturowej, która ostatecznie przyczynia
się do rozwoju nauki. Dla odzwierciedlenia kulturowego bogactwa oraz jego
pozytywnego wpływu na naukę poprosiłem kolegów z różnych części świata o
przetłumaczenie uproszczonego streszczenia na ich język ojczysty.
Uproszczone streszczenie (Polski
/Polish)
Rośliny są żywymi organizmami, które przy użyciu energii słonecznej
przekształcają dwutlenek węgla oraz wodę w cukier. W związku z
wykorzystywaniem wody, rośliny żyjące na powierzchni lądu wykształciły
tkankę naczyniową zwaną ksylemem, która umożliwia pobieranie wody z
ziemi i transportowanie jej wewnątrz organizmu. W przypadku drzew ksylem
tworzy drewno, stanowiące ważne źródło pozyskiwania biomasy dla
przemysłu materiałowego, produkcji masy celulozowej, papieru oraz
biopaliw. Ekologiczne i biologiczne znaczenie drewna, a także jego szerokie
wykorzystanie w różnych metodach technologicznych, zaowocowały
wzmożonymi pracami naukowymi nad zrozumieniem budowy, funkcji i
tworzenia tkanki przewodzącej. W prezentowanej rozprawie badałem
powstawanie komórek ksylemu przewodzących wodę. Komórki przewodzące
wodę otoczone są grubą ścianą komórkowa, która dodatkowo wzmocniona
jest sztywnym polimerem zwanym ligniną.
Taka budowa zapewnia
wytrzymałość i ochronę przed panującym podczas transportu wody
ciśnieniem. W mojej pracy odkryłem, że odkładnie w ścianie komórkowej
lignin wymaga współdziałania ze strony sąsiednich komórek, co dowodzi
występowania współpracy pomiędzy różnymi typami komórek drewna. Co
więcej, komórki ksylemu przewodzące wodę muszą ulec procesowi
promowanej śmierci, dzięki czemu ich wnętrze pozostaje puste. W
konsekwencji tworzone są długie ”rury”, którymi woda jest sprawniej
transportowana. Podczas tego procesu komórki wykorzystują pewnie
niebezpieczne molekularne ”narzędzia”. Częścią mojej pracy było odkrycie
faktu, iż komórki ulegające samozniszczeniu wprowadzają środki ochronne
mające na celu zapewnieni ochrony otaczającym i sąsiadującym z nimi
komórkom. Stanowi to kolejny przykład współpracy międzykomórkowej
podczas rozwoju drewna.
vii
‫ﻣﺭﻛﺯ ﺃﻭﻣﻳﺎ ﻟﻌﻠﻭﻡ ﺍﻟﻧﺑﺎﺕ )ٍ ‪( UPSC‬ﻫﻭ ﻣﻌﻬﺩ ﻓﻌﺎﻝ ﻟﻠﺑﺣﺙ ﻓﻲ ﺑﻳﻭﻟﻭﺟﻳﺎ ﺍﻟﻧﺑﺎﺕ‪ .‬ﻫﺫﺍ ﺍﻟﻣﻌﻬﺩ‬
‫ﻳﺟﻣﻊ ﺑﻳﻥ ﻛﻝ ﻣﻥ ﺟﺎﻣﻌﺔ ﺃﻭﻣﻳﺎ )‪ (Umeå‬ﻭﺍﻟﺟﺎﻣﻌﺔ ﺍﻟﺳﻭﻳﺩﻳﺔ ﻟﻠﻌﻠﻭﻡ ﺍﻟﺯﺭﺍﻋﻳﺔ )‪(SLU‬‬
‫ﻭﻳﻌﻣﻝ ﻓﻳﻪ ﻧﺣﻭ ﻣﺎﺋﺗﻲ ﺷﺧﺹ ﻣﻥ ﺃﻛﺛﺭ ﻣﻥ ﺃﺭﺑﻌﻳﻥ ﺩﻭﻟﺔ ﻣﺧﺗﻠﻔﺔ‪ .‬ﺗﻌﻁﻲ ﻫﺫﻩ ﺍﻟﺗﺷﻛﻳﻠﺔ ﺍﻟﺩﻭﻟﻳﺔ‬
‫ﻓﺭﺻﻪ ﻋﻅﻳﻣﺔ ﺟﺩﺍ ﻟﺗﺑﺎﺩﻝ ﺍﻟﺛﻘﺎﻓﺎﺕ ﺃﻟﻣﺧﺗﻠﻔﺔ ﻭﺑﺎﻟﺗﺎﻟﻲ ﻫﺫﻩ ﺍﻟﺛﺭﻭﺓ ﺍﻟﺛﻘﺎﻓﻳﺔ ﻟﻬﺎ ﺗﺄﺛﻳﺭ ﺍﻳﺟﺎﺑﻲ ﻋﻠﻰ‬
‫ﺗﺷﺟﻳﻊ ﺍﻟﺑﺣﺙ ﺍﻟﻌﻠﻣﻲ‪.‬‬
‫ﻭﻗﺩ ﻁﻠﺑﺕ ﻣﻥ ﺍﻟﺯﻣﻼء ﻣﻥ ﻣﺧﺗﻠﻑ ﺃﻧﺣﺎء ﺍﻟﻌﺎﻟﻡ ﻟﺗﺭﺟﻣﺔ ﻣﻠﺧﺹ ﻣﺑﺳﻁ ﻟﺑﺣﺛﻲ ﻓﻲ ﺍﻟﻠﻐﺔ ﺍﻷﻡ‪.‬‬
‫ﻣﻠﺧﺹ ﻣﺑﺳﻁ )ﺍﻟﻠﻐﺔ ﺍﻟﻌﺭﺑﻳﺔ‪:(Arabic/‬‬
‫ﺍﻟﻧﺑﺎﺗﺎﺕ ﻛﺎﺋﻧﺎﺕ ﺣﻳﺔ ﺗﺳﺗﺧﺩﻡ ﺍﻟﻁﺎﻗﺔ ﺍﻟﺷﻣﺳﻳﺔ ﻟﺗﺣﻭﻳﻝ ﺛﺎﻧﻲ ﺃﻭﻛﺳﻳﺩ ﺍﻟﻛﺭﺑﻭﻥ ﻭﺍﻟﻣﺎء ﺇﻟﻰ ﺳﻛﺭ‬
‫ﺣﺗﻰ ﺗﺳﺗﻁﻳﻊ ﺃﻥ ﺗﻌﻳﺵ‪ .‬ﻭﺑﺎﻟﺗﺎﻟﻲ ﻟﻘﺩ ﻁﻭﺭﺕ ﺍﻟﻧﺑﺎﺗﺎﺕ ﺍﻟﺗﻲ ﺗﻌﻳﺵ ﻋﻠﻰ ﺳﻁﺢ ﺍﻷﺭﺽ ﺃﻧﺳﺟﺗﻬﺎ‬
‫ﺍﻟﻭﻋﺎﺋﻳﺔ ﺑﺣﻳﺙ ﻳﻘﻭﻡ ﻧﺳﻳﺞ ﺍﻟﺧﺷﺏ ﺑﺳﺣﺏ ﺍﻟﻣﻳﺎﻩ ﻣﻥ ﺍﻟﺗﺭﺑﺔ ﻭﻧﻘﻠﻬﺎ ﺩﺍﺧﻝ ﺍﻟﺟﺳﻡ ﺍﻟﻧﺑﺎﺗﻲ‪.‬‬
‫ﻓﻲ ﺃﻷﺷﺟﺎﺭ ﺗﺷﻛﻝ ﺃﻧﺳﺟﺔ ﺍﻟﺧﺷﺏ ﻣﺎ ﻳﺳﻣﻰ ﻋﺎﺩﺓ ﺃﻟﺧﺷﺏ‪ ،‬ﺃﺩ ﻳﻌﺩ ﺍﻟﺧﺷﺏ ﻣﺻﺩﺭﺍ ﻫﺎﻣﺎ ﻣﻥ‬
‫ﻣﺻﺎﺩﺭ ﺍﻟﻛﺗﻠﺔ ﺍﻟﺣﻳﻭﻳﺔ ﻟﻠﻣﻭﺍﺩ ﺍﻟﺗﺎﻟﻳﺔ )ﻟﺏ ﺍﻟﻭﺭﻕ ﻭﺍﻟﻭﺭﻕ ﻭﺇﻧﺗﺎﺝ ﺍﻟﻭﻗﻭﺩ ﺍﻟﺣﻳﻭﻱ(‪ .‬ﻓﺿﻼ ﻋﻥ‬
‫ﺍﻷﻫﻣﻳﺔ ﺍﻟﺑﻳﺋﻳﺔ ﻭﺍﻟﺑﻳﻭﻟﻭﺟﻳﺔ ﻟﻠﺧﺷﺏ‪ ،‬ﻓﻬﻭ ﻳﺳﺗﺧﺩﻡ ﻟﻣﺧﺗﻠﻑ ﺍﻟﺗﻁﺑﻳﻘﺎﺕ‪.‬‬
‫ﺗﺳﻌﻰ ﺍﻟﻌﺩﻳﺩ ﻣﻥ ﺍﻟﺑﺣﻭﺙ ﻟﻔﻬﻡ ﻛﻳﻔﻳﺔ ﺗﺷﻛﻳﻝ ﻫﺫﺍ ﺍﻟﻧﺳﻳﺞ‪ .‬ﻓﻲ ﻫﺫﻩ ﺍﻷﻁﺭﻭﺣﺔ ﺗﻣﺕ ﺩﺭﺍﺳﺔ ﻛﻳﻔﻳﺔ‬
‫ﺗﺷﻛﻳﻝ ﺧﻼﻳﺎ ﻧﺳﻳﺞ ﺍﻟﺧﺷﺏ ﺍﻟﺫﻱ ﻳﻣﺛﻝ ﺍﻟﻘﻧﺎﺓ ﺍﻟﺧﺎﺻﺔ ﻟﻧﻘﻝ ﺍﻟﻣﻳﺎﻩ ‪ .‬ﻫﺫﻩ ﺍﻟﺧﻼﻳﺎ ﺍﻟﻣﻭﺻﻠﺔ ﻟﻠﻣﻳﺎﻩ‬
‫ﺗﺗﻣﻳﺯ ﺑﺑﻧﺎء ﺟﺩﺍﺭ ﺳﻣﻳﻙ ﻣﻥ ﺣﻭﻟﻬﺎ ﺑﻭﺳﺎﻁﺔ ﻣﺎﺩﺓ ﻣﺩﻋﻣﺔ ﺗﺩﻋﻰ ﺑﻭﻟﻳﻣﺭ ﺍﻟﻠﺟﻧﻳﻥ ﻭﺑﺫﻟﻙ ﻳﻣﻛﻧﻬﺎ ﻣﻥ‬
‫ﺗﺣﻣﻝ ﺍﻟﺿﻐﻁ ﺍﻟﻧﺎﺗﺞ ﻣﻥ ﺗﺩﻓﻕ ﺍﻟﻣﻳﺎﻩ‪.‬‬
‫ﺷﺎﺭﻛﺕ ﻓﻲ ﺍﻛﺗﺷﺎﻑ ﺃﻥ ﺗﺭﺳﺏ ﺑﻭﻟﻳﻣﺭ ﺍﻟﻠﺟﻧﻳﻥ ﺍﻟﺫﻱ ﻳﺗﻁﻠﺏ ﻣﺳﺎﻋﺩﺓ ﻣﻥ ﺍﻟﺧﻼﻳﺎ ﺃﻟﻣﺟﺎﻭﺭﺓ‪ ،‬ﻣﻣﺎ‬
‫ﻳﺩﻝ ﻋﻠﻰ ﻭﺟﻭﺩ ﺗﻌﺎﻭﻥ ﺑﻳﻥ ﺧﻼﻳﺎ ﻧﺳﻳﺞ ﺍﻟﺧﺷﺏ ﺍﻟﻣﺧﺗﻠﻔﺔ‪ .‬ﻭﺑﺎﻹﺿﺎﻓﺔ ﺇﻟﻰ ﺫﻟﻙ ﻳﺟﺏ ﻋﻠﻰ ﺧﻼﻳﺎ‬
‫ﺍﻟﺧﺷﺏ ﺍﻟﻣﻭﺻﻠﺔ ﻟﻠﻣﻳﺎﻩ ﺗﻧﻅﻳﻡ ﻋﻣﻠﻳﺔ ﻣﻭﺗﻬﺎ ﻭﺫﻟﻙ ﻟﺗﺭﻙ ﺟﺩﺭﺍﻧﻬﺎ ﻓﺎﺭﻏﺔ‪ ،‬ﻭﺑﺫﻟﻙ ﺗﺷﻛﻝ " ﺃﻧﺎﺑﻳﺏ"‬
‫ﺟﻭﻓﺎء ﻟﻧﻘﻝ ﺍﻟﻣﻳﺎﻩ ﻭﻋﻠﻳﻪ ﺗﻛﻭﻥ ﻋﻣﻠﻳﺔ ﺗﺩﻓﻕ ﺍﻟﻣﻳﺎﻩ ﻋﻠﻰ ﻧﺣﻭ ﺃﻓﺿﻝ‪.‬‬
‫ﺃﺫ ﻋﻧﺩﻣﺎ ﻳﺗﻡ ﺫﻟﻙ‪ ،‬ﻓﺈﻥ ﺧﻼﻳﺎ ﺍﻟﺧﺷﺏ ﺍﻟﻣﻭﺻﻠﺔ ﻟﻠﻣﻳﺎﻩ ﺗﺳﺗﺧﺩﻡ " ﺃﺩﻭﺍﺕ" ﺟﺯﻳﺋﻳﺔ ﺧﻁﺭﻩ ﻟﻐﺭﺽ‬
‫ﺍﻟﻘﻳﺎﻡ ﺑﻌﻣﻠﻳﺔ ﺍﻟﺗﺩﻣﻳﺭ ﺍﻟﺫﺍﺗﻲ‪ .‬ﻭﻗﺩ ﺷﺎﺭﻛﺕ ﻓﻲ ﺍﻛﺗﺷﺎﻑ ﺃﻧﻪ ﺧﻼﻝ ﻋﻣﻠﻳﺔ ﺍﻟﺗﺩﻣﻳﺭ ﺍﻟﺫﺍﺗﻲ‪ ،‬ﺗﻘﻭﻡ ﻫﺫﻩ‬
‫ﺍﻟﺧﻼﻳﺎ ﺍﻟﻣﻭﺻﻠﺔ ﻟﻠﻣﻳﺎﻩ ﺃﻳﺿﺎ ﺑﺗﻧﻔﻳﺫ ﺗﺩﺍﺑﻳﺭ ﻟﺣﻣﺎﻳﺔ ﺍﻟﺧﻼﻳﺎ ﺍﻟﻣﺣﻳﻁﺔ ﺑﻬﺎ‪ ،‬ﻭﻫﻭ ﻣﺎ ﻳﺷﻛﻝ ﻣﺛﺎﻻ ﺁﺧﺭ‬
‫ﻋﻠﻰ ﺍﻟﺗﻌﺎﻭﻥ ﺍﻟﺧﻠﻭﻱ ﺃﺛﻧﺎء ﺗﺷﻛﻳﻝ ﺍﻟﺧﺷﺏ‪.‬‬
‫‪viii‬‬
Vereenvoudigde samenvatting
(Nederlands /Dutch)
Planten zijn levende organismen die met behulp van zonne-energie,
koolstofdioxide en water omzetten in suiker. Om te overleven op het
vaste land moesten de landplanten een vaatstelsel ontwikkelen
(xyleem) om water uit de grond op te nemen en te vervoeren door de
gehele plant. In bomen wordt het xyleem ook wel hout genoemd. Het
hout kan gebruikt worden als bouwmateriaal, maar wordt voor een
groot gedeelte ook gebruikt voor de productie van pulp, papier en
biobrandstoffen. Door de ecologische/biologische belangen en door
het gebruik van het xyleem wordt er veel onderzoek gedaan naar de
ontwikkeling van het xyleem. Deze thesis richt zich op het onderzoek
naar de formatie van het xyleem dat water transporteert. Deze water
transporterende cellen bouwen een dikke celwand om zich heen, wat
wordt verstevigd door een stugge polymeer genaamd lignine. Door
deze versteviging kunnen de water transporterende xyleem cellen
grote druk weerstaan. Ik heb mede ontdekt dat er samenwerking
nodig is tussen de omringende xyleem cellen om de depositie van
lignine mogelijk te maken. Daarbij, moeten de water transporterende
xyleem cellen hun eigen dood in werking stellen, wat de cel leeg
maakt en water transport beter mogelijk maakt. Om deze celdood in
werking te stellen gebruiken de water transporterende xyleem cellen
gevaarlijke moleculaire “gereedschappen” om zichzelf te doden. Ik
was betrokken bij de ontdekking dat tijdens de celdood, de water
transporterende cellen, de omringende cellen beschermen. Dit laat
nog eens zien dat er cellulaire samenwerking is tijdens de xyleem
formatie.
ix
x
Az Umeåi Növénybiológiai Kutatóintézet (Umeå Plant Science Centre)
egy nagyon dinamikus intézmény, mely egyesíti az Umeå Egyetemet
(Umeå University) és a Svéd Agrártudományi Egyetemet (Swedish
University of Agricultural Sciences), több mint kétszáz személyt
foglalkoztatva, a világ több mint 40 országából. Ez a nemzetközi
légkör kiváló lehetőséget biztosít különböző kultúrák megismerésére,
ezáltal is gazdagítva a kutatást. Megkértem a munkatársaimat a világ
különböző részeiről, hogy fordítsák le kutatásom rövid összefoglalóját
a saját anyanyelvükre, ezzel is kifejezve a kulturális sokszínűség
fontos szerepét a tudományban.
Rövid összefoglaló (Magyar/Hungarian)
A növények olyan élőlények, amelyek napenergia felhasználásával a
szén-dioxidot és a vizet cukorrá alakítják. Az evolúció során a
szárazföldön élő hajtásos növényekben kialakult a farész, más néven
xilém, amelyen keresztül a víz eljut a talajból a növény különböző
részeibe. A fák esetében ez a szövet alkotja a faanyagot, amely egy
nagyon fontos biomassza forrás, papír és bioüzemanyag előállításhoz.
A farész biológiai és ökológiai jelentősége, valamint a széleskörű
felhasználása miatt elengedhetelenek az ezen szövet kialakulásának
megismerését célzó kutatások. Az itt bemutatásra kerülő doktori
dolgozat a xilémet alkotó vízszállító sejtek vizsgálatát célozza meg.
Lignin szilárdító polimér épül a sejtfalakba, így a vízszállító sejtek
(tracheidák) sejtfala még jobban megvastagszik, ezáltal képesek
ellenállni a vízszállításból fakadó nyomásnak. Bebizonyítottuk, hogy a
lignin lerakódása a sejtfalban a szomszédos sejtek hozzájárulásával
történik, bizonyítván a különböző xilémsejtek közötti kooperációt.
Továbbá, a vízszállító sejtek véghez kell vigyenek egy „öngyilkossági
programot”,
melynek
eredményeképpen
a
sejt
tartalma
felszámolódik, az üres csövekben (tracheák) a víz akadálymentesen
tud áramlani a növényben. A sejtek veszélyes molekuláris eszközök
arzenálját használják fel az önmegsemmisítéshez. Kutatómunkám
során részt vettem annak a feltárásában, hogy ezek a sejtek miközben
önpusztítást visznek véghez, intézkedéseket tesznek a szomszédos
sejtek megóvásáért, mely egy újabb példa a farész kialakulása során
alkamazott együttműködési stratégiára.
xi
要旨 (Japanese)
植物は太陽エネルギーを利用し二酸化炭素と水を糖に
変換する生物体である。ゆえに、地表に住む植物は地
中から水を汲み上げ、植物内部で輸送するために木部
と呼ばれる維管束組織を進化させた。樹木において、
木部組織はいわゆる木材を形成し、それは木質材料、
パルプ·製紙、バイオ燃料となる重要なバイオマス原
料となっている。生態学的、生物学的、さらにはその
多様な利用価値の重要性から、この組織形成を解明す
る研究の努力がなされてきた。本博士論文には、水の
通道に関わる木部細胞の形成の研究が記されている。
これら水を通道する細胞は、水流による圧力に耐える
ため、リグニンと呼ばれる硬いポリマーによる強化さ
れた厚い細胞壁を形成する。本稿では、リグニンポリ
マーの沈着は隣接した細胞からの助けが必要であり、
そしてそれは異なる木部細胞間での連携が存在すると
いうことの発見を示す。加えて、通道木部細胞は通水
するために細胞を空洞化、つまり自身の死を組織せね
ばならない。その死が起こる際には通道木部細胞は自
身を破壊するための危険な分子道具を使用する。この
自己破壊の間、これらの通道細胞はまた周囲の細胞を
保護するための方策を実行し、つまりそれは木部形成
における細胞間の連携が存在するという別の事象の発
見を本稿でさらに示した。
xii
Sumário simplificado (Português
/Portuguese)
As plantas são organismos que utilizam a energia solar para
converter o dióxido de carbono em açúcares. Desta forma, as
plantas terrestres evoluíram no sentido de desenvolver um
tecido vascular denominado xilema que tem as funções de
absorver a água do solo, transportando-a seguida para todo o
corpo da planta. Nas árvores, o tecido xilémico forma o que é
normalmente denominado de “madeira”. Daqui resulta que o
xilema constitui uma importante fonte de biomassa para
produção de materiais, como papel e aglomerados, tal como
para a produção de biocombustíveis. A relevância biológica e
económica do xilema, bem como as suas várias aplicações,
impulsionou os esforços de investigação para compreender
como se forma este tecido. O trabalho apresentado nesta tese
estuda a formação das células que, no xilema, são as
responsáveis pela condução da água na planta. Estas células
segregam uma espessa parede celular que é reforçada pela
deposição de um polímero rígido, a lenhina, e cuja principal
função é suportar a pressão hidrostática. O presente estudo
demonstrou que a deposição de lenhina nas células condutoras
necessita da ajuda das células adjacentes, evidenciando assim a
existência de cooperação entre os diferentes tipos de células que
constituem o xilema. Adicionalmente, as células condutoras
necessitam de programar a sua própria morte para que se
possam formar os “tubos” ocos através dos quais o fluxo de água
é facilitado, sendo que estas células usam “ferramentas”
moleculares perigosas para se autodestruírem. Estive
igualmente envolvido no estudo que demonstrou que, durante a
sua morte programada, estas células implementam também
medidas de protecção das células vizinhas, o que constitui um
outro exemplo da cooperação celular que existe durante a
formação do xilema.
xiii
Umea Plant Science Centre es un dinámico instituto de investigación en
biología vegetal. Este instituto cuenta con alrededor de 200 personas de más
de 40 diferentes nacionalidades pertenecientes a la Universidad de Umea o a
la Universidad Sueca de Agricultura (SLU). Este ambiente internacional
propicia el intercambio cultural, lo que repercute en la investigación. Para
reflejar esta riqueza cultural y su efecto positivo en la investigación, he
pedido a varios compañeros de diferentes nacionalidades que traduzcan el
resumen de mi tesis doctoral a su lengua materna.
Resumen simplificado (Español
/Spanish)
Las plantas son organismos vivos que usan la energía solar para convertir
dióxido de carbono y agua en carbohidratos. Por esta razón, las plantas que
viven en el medio terrestre han desarrollado un tejido vascular llamado
xilema para captar el agua del suelo y transportarla por el interior. En los
árboles, el xilema forma lo que comúnmente se conoce como madera, hecho
que lo dota de gran importancia al representar una fuente muy importante
de biomasa para la producción de materiales, pulpa, papel y
biocombustibles. La importancia biológica y ecológica del xilema, así como
su uso en varias aplicaciones ha impulsado la investigación para entender
cómo este tejido se forma. El trabajo presentado en esta tesis doctoral
contribuye al estudio de la formación de las células del xilema que conducen
el agua. Estas células en particular forman una pared gruesa alrededor de
ellas mismas que es reforzada por un polímero rígido llamado lignina, lo que
les permite soportar la presión asociada al flujo de agua desde la raíz a la
hoja. He participado en el descubrimiento de que la deposición de lignina
requiere de la ayuda de células vecinas, lo que demuestra la existencia de
cooperación entre diferentes células del xilema. Además, las células
conductoras de agua del xilema deben orquestar su propia muerte de forma
que vacíen sus paredes para dar lugar a “tubos” en los que el agua puede fluir
más fácilmente. Cuando llevan a cabo este proceso, las células conductoras
de agua del xilema usan peligrosas “herramientas” moleculares para
autodestruirse. Estuve también involucrado en el descubrimiento de que
durante su autodestrucción, estas células conductoras de agua también
realizan acciones para proteger a las células del alrededor, lo que constituye
un ejemplo más de la cooperación que se da durante la formación del xilema.
xiv
Umea Bitki Bilm Merkezi bitki biyolojisi bilmi uzerine calisan
dinamik bir enstitu olup, bunyesinde hem Umea Universitesi hem de
Isvec Tarim Bilimleri Universitesi’ni barindiran ve 4o farkli ulkeden
yaklasik 200 kisi calistiran bir kurumdur. Bu uluslararasi cevre farkli
kulturlerin kaynasmasina ve bilime tesvik etmistir. Bu kulturel
zenginligi ve olumlu bilimsel arastirmadaki olumlu etkisini
yansitabilmek icin, farkli ulkelerden olan is arkadaslarima benim
yaptigim arastirmanin ozetini kendi dillerine cevirmeleri icin rica
ettim.
Özet (Türkçe /Turkish)
Bitkiler gunes enerjisini kullanarak karbondioksit ve suyu
sekere donusturen canlilardir. Bu nedenle yer yuzunde yasayan
bitkiler topraktan su alabilmek ve bu suyu gövdelerinde
tasiyabilmek icin xylem adi verilen bir doku olusturmuslardir.
Agaclarda, xylem dokusu odunu olusturur ki bu da material,
kagit sanayi ve yenilenebilir enerji uretimi icin önemli bir
kaynak olusturur. Xylem dokusunun ekolojik, biyolojik ve bir
cok alandaki öneminden öturu xylem dokusunun nasil
olustuguna dair bilimsel calismalar yapilmaktadir. Bu tez
calismasinda, bitkide suyu tasiyan bu xylem dokusunun nasil
olustugu anlatilmaktadir. Suyun tasinmasini saglayan bu
dokuyu olusturan hucreler su basincina dayanikli olabilmek icin
lignin adi verilen bir maddeyi sentezleyerek kalin bir hucre
duvari olustururlar. Benim calismalarim lignin maddesinin
hucre duvarina yerlesmesi icin komsu hucrelerden yardim
aldigini aciga cikarmistir. Bununla beraber, su tasiyan xylem
hucreleri kendi olumlerini organize ederler ve böylece hucrenin
ici bos kalir, bu da suyun hucreden gecisini kolaylastirir. Hucre
ölumunun gerceklesmesi icin, xylem hucrelerinin tehlikeli
molekuller kullanmasi gerekir. Benim calismalarim ayni
zamanda, kendi ölumunu gerceklestiren xylem hucrelerinin
diger hucrelere zarar vermemek icin onlarla irtibata gectigini
ortaya cikarmistir.
xv
‫ﻣﺭﮐﺯ ﺗﺣﻘﻳﻔﺎﺕ ﮔﻳﺎﻫﯽ ﺍﻭﻣﺋﻭ ﻳﮑﯽ ﺍﺯﻣﺅﺳﺳﺎﺕ ﺗﺣﻘﻳﻘﺎﺗﯽ ﻓﻌﺎﻝ ﺩﺭﺯﻣﻳﻧﻪ ﻋﻠﻭﻡ ﮔﻳﺎﻫﯽ ﻣﻳﺑﺎﺷﺩ‪.‬‬
‫ﺍﻳﻥ ﻣﺅﺳﺳﻪ ﺍﺯ ﺩﻭ ﺑﺧﺵ ﻣﺭﮐﺯ ﺗﺣﻘﻳﻔﺎﺕ ﮔﻳﺎﻫﯽ ﺍﻭﻣﺋﻭ ﻭ ﺩﺍﻧﺷﮑﺩﻩ ﮐﺷﺎﻭﺭﺯی ﺳﻭﺋﺩ ﺗﺷﮑﻳﻝ‬
‫ﺷﺩﻩ ﻭ ﺗﻌﺩﺍﺩ ﮐﺎﺭﻣﻧﺩﺍﻥ ﺍﻥ ﺣﺩﻭﺩ ﺩﻭﻳﺳﺕ ﻧﻔﺭ ﺍﺯ ﺑﻳﺵ ﺍﺯ ﭼﻬﻝ ﻣﻠﻳﺕ ﻣﺧﺗﻠﻑ ﻣﻳﺑﺎﺷﻧﺩ‪ .‬ﻭﺟﻭﺩ‬
‫ﺗﻧﻭﻉ ﻓﺭﻫﻧﮕﯽ ﺩﺭ ﺍﻳﻥ ﻣﺭﮐﺯ ﺗﺣﻘﻳﻘﺎﺕ ﺍﻣﮑﺎﻥ ﺗﺑﺎﺩﻝ ﺍﻁﻼﻋﺎﺕ ﻋﻠﻣﯽ‪-‬ﺗﺣﻘﻳﻘﺎﺗﯽ ﺭﺍ ﻓﺭﺍﻫﻡ ﺍﻭﺭﺩﻩ‬
‫ﺍﺳﺕ‪.‬‬
‫ﺑﻪ ﻣﻧﻅﻭﺭ ﻧﺷﺎﻥ ﺩﺍﺩﻥ ﺗﻧﻭﻉ ﻓﺭﻫﻧﮕﯽ ﻭﺳﻳﻊ ﺍﻳﻥ ﻣﺅﺳﺳﻪ ﻭ ﺗﺎﺛﻳﺭﺍﺕ ﻣﺛﺑﺕ ﺍﻥ‪ ،‬ﺍﺯ ﺩﻳﮕﺭ‬
‫ﮐﺎﺭﻣﻧﺩﺍﻥ ﺧﻭﺍﺳﺗﻪ ﺷﺩﻩ ﺍﺳﺕ ﮐﻪ ﺩﺭ ﺻﻭﺭﺕ ﺍﻣﮑﺎﻥ ﭼﮑﻳﺩﻩ ی ﺍﻳﻥ ﭘﺭﻭژﻩ ﺗﺣﻘﻳﻘﺎﺗﯽ ﺭﺍ ﺑﻪ ﺯﺑﺎﻥ‬
‫ﻣﺎﺩﺭی ﺗﺭﺟﻣﻪ ﻧﻣﺎﻳﻧﺩ‪.‬‬
‫ﺧﻼﺻﻪ ﻣﻁﻠﺏ ﺗﺣﻘﻳﻘﺎﺗﯽ)ﻓﺎﺭﺳﯽ‪(Farsi/‬‬
‫ﮔﻴﺎﻫﺎﻥ ﻣﻮﺟﻮﺩﺍﺕ ﺯﻧﺪﻩ ﺍی ﻫﺴﺘﻨﺪ ﮐﻪ ﺑﺎ ﺍﺳﺘﻔﺎﺩﻩ ﺍﺯ ﺍﻧﺮژی ﺧﻮﺭﺷﻴﺪ ‪ ،‬ﺩی ﺍﮐﺴﻴﺪ ﮐﺮﺑﻦ ﻭ ﺍﺏ ﺭﺍ‬
‫ﺗﺒﺪﻳﻞ ﺑﻪ ﻗﻨﺪ ﻣﻴﮑﻨﻨﺪ‪ .‬ﺑﻨﺎﺑﺮﺍﻳﻦ ﮔﻴﺎﻫﺎﻧﯽ ﮐﻪ ﺭﻭی ﺯﻣﻴﻦ ﺯﻧﺪﮔﯽ ﻣﻴﮑﻨﻨﺪ ﻳﮏ ﺑﺎﻓﺖ ﺍﻭﻧﺪی ﺗﺸﮑﻴﻞ‬
‫ﺩﺍﺩﻩ ﺍﻧﺪ ﺑﻪ ﻧﺎﻡ ﺑﺎﻓﺖ ﭼﻮﺑﯽ ﮐﻪ ﺍﺏ ﺩﺭﻭﻥ ﺧﺎک ﺭﺍ ﺑﻪ ﺩﺭﻭﻥ ﺧﻮﺩ ﺍﻧﺘﻘﺎﻝ ﻣﻴﺪﻫﺪ‪ .‬ﺩﺭ ﺩﺭﺧﺘﺎﻥ‪ ،‬ﺍﻳﻥ‬
‫ﺑﺎﻓﺕ ﭼﻭﺑﯽ ﭼﻭﺏ ﻧﺎﻣﻳﺩﻩ ﻣﻳﺷﻭﺩ ﮐﻪ ﻧﻘﺵ ﻋﻣﺩﻩ ﺍی ﺩﺭ ﺗﻭﻟﻳﺩ ﺑﻳﻭﻣﺎﺱ‪ ،‬ﺧﻣﻳﺭ ﭼﻭﺏ‪ ،‬ﮐﺎﻏﺫ ﻭ‬
‫ﺯﻳﺳﺕﺳﻭﺧﺕ ﺩﺍﺭﺩ‪.‬‬
‫ﺍﻫﻣﻳﺕ ﺍﮐﻭﻟﻭژﻳﮑﯽ ﻭ ﺯﻳﺳﺗﯽ ﺍﻭﻧﺩ ﭼﻭﺑﯽ ﻭ ﺑﺎ ﺗﻭﺟﻪ ﺑﻪ ﮐﺎﺭﺑﺭﺩ ﺍﻥ ﺩﺭ ﺯﻣﻳﻧﻪ ﻫﺎی ﮔﻭﻧﺎﮔﻭﻥ‬
‫ﻧﻘﺵ ﭼﺷﻣﮕﻳﺭی ﺩﺭ ﺯﻣﻳﻧﻪ ی ﺗﺣﻘﻳﻘﺎﺕ ﻋﻠﻣﯽ ﭘﻳﺩﺍ ﮐﺭﺩﻩ ﺗﺎ ﺑﺗﻭﺍﻥ ﺩﺭﻳﺎﻓﺕ ﮐﻪ ﺍﻳﻥ ﺑﺎﻓﺕ ﭼﮕﻭﻧﻪ‬
‫ﺷﮑﻝ ﻣﻳﮕﻳﺭﺩ‪.‬‬
‫ﺍﻳﻥ ﭘﺎﻳﺎﻥ ﻧﺎﻣﻪ ی ﺗﺣﻘﻳﻘﺎﺗﯽ ﭼﮕﻭﻧﮕﯽ ﺷﮑﻝ ﮔﻳﺭی ﺳﻠﻭﻝ ﻫﺎی ﺑﺎﻓﺕ ﭼﻭﺑﯽ ﻭ ﻫﺩﺍﻳﺕ ﮐﻧﻧﺩﻩ ﺍﺏ‬
‫ﺭﺍ ﺑﺭﺭﺳﯽ ﻣﻳﮑﻧﺩ‪ .‬ﺳﻠﻭﻝ ﻫﺎی ﻫﺩﺍﻳﺕ ﮐﻧﻧﺩﻩ ﺍﺏ ﻳﮏ ﺩﻳﻭﺍﺭ ﺿﺧﻳﻡ ﺑﻪ ﻧﺎﻡ ﻟﻳﮕﻧﻳﻥ ﺑﻪ ﺩﻭﺭ ﺧﻭﺩ‬
‫ﺗﺷﮑﻳﻝ ﻣﻳﺩﻫﻧﺩ ﮐﻪ ﺑﻪ ﻭﺍﺳﻁﻪ ی ﺍﻥ ﺗﻭﺍﻥ ﭘﺎﻳﺩﺍﺭی ﺩﺭ ﺑﺭﺍﺑﺭ ﻓﺷﺎﺭ ﺍﺏ ﺭﺍ ﺩﺍﺭﻧﺩ‪ .‬ﻣﻥ ﺑﻪ ﺍﻳﻥ‬
‫ﻣﻭﺿﻭﻉ ﭘﯽ ﺑﺭﺩﻡ ﮐﻪ ﺗﻐﻳﻳﺭ ﻓﺭﻡ ﻟﻳﮕﻧﻳﻥ ﺑﻪ ﮐﻣﮏ ﺳﻠﻭﻝ ﻫﺎی ﻣﺟﺎﻭﺭ ﻧﻳﺎﺯ ﺩﺍﺭﺩ ﮐﻪ ﻣﺑﺗﻧﯽ ﺑﺭ‬
‫ﻭﺟﻭﺩ ﻳﮏ ﭘﻝ ﺍﺭﺗﺑﺎﻁﯽ ﺑﻳﻥ ﺳﻠﻭﻝ ﻫﺎی ﭼﻭﺑﯽ ﻣﺧﺗﻠﻑ ﻣﻳﺑﺎﺷﺩ‪ .‬ﻫﻣﭼﻧﻳﻥ ﺑﺎﻓﺕ ﻫﺎی ﭼﻭﺑﯽ‬
‫ﻫﺩﺍﻳﺕ ﮐﻧﻧﺩﻩ ﺍﺏ ﺑﻪ ﺩﻟﻳﻝ ﺧﺎﻟﯽ ﻧﮕﻪ ﺩﺍﺷﺗﻥ ﺩﻳﻭﺍﺭﻩ ی ﺧﻭﺩ ﻭ ﻣﻳﺳﺭ ﻧﻣﻭﺩﻥ ﺟﺭﻳﺎﻥ ﺍﺏ ﺑﺎﻋﺙ‬
‫ﻣﺭگ ﺧﻭﺩ ﺑﻪ ﻭﺳﻳﻠﻪ ی ﻳﮏ ﺍﺑﺯﺍﺭ ﺧﻁﺭﻧﺎک ﻣﻳﺷﻭﻧﺩ‪.‬‬
‫ﺳﻬﻡ ﻣﻥ ﺩﺭ ﺍﻳﻥ ﺯﻣﻳﻧﻪ ﺗﺣﻘﻳﻘﺎﺗﯽ ﻋﻼﻭﻩ ﺑﺭ ﭘﯽ ﺑﺭﺩﻥ ﺑﻪ ﺍﻳﻥ ﺭﻭﺵ ﺧﻭﺩ ﮐﺷﯽ ﺩﺍﻧﺳﺗﻥ ﺍﻳﻥ ﻧﮑﺗﻪ‬
‫ﮐﻪ ﺳﻠﻭﻝ ﻫﺎی ﻫﺩﺍﻳﺕ ﮐﻧﻧﺩﻩ ﺍﺏ ﭼﮕﻭﻧﻪ ﺑﺎﻋﺙ ﺣﻔﺎﻅﺕ ﺍﺯ ﺳﻠﻭﻝ ﻫﺎی ﺍﻁﺭﺍﻑ ﻣﻳﺷﻭﻧﺩ ﮐﻪ ﺧﻭﺩ‬
‫ﻧﻣﻭﻧﻪ ﺍی ﺩﻳﮕﺭ ﺍﺯ ﺍﻳﻥ ﭘﻝ ﺍﺭﺗﺑﺎﻁﯽ ﺳﻠﻭﻝ ﻫﺎ ﺩﺭﭼﮕﻭﻧﮕﯽ ﺍﻳﺟﺎﺩ ﺑﺎﻓﺕ ﭼﻭﺑﯽ ﻣﻳﺑﺎﺷﺩ‪.‬‬
‫‪xvi‬‬
Abstrakt (Svenska)
Den evolutionära framgången hos landväxter har främjats kraftigt av
förvärvandet av xylemets vaskulära vävnader som ansvarar för att
transportera vatten och mineraler ifrån roten uppåt i växten. Xylemvävnad
består i angiospermier primärt av tre celltyper: savtransporterande
kärlelement (KE), fibrer som ger växten mekaniskt stöd och parenkymatiska
celler som ansvarar för vävnadens metabolism. Både KE och fibrer
producerar tjocka sekundära cellväggar (SCWs) som består av cellulosa,
hemicellulosa och lignin. Cellväggar hos KE fungerar som effektiva
vattentransporterande tuber, men först efter att KE har genomgått
programmerad celldöd (PCD) och en fullständig nedbrytning av
cellinnehållet som en del av deras differentiering. I denna avhandling
presenteras studier av hur PCD regleras i KE genom karakterisering av
funktionen hos METACASPASE9 (MC9) i xylemliknande cellkulturer av
Arabidopsis thaliana. Dessa cellkulturer kan induceras till att differentiera
till en blandning av KE och parenchymatiska icke-KE och är därför ett idealt
system för att studera cellulära processer involverade i KE PCD. I detta
system visade det sig att reducera uttryck av MC9 i KE ledde till förhöjda
nivåer av autofagi och orsakade ektopisk celldöd i icke-KE celler. Viabiliteten
hos icke-KE kunde återställas genom en specifik nedreglering av autofagi i
KE cellerna med reducerat MC9 uttryck. Dessa resultat visar att MC9 måste
regleras korrekt under KE PCD för att KE inte blir skadliga för icke-KE.
Detta demonstrerar att det existerar ett cellulärt samarbete mellan KE och
de omgivande parenkymatiska cellerna under pågående KE PCD. Samarbetet
mellan KE och närliggande parenkymatiska celler undersöktes också i
samband med ligninbiosyntes. I avhandlingen presenteras upptäckten av ett
ett cupin-domän innehållande protein, PIRIN2, som kan reglera KE
lignifieringen på ett icke-cell autonomt sätt i Arabidopsis thaliana. PIRIN2
har en negativ effekt på lignin biosyntes genom att motverka positiv
reglering av transkriptionen av ligninbiosyntesgener. Delar av denna
antagonism inkluderar kromatinmodifikationer hos ligninbiosyntesgenerna,
vilket utgör en ny typ av reglering i ligninbiosyntes. Eftersom xylemet utgör
en del av veden i trädarter, kan den i avhandlingen beskrivna icke-cell
autonoma regleringen av lignifiering generera nya sätt att modifiera
ligninbiosyntes och därmed överkomma problem kopplade till lignin i
samband med produktion av nya kemikalier så som biobränslen från
vedbiomassa.
xvii
Abstract (English)
The evolutionary success of land plants was fostered by the acquisition of the
xylem vascular tissue which conducts water and minerals upwards from the
roots. The xylem tissue of flowering plants is composed of three main types
of cells: the sap-conducting tracheary elements (TE), the fibres which
provide mechanical support and the parenchyma cells which provide
metabolic support to the tissue. Both the TEs and the fibres deposit thick
polysaccharidic secondary cell walls (SCWs), reinforced by a rigid phenolic
polymer called lignin. The cell walls of TEs form efficient water conducting
hollow tubes after the TEs have undergone programmed cell death (PCD)
and complete protoplast degradation as a part of their differentiation. The
work presented in this thesis studied the regulation of TE PCD by
characterizing the function of the candidate PCD regulator METACASPASE9
(MC9) in Arabidopsis thaliana xylogenic cell suspensions. These cell
suspensions can be externally induced to differentiate into a mix of TEs and
parenchymatic non-TE cells, thus representing an ideal system to study the
cellular processes of TE PCD. In this system, TEs with reduced expression of
MC9 were shown to have increased levels of autophagy and to trigger the
ectopic death of the non-TE cells. The viability of the non-TE cells could be
restored by down-regulating autophagy specifically in the TEs with reduced
MC9 expression. Therefore, this work showed that MC9 must tightly regulate
the level of autophagy during TE PCD in order to prevent the TEs from
becoming harmful to the non-TEs. Hence, this work demonstrated the
existence of a cellular cooperation between the TEs and the surrounding
parenchymatic cells during TE PCD. The potential cooperation between the
TEs and the neighbouring parenchyma during the biosynthesis of lignin was
also investigated. The cupin domain containing protein PIRIN2 was found to
regulate TE lignification in a non-cell autonomous manner in Arabidopsis
thaliana. More precisely, PIRIN2 was shown to function as an antagonist of
positive transcriptional regulators of lignin biosynthetic genes in xylem
parenchyma cells. Part of the transcriptional regulation by PIRIN2 involves
chromatin modifications, which represent a new type of regulation of lignin
biosynthesis. Because xylem constitutes the wood in tree species, this newly
discovered regulation of non-cell autonomous lignification represents a
potential target to modify lignin biosynthesis in order to overcome the
recalcitrance of the woody biomass for the production of biofuels.
xviii
Preface
This PhD thesis is a book that tells a story, yet it is no fiction. It is no novel,
but it reports novelty. It describes the discovery of new lands in the field of
biology. It is a logbook written by this peculiar type of explorer called a
“scientist”, who uses knowledge, ideas and experiments to navigate in an
ocean of unknown in a quest for the coasts of discovery. Once found, the
coasts of discovery must be precisely placed on the map of universal
knowledge so that others can find this piece of information, verify its validity
and ultimately use it as a resource. The map of universal knowledge is drawn
with words and any addition to it must be a text, an article, a thesis, or more
generally, a story. But not any story. Alfred Hitchcock once asked “what is
drama but life with the dull parts cut out?” Science is no drama. In biology, a
scientific story is life with the uncertainly true and certainly untrue parts cut
out. Should it often seem dull, it at least ought to be true.
Finding the truth, however, is no easy task. The coasts of discovery may sit
still but the search to find them can be troublesome. Science is no drama, but
research can turn to be dramatic. Navigating the ocean of unknown requires
great skills, hard work, patience, organization and cooperation. I must
therefore prove with this thesis that I possess such abilities. This book thus
tells another story: The story of my actions as a researcher, which can only
be read between the lines. This unwritten chapter of personal adventures,
sometimes on the edge of tragedy, often reminiscent of a comedy, always
complex and confusing, has at least made me grow. This period of personal
learning and development shall now find a place on the map of my life. If I
were to imagine the text of my biography, I hope that I could name this
period with only three letters: P, h and D.
xix
Abbreviations (1/3)
(including abbreviations used in the manuscripts)
4CL#: 4-coumarate coenzyme A ligase protein number “#”
ACL5: ACAULIS5 protein
Arabidopsis: Arabidopsis thaliana
At: Arabidopsis thaliana
ATG#: autophagy related protein number “#”
ATP: adenosine triphosphate
bHLH#: basic-helix-loop-helix protein number “#”
C3H#: p-coumarate 3-hydroxylase number “#”
C4H: cinnamate 4-hydroxylase
CAD#: cinnamyl alcohol dehydrogenase number “#”
CCoAOMT#: caffeoyl-Coenzyme A O-methyltransferase number
“#”
CCR#: cinnamoyl-CoA reductase number “#”
CESA#: cellulose synthase protein number “#”
ChIP: chromatin immunoprecipitation
ChIP-qPCR: chromatin immunoprecipitation followed by qPCR
CHS: chalcone Synthase
cLSM: confocal laser scanning microscopy
CoA: Coenzyme A
Co-IP: co-immunoprecipitation
COMT: caffeic acid O-methyltransferase
CSL: cellulose synthase-like protein
CSC: cellulose synthesizing complex
DIC: differential interference contrast
DNA: deoxyribonucleic acid
EXO70: exocyst subunit 70
F5H#: ferulate 5-hydroxylase number “#”
FDA: fluorescein diacetate
FT-IR: Fourier transform infrared
GFP: green fluorescent protein
GRI: GRIM REAPER peptide
GSK3: glycogen synthase kinase 3
GUS: β-glucuronidase
H2Bub1: histone H2B monoubiquitination mark
H3K4me3: lysine 4 trimethylation of histone H3 mark
H3K36me3: lysine 36 trimethylation of histone H3 mark
HA: human influenza hemagglutinin derived-tag (attached to a
protein of interest)
HCT: p-hydroxycinnamoyl-Coenzyme A shikimate/quinate phydroxycinnamoyltransferase
xx
Abbreviations (2/3)
(including abbreviations used in the manuscripts)
His: histidine
HUB#: HISTONE MONOUBIQUITINATION protein number “#”
IP: immunoprecipitation
IRX#: IRREGULAR XYLEM (when knocked-out) protein number
“#”
Leu: leucine
MC #: metacaspase protein number ”#”
MS: Murashige and Skoog medium
MYB: protein containing a DNA binding domain similar to that of
the human MYB proto-oncogene protein (c-myb), called after the
avian myelocytomatosis virus protein b (MYB)
MYC: human c-myc protein derived tag (attached to a protein of
interest), called after the avian myelocytomatosis virus
protein c (MYC)
NST#: NAC SECONDARY WALL THICKENING PROMOTING FACTOR
protein number “#”
OE: overexpressor
OPLS-DA: orthogonal projections of latent structures-discriminant
analysis (also known as: orthogonal partial least squaresdiscriminant analysis)
PA: piperonylic acid
PAL#: phenylalanine ammonia-lyase protein number “#”
PCD: programmed cell death
PCR: polymerase chain reaction
PI: propidium iodide
PLCP: papain-like cysteine protease
PP2AA3: PROTEIN PHOSPHATASE 2A SUBUNIT A3
PRN#: PIRIN protein number “#”
Py-GC/MS: pyrolysis-gas chromatography/mass spectrometry
qPCR: real-time quantitative PCR
RNA: ribonucleic acid
ROS: reactive oxygen species
SCW: secondary cell wall
SND#: SECONDARY WALL-ASSOCIATED NAC DOMAIN protein
number “#”
SSH: suppression subtractive hybridization
STS: silver thiosulfate
T-DNA: transfer-DNA
xxi
Abbreviations (3/3)
(including abbreviations used in the manuscripts)
TDIF: tracheary element differentiation inhibitory factor
TE: tracheary element
TED4: TE differentiation-associated protein 4
Tryp: tryptophan
UBC#: UBIQUITIN CARRIER protein number “#”
UBQ10: (poly)ubiquitin 10
VE: vessel element
VND#: vascular-related NAC domain protein number”#”
VNI2: VND-INTERACTING protein 2
WT: wild-type
XCP#: XYLEM CYSTEINE PROTEASE number “#”
XF: xylem fibre
XND1: xylem NAC domain 1
ZEN1: Zinnia endonuclease 1
Zinnia: Zinnia elegans
xxii
I. Introduction
I.1. General context, scope and relevance of the
research
In one of his correspondences, Darwin (1879) lamented that “[t]he
rapid development […] of all the higher plants within recent
geological times [was] an abominable mystery”, which seemed to
challenge his view that evolution is a slow and gradual process
(Darwin, 1859; Friedman, 2009). Later studies showed that the
diversification of the flowering plants and of other types of
vascular plants had all proceeded at a similarly high pace (Crepet
and Niklas, 2009; Silvestro et al., 2015). Indeed, the different
groups of vascular plants that appeared throughout history have
all been evolutionarily successful thanks to one of their most
defining features: their xylem vascular tissue (Ligrone et al.,
2000), which allows for efficient conduction of water and minerals
from the roots to the leaves (Raven, 1977; Brodribb, 2009), and
which has therefore enabled plants to colonize most (dry) land
habitats (Raven, 1977; Bateman et al., 1998). Nowadays, plants
cover a majority of the global land surface (Latham et al., 2014)
and over ninety percent of the extant plant species are vascular
plants (Crepet and Niklas, 2009). Vascular plants thus have a
tremendous ecological importance, especially when considering
that their vascular xylem represents an important link between soil
and atmosphere within the global water cycle (Van den Honert,
1948).
How xylem transports water upwards in a way that seemingly
defies gravity has long been the subject of numerous reflections
and investigations (for a historical perspective see Brown, 2013).
Most scientists have come to recognize the Cohesion-Tension
theory (attributed to Böhm, 1893; Dixon and Joly, 1894, 1895;
and Askenasy, 1896) as the explanation for the rise of the xylem
sap (Angeles et al., 2004; Brown, 2013). This theory of xylem sap
conduction has inspired engineers to contemplate the possibilities
of creating synthetic xylem-like designs to transport water under
pressure at a null energetic cost for various applications (Stroock
et al., 2014). However, such applications are not yet possible as
further research is needed to reproduce xylem design via
understanding better the biology and the development of the
xylem (Stroock et al., 2014). Studying xylem biology and
1
development has also become increasingly important because
xylem forms wood (xylem comes from the Greek “xylon” which
signifies “wood”) in trees, and therefore represents an important
source of biomass. Woody biomass is already used as construction
material and in the pulp and paper industry, and is predicted to
represent an important source of biofuels, biochemicals and even
food in the future (Pauly and Keegstra, 2010; Ragauskas et al.,
2014; for an optimistic review see Percival Zhang, 2013).
This PhD thesis presents new discoveries and potential applications
related to xylem biology and development in higher plants. More
precisely, the work presented here adds to the basic knowledge of
how xylem vessel cells develop, and identifies genes involved in
xylem biomass composition as potential targets to improve
biomass properties for production of biofuels. This thesis does not
pretend to solve Darwin’s “abominable mystery”, nor does it
explain the world’s ecology or does it solve the food and energy
crisis. This work is merely a demure contribution to solving the
theoretical and practical problems evoked above. If understanding
the vascular xylem of plants paves the way towards great scientific
and technical progresses, this thesis is a single nonetheless
necessary pavement. Or to use of more biological metaphor, the
work presented here is nothing more, and nothing less, than a
single cell within a biological tissue.
I.2. Xylem differentiation in higher plants
I.2.1. Xylem cell types
I.2.1.a. Vascular stem cells
Procambium and cambium
During normal development, all xylem cells differentiate from a
vascular meristem called procambium over primary growth, and
cambium over secondary growth (Torrey et al., 1971; Larson,
1994; Lucas et al., 2013). The (pro)cambial cells maintain a
meristematic identity via intercellular signalling, thus defining the
(pro)cambium as a stem cell niche (Hirakawa et al., 2010;
Hirakawa et al., 2011), while another intercellular signalling is
responsible for specification of xylem cell differentiation (For
review see Hirakawa et al., 2011; Lucas et al., 2013; Milhinhos
and Miguel, 2013). The specified xylem cells are of three types:
2
xylem parenchyma cells, xylem fibres and sap conducting vessels
(Figure 1) and tracheids (Torrey et al., 1971; Fukuda, 1996;
Raven et al., 2005).
3
I.2.1.b. Xylem sap-conducting cells: tracheary elements
Vessels and tracheids
The vessels (in Angiosperms) and tracheids (in Gymnosperms)
transport the xylem sap under negative pressure, which they can
sustain because they deposit patterned secondary cell walls
(SCWs), reinforced by a rigid lignin polymer (Smart and Amrhein,
1985; Eriksson et al., 1988; Yoshinaga et al., 1992). These SCWs
represent an important fraction of the woody biomass of conifer
trees, which do not possess fibres but only tracheids (Bailey and
Tupper, 1918; Raven, 1977). To become functional, tracheids and
vessels undergo programmed cell death (PCD), protoplast
autolysis and partial degradation of their top and bottom cell walls,
called perforation plates, thus forming longitudinally connected
hollow tubes (von Mohl, 1851; O'Brien and Thimann, 1967;
O'Brien, 1970; Torrey et al., 1971; Fukuda, 1996; Kuriyama and
Fukuda, 2002; Motose et al., 2004; Turner et al., 2007). The
tubular arrangement and the ribbed appearance of vessels and
tracheids was found reminiscent of the insect’s respiratory trachea,
leading Malpighi (1675) to collectively name tracheary elements
(TEs) both the vessels and the tracheids. TEs represent one of the
best characterized cell types in plants (Halperin, 1969; Turner et
al., 2007), and their observation yielded most of the current
knowledge on xylem cell specification and differentiation.
I.2.1.c. Cells specialised in mechanical support
Fibres
Differentiation of xylem fibres occurs in Angiosperms and also
involves PCD and autolysis (Stewart, 1966; Courtois-Moreau et al.,
2009; Déjardin et al., 2010; Bollhöner et al., 2012), although
some species such as the model herbaceous plant Arabidopsis
thaliana possess functional living fibres (Bollhöner et al., 2012).
The function of xylem fibres is to provide mechanical support to
the tissue, and subsequently to the entire plant body, thanks to
their lignified SCWs (von Mohl, 1851; Bailey and Tupper, 1918;
Raven, 1977; Bateman et al., 1998; Zhong et al., 2006; Zhong et
al., 2011). It is worth noting that the fibres constitute the main
source of polysaccharidic biomass in angiosperm trees, in great
part due to their SCWs.
4
I.2.1.d. Cells specialised in metabolic support
Parenchyma cells
In contrast to TEs and fibres, the xylem parenchyma cells do not
always deposit a SCW and they can remain alive during up to
several years in trees (Stewart, 1966; O'Brien and Thimann, 1967;
Srivasta and Singh, 1972; Nakaba et al., 2006; Donaldson et al.,
2015). Xylem parenchyma cells are thought to provide metabolic
support to the tissue (Larson, 1994; De Boer and Wegner, 1997
and references therein). More recently, a body of evidence has
accumulated to suggest the involvement of xylem parenchyma in
modulating the flow of xylem sap in the TEs (for review see
Ménard and Pesquet, 2015). The cell wall edification of TEs, and
possibly of fibres, is also influenced by xylem parenchyma cells
(Ryser and Keller, 1992; Fukuda, 1996; Ros Barceló, 2005;
Pesquet et al., 2013; Smith et al., 2013; Paper III). Overall,
xylem parenchyma cells have been mostly studied in relation to
their functions and interactions with other xylem cell types and
little is known about the molecular and cellular aspects of their
differentiation. Interestingly, xylem parenchyma cells can transdifferentiate into TEs upon adverse conditions such as pathogen
infection (Reusche et al., 2012), or wounding (Vöchting, 1892;
Simon, 1908) which thus represents an experimental system to
induce and study TE differentiation (Sinnott and Bloch, 1945).
I.2.2. Specification of xylem cell fate
I.2.2.a. Molecular factors involved in xylem specification
Mobile molecular factors specifying xylem cell fate
The specification of xylem cell differentiation involves a variety of
(mobile) molecular factors including micro RNAs (Emery et al.,
2003; Carlsbecker et al., 2010), class III homeodomain leucine
zipper transcription factors (Zhong and Ye, 1999; Emery et al.,
2003; Ohashi-Ito et al., 2005; Carlsbecker et al., 2010),
arabinogalactan proteins (Motose et al., 2004; Kobayashi et al.,
2011), signal peptides (Matsubayashi et al., 1999; Ito et al., 2006;
Kondo et al., 2011; Kondo et al., 2014) and the plant hormones
ethylene (Pesquet and Tuominen, 2011), gibberellins (Eriksson et
al., 2000; Biemelt et al., 2004; Tokunaga et al., 2006),
brassinosteroids (Iwasaki and Shibaoka, 1991; Yamamoto et al.,
1997; Caño-Delgado et al., 2004), cytokinins (Fosket and Torrey,
1969) and auxin (Jacobs, 1952, 1954). While most of these factors
5
have been reviewed elsewhere (Torrey et al., 1971; Fukuda, 1996;
Turner et al., 2007; Lucas et al., 2013; Milhinhos and Miguel,
2013; Schuetz et al., 2013), the roles of auxin, cytokinins and
brassinosteroids are further described below because these
hormones have been instrumental in establishing experimental
systems where they are used to trigger the differentiation of TEs
(Halperin, 1969; Fosket, 1970; Basile et al., 1973; Kohlenbach
and Schmidt, 1975; Fukuda and Komamine, 1980; Kubo et al.,
2005; Oda et al., 2005; Kwon et al., 2010; Pesquet et al., 2010;
Kondo et al., 2015).
I.2.2.b Hormonal induction
experimental systems
of
TE
differentiation
in
Experimental systems and the discovery of a xylogenic role
for auxin and cytokinins
TE formation induced by wounding of Coleus stems constituted the
experimental system used to demonstrate for the first time that
auxin is a necessary factor for TE differentiation (Jacobs, 1952).
Indeed, wounding-induced TE differentiation was inhibited when
the auxin-producing leaf above the wound was removed, while
applying auxin through the petiole stump restored TE
differentiation (Jacobs, 1952). TE differentiation was also
promoted by addition of cytokinins to cultured cytokinin-deficient
soybean calli, thus demonstrating a xylogenic role for cytokinins
(Fosket and Torrey, 1969).
Experimental systems using auxin and cytokinins
A combination of cytokinin and auxin was later found to induce TE
differentiation in various experimental conditions such as Coleus
stem explants (Fosket, 1970), lettuce leaf disks (Basile et al.,
1973) and Zinnia elegans (hereafter Zinnia) cell suspensions
(Kohlenbach and Schmidt, 1975). Fukuda and Komamine (1980)
upgraded the Zinnia cell suspensions into an optimized hormoneinducible in vitro TE differentiation system. This Zinnia system
enabled for better characterizing the cell biology of TEs by getting
rid of the influence (and experimental hinders) from the
surrounding tissues (Turner et al., 2007).
The discovery of a xylogenic role for brassinosteroids
In particular, the Zinnia system allowed uncovering a potential role
for brassinosteroids in TE differentiation as no TEs formed in Zinnia
cells treated with uniconazole (Iwasaki and Shibaoka, 1991;
6
Yamamoto et al., 1997) – an inhibitor of brassinosteroid and
gibberellin biosynthesis (Iwasaki and Shibaoka, 1991; Yokota et
al., 1991) – unless the cells simultaneously received exogenous
brassinosteroids (Iwasaki and Shibaoka, 1991; Yamamoto et al.,
1997).
Experimental systems using brassinosteroids
Exogenous brassinosteroids alone or in combination with auxin and
cytokinin have since been used to induce TE differentiation in
experimental systems consisting of Arabidopsis thaliana (hereafter
Arabidopsis) tissue cultures (Kwon et al., 2010) or cell suspensions
(Kubo et al., 2005; Oda et al., 2005; Pesquet et al., 2010).
Furthermore, parenchyma cells of Arabidopsis leaf disks can also
efficiently and synchronously differentiate into TEs when treated
with a mix of auxin, cytokinin and bikinin (Kondo et al., 2015),
which is thought to mimic the brassinosteroid signalling cascade
(De Rybel et al., 2009). Noteworthy, bikinin functions by inhibiting
the glycogen synthase kinase 3 (GSK3), a family of enzymes which
blocks the brassinosteroid signalling cascade in absence of
brassinosteroids (De Rybel et al., 2009), and/or in presence of the
tracheary element differentiation inhibitory factor (TDIF; Ito et al.,
2006) signal peptide (Kondo et al., 2014).
The xylogenic mode of action of auxin, cytokinins and
brassinosteroids
The apparent overlap between the brassinosteroid and TDIF
signalling cascades raises the question of whether brassinosteroids
are essential for xylem cell specification in normal physiological
conditions, or if brassinosteroids are merely inessential, however
able to promote xylem differentiation. Auxin and cytokinin are
essential for xylogenesis and a mechanism for their action has
recently been proposed: a comprehensive approach combining
experimental biology and mathematical modelling focusing on the
early development of the vasculature of Arabidopsis suggested
that auxin and cytokinin interact to define a cytokinin-responsive
domain of cell divisions and an auxin-accumulating domain of TE
differentiation (De Rybel et al., 2014).
7
I.2.3. The four modules of the xylem differentiation
programmes
I.2.3.a. The differentiation programmes of TEs and fibres
consist of four modules
The four major processes of TE and fibre differentiation
represent modules that are molecularly and evolutionarily
partially independent
After TEs and fibres have elongated and enlarged, their core
differentiation programmes seem similar because they consist of
the same four modules: SCW deposition, lignification, PCD and
autolysis (for recent reviews see Schuetz et al., 2012; Lucas et al.,
2013; Escamez and Tuominen, 2014). The need to distinguish
between these four modules is dual: firstly, they are anatomically
distinct and their respective ontogenies do not fully overlap in time
(for review see Escamez and Tuominen, 2014). Secondly, despite
some degree of regulatory and molecular interconnection, each of
these processes has been successfully decoupled from the others
in some Arabidopsis mutants (Turner and Hall, 2000; Avci et al.,
2008; Muñiz et al., 2008; Bollhöner et al., 2013; Smith et al.,
2013) or by pharmacological treatment in Zinnia TE cell
suspensions (Woffenden et al., 1998; Pesquet et al., 2013). The
distinction between the four modules is further justified if
considered in an evolutionary perspective (Figure 2), with the
appearance of PCD and autolysis predating that of SCW deposition
and lignification in the water conducting cells of land plants
(Friedman and Cook, 2000; Ligrone et al., 2000, 2012; Escamez
and Tuominen, 2014).
Similarities and differences between the differentiation of
TEs and fibres
Interestingly, the fibres of some species such as Arabidopsis do
not undergo PCD or autolysis before monocarpic senescence
(Bollhöner et al., 2012), and observations in poplar trees suggest
different molecular mechanisms for PCD and autolysis between TEs
and fibres (Courtois-Moreau et al., 2009). Furthermore, fibres
have SCWs covering nearly all of the cell surface, while TEs display
SCW thickenings with annular or spiral patterns for the first
formed primary xylem vessels (protoxylem), reticulate, scalariform
or pitted patterns for the metaxylem, and in the secondary xylem,
8
the vessels harbour pitted SCWs (Bailey and Tupper, 1918;
Wooding and Northcote, 1964; Turner et al., 2007; Muñiz et al.,
2008; Déjardin et al., 2010; Pesquet et al., 2010; Oda and
Fukuda, 2012a).
9
The cell walls of TEs and fibres often differ in their lignin contents,
as well as in the abundance of different monomers within the lignin
heteropolymer (Eriksson et al., 1988; Saka and Goring, 1988;
Donaldson, 2001; Gorzsás et al., 2011; paper III). Hence, the
four modules of TE and fibre differentiation programmes are
grossly similar in their purposes and in their outcomes, but the
underlying molecular mechanisms must differ at least in part
between the two cell types. Most of the current knowledge on the
cell biology and the molecular regulation of xylem differentiation
accumulated over the study of TEs, and only to a limited extent
over the study of fibres. Therefore, most of the descriptions of the
molecular and cellular events of xylem differentiation presented
below (sometimes implicitly) refer to TEs, while specific
descriptions of fibre differentiation are explicitly exposed when the
corresponding knowledge exists.
I.2.3.b. The molecular master switches controlling the xylem
differentiation programmes
TE differentiation is triggered by NAC transcription factors
The differentiation of TEs can be activated by transcriptional
master switches from the NAC (NAM/ATAF/CUC; Aida et al., 1997)
transcription factor family, which function downstream of the
hormonal signals that drive xylem specification (Kubo et al.,
2005). In Arabidopsis TE differentiation is controlled by
VASCULAR-RELATED NAC DOMAIN 6 (VND6) and VND7, which
were first identified based on their expression profile in TEdifferentiating Arabidopsis cell suspensions (Kubo et al., 2005).
Arabidopsis plants, tobacco and Arabidopsis cell suspensions as
well as poplar leaves overexpressing VND7 or VND6 displayed
ectopic differentiation of TEs, including SCW deposition,
lignification and PCD (Kubo et al., 2005; Oda et al., 2010; OhashiIto et al., 2010; Yamaguchi et al., 2010b; Schuetz et al., 2014).
The trans-differentiating TEs induced by overexpression of
Arabidopsis VND7 or VND6 in Arabidopsis plants and poplar leaves
harboured SCW patterns reminiscent of protoxylem or metaxylem,
respectively (Kubo et al., 2005). Conversely, differentiation of
metaxylem or protoxylem was inhibited in Arabidopsis seedlings
expressing a dominant repressor version of VND6 or VND7,
respectively (Kubo et al., 2005). These observations lead to the
suggestion that VND7 specifically triggers the differentiation of
protoxylem while VND6 triggers the differentiation of metaxylem
(Kubo et al., 2005), although VND7 was later shown to be able to
10
induce the differentiation of all types of TEs in Arabidopsis
(Yamaguchi et al., 2008). In addition, infection of Arabidopsis
xylem vessels by the pathogen Verticillium longisporum induces
the surrounding parenchyma cells to trans-differentiate into TEs in
a VND7-dependent manner (Reusche et al., 2012). VND7 may
interact with the Arabidopsis VND2 to VND5 (Yamaguchi et al.,
2008), which also have the ability to trigger TE differentiation
when overexpressed, as do their Arabidopsis homologue VND1 and
VND homologues in other species such as poplar or banana
(Ohtani et al., 2011; Zhou et al., 2014; Negi et al., 2015).
However, the importance of VND1-5 compared to VND6 and VND7
during normal xylem development remains unclear.
VND6 and VND7 appear as the transcriptional master
switches of TE differentiation
Xylem development can be hindered by overexpressing the
Arabidopsis XYLEM NAC DOMAIN1 (XND1) or its poplar or cotton
homologues (Zhao et al., 2008; Grant et al., 2010; Li et al.,
2014), suggesting that XND1 is a negative regulator of TE
formation. On the other hand, only the size of the TEs, but not the
overall xylem formation, was reduced in the xnd1 loss of function
mutants, suggesting that physiological levels of XND1 normally
retard the onset of SCW deposition during TE cell expansion,
possibly by counteracting VND6 and VND7 (Zhao et al., 2008).
Similarly, the NAC VND-INTERACTING2 (VNI2) acts as a
transcriptional repressor of xylem differentiation, as illustrated by
the similar vascular defects in Arabidopsis plants expressing a
dominant repressor version of VND7 or VNI2, or overexpressing
VNI2 (Yamaguchi et al., 2010a). In summary, several NACs can
influence xylem differentiation but only VND6 and VND7 seem to
represent the molecular master switches that are sufficient to
integrate a diversity of upstream signals into a trigger for the TE
differentiation programme.
NAC transcription factors control the fibre SCW biosynthesis
and possibly the entire fibre differentiation
Regarding the fibres, it remains unclear whether their
differentiation is also controlled by molecular master switches. The
transcription factors NAC SECONDARY WALL THICKENING
PROMOTING FACTOR (NST3)/SECONDARY WALL-ASSOCIATED
NAC DOMAIN PROTEIN (SND1) and NST1, which are
phylogenetically related to VNDs (Zhong et al., 2006; Mitsuda et
al., 2007; Zhong et al., 2010a), are expressed in the xylem fibres
11
and in the interfascicular fibres of Arabidopsis, and can therefore
be regarded as candidate regulators of fibre differentiation. Ectopic
overexpression of NST1 and NST3/SND1 activates the
transcriptional machinery, and subsequent formation, of TE-like
SCW
thickenings,
but
apparently
not
PCD,
in
nonsclerenchymatous cell types (Mitsuda et al., 2005; Zhong et al.,
2006). Furthermore, double mutants for NST1 and NST3/SND1 or
expression of a dominant repressor version of NST3/SND1
triggered the loss of functional fibres in Arabidopsis stems (Zhong
et al., 2006; Mitsuda et al., 2007; Zhong et al., 2010a, b). This
loss of functional fibres could correspond to a loss of the fibre cell
type, or alternatively to a failure of existing fibre cells to fulfil their
function due to insufficient SCW biosynthesis. Hence, NST1 and
SND1/NST3 can clearly be identified as the master regulators of
fibre SCW biosynthesis (Mitsuda et al., 2005; Zhong et al., 2006;
Mitsuda et al., 2007; Zhong et al., 2008; Zhong et al., 2010a, b),
but the current data does not clarify whether NST1 and
SND1/NST3 are more generally master switches of the entire fibre
differentiation programme in Arabidopsis. This ambiguity is due to
the fact that the differentiation of Arabidopsis fibres involves SCW
biosynthesis but not PCD. Hence, the observation of PCD cannot
be used to distinguish between the biosynthesis of the fibre SCWs
and the entire fibre differentiation programme in Arabidopsis. On
the other hand it is possible that the fibre differentiation and the
SCW biosynthesis programmes are one and the same in
Arabidopsis, rendering NST1 and SND1/NST3 potential master
switches of fibre differentiation.
I.2.3.c. Polysaccharidic Secondary Cell Wall deposition
Cellulose and hemicelluloses
The secondary walls of the xylem cells are mainly composed of a
cellulose framework and a matrix of hemicelluloses, two different
types of polysaccharidic polymers which are deposited before the
cell wall reinforcement by lignin (Harlow and Wise, 1928; Timell,
1967; Northcote, 1972; Taylor et al., 2003; Somerville, 2006;
Scheller and Ulvskov, 2010). More precisely, hemicelluloses consist
of ramified polysaccharidic β-(14)-linked backbones most often
composed of combinations of pentoses such as xylose and
arabinose, and hexoses such as mannose, glucuronic acid and
glucose (for review see Timell, 1967; Scheller and Ulvskov, 2010).
Cellulose, in contrast, is solely composed of β-(14)-linked
glucose chains which further organize through hydrogen bonds and
12
Van der Waals interactions in order to form crystalline microfibrils
(for review see Brett, 2000; Somerville, 2006). In contrast with
primary cell walls, the cellulose microfibrils of the secondary walls
are organized into parallel strata, whose orientation allows
distinguishing three SCW layers (S1, S2 and S3), which may also
differ in their hemicellulose contents and compositions (Wardrop
and Bland, 1959; Timell, 1967; Turner et al., 2007; Kaneda et al.,
2010).
Biosynthesis and deposition of SCW polysaccharides
The locations and processes associated with the biosynthesis of
cellulose and hemicelluloses differ. How the various hemicelluloses
are produced is not fully understood but the current knowledge
indicates that they are synthesized by membrane-bound cellulose
synthase-like (CSL) glycosyl transferases in the Golgi apparatus,
and subsequently exported to the cell walls (reviewed by Scheller
and Ulvskov, 2010; Rennie and Scheller, 2014). Both the primary
wall cellulose and the cellulose of the secondary cell walls are
synthesized and deposited at the plasma membrane by rosetteshaped cellulose synthesizing complexes (CSCs) containing
cellulose synthase (CESA) enzymes (Mueller and Brown, 1980;
Herth, 1983; Kimura et al., 1999; Taylor et al., 2003; Somerville,
2006; Turner et al., 2007; Watanabe et al., 2015). The study of
TEs in cell suspensions enabled observing that TE SCW patterning
requires the CSCs to be restricted to specific domains of the
plasma membrane by the cortical microtubules associated with
specific interactors (Oda et al., 2010; Pesquet et al., 2010; Oda
and Fukuda, 2012b; Watanabe et al., 2015). The CSCs depositing
SCW cellulose in spermatophytes comprise a specific subset of
three CESAs (Taylor et al., 2003; Harholt et al., 2012) which were
first identified by a mutagenesis approach in Arabidopsis, and
named based on their collapsed xylem phenotype: IRREGULAR
XYLEM1 (IRX1)/CESA8, IRX3/CESA7 and IRX5/CESA4 (Turner and
Somerville, 1997; Taylor et al., 1999; Taylor et al., 2000; Taylor
et al., 2003; Somerville, 2006; Turner et al., 2007; Harholt et al.,
2012).
The polysaccharides of xylem SCWs as a source of biofuels
Cellulose and to a lesser extent hemicelluloses represent a source
of fermentable sugars for the production of biofuels (Pauly and
Keegstra, 2010; Rennie and Scheller, 2014). Therefore, the thick
cell walls of xylem TEs and fibres can be seen as a major source of
biofuels, but their deconstruction and conversion into biofuels is
13
hindered by the SCW cross-linked hemicelluloses which have a
high content of low fermentable pentoses (Petersen et al., 2012;
Rennie and Scheller, 2014). Interestingly, the amount of the
hemicellulose xylan could be specifically decreased in the fibres of
Arabidopsis while maintained in the TEs by expressing a xylan
biosynthetic gene downstream of the promoter of VND6 or VND7
in knock-out plants for this xylan biosynthetic gene (Petersen et
al., 2012). Some of these Arabidopsis plants with normal TE cell
walls and xylan-deficient fibre cell walls had no defects in growth
or stem strength, while their release of fermentable sugar was
significantly increased (Petersen et al., 2012). This increased
saccharification was attributed to a decrease in hemicellulosemediated tethering of cellulose microfibrils, to an increase in the
amount of easy fermentable hexoses in the walls and to a
decrease in lignin contents (Petersen et al., 2012). Indeed, lignin
and hemicelluloses are considered to bind together in order to
increase the strength of the xylem cell walls (Meshitsuka et al.,
1982; Boerjan et al., 2003; Zhou et al., 2010; Balakshin et al.,
2011). In this accepted model of secondary cell wall architecture,
the lignin plays a major biological role in cell wall reinforcement
(Stewart et al., 1953; Timell, 1967; Amrhein et al., 1983; Smart
and Amrhein, 1985; Boerjan et al., 2003; Bonawitz and Chapple,
2013) which also represents a major hinder for wall deconstruction
(Boerjan et al., 2003; Yang and Wyman, 2004; Bonawitz and
Chapple, 2013; Wilkerson et al., 2014; Anderson et al., 2015).
I.2.3.d. Lignification
Lignin deposition in xylem cell walls
Lignin is a complex heteropolymer composed of different
monomers from the phenylpropanoid pathway, with a
predominance of p-coumaryl, coniferyl, and sinapyl alcohols, which
differ in their degree of methoxylation (Boerjan et al., 2003;
Bonawitz and Chapple, 2010; Ralph, 2010; Vanholme et al., 2010;
Hao and Mohnen, 2014). The biosynthetic pathway leading to the
production of these monomers, referred to as monolignols, is well
understood (Boerjan et al., 2003; Bonawitz and Chapple, 2010;
Ralph, 2010; Vanholme et al., 2010; Vanholme et al., 2013; Hao
and Mohnen, 2014; Wang et al., 2014) and the enzymatic kinetics
of the lignin biosynthetic enzymes have been thoroughly
investigated and modelled in the tree species Populus trichocarpa
(Wang et al., 2014). These monolignols are thought to be exported
by ATP-dependent transporters (Kaneda et al., 2008; Miao and
14
Liu, 2010; Alejandro et al., 2012; Schuetz et al., 2013; Schuetz et
al., 2014) to the cell walls where they undergo oxidative coupling
reactions collectively named lignification (Boerjan et al., 2003 and
references therein). Whether lignification requires dirigent proteins
to guide the polymerization or not remains a point of debate
(Davin and Lewis, 2000; Boerjan et al., 2003; Ralph, 2010) in
which the former mechanism is considered unlikely (see for review
Boerjan et al., 2003; Ralph, 2010). In any case, the supply of
reactive oxygen species (ROS) by enzymes such as laccases (Liu
et al., 1994; Brown et al., 2005; Berthet et al., 2011; Zhao et al.,
2013; Schuetz et al., 2014) and peroxidases (Lee et al., 2013;
Shigeto et al., 2015) is required for the oxidative coupling of
monolignols. Part of the monolignols and of the ROS required for
their polymerization are provided to the xylem fibres and to the
TEs by the surrounding cells, meaning that lignification is a
partially non-cell autonomous and sometimes occurs post-mortem
(Stewart et al., 1953; Stewart, 1966; Pesquet et al., 2013; Smith
et al., 2013).
Heterogeneity of xylem lignin
Once incorporated into the lignin polymer, the aforementioned
monolignols constitute p-hydroxyphenyl (H), guaiacyl (G) and
syringyl (S) units, the two latter being the most common units in
the xylem of angiosperms (For review see Donaldson, 2001
Boerjan et al., 2003; Bonawitz and Chapple, 2010; Ralph, 2010;
Vanholme et al., 2010; Hao and Mohnen, 2014). The proportion of
each unit varies between species (see for review Vanholme et al.,
2010), between cell types (Saka and Goring, 1985; Eriksson,
1988; Donaldson, 2001; Gorzsás et al., 2011) as well as between
different parts of the same cell wall such as the middle lamella, the
primary wall and the different layers of the SCWs (Terashima and
Fukushima, 1988; Donaldson, 1994). Especially, the xylem vessels
generally deposit more lignin than the fibres do, and the lignin of
vessels is enriched in G units while that of fibres contains more S
units (Saka and Goring, 1985; Donaldson, 2001 and references
therein). Furthermore, S lignin is thought to be deposited later
than G lignin (Terashima and Fukushima, 1988; Terashima et al.,
1993; Donaldson, 2001), which, together with the former
observations, suggests a complex regulation of lignin biosynthesis
(Terashima and Fukushima, 1988).
15
Transcriptional regulation of lignification by transcription
factors
Campbell and Sederoff (1996) proposed that the regulation of
lignin biosynthetic genes’ transcription could represent an
additional important regulatory step in the spatial, temporal and
compositional control of lignification. For example, the
characterization of the promoter activity of the parsley gene
encoding for 4-coumarate coenzyme A ligase (4CL)-1, and of the
bean gene encoding for phenylalanine ammonia-lyase (PAL)-2 in
transgenic tobacco revealed the existence of cis-elements involved
in controlling the spatial expression of the corresponding genes
(Leyva et al., 1992; Hauffe et al., 1993; Hatton et al., 1995). The
sequence of some AC-rich cis-elements (AC elements) controlling
the spatial and developmental pattern of the bean PAL-2 promoter
activity suggested that the expression of this gene, and by
extension the expression of lignin biosynthetic genes in general,
could be specifically regulated by MYB-family transcription factors
(Hatton et al., 1995). Later studies identified several MYB
transcription factors able to directly and specifically regulate the
transcription of monolignol biosynthetic genes in Arabidopsis
(Zhong et al., 2008; Zhou et al., 2009; Öhman et al., 2013), in
maize (Fornalé et al., 2010) and in pine (Patzlaff et al., 2003b;
Patzlaff et al., 2003a). In addition to MYBs, the tobacco Pal-box
binding protein LIM1 was identified as a specific transcriptional
activator for three key monolignol biosynthetic genes (Kawaoka et
al., 2000). Recently, a system biology approach aiming to unravel
the gene regulatory networks for SCW biosynthesis in Arabidopsis
suggested the existence of other specific regulators of lignin
biosynthetic gene expression, most of which are MYBs (TaylorTeeples et al., 2015). This over-representation of MYBs in the
specific regulation of monolignol biosynthesis is interesting,
especially considering that MYBs are not part of the transcription
factors over-represented in the overall SCW-related regulatory
network (Taylor-Teeples et al., 2015). It is thus possible that
“regulatory sub-networks” specifically control the expression of
lignin-related genes, consistent with the fact that lignin represents
a “late” evolutionary innovation in the design of water conducting
cells in land plants (Friedman and Cook, 2000; Escamez and
Tuominen, 2014). Furthermore, the expression of lignin
biosynthetic genes is also regulated by more general transcription
factors such as the Arabidopsis VND7 (Zhong et al., 2010b;
Yamaguchi et al., 2011), NST3/SND1 (Zhong et al., 2010a) and
their downstream direct targets MYB46 and MYB83, which are
16
considered to orchestrate the polysaccharidic SCW biosynthesis
and the cell wall lignification (Zhong et al., 2007; Zhong et al.,
2008; Ko et al., 2009; McCarthy et al., 2009; Zhong and Ye,
2012).
These
examples
demonstrate
that
the
(direct)
transcriptional regulation of monolignol biosynthetic genes is
multilayered, including feed-forward loops (Taylor-Teeples et al.,
2015) starting with factors that promote the entire differentiation
of the lignifying xylem cells through factors that orchestrate the
coordinated biosynthesis of complex SCWs down to lignin-specific
transcription factors.
Transcriptional regulation of lignification by chromatin
modifications
Recently, a genome-wide mapping of the transcriptional activation
chromatin mark lysine 4 trimethylation of histone H3 (H3K4me3)
in the xylem tissue of eucalyptus suggested a potential
transcriptional regulation of monolignol biosynthetic genes at the
chromatin level (Hussey et al., 2015). In plants, chromatin regions
associated with active transcription bear a combination of the
activation marks H3K4me3, lysine 36 trimethylation of histone H3
(H3K36me3) and Histone H2B monoubiquitination (H2Bub1)
(Roudier et al., 2011; Sequeira-Mendes et al., 2014). While
mammals and yeasts require the presence of H2Bub1 marks for
the establishment of the H3 methylations associated with active
transcription (for review see Cao and Ma, 2011), the situation is
different in plants: The genome-wide abundance of H3K4me3
remains roughly unchanged in Arabidopsis loss-of-function
mutants for the two E2 enzymes UBIQUITIN CARRIER PROTEIN1
(UBC1) and UBC2, or either of the E3 ligases HISTONE
MONOUBIQUITINATION1 (HUB1) and HUB2, which associate into a
complex to perform H2B monoubiquitination (Fleury et al., 2007;
Cao et al., 2008; Dhawan et al., 2009; Schmitz et al., 2009;
Himanen et al., 2012). Interestingly, loss of function in the
machinery writing H2Bub1 marks was found to affect H3
methylations and subsequent gene expression of a subset of genes
related to flowering (Cao et al., 2008; Dhawan et al., 2009;
Schmitz et al., 2009) and to the circadian clock (Himanen et al.,
2012). The gene specific effect of H2Bub1 (and/or its writing
machinery) on the level of H3K4me3, and potentially of
H3K36me3, implies the existence of additional factors that drive
this specificity. Specific regulation of the levels of H3K4me3 marks
in lignin biosynthetic genes in relation to H2Bub1 marks (and/or
17
the proteins writing them) has not yet been reported in the
literature but is presented later in this thesis (paper III).
Biotechnological
modifications
of
lignin
to
reduce
lignocellulosic biomass recalcitrance
The transcriptional and enzymatic regulations of monolignol
biosynthesis are better characterized than the export and the
polymerization of monolignols, which has led to numerous
attempts to target these former regulatory steps for the
improvement of sugar release from lignocellulosic biomass
(Bonawitz and Chapple, 2013). However, many of the efforts to
reduce monolignol biosynthesis and/or alter lignin composition by
knocking-down monolignol biosynthetic genes have resulted in
plants with growth defects that outweigh the gains from improved
sugar release (for review see Bonawitz and Chapple, 2013). One
apparent exception was reported in greenhouse grown poplars
with reduced expression of 4CL-1 and therefore reduced lignin
content which was associated with increased growth and higher
cellulose content compared with wild-type trees (Hu et al., 1998).
However, longer-term experiments on 4CL-1 down-regulated trees
suggested that their decreased lignin content was associated with
increased susceptibility to vascular embolism and with a negative
impact on xylem hydraulics (Voelker et al., 2011). Recently,
elaborate metabolic engineering based on “gain-of-function”
strategies successfully reduced xylem lignin content and/or
recalcitrance to degradation in Arabidopsis (Zhang et al., 2012;
Eudes et al., 2014) and poplar (Wilkerson et al., 2014) without
affecting the amount of plant biomass. Using pine xylogenic cell
suspensions, Wagner et al. (2015) also provided a proof of concept
that the less recalcitrant S-lignin (Li et al., 2010) could be
introduced in gymnosperms which otherwise mainly contain Glignin. In Arabidopsis the knowledge of the transcriptional
regulation of TE and fibre differentiation allowed to perform “gene
rewiring” to increase the amount of polysaccharides and drastically
reduce the amount of lignin in the SCWs of fibres, without
affecting the TEs (Yang et al., 2013). If applied to woody species,
this approach is thus expected to maintain xylem hydraulics and
normal plant growth while simultaneously increasing the amount
of extractable sugars and decreasing the recalcitrance of fibre cell
walls. However, the use of a master switch transcription factor
such as NST1 or its orthologues to create a fibre-specific artificial
feed forward loop (Yang et al., 2013) in another species than
Arabidopsis may result in the premature induction of fibre
18
programmed cell death. In the TEs of Arabidopsis, the occurrence
of premature cell death has been shown to prevent SCW
deposition (Muñiz et al., 2008) and a similar output could be
expected in the fibres of other species. Hence, it is important to
identify and use specific regulators of lignin biosynthesis and of
polysaccharidic SCW deposition in order to implement such an
elegant “gene rewiring” strategy (Yang et al., 2013) in wood,
without taking the risk of inducing premature wood fibre PCD.
I.2.3.e. Programmed Cell Death
Definition of programmed cell death
Programmed cell death is an important mechanism for the
development and the health of multicellular organisms, as well as
for the fitness of populations of unicellular organisms (Lockshin
and Zakeri, 2001; Ameisen, 2002; Fendrych et al., 2014; Bidle,
2015). Lockshin and Williams (1964) coined the term
“programmed cell death” to describe developmentally predictable,
non-accidental sequences of events during cell death that had
been observed since the 19th century (for a historical perspective
see Clarke and Clarke, 1996; Lockshin and Zakeri, 2001). Studies
in the metazoan model organism Caenorhabditis elegans lead to
the discovery of a genetic regulation of PCD (Horvitz et al., 1983),
which was confirmed by the identification of a caspase (cysteinedependent aspartate proteinease) as the first characterized
positive regulator of developmental cell death (Yuan et al., 1993;
Alnemri et al., 1996; Lockshin and Zakeri, 2001, 2002). The
developmental cell deaths of TEs and fibres are both considered to
be (genetically) programmed because poplar fibres die in a
coordinated fashion (Courtois-Moreau et al., 2009) and TE cell
death is associated with a conserved sequence of physiological
events (for review see Escamez and Tuominen, 2014).
Furthermore, in the case of TEs, cell death is a functionalizing
event which contributes to the biological, post-mortem function of
these cells (Raven, 1977; Friedman and Cook, 2000; Ménard et
al., 2015).
Programmed cell death and autolysis are different
processes
Fully differentiated xylem TEs and Fibres can be clearly identified
as dead because they have undergone complete protoplast
autolysis, defined as the degradation a cell’s content by its own
enzymes (O'Brien and Thimann, 1967; Torrey et al., 1971;
19
Wodzicki and Brown, 1973; Shininger, 1979; Fukuda, 1996;
Escamez and Tuominen, 2014). Although fully autolyzed cells
ought to be dead, this does not necessarily imply that
programmed cell death and autolysis are part of the same cellular
process (for review see Escamez and Tuominen, 2014). O’Brien
and Thimann (1967) had already proposed that TE protoplast
autolysis could involve different mechanisms from TE PCD. More
recently, TE PCD and autolysis were proven to be genetically
distinct in Arabidopsis mutants in which loss-of-function of the
cysteine proteases XYLEM CYSTEINE PROTEASE 1 (XCP1) or both
XCP1 and XCP2, or METACASPASE9 (MC9) impaired autolysis
without affecting PCD (Avci et al., 2008; Bollhöner et al., 2013).
As a consequence, the occurrence of PCD cannot be deduced
based on the sole observation of autolytic features, but rather
requires the observation of a clearly established sequence of
morphological, physiological and molecular events (Escamez and
Tuominen, 2014).
Physiological events associated with TE PCD
The instant of death in TEs is marked by the rupture of the
tonoplast and the concomitant demise of cyclosis (Groover and
Jones, 1999; Bollhöner et al., 2012), because it constitutes a point
of no-return in the progression of the TE cell death (Fukuda,
1996). Therefore, any event that is part of the cell death
programme must take place before the vacuolar collapse, while
later events should be considered as post-mortem (Escamez and
Tuominen, 2014). To date, only few pre-mortem physiological
events have been associated with TE PCD. During the most studied
type of metazoan PCD, apoptosis (Kerr et al., 1972), the integrity
of the mitochondrial envelope is compromised, leading to the
release of the cytochrome c into the cytoplasm, triggering a PCD
signalling cascade (Cain et al., 2002; Garrido et al., 2006). The
existence of such mechanism during TE PCD was investigated in
differentiating Zinnia elegans cell suspensions, in which
mitochondria undergo subtle morphological changes and loss of
membrane potential before TE cell death (Yu et al., 2002). In this
system,
TE
vacuolar
collapse
could
be
induced
by
pharmacologically triggering the loss of mitochondrial membrane
potential, but not by triggering the release of cytochrome c (Yu et
al., 2002). Thus, in contrast with metazoan apoptosis, TE PCD
does not rely on a cytochrome c-dependent signalling although it is
affected by mitochondrial membrane integrity (Yu et al., 2002).
Shortly before TE cell death, altered tonoplast permeability was
20
observed in differentiating Zinnia cell suspensions (Kuriyama,
1999; Obara et al., 2001). In addition, vacuolar collapse could be
induced upon treatment with probenecid to affect anion transport
across the tonoplast (Kuriyama, 1999). Finally, Zinnia TE PCD
could be induced by calcium influx, probably in response to
extracellular proteolysis, as demonstrated by exogenous
application of pharmacological compounds that promoted or
inhibited these processes (Groover and Jones, 1999). From the
previous observations, it seems that the molecular regulations of
mitochondrial membrane and tonoplast integrity, and of calcium
influx, in turn regulate TE PCD.
Molecular factors associated with the positive regulation of
TE PCD
The induction and execution of plant PCD have been proposed to
rely on the action of proteases in the same way as caspases, which
mediate proteolytic signalling cascades during apoptosis (Lam,
2005; Woltering, 2010). Caspase-like activities have been reported
in differentiating xylem in poplar trees (Han et al., 2012) and in
xylogenic Zinnia cell suspensions (Twumasi et al., 2010), in which
treatment with caspase inhibitors decreased TE differentiation
(Lakimova and Woltering, 2009; Twumasi et al., 2010). However,
caspase homologues cannot be found in plants and the caspaselike activity detected in differentiating xylem (Twumasi et al.,
2010; Han et al., 2012) could not be unambiguously linked to TE
PCD. Plants possess structural analogues of caspases called
metacaspases (MCs) which display a different substrate specificity
(Uren et al., 2000), but which were anyway proposed to function
in promoting PCD. Consequently, MC9, the only metacaspase
whose expression is induced during Arabidopsis in vitro TE
differentiation was proposed as a positive regulator of TE PCD
(Turner et al., 2007). Similarly, numerous other TE expressed
cysteine-dependent proteases (Ye and Varner, 1996; Beers and
Freeman, 1997; Zhao et al., 2000; Bozhkov and Jansson, 2007;
Turner et al., 2007), and a serine-dependent protease (Groover
and Jones, 1999), were proposed to be positive effectors of TE
PCD. However, only three of these proteases were genetically
characterized, revealing that they function in post-mortem
autolysis (Avci et al., 2008; Bollhöner et al., 2013), while none of
the above proteases has so far been demonstrated to be a positive
regulator of PCD per se. Even though a function for MC9 could not
clearly be established in TE PCD (Bollhöner et al., 2013), MC9 has
been shown to control the ROS-induced spreading of cell death in
21
Arabidopsis leaves by activating the pro-death signalling GRIM
REAPER (GRI) peptide (Wrzaczek et al., 2015). It is therefore
possible that MC9 controls a yet unknown form of cell death
signalling. Another reason for MC9 to remain a candidate positive
regulator of TE PCD is the fact that a MC9 homologue has been
shown to regulate the mode of cell death during spruce embryo
suspensor PCD, via regulating autophagy in these cells (Minina et
al., 2013).
Potential participation of autophagy in TE PCD
The cellular process of autophagy (De Duve and Wattiaux, 1966)
has been proposed as a mechanism that promotes plant PCD in
general (van Doorn et al., 2011) and fibre PCD (Courtois-Moreau
et al., 2009) and TE PCD (Kwon et al., 2010) in particular.
Autophagy was originally discovered by the observation of vesicles
named autophagosomes that could engulf some cytoplasmic
content in a cell in order to target it for degradation in lytic
compartment such as lysosomes or vacuoles (De Duve and
Wattiaux, 1966). This vesicular pathway for degradation has later
been renamed macroautophagy on account of the discovery of
morphologically
distinct
but
mechanistically
overlapping
autophagic (“self-digestion”) pathways such as microautophagy,
chaperone-mediated autophagy and organelle-specific autophagy
(For review see Liu and Bassham, 2012). Macroautophagy is the
best characterized autophagic pathway owing to the discovery in
yeasts of specific genes involved in its regulation (ATG genes;
Klionsky et al., 2003), most of which are conserved in animals and
plants (Yang and Klionsky, 2010; Liu and Bassham, 2012). Due to
its predominance in the literature, the process of macroautophagy
is usually referred to (including below) as autophagy (Figure 3).
Autophagy was originally recognized to function in nutrient
recycling during starvation (Mortimore and Pösö, 1987; Mizushima
et al., 2004; Thompson et al., 2005; Yang and Klionsky, 2010)
through bulk degradation of cytoplasmic content. Autophagy is
now known to also operate in favourable trophic conditions in
Arabidopsis (Guiboileau et al., 2012). Furthermore, some instances
of selective autophagic degradation of specific targets have been
reported (Yang and Klionsky, 2010; Derrien et al., 2012; Michaeli
et al., 2015; Wurzer et al., 2015). Specific target degradation by
autophagy has been linked to the existence of adaptor proteins
which bind to both the autophagosome membrane-bound ATG8
protein (considered a marker for autophagosomes; Klionsky et al.,
2012) and the targeted cargo (Yang and Klionsky, 2010; Svenning
22
et al., 2011; Wurzer et al., 2015). The fact that autophagy does
not solely function in bulk cytoplasmic degradation but that it also
targets specific proteins for degradation implies a potential
regulatory role for autophagy in cellular processes, including PCD.
However, there is still a debate in both animal (Clarke and Puyal,
2012; Denton et al., 2012; Shen et al., 2012; Gump et al., 2014;
Liu and Levine, 2014) and plant research (van Doorn et al., 2011;
Hackenberg et al., 2013; Minina et al., 2013; Lv et al., 2014;
Minina et al., 2014) regarding whether autophagic cell death
exists, whether increased autophagy is merely present in some
cells undergoing PCD without contributing to the cell death
processes, or whether autophagy indirectly contributes to PCD by
maintaining the organization of the dying cells which therefore die
in a controlled rather than necrotic fashion.
23
Before this thesis (Paper II), only one published study addressed
the role of autophagy in the TEs, claiming the occurrence of
autophagic cell death during Arabidopsis TE differentiation (Kwon
et al., 2010). However, this study relied on constitutive downregulation or constitutive, ectopic induction of autophagy which
both affected xylem differentiation rates (Kwon et al., 2010),
rendering impossible the interpretation of the results in terms of
TE PCD. In the case of fibre PCD, the contribution of autophagy to
the cell death processes was hypothesized based on induction of
ATG genes and based on morphological features of cytoplasmic
degradation, but autophagic cell death was not demonstrated
(Courtois-Moreau et al., 2009). Indeed, autophagic cell death is
difficult to demonstrate. In theory, demonstrating autophagic cell
death requires at least the observation of an increased level of
autophagy in the dying cells, as well as an effect of autophagy
inhibition on the observed PCD process (as can be derived from
the recommendations from the Nomenclature Committee on Cell
Death; Galluzi et al., 2012). Therefore, to date there is not a
single cellular process or molecular effector whose role is
unambiguously characterized as to specifically trigger or execute
PCD in xylem.
Negative regulation of TE PCD
The absence of any known positive regulator of PCD in xylem could
come from the fact that evolution co-opted existing genes with a
non-death related function to regulate PCD (Ameisen, 2002;
Galluzzi et al., 2008); genes which likely also retained all or part of
their ancestral pro-life function (Ameisen, 2002; Gallusi et al.,
2008), hindering the unambiguous identification of their role in
PCD. In addition, the potential functional redundancy of positive
PCD regulators or the probable deleterious effect of impaired PCD
on health and development likely hinder the discovery of factors
that promote PCD. Alternatively, it could be that in at least some
types of PCD, no positive regulator exists: In theory, PCD need not
be mediated by specific positive regulators, but may solely require
the presence of cell death repressors whose action is stopped in a
genetically controlled fashion. Such a negative regulator of TE PCD
could be the tetra-amine synthase ACAULIS5 (ACL5), or its
product thermospermine, based on the observation that TEs
undergo premature cell death in Arabidopsis acl5 mutants (Muñiz
et al., 2008). On the other hand, protoxylem TEs are not affected
by the loss of ACL5 function in Arabidopsis (Muñiz et al., 2008),
suggesting that either there exist a different type of PCD in
24
protoxylem TEs from other TEs or that ACL5 is involved in a more
complex (PCD-related) regulatory mechanism. It is also unclear
whether ACL5 functions in TEs via its product because exogenous
application of thermospermine in TE-differentiating Zinnia cell
suspensions was shown to inhibit the entire differentiation process
(Kakehi et al., 2008). Hence, despite a great amount of available
data and clever hypotheses, the regulation of TE PCD remains
elusive, possibly because it has been investigated too often within
the conceptual framework of animal PCD, or without a careful
distinction between PCD and cellular autolysis.
I.2.3.f. Programmed autolysis
Protoplast autolysis and cell wall autolysis
The occurrence of protoplast autolysis in TEs and fibres has been
deduced from the observation that their protoplasts become fully
degraded while their thick cell walls prevent protoplast elimination
by neighbouring cells (Esau, 1943; O'Brien and Thimann, 1967;
O'Brien, 1970; Wodzicki and Brown, 1973; Groover et al., 1997;
Avci et al., 2008; Courtois-Moreau et al., 2009; Bollhöner et al.,
2012; Bollhöner et al., 2013). Another dimension of TE autolysis is
the partial degradation of the side walls and of the end walls, the
latter leading to the creation of the perforation plate whose
structure greatly influences the xylem hydraulics (O'Brien and
Thimann, 1967; O'Brien, 1970; Carlquist, 2012; Feild and
Brodribb, 2013). Despite the long standing observation of this
partial wall degradation and of its impact on xylem hydraulics, its
regulation and molecular effectors remain unknown. The
underlying mechanism has been proposed, but not proven, to rely
on the release of cell wall polysaccharide-degrading enzymes by
the dead TEs during the bulk autolysis of their protoplast (O’Brien
and Thimann, 1967; O’Brien, 1970).
A genetic programme for autolysis
The complete autolysis of protoplasts during the development of
TEs and fibres has three implications: Firstly, the completion of full
protoplast autolysis can only be a post-mortem event, although it
cannot be excluded that autolytic processes start before the
instant of death. Secondly, the preparation and execution of the
protoplast autolysis must be coordinated with the other modules of
the differentiation programme for the cells to become functional.
Thirdly, there must exist a genetic programme for autolysis, for
these cells to build up their autolytic machinery (Escamez and
25
Tuominen, 2014). Consistently, the expression or activity of
proteases, endonucleases and ribonucleases have been detected in
differentiating xylem in Zinnia, Arabidopsis and poplar plants as
well as in TE-differentiating Arabidopsis and Zinnia cell
suspensions (Thelen and Northcote, 1989; Minami and Fukuda,
1995; Ye and Droste, 1996; Ye and Varner, 1996; Beers and
Freeman, 1997; Aoyagi et al., 1998; Woffenden et al., 1998; Zhao
et al., 2000; Demura et al., 2002; Funk et al., 2002; Ito and
Fukuda, 2002; Pesquet et al., 2004; Ehlting et al., 2005; Kubo et
al., 2005; Pesquet et al., 2005; Turner et al., 2007; Avci et al.,
2008; Farage-Barhom et al., 2008; Courtois-Moreau et al., 2009;
Yamaguchi et al., 2011; Han et al., 2012; Bollhöner et al., 2013
Paper II). In order to protect the cells from a premature demise
by their own hydrolytic machinery, it was proposed that these
enzymes could be stored in the vacuole from which they would be
released upon death by the vacuolar rupture (Wodzicki and Brown,
1973; Fukuda, 1997; Groover et al., 1997; Obara et al., 2001).
This vacuolar localization of autolytic hydrolases is supported by
the fact that several of them harbour an endomembrane-targeting
signal peptide (Aoyagi et al., 1998; Avci et al., 2008).
Furthermore, several of these hydrolases have an acidic optimal
pH for their activity (Thelen and Northcote, 1989; Ye and Varner,
1996; Vercammen et al., 2004), supporting their localization to
the vacuole (Fukuda, 1997) and the proposed role of the vacuole
rupture in activating the bulk autolysis of the protoplast (Wodzicki
and Brown, 1973; Fukuda, 1997; Groover et al., 1997; Obara et
al., 2001; Avci et al. 2008; Bollhöner et al., 2013). On the other
hand,
plant
cells
contain
other
acidic
endomembrane
compartments which could also be responsible for cytoplasmic
acidification at the end of PCD (Fendrych et al., 2014; Luo et al.,
2015) and to which some of these proteases could also localize
before the onset of the bulk autolysis (Ménard et al., 2015).
Molecular executors of the TE autolysis
Among the different hydrolases thought to be associated with TE
protoplast autolysis, only a few have been further characterized.
The Zn2+-dependent S1-type Zinnia endonuclease 1 (ZEN1) is the
only nuclease that has been shown to have the ability to degrade
nuclear DNA in vitro and to contribute to nuclear degradation
during TE autolysis in Zinnia cell suspensions (Ito and Fukuda,
2002). TE autolysis was also impaired by pharmacological
treatment of Zinnia cells with a cysteine protease and proteasome
inhibitor, but not with a specific proteasome inhibitor, providing
26
the first piece of evidence for the involvement of cysteinedependent proteases in TE autolysis (Woffenden et al. 1998). TE
autolysis was indeed found incomplete when observed by electron
microscopy in Arabidopsis loss-of-function mutants for the papainlike cysteine protease (PLCP) XCP1, for both XCP1 and XCP2 (Avci
et al., 2008), as well as for the metacaspase MC9 (Bollhöner et al.,
2013). Interestingly, MC9 was detected in the cytoplasm of TEs
and was therefore assumed to remain inactive until the
acidification of the cytoplasm by the vacuolar rupture (Bollhöner et
al., 2013); although this thesis identifies a pre-mortem role for
MC9 as well as an additional vacuolar localization (Paper II).
XCP1 and XCP2 were found to localize mainly inside the vacuole of
the TEs, where they are thought to contribute to intra-vacuolar
hydrolytic processes before the onset of the bulk protoplast
autolysis (Avci et al., 2008). The apparent overlap between the
functions of XCP1, XCP2 and MC9 prompted Bollhöner et al. (2013)
to investigate their potential relationship, especially because
proteolytic cascades can lead to the activation of such papain-like
cysteine proteases (van der Hoorn et al., 2004; Richau et al.,
2012). However XCP2 behaved as a target for degradation by MC9
in vitro, and the analysis of papain-like cysteine protease (PLCP)
activity in mc9 xcp1 xcp2 triple mutants clearly indicated that MC9
rather activates at least one other unidentified PLCP (Bollhöner et
al., 2013). The identification of PLCPs as executioners of bulk TE
autolysis is not surprising because this type of hydrolases is known
to be remarkably stable in proteolitically harsh conditions (Richau
et al., 2012). In contrast no serine-dependent protease has been
shown to contribute to autolysis, but some of them instead seem
to act in the early signalling of TE differentiation (for review see
Petzold et al., 2012) or probably in the signalling of TE PCD
(Groover and Jones, 1999).
The programme for autolysis includes safeguards
The process of TE autolysis itself can be expected to have
consequences beyond the autolyzing TE itself because it likely
triggers the release of a number of molecules. Observations of
xylem differentiation within in vitro tobacco explants lead to the
hypothesis that autolyzing TEs could release auxin and cytokinins,
thus providing positional information for TEs to differentiate endto-end and for the cambium to produce more xylem cells
(Sheldrake and Northcote, 1968). Furthermore, anatomical
characterization of xylem differentiation in oat coleoptiles
suggested that autolyzing TEs release some hydrolases (O’Brien
27
and Thimann, 1967; O’Brien 1970). However, the protoplasts and
the cell walls of xylem parenchyma cells adjacent to autolyzing TEs
remain apparently undamaged in oat and in maize, suggesting
that mechanisms must exist to protect the neighbouring cells
during TE autolysis (O'Brien and Thimann, 1967; O'Brien, 1970;
Srivasta and Singh, 1972). One such mechanism of “programmed
safeguard” relies on the protein TED4 which was identified based
on its expression during TE differentiation of Zinnia cell
suspensions and in the xylem of Zinnia plants (Demura and
Fukuda, 1993; Demura and Fukuda, 1994). Both TEs and non-TE
cells secrete the TED4 protein during xylem differentiation in order
to inhibit the otherwise harmful activity of the proteasome 20S
subunit released by autolyzing TEs (Endo et al., 2001). The
secretion of TED4 by immature xylem cells in anticipation of TE
autolysis (Endo et al., 2001) further exemplifies the programmed
nature of TE autolysis. In addition, the fact that both TEs and nonTEs seem to implement safeguards against potential adverse
effects of TE autolysis suggests that xylem cells cooperate in the
spatial restriction of autolysis, in addition to their known
cooperation during lignification.
28
I.3. Aims of the research
The general aim of the work presented in this thesis was to
discover new regulations of lignification and TE programmed cell
death during xylem development. Such new regulations were
indeed expected to provide novel knowledge of xylem
development as well as trails for future (bio)technological
applications.
To discover new regulations of lignification, my colleagues and I
first aimed to identify new regulators of TE lignification by
performing a screen in Zinnia elegans xylogenic cell suspensions
as an experimental system (Paper I). The next aim consisted in
testing the potential involvement of candidate genes in controlling
lignification by using a reverse genetic approach in the model plant
Arabidopsis thaliana (Paper I, Paper III). Finally, this work
aimed to unravel the function of at least one promising candidate
gene specifically involved in non-cell autonomous lignification in
order to unravel new regulations of lignification (Paper III, Paper
IV).
To discover new regulations of TE PCD, the role of the candidate
PCD regulator Arabidopsis METACASPASE9 was investigated at the
cellular level using Arabidopsis xylogenic cell suspensions as an
experimental system (Paper II).
29
II. Results and discussion
II.1. New regulations of xylem lignification
II.1.1. Identification of new regulators of non-cell
autonomous TE lignification
II.1.1.a. Screening for candidate genes potentially regulating
lignification
The existence of a post-mortem lignification demonstrates
the partial non-cell autonomy of lignification
Observations of the xylem chemistry along stem cross sections in
trees revealed that the amount of lignin in the cell walls of xylem
TEs and fibres increased after the death of these lignifying cells
(Stewart et al., 1953; Stewart, 1966). In addition, the xylem
parenchyma cells were shown to provide the neighbouring, dead,
lignifying cells with ROS in the stems of Zinnia elegans,
presumably to allow post-mortem oxidative coupling of
monolignols (Ros Barceló, 2005)0. We confirmed that lignification
occurs partially post-mortem by two approaches: First, in the
stems of Zinnia plants, our observations of xylem lignification in
cross sections of consecutive internodes showed that TE
lignification had progressed beyond the TE life span (Paper I, Fig.
2). Second, in xylogenic Zinnia cell suspensions, dead, nonlignified TEs that had been treated with the inhibitor of monolignol
biosynthesis piperonylic acid (PA) could lignify post-mortem
following the exogenous supply of monolignols (Paper I, Fig. 1).
Consequently, we deduced that TE lignification occurs at least
partially in a non-cell autonomous manner and we confirmed that
the parenchymatic non-TE cells in Zinnia xylogenic cell
suspensions provide the dead, lignifying TEs with both ROS and
monolignols (Paper I, Fig. 7). Furthermore, these observations
established the xylogenic Zinnia elegans cell suspensions as a
suitable system to screen for candidate regulators of lignification,
including potential regulators of the non-cell autonomous
component of TE lignification.
Differentially expressed genes
during inhibition of
lignification in xylogenic Zinnia cell suspension
We discovered that the ethylene signalling inhibitor silver (Beyer et
al., 1976), in the form of thiosulfate (silver thiosulfate: STS), could
30
arrest both TE lignification and PCD in Zinnia xylogenic cell
suspensions (Paper I, Fig. 3). Hence, differentiating Zinnia cell
suspensions were treated with 60 µM STS, and screening for
differentially expressed genes between STS-treated and untreated
conditions was performed by using suppression subtractive
hybridization (SSH) (Paper I, Supplementary Data Set 1
online). Several genes known or thought to be involved in lignin
biosynthesis were differentially expressed upon STS treatment
(Paper I), strongly suggesting the existence of other regulators of
lignification within the genes differentially expressed in response to
STS.
II.1.1.b. Confirmation of candidate genes as regulators of
lignification
Candidate genes identified in Zinnia have orthologues which
control lignification in Arabidopsis
The next step in finding new regulators of lignification consisted in
identifying homologues for the Zinnia candidates in the genome of
Arabidopsis in order to test their potential lignin-related role by
reverse genetics. T-DNA insertion lines for fifty-four different
Arabidopsis genes, homologous to the Zinnia candidates, were
obtained and the cell wall properties of their xylem-rich hypocotyls
were
analyzed
by
pyrolysis-gas
chromatography/mass
spectrometry (Py-GC/MS) (Paper I, Fig. 9; Supplemental Data
Set 2 online; Paper III, Fig. 2). The relative contents of total
lignin, G-type, S-type or H-type lignin, or the ratio of S lignin to G
lignin, were significantly different from wild-type levels (Welchcorrected T-test) in the T-DNA lines of sixteen of the candidate
genes. Further multivariate data analysis of the cell wall
components by orthogonal projections of latent structuresdiscriminant analysis (OPLS-DA) (Bylesjö et al., 2006) revealed
significantly different cell wall properties between the wild-type
and the T-DNA lines for twenty of the candidate genes. The T-DNA
lines for nine of the candidate genes showed significant differences
from wild-type using both Welch-corrected T-test and OPLS-DA
analysis (Figure 4), indicating that these genes are putative new
regulators of lignification.
31
Relative lignin contents in hypocotyls in percent of
WT lignin contents
Total lignin
Target gene
Mutant
At2g43120
prn2-2
At2g43120
prn2-1
At5g64970
SAIL_ 580_C07
At5g61750
At3g25640
SALK_099748
Ɨ
At2g38870
SALK_138287
Ɨ
SALK_118377
At1g26520
Ɨ
SALK_142598
At1g01620
Ɨ
rcd1-2
At1g32230
Ɨ
rcd1-1
At2g30490
Ɨ
c4h-3
At1g15950
Ɨ
ccr1-3
*
*
*
*
*
0%
H lignin
OPLS-DA Expression
TEs
“significance”
non-TEs
Q2(cum)
*
*
*
*
*
SALK_051107
At1g32230
S lignin
*
SALK_026189
At2g25660
G lignin
*
*
100% 200%
0%
*
*
100% 200%
*
*
*
0%
*
100% 200%
*
0% 100% 200% 300% 0
0,5
1
1
100
10000
Figure 4: Screening for regulators of non-cell autonomous lignification.
Relative lignin contents in Arabidopsis T-DNA insertion mutants for potential regulators
of lignification were analyzed by MS-Py (Paper I) and are displayed as percentages of
the corresponding relative lignin contents in wild-types. The displayed candidate
regulators were selected based on two criteria: (i) The corresponding T-DNA lines
displayed relative lignin contents that were significantly di fferent from wild-type levels
according to a Welch-corrected T-test (n=3), as indicated by an asterisk (p<0,05). (ii)
Pairwise comparison of pyrolysates by orthogonal projections to latent structures discriminant analysis (OPLS-DA) indicated a significant di fference (”Q2(cum)”>0,5) in
the cell wall structures of the T-DNA lines compared with wild-type. The
SAIL_580_C07 mutant was included because eventhough its relative contents of total,
G, S or H lignin did not significantly differ from the wild-type, its ratio of S-type to
G-type lignin was significantly different from the wild-type (data not shown). The
previously characterized mutants c4h-3 (Ruegger and Chapple, 2001) and ccr1-3
(Derikvand et al., 2008) were included as controls, which displayed the expected
changes in lignin properties compared to wild-type.
For each analyzed T-DNA line, the expression of the corresponding wild-type gene
was recovered from cell-type specific micro-array analysis from Arabidopsis root cells
(Brady et al., 2007) that were later analyzed and organized to represent spatio temporal expression data (Cartwright et al., 2009). The average expression of each
gene in “old” xylem vessels (sections 7-12 from Brady et al., 2007) was displayed as
“TEs” while “non-TEs” represent the average expression in stele cells (including
procambial and parenchymatic cells) from the same sections. The expression of
At2g43120 was not measured due to the absence of a corresponding probe on the
micro-array chip used by Brady et al. (2007). Ɨ marks indicate genes whose expression
in Arabidopsis xylogenic cell suspensions remained at least 75% of their maximum
expression after TE cell death (after data from Kubo et al., 2005), indicating a possible
involvement in post-mortem lignification (Paper I, Supplemental Data Set 2 online ).
32
II.1.1.c. Identifying genes regulating non-cell autonomous
lignification
Several regulators of lignification are expressed in
lignifying and non-lignifying xylem cells
In order to identify new regulations of lignification, the sole
identification of new regulators is not sufficient because these
regulators may simply represent new intermediates in already
known regulatory pathways. The non-cell autonomous component
of lignification had remained poorly characterized before the work
presented in this thesis. We therefore reasoned that the functional
characterization of candidate regulators of the non-cell
autonomous lignification would shed light on new regulations of
lignification. To determine if a gene could function in non-cell
autonomous lignification, we looked for its potential expression in
non-lignifying cells using publicly available expression data. We
found that five out of the nine aforementioned regulators of
lignification maintained a high level of expression after TE PCD in
Arabidopsis xylogenic cell cultures (Kubo et al., 2005), indicating
that these genes were (also) expressed in parenchymatic non-TE
cells (Figure 4; Paper I, Supplemental Data Set 2 online).
Furthermore, three out of the nine genes had previously been
found to be expressed in both the TEs and their neighbouring nonlignifying non-TE cells in the main root of Arabidopsis seedlings
(Figure 4) (Brady et al., 2007; Cartwright et al., 2009). These
three genes (namely At5g61750, At3g25640 and At2g38870) were
clearly more expressed in non-TEs than in TEs, suggesting that
they could mainly function in non-cell autonomous lignification.
However, the transcripts of some of the genes expressed beyond
TE cell death in xylogenic Arabidopsis cell suspensions (Kubo et
al., 2005) had not been detected in non-TEs in Arabidopsis roots
(Brady et al., 2007; Cartwright et al., 2009), and conversely.
Hence, the available expression data (Kubo et al., 2005; Brady et
al., 2007; Cartwright et al., 2009), however useful, seems
incomplete and it cannot be used alone to infer a role in non-cell
autonomous lignification. Based on this available expression data
and on the histochemical β-glucuronidase promoter-reporter assay
for one of the candidate genes (At1g32230; Paper I, Fig. 8), we
could therefore identify promising candidates probably involved in
non-cell autonomous lignification. On the other hand, it remained
unclear whether they could be specific regulators of the non-cell
autonomous part of lignification because none of these genes
could be shown to be specifically expressed in the non-lignifying
33
xylem cells. Nevertheless, the available expression data had been
obtained by micro-array using a chip that did not allow for
detection of the transcripts from one of the nine genes of interest;
the PIRIN2 (At2g43120) gene, which could therefore still represent
a specific regulator of non-cell autonomous lignification.
PIRIN2 is a regulator of lignification which is not expressed
in lignifying TEs
The Arabidopsis PIRIN2 (PRN2) was the only Arabidopsis PRN, out
of the four orthologues (Paper III, Supplementary Fig. 1) of
the candidate lignin regulator Zinnia PIRIN (Paper I), whose
knock-out lines showed altered lignin properties compared with
wild-type in Py-GC/MS analyses (Figure 4; Paper III, Fig.2). The
spatio-temporal expression pattern of PIRIN2 was studied by
monitoring the activity of its promoter, using the histochemical βglucuronidase promoter-reporter assay (Paper III, Fig. 1). We
found the promoter of PIRIN2 to be active in xylem parenchyma
cells but not in the lignifying xylem tracheary elements (Paper
III, Fig. 1), indicating that PRN2 regulates specifically the non-cell
autonomous component of TE lignification. Therefore we undertook
characterizing the function of PRN2 during non-cell autonomous
lignification as a mean to uncover new regulations of lignification.
II.1.2.
Transcriptional
autonomous lignification
regulation
of
non-cell
II.1.2.a. Transcriptional regulation of lignin biosynthetic genes
at the chromatin level
PIRIN2 works as a transcriptional regulator of lignin
biosynthetic genes
Detailed analyses of the cell wall composition in the hypocotyls of
prn2 knock-out mutants and in PRN2 overexpressing plants
(Zhang et al., 2014) by Py-GC/MS and by Fourier transform
infrared (FTIR) microspectroscopy revealed that PRN2 functions as
a negative regulator of S lignin biosynthesis, and to a lesser extent
as a negative regulator of G lignin biosynthesis (Paper III, Figs.
2,3,5). Earlier reports had determined that the human PIRIN
orthologue could function as a transcriptional co-regulator
(Wendler et al., 1997; Dechend et al., 1999). We therefore
hypothesized that the Arabidopsis PIRIN2 could control lignin
biosynthesis by regulating the expression of lignin biosynthetic
34
genes. Indeed, real-time quantitative PCR (qPCR) analyses showed
increased expression of several lignin biosynthetic genes in the
hypocotyls and the stems of prn2 mutants compared with wildtype levels, while PRN2 overexpressors consistently displayed
opposite expression patterns (Paper III, Figs. 3, 6). Hence,
PRN2 functions as a transcriptional regulator of lignification.
However, PRN proteins have never been shown to directly bind to
chromatin, which suggested that the Arabidopsis PRN2 is a
transcriptional co-regulator like its orthologue in human.
PIRIN2 interacts with the transcriptional regulator HUB2
To determine whether PIRIN2 could function as a transcriptional
co-regulator, we identified potential interactors of the PIRIN2
protein
by
yeast-two-hybrid
screening
(Paper
III,
Supplementary Table 1). Next, the cell wall properties were
analyzed by Py-GC/MS in the hypocotyls of T-DNA insertion lines
for the eighteen potential candidate interactors (Paper III,
Supplementary Fig. 6), revealing a decrease in the proportion of
S lignin in the T-DNA lines for the HISTONE H2B
MONOUBIQUITINATION2 (HUB2)(Fleury et al., 2007; Cao et al.,
2008). The Arabidopsis HUB2 had earlier been shown to function in
a protein complex that binds to chromatin and performs
monoubiquitination of histone H2B (H2Bub1) (Fleury et al., 2007;
Cao et al., 2008). The H2Bub1 chromatin marks have been
associated with increased trimethylation of lysine 4 and possibly of
lysine 36 of histone 3 (H3K4me3 and H3K36me3, respectively) at
specific loci, triggering the subsequent up-regulation of the
corresponding target genes (Cao et al., 2008; Schmitz et al.,
2009; Himanen et al., 2012). Hence, we hypothesized that HUB2
could function as a positive transcriptional regulator of S lignin
biosynthesis, and that PRN2 could interact with and antagonize the
function of HUB2. The ability of HUB2 and PRN2 to interact
together was confirmed by yeast-two-hybrid assay and by coimmunoprecipitation (Co-IP) (Paper III, Fig. 4). The possible
physiological interaction between HUB2 and PRN2 was also
supported by histochemical β-glucuronidase promoter-reporter
assay, which revealed HUB2 promoter activity in xylem, including
the non-TE cells (Paper III, Fig. 5). In addition, we found that
lignin biosynthetic genes were down-regulated in the hypocotyls of
hub2 T-DNA knock-out mutants (Paper III, Fig. 5), consistent
with our hypothesis that HUB2 is a positive transcriptional
regulator of S-type lignin biosynthesis.
35
PIRIN2 tunes down the HUB2-dependent transcriptional
activation of lignin biosynthetic genes
To verify if PRN2 could antagonize the positive regulation of S
lignin biosynthesis by HUB2, we performed Py-GC/MS analysis to
compare the proportion of S lignin between the hypocotyls of wildtype, prn2 and hub2 single mutants, and prn2 hub2 double
mutants. While the hub2 mutants displayed a trend towards a
lower proportion of S-type lignin than wild-type, both the prn2 and
prn2 hub2 plants showed increases in S lignin compared to wildtype (Paper III, Fig. 5), suggesting that PRN2 and HUB2 function
within the same pathway. Furthermore, Py-GC/MS analysis of stem
samples revealed that the proportion of S lignin was further
decreased in PRN2 overexpressing plants than in hub2 knock-outs,
while hub2 mutants overexpressing PRN2 contained the same
proportion of S lignin than hub2 mutants alone (Paper III, Fig.
5). Therefore, the negative regulation of lignin biosynthesis by
PRN2 requires the presence of HUB2. Together, our data indicates
that PRN2 functions as an antagonist of the up-regulation of lignin
genes by HUB2. HUB2 has never been shown to function as a
transcription factor, but rather to up-regulate specific target genes
by promoting chromatin modifications associated with active
transcription such as H3K4me3 marks (Cao et al., 2008; Himanen
et al., 2012). H3K4me3 marks have recently been proposed to
play a role in the regulation of lignin biosynthetic genes in the
developing wood of eucalyptus (Hussey et al., 2015). Hence, we
hypothesized that PRN2 could interact with HUB2 in order to
repress the downstream establishment of the H3K4me3 marks at
the loci of lignin biosynthetic genes.
PIRIN2 modulates chromatin marks
To test if PRN2 could affect the amount of H3K4me3 marks in the
chromatin of lignin biosynthetic genes, we undertook an approach
that relies on chromatin immunoprecipitation (ChIP) using an antiH3K4me3 antibody followed by qPCR for quantification of the
immunoprecipitated chromatin. Due to the amount of material
required for these ChIP-qPCR analyses, hypocotyls could not be
used. Instead, the lower parts of the main stem from fifteen plants
had to be pooled to create each biological replicate. We first
verified that the expression of lignin biosynthetic genes was upregulated in the stems of prn2 plants compared with wild-type
(Paper III, Fig. 6), as it was the case in the hypocotyls. Next,
ChIP-qPCR analysis of five lignin biosynthetic genes indicated a
clear trend towards an increased amount of H3K4me3 in prn2
36
plants compared with wild-type (Paper III, Fig. 6). Refined ChIPqPCR analysis of four loci within the gene encoding for the S ligninrelated caffeic acid O-methyltransferase (COMT) confirmed that
H3K4me3 marks were more abundant in prn2 compared with wildtype (Paper III, Fig. 6). Furthermore, none of the four loci within
the COMT gene displayed changes in H2Bub1 (Paper III, Fig. 6),
demonstrating that PRN2 modulates the abundance of H3K4me3
downstream of HUB2 rather than the abundance of H2Bub1
controlled by HUB2. Collectively, these observations indicate that
there exists a transcriptional regulation of the non-cell
autonomous component of lignification. This transcriptional
regulation takes place at least in part at the chromatin level and
involves PRN2 and HUB2. HUB2 is involved in establishing all
H2Bub1 marks genome-wide in Arabidopsis (Cao et al., 2008) but
loss of function of the protein complex that establishes H2Bub1
only affects the abundance of H3K4me3 in specific subsets of
genes (Cao et al., 2008; Himanen et al., 2012). Consequently,
factors must exist that drive this specificity and our data strongly
suggest that PRN2 is one such co-regulator involved in the specific
regulation of lignin biosynthetic genes by a HUB2-containing
protein complex. PRN2 works as a negative regulator of H3K4me3
establishment downstream of HUB2, which implies the existence of
yet unidentified factors that establish HUB2-dependent H3K4me3
marks in specific target genes. Interestingly, H2Bub1 and the
downstream H3K4me3 marks regulate the expression of circadian
clock genes in Arabidopsis (Himanen et al., 2012) and H2Bub1
marks are involved in recruiting transcription factors that regulate
circadian clock genes in mammalian cells (Tamayo et al., 2015).
By extension, it is possible that the HUB2-containing protein
complex and/or the H2Bub1 marks recruit proteins that establish
H3K4me3 marks as well as transcription factors that regulate the
expression of lignin biosynthetic genes in Arabidopsis.
II.1.2.b. Transcriptional regulation of lignin biosynthesis by
protein complexes
Identification of a PIRIN2-interacting transcription factor
By analogy with the human PIRIN which interacts with several
different transcriptional regulators (Wendler et al., 1997; Dechend
et al., 1999), we hypothesized that the Arabidopsis PIRIN2 could
also modulate the activity of other transcriptional regulators of
lignin biosynthetic genes than the sole HUB2. Among the eighteen
potential PRN2 interactors that we had identified by yeast-two37
hybrid screening (Paper III), one was a putative transcription
factor: the basic-helix-loop-helix (bHLH) family protein bHLH62
(At3g07340). The ability of bHLH62 to interact with PRN2 was
confirmed by yeast-two-hybrid assay and by Co-IP (Paper IV,
Fig. 1). Furthermore, gene expression analysis from individual cell
types in the main root of Arabidopsis seedlings (Brady et al.,
2007; Cartwright et al., 2009) indicated the presence of bHLH62
transcripts in both the lignifying TEs and in the non-TEs in which
PRN2 is also expressed. Hence, bHLH62 and PRN2 can interact in a
physiological context.
bHLH62 is a regulator of polysaccharidic SCW biosynthesis
and lignification
For PRN2 to function as a co-regulator of bHLH62 in the
transcriptional control of lignin biosynthesis, bHLH62 must itself be
a regulator of lignin biosynthesis. At first, this did not seem to be
the case because analysis of the cell wall composition by PyGC/MS in the hypocotyls of a T-DNA insertion line for bHLH62 did
not reveal any difference in lignin composition compared with wildtype (Paper III, Supplementary Fig. 6). However, careful
characterization of the used T-DNA insertion line (SALK_091124)
revealed that despite the absence of full-length bHLH62 transcript,
this line could produce a partial bHLH62 transcript including the
bHLH-domain (data not shown). To better investigate the potential
role of bHLH62 in regulating lignification, we obtained another TDNA insertion line which we confirmed to be a knock-out mutant
(hereafter bhlh62-1) suitable for reverse genetic analysis (Paper
IV, Fig. 2). Analysis of cell wall contents by Py-GC/MS of the
lower main stem of bhlh62-1 showed a significant decrease in S
lignin, and to a lesser extent in G lignin, compared with wild-type
(Paper IV, Fig. 3). Similar trends were observed in hypocotyls
although these differences were not significant, thus justifying to
conduct further analyses in stems rather than hypocotyls. Stem
cross-sections stained for lignin with phloroglucinol indicated that
the differences in lignin contents between wild-type and bhlh62-1
did not arise from a difference in xylem anatomy and/or in the rate
of xylem differentiation, but rather from differences in SCW
lignification (Paper IV, Fig. 3). Consistently, additional Klason
analysis of cell wall residues from the lower main stem of bhlh62-1
showed decreased lignin content compared with wild-type (Paper
IV, Fig. 4). In addition, the amounts of cell wall polysaccharides
seemed lower in bhlh62-1 than in wild-type (Paper IV, Fig. 4).
Together, our results attribute a role for bHLH62 in the positive
38
regulation of
lignification.
polysaccharidic
SCW
edification
and
of
SCW
PIRIN2 is an antagonistic interactor of bHLH62 in the
regulation of lignification
To determine the potential relation between PRN2 and bHLH62, we
compared the cell wall contents of the lower main stems of wildtype, bhlh62-1, prn2-2, and bhlh62-1 prn2-2 double mutants. The
total lignin content of the prn2-2 mutant did not seem to differ
from that of the wild-type, while both bhlh62-1 and bhlh62-1
prn2-2 plants displayed a similar decrease in lignin content
compared with wild-type (Paper IV, Fig. 4). Hence, bHLH62 and
PRN2 seem to function together in the regulation of lignification.
This regulation of lignification was hypothesized to take place at
the transcriptional level, based on the suspected function of PRN2
as a transcriptional co-regulator and based on the predicted
function of bHLH62 as a transcription factor. The expression of
lignin biosynthetic genes measured by qPCR in the lower main
stem of the aforementioned lines showed a great variability,
leading to the absence of significant difference between the
bhlh62-1 mutant and the wild-type (Paper IV, Fig. 4). As could
be expected, the expression of most of the monitored lignin
biosynthetic genes tended to show an increased expression in the
prn2-2 mutant compared with the wild-type (Paper IV, Fig. 4).
Interestingly, the bhlh62-1 prn2-2 double mutants displayed
expression levels similar to those in the bhlh62-1 mutants rather
than in the prn2-2 mutants for most of the lignin biosynthetic
genes (Paper IV, Fig. 4), indicating that PRN2 functions as an
antagonistic co-regulator of bHLH62. Hence, while bHLH62 could
be involved in regulating polysaccharidic SCW biosynthesis and
cell-autonomous lignification without PRN2, PRN2 and bHLH62
seem to function as an antagonistically in the non-TEs, in order to
modulate the non-cell autonomous component of lignification
(Paper IV, Fig. 5). The non-cell autonomous lignification is also
transcriptionally regulated by a PRN2- and HUB2-containing
protein complex and it remains to be determined whether or not
bHLH62 belongs to the same complex in non-TEs. In any case, it is
clear that there exists at least one PRN2-containing protein
complex (see Figure 5 for a plausible complex) which
transcriptionally regulates the non-cell autonomous lignification.
Although it is a common feature across the tree of life that most
proteins do not function as single units but as multimers, most of
these multimers are homodimers and the occurrence of high order
39
heteromultimers is very rare (Lynch, 2013). Hence, the existence
of a potential complex involving at least HUB2 and its obligatory
interactors (Cao et al., 2008) as well as PRN2 would represent a
somewhat idiosyncratic situation which may be specific to the
regulation of non-cell autonomous lignification.
Figure 5: Plausible protein complex regulating lignin gene expression.
Information on protein-protein interaction were obtained from the Arabidopsis Interactome Mapping Consortium (Dreze et al., 20 11; http://interactome.dfci.harvard.edu) and
from the work presented in this thesis to assemble a plausible protein complex that
could regulate the expression of lignin biosynthetic genes. According to this model,
PRN2, HUB2 and bHLH62 could function within a unique protein complex which links
the establishment of H2Bub1 chromatin marks to the dowstream H3K4me3 and to the
subsequent transcriptional activation of the target genes.
40
II.1.3. Conclusions and perspectives
regulations of xylem lignification
on
new
The work presented above experimentally confirmed that
lignification of xylem TEs is partially non-cell autonomous and
occurs partially post-mortem in the Zinnia xylogenic cell
suspensions as well as in whole Zinnia and Arabidopsis plants.
These systems subsequently enabled identifying the Arabidopsis
PIRIN2, which is not expressed in lignifying xylem cells, but which
nevertheless regulates lignification, and therefore constitutes a
specific regulator of non-cell autonomous lignification. The
functional characterization of PRN2 revealed that the non-cell
autonomous component of lignification is transcriptionally
regulated, including by the modulation of chromatin marks
associated with active transcription. Transcriptional regulation of
lignification at the chromatin level had previously been
hypothesized (Hussey et al., 2015) but the work presented here
provides the first demonstration of a direct regulation of lignin
biosynthetic genes via chromatin modifications. Furthermore, the
non-cell autonomous lignification is regulated by at least one
PRN2-containing protein complex (Figure 5), or by two separate
complexes in which PRN2 would act as an antagonist of the
positive transcriptional regulators HUB2 and bHLH62. Finding and
characterizing other potential protein interactors for PRN2, HUB2
or bHLH62 could shed light on whether PRN2 functions as a
general negative transcriptional co-regulator in different protein
complexes or whether PRN2, HUB2 and bHLH62 operate within one
single complex. Either way, the existence of non-cell autonomous
lignification and of its specific regulation(s) constitutes one
example of cooperation between the different xylem cell types
during xylem development. Such cell cooperation likely involves
some form of coordination between the cooperating cells. Indeed,
mobile factors such as proteins, peptides, micro-RNAs and
hormones have already been shown to be involved in the
coordination of early xylem differentiation with other tissues, or
within the same tissue (Jacobs, 1952; 1954; Fosket and Torrey,
1969; Iwasaki and Shibaoka, 1991; Yamamoto et al., 1997;
Matsubayashi et al., 1999; Zhong and Ye, 1999; Eriksson et al.,
2000; Emery et al., 2003; Biemelt et al., 2004; Caño-Delgado et
al., 2004; Motose et al., 2004; Ohashi-Ito et al., 2005; Ito et al.,
2006; Carlsbecker et al., 2010; Kobayashi et al., 2011; Kondo et
al., 2011; Pesquet and Tuominen, 2011; De Rybel et al., 2014;
Kondo et al., 2014). Investigating the intercellular signalling
41
between differentiating cells at later stages of xylem formation
could reveal factors coordinating the cooperation between the cells
involved in non-cell autonomous lignification. Regarding potential
applications, the knowledge that parenchyma cells are involved in
the lignification of xylem TEs and fibres means that these xylem
parenchyma cells represent a new target cell type for
biotechnological strategies to overcome lignocellulosic biomass
recalcitrance. For example, orthologues of the Arabidopsis PIRIN2
could be targeted to reduce lignification or alter lignin composition
in species that are relevant for biomass production, in order to
ease the conversion of lignocellulosic biomass into biofuels (see
patent by Boerjan, W., Vanholme, R.M.I., Turumtay, H.,
Tuominen, H. Escamez, S. and Zhang, B.; 2015).
II.2. New regulations of xylem cell death
II.2.1. Restriction of cell death to the target cell type
II.2.1.a. Dying TEs implement safeguards to protect the
surrounding cells
Xylogenic cell suspensions as a relevant system to
functionally characterize METACASPASE9
The potential involvement of the Arabidopsis METACASPASE9
(MC9) in TE PCD had earlier been hypothesized (Turner et al.,
2007; Bollhöner et al., 2013) based on two grounds: (i) Because
several metacaspases (MCs) had been linked to the regulation of
several types of plant PCD (He et al., 2008; Coll et al., 2010;
Watanabe and Lam, 2011) and (ii) because MC9 is the only MC
gene whose expression is induced during TE differentiation in
Arabidopsis cell suspensions (Turner et al., 2007). Comparison of
Arabidopsis mc9 loss-of-function mutants with wild-type seedlings
did not reveal any significant change in the distance of protoxylem
TE PCD from the root tip which suggested that MC9 did not control
PCD (Bollhöner et al., 2013). On the other hand, such analyses
showed a great variability between and within experiments,
possibly due to the difficulties associated with live imaging of
xylem in planta and because very few TEs die at any given time.
To overcome these technical hindrances we undertook the
functional characterization of MC9 using a reverse genetic
approach in Arabidopsis xylogenic cell suspensions (Pesquet et al.,
2010) (Paper II). First, we confirmed the expected TE-specific
42
expression of MC9 by monitoring a MC9:GFP fusion expressed
under the transcriptional control of MC9 endogenous promoter in
differentiating cell suspensions (Paper II, Fig. 1). Next, we
generated cell lines with down-regulated MC9 expression using
RNAi (hereafter MC9-RNAi) and we verified that autolysis was
impaired in MC9-RNAi TEs (Paper II, Fig. 1), as expected from
the known role of MC9 in TE autolysis in whole plants (Bollhöner et
al., 2013). Hence, the Arabidopsis xylogenic cell suspension
system appeared as a suitable system to further characterize the
role of MC9 in TEs, including its potential involvement in
programmed cell death.
METACASPASE9 operates in dying TEs to prevent ectopic
death of non-TEs
To monitor TE PCD, differentiating wild-type and MC9-RNAi cell
suspensions were stained with the viability dye fluorescein
diacetate (FDA) and observed at different times over the course of
their differentiation. The proportion of dead TEs did not
significantly differ between wild-type and MC9-RNAi cell
suspensions at any time point (Paper II, Fig. 1), suggesting the
absence of a role for MC9 in regulating TE cell death. Instead,
observations with the FDA staining revealed the occurrence of
ectopic cell death of parenchymatic non-TEs in differentiating MC9RNAi lines (Paper II, Fig. 2). Because MC9 is specifically
expressed in the TE cell type, we hypothesized that the MC9-RNAi
TEs were responsible for the ectopic cell death of the non-TEs.
Indeed, earlier work in Zinnia xylogenic cell suspensions have
shown that autolyzing TEs could release potentially harmful
cellular material, whose deleterious effect had to be prevented by
the secretion of a protective inhibitor (Endo et al., 2001).
Consistent with a harmful effect of dying MC9-RNAi TEs on nonTEs, the temporal progression of non-TE cell death correlated with
the temporal progression of TE PCD in MC9-RNAi lines (Paper II,
Fig. 2). Inducing the differentiation of a mix of wild-type and MC9RNAi cells (1:1) suppressed the ectopic cell death of the non-TEs,
while purposely reducing the differentiation of TEs in MC9-RNAi cell
suspensions also reduced the ectopic cell death, further suggesting
that MC9-RNAi TEs caused the ectopic non-TE death. Therefore,
the collective evidence strongly suggests that MC9 operates within
the differentiating TEs where it implements safeguards to prevent
the dying TEs from becoming harmful for the non-TEs.
43
II.2.1.b. The spatial restriction of cell death requires tight
regulation of autophagy in TEs
METACASPASE9 regulates the level of autophagy in TEs
In a review article Bozhkov and Jansson (2007) had proposed that
the restriction of cell death to the target cell type during PCD in
plants relies on the cellular process of autophagy. Interestingly,
autophagy was associated with the progression of PCD in xylem
fibres (Courtois-Moreau et al., 2009) and in TEs (Kwon et al.,
2010). Furthermore, during the PCD that occurs in developing
spruce somatic embryos, a metacaspase regulates the level of
autophagy and thereby enables the correct progression of cell
death (Minina et al., 2013). Hence, we hypothesized that MC9
could regulate autophagy in TEs in order to spatially restrict cell
death. Cell type-specific quantification of autophagy by differential
interference contrast (DIC) microscopy observations after
inhibition of autophagic body degradation by Concanamycin A
(Yoshimoto et al., 2004) revealed a higher level of autophagy in
TEs than in non-TEs (Paper II, Fig. 3). Interestingly, we
observed a higher level of autophagy in MC9-RNAi TEs than in
wild-type TEs, while the level of autophagy remained similar in the
non-TEs of both genotypes. Hence, MC9 appears to tune-down the
level of autophagy in TEs. This implies that MC9 functions in part
before TE PCD, and not solely during post-mortem autolysis as
previously hypothesized based on the cytoplasmic localization of
MC9 in whole plants and because MC9 is active at low pH
(Bollhöner et al., 2013). The acidic pH requirement for MC9
activity and our observation that MC9 functions pre-mortem led us
to hypothesize that MC9 could also be localized in the vacuoles of
TEs, an acidic organelle where part of the autophagic process
takes place. To test this hypothesis, we observed a MC9:GFP
fusion protein expressed under the control of MC9 promoter in the
TEs of Arabidopsis roots by using confocal laser scanning
microscopy (cLSM). GFP signal was mainly detected in the
cytoplasm of TEs, although GFP-positive punctuates could also be
observed in the vacuolar compartment (Paper II, Fig. 4). The
number of vacuolar MC9:GFP punctuates was significantly
increased after treating the seedlings with Concanamycin A, which
inhibits vacuolar degradation of autophagic bodies as well as
quenching of the GFP by the acidic pH (Paper II, Fig. 4).
Simultaneous treatment of the seedlings with Concanamycin A and
the inhibitor of autophagosome formation wortmannin (Blommaart
et al., 1997) significantly decreased the amount of vacuolar
44
MC9:GFP punctuates compared with Concanamycin A treatment
alone (Paper II, Fig. 4), which suggests that these punctuates
could represent autophagic bodies. Importantly, the vacuolar
localization of MC9 indicates that MC9 can function before TE PCD,
making it possible for MC9 to regulate the level of autophagy in
living TEs.
The regulation of TE autophagy by MC9 restricts cell death
to the target cell type
To test whether MC9 could restrict cell death to the TEs by tuning
down the level of autophagy in this cell type, we undertook
repressing autophagy specifically in MC9-RNAi TEs. For this
purpose we created an RNAi fragment targeting the autophagy
gene ATG2 (ATG2-RNAi), which is known to be essential for
autophagy in Arabidopsis (Inoue et al., 2006; Hackenberg et al.,
2013). The ATG2-RNAi was placed under the transcriptional control
of the promoter of the SCW-specific cellulose synthase gene
IRREGULAR XYLEM1 (IRX1; Turner and Somerville, 1997; Taylor et
al., 2000), which is active in the TEs but not in the parenchymatic
non-TEs. Next, we generated MC9-RNAi proIRX1::ATG2-RNAi
double transgenic cell lines in which we could verify that MC9 was
still down-regulated (Paper II, Supplementary Fig. 1), and that
the level of TE autophagy was lower than in MC9-RNAi cell
suspensions (Paper II, Fig. 5). Interestingly, decreasing the level
of autophagy in MC9-RNAi TEs by the additional proIRX1::ATG2RNAi autophagy knock-down suppressed the ectopic death of the
non-TE cells (Paper II, Fig. 5). Therefore, tight regulation of the
level of autophagy in TEs by MC9 is required to confine cell death
to the TE cell type (Figure 6), at least in the Arabidopsis xylogenic
cell suspension system. Because of the numerous plant systems in
which metacaspase activity and autophagy have been detected
during programmed cell death (For review see Minina et al., 2014;
Van Durne and Nowack; 2016), it is tempting to hypothesize that
MC-dependent regulation of autophagy during PCD could represent
a general mechanism for protecting the surrounding cells.
45
Living non-TE
Wild-type xylogenic cells
Developing TE
Extra-cellular medium
Autolytic TE
Living non-TE
Developing TE
MC9-RNAi xylogenic cells
Dead non-TE
Autolytic TE
(partially impaired
autolysis)
A
Autophagosome
AB
Autophagic body
Extra-cellular medium
Degraded autophagic
body
Lytic vacuole
MC9
METACASPASE9
Harmful molecules
Safeguard molecules
Figure 6: Plausible model for the restriction of cell death to TEs by the MC9-mediated
control of TE autophagy. In this model, the level of autophagy in TEs is tuned down by
MC9 to prevent the excessive release of harmful molecules (e.g. toxins, lytic enzymes
or pro-death signal peptides) and/or to foster the release of safeguard molecules
(e.g.inhibitors of lytic enzymes or pro-life signal peptides) from the TEs.
46
II.2.2. Conclusions and perspectives
regulations of xylem cell death
on
new
The work presented above relied on the functional characterization
of the candidate xylem PCD regulator METACASPASE9 to discover
new regulations of the programmed cell death of tracheary
elements. The quantification of TE PCD at the level of the cell
population in Arabidopsis xylogenic cell cultures (Paper II, Fig.
5), together with the previous observation of TE PCD in the mc9
loss-of function mutant plants (Bollhöner et al., 2013), did not
suggest a direct involvement of MC9 in the execution of TE PCD
per se. Instead, MC9 regulates the level of autophagy in TEs to
prevent them from becoming harmful for the surrounding cells.
Indeed, TEs must accumulate a molecular arsenal which executes
the TE post-mortem autolysis and which could contribute to the
execution of the cell death programme. It is thus conceivable that
while preparing for cell death and autolysis, as well as during the
post-mortem autolysis, TEs could release harmful molecules
against which safeguards must be implemented during the
progression of TE PCD (as exemplified by Endo et al., 2001). The
implementation of such safeguards relies at least partially on the
regulation of autophagy by MC9 in TEs, which therefore represents
a form of cell death regulation in that it spatially restricts cell
death. This newly discovered regulation of xylem cell death also
represents an example of cell cooperation between the TEs and the
non-TEs because the non-TEs are protected at least in part by the
TEs themselves. Investigating the molecules released by the TEs
with different levels of MC9 expression or different levels of
autophagy, for example by analyzing the extracellular medium of
xylogenic cell suspensions, could shed light on the intercellular
signalling that restricts xylem cell death to the target cell types
downstream of autophagy. The results of investigations on the role
of autophagy during PCD have often been difficult to interpret,
leading to conflicting views on whether autophagy is a “pro-life” or
a “pro-death” process, both in plant systems (Lv et al., 2014;
Minina et al., 2014) and in animal systems (Denton et al., 2012;
Clarke and Puyal, 2012; Shen et al., 2012; Liu and Levine, 2014).
Part of the difficulty in interpreting the wealth of data on
autophagy during PCD may arise from the fact that most studies
relied on the constitutive promotion or constitutive inhibition of
autophagy, rather than on alterations of autophagy specifically in
the cells undergoing PCD. In contrast, the present study relied on
cell-type specific down-regulation of autophagy and provided
47
evidence that autophagy in certain cells can dramatically affect the
surrounding cells, consistent with recent discoveries that
autophagy can influence protein secretion (Torisu et al., 2013;
Bhattacharya et al., 2014; Cinque et al., 2015; Kang et al., 2015;
Kraya et al., 2015; Nowag and Münz, 2015). It is therefore
possible that modulation of autophagy could be used in particular
target cells to affect or kill the surrounding cells for therapeutic or
biotechnological applications.
48
Acknowledgements
A bit more than five years have passed since I started working on my PhD
project. Such a long period. An entire slice of life, so rich that there would
have been enough material for me to write another book, had I not been so
busy producing this one. So many things have happened during and before
this PhD, that led to the completion of this work and which I must be
thankful for.
In the preface of his book Protein Biochemistry and Proteomics, Hubert
Rehm writes that “only luck, diligence, ingenuity, good mentoring and
intelligence – in this order—bring success at the lab bench.” Hence, one
would only have to be thankful to one’s mentor(s) and for one’s luck, while
all the rest would be individual skills and individual merit. That can only
seem true if one forgets the importance of formal and informal cooperation
in research as in any collective achievement. That can only seem true if one
tries to claim more individual merit, and possibly more resources, than one
could legitimately afford in a fairly assessed situation. In any scientific book
and in any PhD thesis that I have read, it is no mistake that many more
persons are thanked than just one’s mentor(s) and one-self.
My first acknowledgements must go to my family in general, and to my
brothers, parents and grand-parents in particular, for having cultivated my
curiosity and raised me with the caring love that one needs to build enough
confidence to cope with the challenges of pursuing a scientific carrier. I am
especially thankful to my parents and grand-parents for the sacrifices they
made to provide me with financial support over the long period of my
studies, while themselves living most of the time under the poverty line.
The sole fact that someone born and raised in a poor family had a chance to
make it this far as to get a PhD also calls for indirect, however important,
acknowledgements: I want to thank the social welfare system in France
which gave me access to a good, nearly free, public education up to the
master’s degree. I especially want to thank all the persons involved in
implementing this system and those making it work nowadays, such as my
teachers.
I want to thank my supervisor, Hannele Tuominen for having given me
the chance to work in her team, especially considering how young (and
therefore potentially immature) I was when she recruited me, and given how
poor my English was. I am also thankful for all the times when you trusted
49
me enough to associate me to important tasks such as organizing
international conferences. I am thankful for the relative freedom that you let
me have in conducting my work. I am thankful for the support and for the
scientific and carrier mentoring that you offered me. Finally, I am sorry that
my Swedish did not improve as much as I said it would. The research part of
the work turned out more difficult than I had imagined and getting over the
numerous obstacles that I faced drained all my efforts and energy.
On this particular point, I would like to address some very special
acknowledgements to SIGMA-ALDRICH for having repeatedly provided
me with the wrong type of synthetic brassinosteroids during about a year.
Seeing all my experiments fail during a period of nine month really taught
me the perseverance and tenacity that I believe will make the rest of my life
easier. Also, thank you Sigma for not offering any refund in the first place,
wasting even more of my time into fighting in order to be less than fairly
compensated.
I want to thank my co-supervisor Edouard Pesquet for providing me with
his xylogenic cell culture system and for teaching me how to use it. I am
grateful for the time you managed to spare to support me with this system, to
help me with the experimental design and to give me feedback on my
research and on my English. Although I cannot match your great British
accent, my English has clearly improved, in part thanks to you.
I want to thank Delphine Ménard, Henrik Serk, Raphaël Decou,
Thomas Dobrenel and Carole Dubreuil for theoretical and technical
help with the cell culture system, as well as for our interesting discussions.
More generally, I want to thank all my colleagues who helped me with
technical tips, reagents, equipment and useful comments. To avoid writing
down a long list of persons whereby I am too afraid to forget or misspell
someone’s name, I would summarize by telling that this includes nearly
anyone who has worked at the Umeå Plant Science Centre during the past
five years, as well as a number of colleagues at the Joint BioEnergy Institute
(California, USA) and at the University of British Columbia (Vancouver,
Canada).
In particular, I want to express my gratitude to my Canadian colleagues of
the Working on Walls project. Thank you for the opportunity to visit you for
an inspiring symposium and for the success of our collaborative efforts to
organize a second symposium in Umeå. Especially I wish to acknowledge
Brian Ellis, Margaret Ellis, Carl Douglas, Ryan Eng, Teagen
Quilichini, and Lan Tran.
50
I also want to warmly thank my colleagues at the joint BioEnergy Institute
for welcoming me and helping me during my work there. Special thanks to
Henrik Vibe Scheller, Ai Oikawa, Irina Silva, Mary Agnitsch,
Aymerick Eudes, Gabriella Papa and Noppadon (Tik)
Sathitsuksanoh.
At UPSC, I want to express special acknowledgements to those of my
colleagues who supported me much more than they had to with technical
work, theoretical discussions and psychological support. Thank you Bo
Zhang for supervising me at the bench, for teaching me most of the
molecular biology I know and for spending so much time helping me. Thank
you Jakob Prestele for all the technical and psychological help and support
and especially for the numerous inspiring scientific discussions that you
offered me. Thank you Lorenz Gerber and Junko Tahahashi-Schmidt
for all your help with Py-GC/MS analyses. Thank you Benjamin
Bollhöner, Maribel García-Lorenzo, Hardy Hall, Yin Wang,
Berndette Sztojka, Veronica Bourquin, Marlene Karlsson, Paige
Cox, Bernard Wessels, Andreia Matos and Ana Milhinhos for your
solidarity and for making it a pleasure to work with you in Hannele
Tuominen’s team.
I am grateful to the former students Domenique André and Hendrik
Reuper who worked with me and whose skills have impressed me.
I am grateful to Ewa Mellerowicz for having accepted to organize and run
a literature course on The vascular cambium.
I want to thank my colleagues PhD students with whom I have been
teaching, always with pleasure, knowing that we could trust each-other to
make everything work optimally: Benjamin Bollhöner, Bernard
Wessels, Malgorzata Pietrzykowska and Barbara Terebienec.
I want to express my gratitude to the members of my reference group for the
feedback and help with scientific and carrier guidance: Stéphanie Robert,
Totte Niittylä and Vaughan Hurry.
I want to thank the persons that have prevented, and keep on preventing, the
department of Plant Physiology at UPSC from collapsing due to bureaucratic
and technical/logistic issues: Siv Sjödin, Monica Iversen, Inger
Wänglund, Karin Degerman, Ylva Johansson, Jenny Bagglund,
Jan Karlsson, Susanne Larsson, Rosie Forsgren, Hans Isaksson
and Thomas Hiltonen. If plant biologists were investigating your
importance in the same way as they determine the importance of a gene (by
51
stopping its function and see what does not work anymore), then one would
likely conclude that you represent the most important “genes” of our
department.
Plant biologists, such as I, also need to grow plants of course, which requires
growth facilities and personnel. I therefore would like to thank all the
personnel who keeps the greenhouses and transformation facilities working.
In particular, I must thank those of you who were directly involved in poplar
transformation related to my research and who helped keeping my plants
healthy, or who helped me screen, measure, harvest and process them:
Britt-Marie Åkerman, Inga-Lill Darstedt, Verena Fleig, Veronica
Bourquin, Jenny Lönnebrink, Britt-Marie Wrede and Marlene
Karlsson.
I want to thank my international friends and colleagues for translating the
simplified summary of my research into so many languages, allowing my
thesis to display the great cultural wealth of UPSC: Bo Zhang, Ana
Milhinhos, Domenique André, Alfredo Zambrano, Pieter
Nibbering, Berndette Sztojka, Umut Rende, Veronica Bourquin,
Marta Derba-Maceluch, Haleh Hayatgheibi, Junko TakahashiSchmidt and Sanaria Abbas Alallaq. Also, thank you Daniel Decker
and Hannele Tuominen for the translation of the abstract of my research
into Swedish.
I want to express my gratitude to Domenique André for helping me with
correcting mistakes in the text of this thesis.
There are so many great personalities that I have met and become friend
with. To all of you my friends I want to express my gratitude. Your
friendship has helped me maintain my motivation and efforts, especially
during the numerous difficult times that I had during this PhD. For this
thesis is about professional rather than personal life, I cannot write down all
your names and detail all the good moments we shared but I want you to
know that even in such a rush time I am sparing some thoughts for you.
Finally, I must emphasize the paramount importance of the woman who
shares my life, Domenique André. There is so much for me to be grateful
to you that it would never fit into this thesis, but I can at least write this:
Thank you!
52
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