The birch genes BpMADS1 and BpMADS6 and their use in the

University of Joensuu, PhD Dissertations in Biology
No:23
The birch genes BpMADS1 and BpMADS6
and their use
in the modification of flowering
by
Juha Lemmetyinen
Joensuu
2003
Lemmetyinen, Juha
The birch genes BpMADS1 and BpMADS6 and their use in the modification of flowering.
- University of Joensuu, 2003, 85 pp.
University of Joensuu, PhD Dissertations in Biology, n:o 23. ISSN 1457-2486.
ISBN 952-458-386-0
Key words: AGAMOUS, BARNASE, Betula pendula, flowering, SEPALLATA3, sterility
Genetic modification gives many possibilities in plant breeding. Although its applications in
forest trees still wait for their full scale coming, much work is being done for it. Before transgenic
plants can be tested in field tests and used, it has to be ensured that they do not spread their
transgenes to the wild populations. Therefore, a method to prevent flower formation in birches
and other plants has been the main aim of this study. For the testing of various gene constructs
for flower development in a reasonably short time in birch, early-flowering birch clones were
selected and their suitability was studied.
In order to prevent flowering, two birch genes regulating flower development were isolated
and characterised: BpMADS1, most similar to SEPALLATA3 in Arabidopsis, and BpMADS6,
most similar to AGAMOUS in Arabidopsis. BpMADS1 was expressed in inflorescence meristems
and later in stamens and carpels. The overexpression of BpMADS1 in tobacco resulted in
accelerated flowering, which shows that it is capable of accelerating the formation of the
inflorescence meristem. In Arabidopsis, the overexpression of BpMADS1 resulted in a reduced
number of floral organs or whorls. In addition, it caused a mixed identity of floral organs in the
three outer whorls, e.g. sepals with ovules. In birch, the suppression of BpMADS1 resulted in the
formation of some inflorescences in which leaves instead of stamens were formed and these
leafy and sterile inflorescences often continued their development as branches. This suggests
that BpMADS1 is an important gene in flower development and that it is needed at least for
stamen formation. It might also play a role in inflorescence development. BpMADS6, similarly
to BpMADS1, was expressed in stamens and carpels and its overexpression accelerated the
flowering in tobacco. In addition, its overexpression in tobacco resulted in changes in sepals
and petals. In Arabidopsis, it caused extremely early flowering. In birch, the suppression of
BpMADS6 occasionally resulted in flowers without stamens or carpels, but with increased tepals.
The tissue-specific ablation was used to induce sterility. This is based on the use of the
inflorescence-specific promoter of BpMADS1 ligated to the RNAse gene BARNASE. The gene
construct was first tested in tobacco and Arabidopsis. In both of them, the gene construct prevented
flower formation partially or completely resulting in sterility. The growth of the sterile plants
was increased. In tobacco, the increased growth was mainly caused by the accelerated formation
of axillary branches after an unsuccessful effort to form inflorescences and flowers. In our earlyflowering birch clones, the construct prevented inflorescence formation in seven lines without
considerable effects on early vegetative growth. The most noteworthy one of the vegetative
effects observed after the start of flowering was occasional dichotomic branching apparently
caused by the attempts to form terminal inflorescences. In many other lines with or without
inflorescences, there were also considerable disturbances in vegetative growth apparently caused
by unspecific expression. However, the results show clearly that the flowering of birch can be
completely prevented, and therefore they are important steps in the control of flower formation
and controlled containment of transgenes in genetically modified trees.
Juha Lemmetyinen, Department of Biology, University of Joensuu, P.O.Box 111, FIN-80101
Joensuu, Finland
3
ABBREVIATIONS
AG
AGL
BpMADS1
BpMADS6
GUS
kb
MUG
SEP1,2,3
AGAMOUS
AGAMOUS-LIKE
Betula pendula MADS1
Betula pendula MADS6
β-glucuronidase
kilo base
4-methylumbelliferyl-β-glucuronide
SEPALLATA1,2,3
4
CONTENTS
LIST OF ORIGINAL PUBLICATIONS
6
1. INTRODUCTION
7
1.1. General background
7
1.2. Regulation of flowering in Arabidopsis
7
1.2.1. Flowering induction
8
1.2.2. Inflorescence meristem
10
1.2.3. Floral meristem
10
1.2.4. Floral organs
11
1.2.5. AGAMOUS and SEPALLATA genes
11
1.3. Flower development-related genes from trees
12
1.4. Birch
14
1.5. Biotechnical modification of flowering
15
1.5.1. Prevention of flowering
15
1.5.2. Acceleration of flowering
16
2. AIMS OF THE STUDY
17
3. MATERIAL AND METHODS
17
3.1. Plant material
17
3.2. Isolation and analysis of BpMADS1 and 6
18
3.3. Gene constructs
18
4. RESULTS
18
4.1. Early-flowering birch clones
18
4.2. BpMADS1 and BpMADS6
19
4.2.1. Isolation of MADS-box genes
19
4.2.2. Expression
20
4.2.3. Effects of overexpression and antisense constructs
20
4.2.4. Analysis of the BpMADS1 promoter
21
4.3. Prevention of flowering using the BpMADS1::BARNASE construct
5. DISCUSSION
22
23
5.1. Advantages and disadvantages of the early-flowering birches
23
5.2. BpMADS1
25
5.3. BpMADS6
27
5.4. Suitability of BpMADS1 and BpMADS6 for the prevention of flowering
27
5.5. Suitability of BpMADS1 and BpMADS6 for the acceleration of flowering
29
6. GENERAL CONCLUSIONS
29
ACKNOWLEDGEMENTS
30
REFERENCES
30
5
LIST OF ORIGINAL PUBLICATIONS
This thesis is mainly based on the following publications but it also includes some previously
unpublished results. In the text, the publications are referred to by the Roman numerals I-IV.
I
Lemmetyinen, J., Keinonen-Mettälä, K., Lännenpää, M., von Weissenberg, K. and
Sopanen, T. 1998. Activity of the CaMV 35S promoter in various parts of transgenic
early-flowering birch clones. Plant Cell Rep. 18: 243-248.
II
Lemmetyinen, J., Hassinen, M., Elo, A., Porali, I. Keinonen, K., Mäkelä, H. and Sopanen,
T. Functional characterisation of SEPALLATA3 and AGAMOUS orthologues in silver
birch. Physiology Plantarum (in press).
III
Lemmetyinen, J., Pennanen, T., Lännenpää, M. and Sopanen, T. 2001. Prevention of
flower formation in dicotyledonous. Molecular Breeding 7:341-350
IV
Lemmetyinen, J., Keinonen, K. and Sopanen, T. Prevention of flowering of a tree, silver
birch. Molecular Breeding (in press).
All publications are reprinted with permission from publishers. Copyrights for I by SpringerVerlag GmbH & Co. KG, for II by Physiologia Plantarum and for III and IV by Kluwer Academic
Publishers B.V.
6
1. INTRODUCTION
including forest trees (Lemmetyinen and
Sopanen, in press).
Flowering is a very complex process which
can be divided into several phases: flowering
induction and the formation of inflorescence
meristem, floral meristem and floral organs.
Many of the genes regulating these processes
are already known in Arabidopsis. Much
information has been gained through
suppressing or by expressing many of these
genes ectopically in transgenic plants.
However, several interactions are still quite
unclear and probably many genes are unknown.
In trees, only a small number of genes
regulating flower development have been
isolated and only a few of them have been
studied by over-expressing or suppressing them
in trees. This is partly due to problems caused
by the long delay before flowering, the long
development time of flowers, and the very large
size of trees when flowering occurs. This
makes trees in many ways labourious or almost
impossible for controlled laboratory studies
and genetic modifications. Because many trees
start to flower when about 10 to 40 years old
(Clark, 1983), it takes a very long time before
the effects of a transgene on flowering can be
detected. Recently, some progress has been
made to speed up the flowering of trees using
genetic modification (Lemmetyinen and
Sopanen, in press), which will probably be
useful in future studies on flower development.
1.1. General background
The genetic modification of trees would be
especially useful because the breeding of trees
using conventional techniques is very slow.
However, when fertile transgenic plants are
cultivated in nature, transgenes can spread
uncontrollably via pollen and seeds. This is a
problem especially with native trees spreading
the transgenes into wild trees in neighbouring
populations or in the case of wind-pollinated
trees even further away. Because the ecological
consequences are not known, at least at the
present, the uncontrollable spread of
transgenes is not accepted by the authorities.
Therefore, a method to prevent flowering or
at least to induce sterility is needed. If for some
reason the transferred gene would, for example,
cause some undesirable environmental effects,
it would still be possible to remove the
transgene from the ecosystem by cutting down
the transgenic trees. Our group is developing
methods to prevent the spread of transferred
genes in birch by making them non-flowering
and in this way also sterile.
Because in many cases the genetic
background of desirable properties is not
known, the application of gene transfer is not
always possible in breeding. Therefore,
traditional breeding is still needed for a long
time. Particularly, because trees have a long
generation time, their breeding is extremely
slow and tedious. Therefore, transient
acceleration of flowering during breeding
would be most desirable. Methods to accelerate
flowering by genetic modification have been
another goal of our group.
Although flowering is crucial for plant
evolution and for the survival of plant species,
little has been known about its genetic
background until about the last ten years. Most
of the recent advances in clarifying the genetic
networks that interact to control flowering and
flower development have come from studies
using the model plant species, Arabidopsis
thaliana (Simpson et al., 1999; Araki, 2001).
Much work has also been done with
Antirrhinum (see, e.g. Saedler et al., 2001).
Little by little it has been possible to apply this
information to economically important plants
1.2. Regulation of flowering in Arabidopsis
Arabidopsis thaliana has many advantages
(see, e.g. Bowman 1994) which explain why it
has become a useful model plant. It is small in
size and therefore it can be cultivated in large
numbers in a small space. This has allowed a
relatively easy identification of a great number
of mutants. It has a rapid regeneration time (56 weeks under optimum growth conditions)
making it suitable for genetic studies. It has a
small genome, which has been completely
sequenced (The Arabidopsis Genome
Initiative, 2000). This is of great help in the
identification and isolation of new genes.
When the studies are focused on a single
model species, different studies complement
and support each other and a model can be
constructed where the genes can be presented
7
with their relations to each other. For recent
reviews for flowering time see e.g. Koornneef
et al., 1998; Simpson et al., 1999; Samach and
Coupland, 2000; Araki, 2001; Battey and
Tooke, 2002; Mouradov et al., 2002; Sung et
al., 2003 and for flower development e.g. Jack,
2001; Theissen, 2001; Kieffer and Davies,
2001.
gibberellin promotive pathways (Simpson et
al., 1999). Several genes are functioning in
each pathway either by repressing or activating
other genes on the same or on different
pathways.
In Arabidopsis, several environmental and
endogenous factors control the transition to
flowering (Fig. 1). The proper combinations
of these factors lead to the induction of
flowering and inflorescence formation. Lack
of one factor can usually be compensated by
other factors. For this reason, single mutations
do not usually prevent flowering completely.
A large number of genes influencing flowering
makes fine adjustment possible and ensures
that the switch from vegetative to reproductive
growth takes place at the most appropriate time
with respect to a variety of abiotic and biotic
factors. During vegetative growth, flowering
is prevented by repressor genes. The induction
of flowering is partly a consequence of the
downregulation of these genes (Koornneef et
al., 1998). The downregulation of these genes
results in the activation of genes that are
needed, either directly or indirectly, for
inflorescence meristem formation. In
inflorescence meristems, the genes needed for
floral meristem formation are activated and,
in floral meristems, the genes needed for the
formation of floral organs are activated.
Many genes repressing flowering have been
isolated and characterized from Arabidopsis.
Perhaps the most dramatic genes characterized
so far are EMBRYONIC FLOWER 1 and 2
(EMF1 and EMF2) (Aubert et al., 2001;
Yoshida et al., 2001). In emf1 and emf2
mutants, flowering starts immediately after
germination, completely bypassing the rosette
shoot development. Although the exact places
of EMF1 and 2 in the regulatory pathways of
flower development are not known, it seems
that they repress at least several flower organ
identity genes (see later) (Moon et al., 2003).
Similarly, TERMINAL FLOWER 1 (TFL1)
participates in the phase transition but in the
tfl1 mutants there is some vegetative
development (Ratcliffe et al., 1998). Actually,
the phenotype of tfl1 is similar to a moderate
repression of EMF1 or 2 and it has been
suggested that EMF1 and 2 are required for
TFL1 activity (Aubert et al., 2001; Yoshida et
1.2.1. Flowering induction
After seed germination the plant is in a juvenile
phase, during which it is unable to respond to
factors inducing flowering. After some time, it
changes into the adult phase achieving the
ability to respond to inducing factors. At the
same time, the plant shows many features
typical to this phase, e.g. abaxial trichomes
(Telfer et al., 1997). It is still largely unclear
how these characters are linked to flowering.
Very little is known on the genetic regulation
of this change in Arabidopsis or any other plant,
although some genes are known to be involved,
e.g. HASTY (Telfer and Poethig, 1998). Before
flowering the plant is said to be in the
vegetative phase (either in juvenile or nonflowering adult phase); when flowering has
been induced, the plant changes into the
reproductive phase. Juvenile ÷ adult and
vegetative ÷ inflorescence ÷ floral phase
transitions are distinct processes that affect
each other (Battey and Tooke, 2002).
The induction of flowering is a very
complicated process in which several factors
are involved either alone or together. The
factors that determine the flowering time
depend on the species both qualitatively and
quantitatively. In different species there are
different optima on the flowering time (need
of special pollinators, long development of
seeds etc.). In some species, environmental
factors primarily determine when it is the right
time to flower and in some species the
flowering time is determined by intrinsic
factors. In Arabidopsis, genetic analyses of
mutants and natural variation in various
ecotypes have led to the identification of at
least 80 loci that affect the flowering time
(Levy and Dean, 1998). In Arabidopsis, there
are at least four different or partly different
pathways leading to flowering: the
photoperiod, autonomous, vernalization, and
8
LIGHT
SUCROSE
PHYA PHYB
CRY1
CRY2
GROWTH
VERNALIZATION GIBBERELLINS
GA1
LD
FCA
FRI
ESD4
VIP4
TFL1
TFL2
FLC
MAF1-4
MAF5
SPY
CO
GAI
EMF1
EMF2
FT
SOC1
AGL24
LFY
WUS
UFO
X
TFL2
CLF
KNAT2
WLC
LUG
*
FUL
AP1
CAL
AP2
FLORAL ORGANS
WHORLS
AG*
AP3*
PI*
ANT
SEP1
SEP2
SEP3*
SEPALS
PETALS
STAMENS
CARPELS
1
2
3
4
B (AP3, PI)
FUNCTIONS
A (AP1, AP2)
C (AG)
Figure 1. Arabidopsis genes regulating flowering time and flower development. Originally the figure was mainly
based on the figure of Blázquez (2001), modified in Lemmetyinen and Sopanen (in press), but more recent results
have also been incorporated, based on the references cited in text, except results with ESD4 (Reeves et al., 2002),
VIP4 (Zhang and van Nocker, 2002) and MAF1-5 (Ratcliffe et al., 2003). The arrowheads indicate activation and the
bars indicate repression. Genes involved, at least partly, in the same functions are boxed. In many cases, the effect
may be indirect and the line connecting two genes may represent a chain of events. The figure shows how several
external and internal factors lead to the activation of floral meristem genes and how they then participate in the
activation of genes determining the identity of the floral organs. The ABC model at the bottom of the figure shows
how the A, B and C functions, representing various combinations of organ identity genes, determine the identity of
floral organs. However, e.g. SEP genes are also involved in the process.
9
al., 2001).
One of the most important factors
controlling flowering time is the length of the
day. Arabidopsis is a facultative long-day plant
and many genes regulate the exact timing of
flowering (Simpson et al., 1999; Samach and
Coupland, 2000; Mouradov et al., 2002). The
photoreceptor genes, as well as the genes
involved in the functioning of the circadian
clock (Mazzella et al., 2001), function
upstream of CONSTANS (CO), which has a
central role in mediating between the circadian
clock and flowering (Suárez-López et al.,
2001). CO is the latest known gene specific to
the photoperiod pathway (Mouradov et al.,
2002). The genes downstream of CO are
FLOWERING LOCUS T (FT), SUPPRESSOR
OF OVEREXPRESSION OF CO1 (SOC1, also
called AGAMOUS-LIKE 20 [AGL20])
(Samach et al., 2000) and AGL24 (Yu et al.,
2002). These genes are also parts of other
pathways.
The autonomous pathway includes the genes
regulating flowering time independently of
day-length, but FLOWERING LOCUS C (FLC)
and the genes downstream are also responsible
for vernalization effects. Vernalization is an
important phenomenon especially for the
winter annual varieties of Arabidopsis. In these
varieties, exposure to low temperature for
several weeks accelerates flowering and may
be mediated through changes in DNA
methylation (Finnegan et al., 1998; Sheldon et
al., 1999; Sheldon et al., 2000). In Arabidopsis,
the two major determinant genes of the
vernalization response are FLC and FRIGIDA
(FRI) (Michaels and Amasino, 1999; Johanson
et al., 2000). FLC represses the floral meristem
identity genes LEAFY (LFY) and APETALA1
(AP1). The repression of AP1 is probably,
however, mediated via the repression of SOC1
(Lee et al., 2000).
The gibberellin (GA)-dependent pathway is
especially important in short days. In short
days, the ga1 mutant, defective in GA
synthesis, does not flower at all or flowers very
late (Blázquez et al., 1998). The GA-dependent
pathway functions by regulating the expression
of the floral meristem identity gene, LFY
(Blázquez et al., 1998; Nilsson et al., 1998a).
1.2.2. Inflorescence meristem
After induction, the vegetative meristem, which
has been producing leaf initials on its flanks,
changes into an inflorescence meristem. It is
broader and flatter than the vegetative
meristem, and starts to produce initials of
secondary inflorescences or flowers on its
flanks. The bolting starts and the flowering
shoot is formed. Several genes are involved in
the formation of the inflorescence meristem,
e.g. LFY and FRUITFULL (FUL, also called
AGAMOUS-LIKE8 [AGL8]) (Blázquez et al.,
1997; Hempel et al., 1997; Ferrándiz et al.,
2000), but it is not clear whether there are some
genes which specifically determine the identity
of the inflorescence meristem. FUL is
expressed in the inflorescence meristem very
early, but its role in the formation of
inflorescence is redundant (Ferrándiz et al.,
2000). TFL1 plays an important role in the
inflorescence meristem by repressing the
activity of LFY and AP1 in the apex of the
inflorescence meristem thus preventing the
formation of a terminal flower, which would
stop the growth of the meristem (Shannon and
Meeks-Wagner, 1993).
In Antirrhinum, DefH28 is expressed early
in the inflorescence meristem and its
overexpression in Arabidopsis leads to early
flowering (Müller et al., 2001). In birch, we
have isolated a closely related gene BpMADS4
(Elo et al., 2001), which also is expressed at
the earliest stage in inflorescence meristem (M.
Hassinen and T. Sopanen, unpublished results).
In antisense orientation it completely prevents
the formation of inflorescences and if
overexpressed, it can cause extremely early
flowering (A. Nowak and T. Sopanen,
unpublished results). Therefore, this gene could
be involved in the inflorescence function. In
Arabidopsis, no gene similar to DefH28 and
BpMADS4 has been characterised.
1.2.3. Floral meristem
Floral meristems start to form as a bulge on
the flanks of the inflorescence meristem. The
first genes (FLORICAULA and SQUAMOSA)
involved in flower formation were isolated
from Antirrhinum mutants, in which shoots had
developed instead of flowers (Coen et al.,
1990; Huijser et al., 1992). Related genes, LFY
10
and AP1, have been isolated from Arabidopsis
(Weigel et al., 1992; Mandel et al., 1992). Both
genes are expressed in the floral meristems
from very early stages of flower formation. The
overexpression of these genes results in very
early flowering and the formation of a terminal
flower (Mandel and Yanofsky, 1995; Weigel
and Nilsson, 1995). These genes are crucial
for flower formation and all the pathways
inducing flowering lead to the upregulation of
at least one of them. In addition, the
upregulation of LFY also leads to the
upregulation AP1 and vice versa (Liljegren et
al., 1999; Wagner et al., 1999).
cause for innovations in reproductive
development during terrestrial plant evolution,
such as flower, fruit and seed formation
(Theissen et al., 2000). Although the first
MADS box genes in plants were identified as
regulators of floral organ identity, many MADS
box genes are expressed vegetatively and have
functions beyond flowering (Alvarez-Buylla et
al., 2000).
A typical plant MADS domain protein has
a highly conserved MADS domain, a less
conserved I region, a conserved K-box and a
variable C terminal region (Riechmann and
Meyerowitz, 1997). In addition, AG and its
closest relatives have a highly variable N
terminal region. MADS domain proteins bind
to DNA either as homo- or heterodimers
recognizing various CC(A/T)6GG consensus
sequences called CArG-boxes with their
MADS domains (Shore and Sharrocks, 1995;
Riechmann and Meyerowitz, 1997; West et al.,
1997). In addition to DNA binding, MADS
domain is also involved in dimerisation and
accessory factor binding functions (Shore and
Sharrocks, 1995). The I region is a key
determinant for the dimerisation specificity and
the K domain is supposed to promote
dimerisation through interactions between K
domains (Riechmann and Meyerowitz, 1997).
It has been shown that the C terminus is
involved in the formation of higher order
complexes and it also has the transcriptionalactivator domain (Egea-Cortines et al., 1999;
Honma and Goto, 2001).
1.2.4. Floral organs
Soon after formation of the floral meristem,
initials of floral organs start to form. The
identity of the floral organs is determined by
the so-called ABC function genes (Coen and
Meyerowitz, 1991). According to the ABC
model, the sepals develop in whorl 1 as a
consequence of the activity of A function genes
(AP1 and AP2), the petals in whorl 2 as a
consequence of A and B function genes (AP1,
AP2, AP3 and PI), the stamens in whorl 3 as a
consequence of B and C function genes (AP3,
PI and AG) and finally the carpels in whorl 4
as a consequence of a C function gene (AG).
Later this “classical ABC model” has been
refined and even extended to an ABCDE model
(Theissen, 2001). In the ABCDE model, the D
function (AGL11) is needed for ovule identity
and the E function (SEP1-3) for petal, stamen
and carpel identities. The molecular basis of
the ABC model is the formation of ternary and
quaternary complexes of proteins (Honma and
Goto, 2001).
All the genes in the ABC model except AP2
are MADS box genes. As their name suggests,
MADS box genes exist in organisms of
different kingdoms: MCM1 (from yeast),
AGAMOUS (from plants), DEFICIENS (from
plants) and SRF (serum response factor from
human) (Schwarz-Sommer et al., 1990). In
Arabidopsis thaliana, the MADS box gene
family consists of over 80 different potential
genes which encode transcription factors
(Riechmann et al., 2000). It has been suggested
that changes in MADS box gene structure,
expression and function have been the major
1.2.5. AGAMOUS and SEPALLATA genes
The first floral homeotic gene to be isolated
from Arabidopsis was AG (Yanofsky et al.
1990. As many other genes, AG was identified
utilising mutants. In ag mutants, petals instead
of stamens are formed in the third whorl and a
new flower instead of a carpel is formed in the
fourth whorl resulting an overall phenotype
with (sepal, petal, petal)n flowers (Bowman et
al. 1989). Therefore, AG is required for both
floral meristem determination and organ
identity specification. The overexpression of
AG resulted in precocious flowering and the
formation of terminal flowers Mizukami and
Ma, 1997).
Several genes regulate the expression of AG,
11
for example, LFY, WUSCHEL (WUS) and
KNAT2 activate (Lohmann et al., 2001; Pautot
et al., 2001) whereas AP2 (Bowman et al.,
1991), LEUNIG (LUG) and AINTEGUMENTA
(ANT) (Krizek et al., 2000; Liu et al., 2000),
EMF1and 2 (Moon et al., 2003) and CLF
(Goodrich et al., 1997) suppress. In contrast,
AG suppresses AP1 (Gustafson-Brown et al.,
1994) and WUS (Lohmann et al., 2001) and
activates SHATTERPROOF (SHP2, also called
AGL5) (Savidge et al. 1995). The suppression
of AP1 by AG leads to the restriction of AP1
activity in the two outer whorls and the
suppression of AG by AP2 leads to the
restriction of AG in the two inner whorls.
Phylogenetical data indicates that AG is an old
gene (more than 300 million years) and its
homologues can also be found in
gymnosperms, where they are expressed in
male and female reproductive organs (Theissen
et al., 1996; 2000).
In Arabidopsis there are three highly
redundant SEPALLATA genes that are also
phylogenetically quite close to each other
(Pelaz et al., 2000; Parenicova et al., 2003).
As single mutants they have only very slightly
altered phenotypes. However, the functions of
SEP genes were greatly clarified when a triple
mutant was constructed (Pelaz et al. 2000). In
triple sep mutants, the petals, stamens and
carpels are replaced by sepaloid organs, which
indicates that the SEP genes are necessary for
the development of the three inner whorls.
They are possibly less redundant in some other
plant species.
The mechanism of the function of SEP3 is
based on the formation of quaternary
complexes, in which SEP3 is able to function
as a transcription factor. Such suggested
complexes include, for example, AP1-SEP3AP3- PI and SEP3-AP3-PI-AG (Honma and
Goto, 2001). The simultaneous ectopic
expression of, for example, SEP3, AP3, PI
and AG, is enough to result in the conversion
of leaves into staminoid organs (Honma and
Goto 2001). The regulation of SEP genes is
still largely unknown, but it has been shown
that at least EMF1 is able to repress them
(Moon et al. 2003).
1.3. Flower development-related genes from
trees
In trees, the economic importance of a species
has been the main argument in the selection of
species that have been studied for the genetic
regulation of flower development. Therefore,
among broad-leaved forest trees poplars,
eucalypts and birches have been the main
species studied (Table 1). Gymnosperms have
been more difficult because there are workable
protocols for micropropagation and gene
transfer for only a few species in gymnosperms.
However, some progress has been made,
especially in the isolation of genes regulating
flowering (Table 1).
Poplars have become very important in
biotechnological and genetic research. Poplars
are fast-growing trees, can grow on marginal
soils and are widely adaptable (Klopfenstein
et al., 1997). Despite their fast growing, the
time to first flowering can take up to 20 years
(Bhalerao et al., 2003). In poplars, male and
female inflorescences are on separate trees
(Sheppard et al., 2000).Therefore, the
production of sterile lines may be even easier
with poplars than with monoecious trees,
because it is necessary to prevent only stamen
or carpel formation, not necessarily both. Some
genes, which may be suitable for speeding up
or for preventing flowering, have already been
isolated from poplars, e.g. including the
homologues of AG, AP1, AP3 and LFY (Table
1). Interestingly, the AG homologues from
Populus trichocarpa, PTAG1 and 2, have some
expression in vegetative tissues in addition to
stamens and carpels and therefore differ from
the other known AG homologues (Brunner et
al., 2000).
Eucalyptus species are especially important
in Australia, but because of their fast growth
and good fiber quality, their cultivation
elsewhere is increasing. Many of the eucalyptus
genes isolated and analysed (Table 1) are
potentially suitable for the modification of
flowering. The genes isolated include, for
example, the homologues for the putative floral
meristem genes, LFY and AP1, as well as for
the homeotic genes, AG, PI and SEP genes.
In apple trees, the ability to regulate
flowering is important in breeding, in order to
accelerate flowering or to understand fruit
12
Table 1. List of isolated genes apparently regulating flower development in trees. The list also shows the most
similar genes or group of genes in Arabidopsis (except TM3, which is in tomato), but it should be noted that in most
cases a functional similarity has not been confirmed. Most, but not all genes in the list have been mentioned in the
text. The presence of some genes is based on unpublished results or mentions in abstracts, and therefore the sequences
are not yet available in the sequence databases.
tree genes
Dicots:
PTAG1 and 2
PTAP1-1 and 2
PTD
PTLF
two TFL1 homologs
EAP1 and 2
ETL
ELF1 (and 2)
EGM1 and 3
EGM2
AG homolog
TFL1 homologs
BpMADS1
BpMADS2
BpMADS3-5
BpMADS6
BpMADS7
BpMADS8
BpFLO
BpSPL
MdMADS1
MdMADS2
MdMADS3 and 4
MdMADS5
MdMADS6-9
MdMADS10
MdMADS11
MdPI
MdH1
CaMADS1
LAG
PlaraLFY
Conifers:
CjMADS1 and 2
DAL1
DAL2
DAL3
DAL10
DAL11-13
PaAP2L1 and 2
SAG1
NLY (NEEDLY)
PrDGL
PrMADS2 and 3
PrMADS4-9
PRFLL
PMADS1
DFL1 and 2
plant species
similar gene(s) in Arabidopsis
References
Populus trichocarpa
AG (AGAMOUS)
Brunner et al. 2000
Populus
AP1 (APETALA1)/ FUL (FRUITFULL) Skinner et al. 2000
Populus trichocarpa
AP3 (APETALA3)
Sheppard et al. 2000
Populus trichocarpa
LFY (LEAFY)
Rottmann et al. 2000
Populus
TFL1 (TERMINAL FLOWER 1)
Dye et al. 2001
Eucalyptus globulus
AP1/ FUL
Kyozuka et al. 1997
Eucalyptus globulus
AGL14/ TM3
Decroocq et al. 1999
Eucalyptus globulus
LFY
Southerton et al. 1998a
Eucalyptus grandis
SEP1, 2 and 3 (SEPALLATA1, 2 and 3) Southerton et al. 1998b
Eucalyptus grandis
PI (PISTILLATA)
Southerton et al. 1998b
Eucalyptus
AG
Southerton et al. 2001
Eucalyptus
TFL1
Collins and Gampbell 2001
Betula pendula
SEP1, 2 and 3
Paper III
Betula pendula
PI
Järvinen et al. 2003
Betula pendula
AP1/ FUL
Elo et al. 2001
Betula pendula
AG
Paper II
Betula pendula
AGL11
unpublished
Betula pendula
AP3
unpublished
Betula pendula
LFY
unpublished
Betula pendula
SPL
Lännenpää et al., in press
Malus x domestica
SEP1, 2 and 3
Sung and An 1997
Malus x domestica
AP1/ FUL
Sung, Yu and An 1999
Malus x domestica
SEP1, 2 and 3
Sung et al. 2000
Malus x domestica
AP1/ FUL
Yao et al. 1999
Malus x domestica
SEP1, 2 and 3
Yao et al. 1999
Malus x domestica
AGL11
Yao et al. 1999
Malus x domestica
AGL6
Yao et al. 1999
Malus x domestica
PI
Yao, Dong and Morris 2001
Malus x domestica
BEL1
Dong et al. 2000
Corylus avellana
AG
Rigola et al. 1998
Liquidambar styraciflua AG
Liu et al. 1999
Platanus racemosa
LFY
Frohlich and Parker 2000
Cryptomeria japonica
Picea abies
Picea abies
Picea abies
Picea abies
Picea abies
Picea abies
Picea mariana
Pinus radiata
Pinus radiata
Pinus radiata
Pinus radiata
Pinus radiata
Pinus resinosa
Pseudotsuga menziesii
PI/AP3
AGL6
AG
AGL14/ TM3
?
PI/AP3
AP2 (APETALA2)
AG
LFY
PI/AP3
AGL6
AGL14/ TM3
LFY
AGL6
LFY
13
Fukui et al. 2001
Tandre et al. 1995
Tandre et al. 1995; 1998
Tandre et al. 1995
Sundström 2001
Sundström et al. 1999
Vahala et al. 2001
Rutledge et al. 1998
Mouradov et al . 1998a
Mouradov et al . 1999
Mouradov et al . 1998b
Walden et al. 1998
Mellerowicz et al. 1998
Liu and Podila 1997
Strauss et al. 1995
development better. Several genes apparently
regulating flower/fruit development have been
isolated from the apple tree (Table 1.). AG
homologues have also been isolated from
Corylus avellana and Liquidambar styraciflua
(Rigola et al., 1998; Liu et al. 1999) and the
LFY homologue from Platanus racemosa
(Frohlich and Parker, 2000).
The reproductive organs of conifers differ
much from those of angiosperms (Rutledge et
al., 1998). Thus, it is interesting that
homologues for many genes regulating the
flower development in angiosperms can also
be found in conifers (Table 1.). Conifers seem
to have their B and C function gene
counterparts, but until now no genes similar to
AP1 have been found. However, two AP2-like
genes have been isolated for Picea abies
(Vahala et al., 2001). In Picea abies, there are
three different B function genes, DAL11-13,
which cannot be divided into two distinct
families as those in angiosperms (Sundström
et al., 1999). It has been postulated that even
at the very early stages in the evolution of
vascular plants, the B function genes were
involved in the specification of the male organs
(Theissen et al., 2000). When the C function
genes, SAG1 from Picea mariana (Rutledge
et al., 1998) and DAL2 from Picea abies
(Tandre et al., 1998), were ectopically
expressed in Arabidopsis, they resulted in the
conversion of petals into stamens and sepals
into carpels, as well as in the loss of the
indeterminacy of the inflorescence meristem.
This effect was quite similar to the ectopic
expression of AG in Arabidopsis (Mizukami
and Ma, 1992). This suggests that the basic
functions of the C class genes in the
determination of the male and female
reproductive organs are present already in
conifers.
different bush-like dwarf birch (Betula nana
L.). Silver and downy birches are used, for
example, for making paper, plywood and
furniture. Through breeding, the stem volume
of silver birch has been increased by about 30%
(Hagqvist and Hahl, 1998). In Finland, birches
are studied largely for their physiology and
economical quality. Birches offer many
advantages for studies on the effects of
transgenes. Birches are easy to micropropagate
(Simola, 1985; Ryynänen and Ryynänen, 1986)
and relatively easy to transform (Keinonen
1999). In Finland, many research groups are
studying the possibilities to improve the
properties of birch by using gene technology,
e.g. freezing, drought and ozone tolerance or
insect and pathogen resistance as well as wood
quality. Field tests are often needed for the
testing of the performance of the transgenic
trees. However, one problem in testing is that
the tests have to be finished before the birches
start to flower because of the legislation. This
restricts the possibilities to study transgene
effects in adult trees.
When the flower development is studied
using transgenic techniques, late flowering is
a great disadvantage. With a normally
flowering birch clone, it takes from 5 till 10
years to establish the effect of a transgene
construct on flower development in normal
growth conditions. Therefore, a method to
shorten the juvenile phase and accelerate the
onset of flowering is needed to speed up the
testing of gene constructs that are used to study
the functions of genes related to flowering or
to study the prevention of flowering. Another
beneficial feature for good research material
would be the onset of flowering when the plant
is still small. The testing of a gene construct,
e.g. for the prevention of flowering, needs
many transgenic lines. The testing of them
would be impractical in the greenhouse
because of the large size of the plants. The
testing of plants must be done in the greenhouse
in order to ensure that the transferred genes
cannot escape.
Fortunately, Stern (1961) has previously
produced birches which flower very early. This
was achieved using conventional breeding
during three generations. Such early flowering
birches are very important when studying the
1.4. Birch
Birches are economically important broadleaved forest tree species in temperate regions
and the most important ones in Finland where
there are three species of birches. Economically
the most important is silver birch (Betula
pendula Roth). The two other species are
downy birch (Betula pubescens Ehrh.), which
greatly resembles silver birch, and the quite
14
effects of transgenes on flower development.
An additional advantage of birch is that the
flowering of ordinary birches can be
considerably accelerated, in contrast to, e.g.
poplars (Meilan 1997), if grown in the
greenhouse in long-day conditions (Longman
and Wareing 1959) or under continuous light
and high CO2 (Holopainen and Pirttilä 1978).
Even in an ordinary greenhouse with no heating
in winter and without supplementary light,
birches usually start flowering at the age of two
or three years (Viherä-Aarnio and Ryynänen,
1995).
In birch, the male and female flowers are
on the same tree, but on separate
inflorescences, termed catkins (Atkinson
1992). The male inflorescences develop at the
ends of long shoots, whereas the female
inflorescences develop at the ends of short side
shoots. The flowers are in groups of three in
the axils of three fused scales. The male flowers
consist of a reduced perianth with 2-3 reduced
tepals and two stamens. The female flowers
are more simple and they consist of only a
single pistil with two stigmata. The male
inflorescences start to develop in May about
one year before anthesis and emerge in June.
The female inflorescences start to develop
about two months later in July but do not
emerge before the following spring shortly
before flowering.
Genes regulating the development of birch
(Betula pendula) inflorescences and/or flowers
are studied in our group. At present, our group
has isolated 8 different MADS box genes and/
or their cDNAs (Table 1). In this study I have
concentrated on two of them, BpMADS1 (IIIV) and BpMADS6 (II). In addition, we have
isolated three genes, BpMADS3-5 (Elo et al.,
2001), similar to FUL and AP1, BpMADS2
(Järvinen et al., 2003), similar to PI, BpMADS7
(P. Järvinen, J. Lemmetyinen and T. Sopanen,
unpublished results), similar to AGL11 and
BpMADS8 (S. Parkkinen, J. Lemmetyinen and
T. Sopanen, unpublished results), similar to
AP3. In addition, a SBP-box gene BpSPL1
(Lännenpää et al., in press) and a homologue
to LFY have been isolated (Table 1). The
ongoing birch EST projects are also likely to
provide much information of genes related to
flowering.
1.5. Biotechnical modification of flowering
1.5.1. Prevention of flowering
Prevention of flowering, or at least sterility, is
needed for environmentally safe testing and
cultivation of transgenic plants in nature when
they have possibilities to spread uncontrollably
or to spread the transgenes via pollen to native
populations. In addition, the prevention of
flowering may allow the trees to allocate more
resources to vegetative growth. The prevention
of flowering could also be used to reduce the
amount of allergenic pollen, especially near the
habitation. It has been estimated that birch
pollen causes allergenic reactions to 100
million individuals (Vrtala et al. 2001). The
prevention of flowering would be beneficial
for many other reasons, e.g. the reduction of
fruit litter of sweetgum (Brunner et al., 1998).
Two different principles have been
employed in studies aiming at the prevention
of flowering: tissue-specific ablation (Fig. 2)
and suppression of gene(s) essential for flower
formation, e.g. by using the antisense technique
Bacillus amyloliquefaciens
Birch
Promoter
Coding region
Flower-specific gene
Promoter
Coding region
BARNASE gene
Flower-specific
BARNASE gene
X
X
Non-flowering birch
Figure 2. Strategy for the prevention of flowering
using the inflorescence/flower-specific BARNASE
construct.
15
Non-transgenic birch
Necessary gene for
flowering
mRNA
Transgenic birch
Necessary gene for
flowering
suppress a target gene (Brunner et al., 1998).
The antisense technique (Fig. 3) is much used
but, recently, another more effective method,
RNA interference (RNAi) has been developed
(Waterhouse et al., 1998; Wesley et al., 2001;
Hannon, 2002).
The same gene in
reverse orientation
(antisense)
mRNA
Antisense RNA
douple-stranded RNA
Necessary protein for
flowering
Flowers are formed
1.5.2. Acceleration of flowering
For accelerated breeding with conventional
techniques, the transient acceleration of
flowering can be achieved by two principles.
In some species, the flowering can be
accelerated by using various treatments, e.g.
using hormones or growth retardants (Meilan,
1997). For some species, there is not yet any
known treatment and in most cases more
effective acceleration is needed. Fortunately,
it is possible to use transgenic techniques to
resolve this problem by transferring to the
target plant a gene that accelerates flowering.
This would dramatically reduce the time
needed for completing a breeding programme,
especially when it is possible to use molecular
markers. The use of markers makes it possible
to determine in a young seedling if the desired
genes are present in offspring. After the
breeding program the flowering-accelerating
gene can be crossed out and the final plant
would be non-transgenic. Alternatively, the
transgene could be eliminated, e.g. by using
an inducible gene excision system such as Cre/
loxP (Zuo et al., 2001).
The possibility to use gene technology has
given many possibilities to study the
mechanisms of flower development and its
regulation. However, in studies on flowering
of trees by means of modern methods, a long
juvenile phase also causes problems and makes
it impractical to study many features, e.g. the
effects of the overexpression or suppression
of genes involved in flowering. As an
exception, the gene constructs resulting in
accelerated flowering can be studied in a
reasonably short time.
There are only a few reports on the use of
gene transfer in the acceleration of flowering
in trees. Generally, the aspen starts flowering
when 8-20 years old, but the overexpression
of the Arabidopsis floral meristem gene LFY
caused flower formation in hybrid aspens in
only a few months in tissue culture and in 6-7
Degraded RNA
No flowers
Figure 3. Strategy for the prevention of flowering
using antisense technique.
(Fig. 3).
For tissue-specific ablation, the BARNASE
gene has been successfully used several times
(Koltunow et al., 1990; Goldman et al., 1994).
BARNASE is an RNase gene isolated from
Bacillus amyloliquefaciens, in which the
protein is secreted out of the cell (Hartley,
1988). For ablation, the signal sequence is
removed and therefore the enzyme stays in the
cytoplasm and hydrolyses the cytoplasmic
RNAs, which leads to cell death. The other
cytotoxin gene used is Diphtheria Toxin A gene
(DTA) (Koltunow et al., 1990; Thorsness et al.,
1993; Nilsson et al., 1998b). DTA encodes an
enzyme which inactivates Elongation Factor 2
(EF-2) by attaching an ADP ribosyl group and
thus prevents protein synthesis. In trees, some
attempts for tissue-specific ablations have been
reported. In poplars, the DTA and BARNASE
constructs with the anther-specific TA29, SLG
or TTS promoters have been used, but they also
decreased vegetative growth (Skinner et al.,
2000) suggesting that these promoters as such
are not useful for the prevention of flowering.
Another possibility to prevent flowering is
the suppression of at least one of the genes
necessary for flower formation (for reviews,
see e.g. Strauss et al., 1995; Lemmetyinen and
Sopanen, in press). For this, however, much
more information about the genes necessary
for flowering and long testing is needed to
ensure that the plants do not flower, e.g. due
to an alternative pathway of the induction.
There are several alternative methods to
16
months of growth in soil (Weigel and Nilsson,
1995). In this case, however, the flowers were
sterile. In addition, the rooting of these plants
was poor. The overexpression of PTLF in
poplars was not as effective as the
overexpression of LFY, because it resulted in
accelerated flowering only in one transgenic
line among many lines tested (Rottmann et al.,
2000). The fertility was reduced in these cases,
too. However, in Citrus, the overexpression of
AP1 or LFY resulted in the production of
normal, fertile flowers within a year, and the
flowers also produced normal fruits (PeZa et
al., 2001). Normally the Citrus flowers at the
age of 6-10 years. This result showed that by
using the right gene, flowering can be
accelerated for the breeding period. It also
showed that different species behave in
different ways when the same gene is
overexpressed.
of the isolated genes can be used in the
acceleration of flowering.
The specific aims of this thesis have been:
1. To develop a system to test
experimentally the effects of gene constructs
on birch flower development. Birches usually
start to flower for the first time when they are
five to ten years old. This makes the use of
some experimental studies very slow.
Therefore, the aim was to select earlyflowering birch clones and to study whether
they were suitable for studies on the transgene
effects on flower development.
2. To isolate two genes involved in flower
development and to characterise them. In the
characterisation, the main aims were to find
out the functions of these genes and their
similarities and differences to related genes
from other plants. Specific attention was paid
to properties related to the possible
biotechnological use of these genes.
3. To test the suitability of these genes or
their promoters in the prevention of
inflorescence or flower formation, especially
in birch but also in other plant species.
4. To test the suitability of these genes in
the acceleration of flowering in birches and in
other plants.
2. AIMS OF THE STUDY
The inability to regulate flowering is one of
the restraints in the breeding and genetic
modification of trees. In conventional
breeding, the time between generations
fundamentally determines the speed of
progress. When transgenic plants are cultivated
on field, the spread of transgenes must be
prevented. Therefore, the understanding of the
regulation of flowering and flower
development is important, because it allows
various possibilities for the genetic
manipulation of flowering.
In our group, the main aim has been to
develop a method to prevent flower formation.
Therefore, several genes have been isolated
and characterized in order to find suitable
candidate genes or promoters to be used in the
prevention of flowering. Because the final
testing of the constructs in field trials takes
many years, it is necessary to test several
different constructs at the same time. We have
also made studies in order to get a better
understanding of birch flower development and
to find out whether there is a gene which is
absolutely necessary for flower development
and which could be used for the prevention of
flowering. We have also studied whether some
3. MATERIAL AND METHODS
3.1. Plant material
Seeds of early-flowering silver birches (Betula
pendula Roth) (Stern 1961) were obtained
from Dr. Heide Glock (Institut für Forstgenetik
und Forstpflanzenzüchtung, Forsliche
Biometrie und Informatik der Universität
Göttingen, Germany). These seeds were sown
and the seedlings were grown in the greenhouse
(I). The first plants producing inflorescences
were selected and they were micropropagated
using the woody plant medium (WPM) (Lloyd
and McCown 1980; I). The preference of the
clones for the culture medium was tested using
WPM, Murashige and Skoog (1962) medium
(MS) and half-strength (½) MS (I). The
micropropagated birches were rooted on ½ MS
with 2.9 FM indolylacetic acid. The genes were
transferred to birch with Agrobacterium
(C58C1 with pGV3850 or pGV2260) as
17
described in (I) and (Keinonen, 1999).
Transgenic tobacco (Nicotiana tabacum L. cv.
Petit havana SR1) lines were obtained by
transferring the gene construct by
Agrobacterium to leaf tissues and Arabidopsis
thaliana (Wassilewskija) plants were
transformed by vacuum infiltration of
Agrobacterium. The transformations and
cultivation are described in papers II and III.
fluorometric assay (MUG) (Jefferson et al.,
1987; I). The BpMADS1::BARNASE
construct was generated by replacing the GUS
region with the combination of the BARNASE
and the BARSTAR genes (III). The gene
constructs used for overexpression or
suppression with antisense (II) were done in
pHTT602 plasmid as described in Elo et al.
(2001).
3.2. Isolation and analysis of BpMADS1 and 6
A partial cDNA clone of BpMADS1 was first
isolated using PCR with degenerative primers
as described in paper III and then the full-length
cDNA clone was isolated using the partial
cDNA clone as the probe in the screening of a
λZAPII cDNA library (III). BpMADS6 was
isolated by the screening of the same library
as for BpMADS1 but using an AG fragment
(pCIT565) (Yanofsky et al., 1990) as the probe
(II). The promoter region of BpMADS1 (3.1
kb) was first isolated by the screening of a
λFixII genomic library using the 3' end of
BpMADS1 as the probe and then subcloned
using PCR (III).
The expression of the genes was studied by
using northern hybridizations and in situ
hybridization analyses (II and III). The copy
number of the genes was studied using
Southern hybridization. The presence of the
transgene was studied using either PCR and/
or Southern hybridization (I-IV). The sequence
comparisons and phylogenetic analyses (II)
were mainly done using the GCG software
package (versions 8-10, Wisconsin) and
Neighbor-Joining algorithm (Saitou and Nei,
1987) with Clustalx software (Thompson et al.
1997). The microscopy was done as described
in papers I-IV.
4. RESULTS
4.1. Early-flowering birch clones (I)
Because the early-flowering birches developed
by Stern (1961) seemed to be very useful for
testing the effects of transgenes on flower
development, seeds of them were acquired. The
germination of the seeds was poor (less than
1%) but about 150 seedlings were obtained.
The seedlings were tested for flowering and
the twelve most early flowering among the 150
plants were selected for cloning and named
(BPM1-12) according to the order of their
flowering time. In the conditions used, the first
inflorescences appeared in these clones about
a year after the germination. At that time, most
of the plants were about 1.2 m high. Ten of
these clones survived the continuous in vitro
culturing. Two lines (BPM2 and 5) were
selected for further use because they showed
the best combination of early flowering,
abundant production of inflorescences and a
growth habit most similar to that of ordinary
birches. The two clones differed in the places
where the first inflorescences appeared. In
BPM2, the first inflorescences generally
appeared in the axis of the leaves, whereas in
BPM5, they generally appeared at the ends of
shoots or branches. Later these BPM clones
proved to be especially competent in gene
transfer (Keinonen, 1999).
Because special conditions (elevated CO2
level, strong continuous illumination) can be
used to accelerate the flowering of birch
(Holopainen and Pirttilä 1978), these
conditions were used when the flowering time
of the selected twelve clones was studied for
the second time. The cloned plants started
inflorescence formation earlier than the
seedlings from which they originated. After
micropropagation, the same clones started
3.3. Gene constructs
For the testing of the CaMV 35S promoter
function in birch, a GUS reporter gene in
CaMV 35S-GUS INT construct (Vancanneyt
et al. 1990) was used (I). The same construct
in tobacco was also used as a positive GUS
staining control. For testing, the BpMADS1
promoter was ligated into the pBI101 vector
resulting in the BpMADS1::GUS construct
(III). The expression of GUS was assayed
either using histochemical GUS staining or a
18
flowering at the age of about four months
instead of one year. In this study, the
comparison between normal growth conditions
and the conditions accelerating flowering was
not made. However, later we have found that
BPM2 and 5 can start inflorescence formation
at the age of four to six months even when they
are cultivated in normal growth conditions in
the greenhouse (unpublished results).
Therefore, the special growth conditions are
not necessary for the early flowering of those
clones. However, for normally flowering birch
clones (e.g. JR1/4) the conditions accelerating
flowering seem to be more beneficial. Although
a single ramet of JR1/4 once produced
inflorescences in the greenhouse, the
inflorescence production has been more regular
in the conditions for accelerated flowering
(unpublished observations). Because
continuous light is used both in the ordinary
greenhouse and in the accelerated growth, the
long days do not seem to be the only reason
for the more regular inflorescence formation
in JR1/4.
In our normal growth conditions (a
greenhouse with natural day light
supplemented with lamps), the start of
inflorescence formation is somewhat variable
and it is not strictly linked to the age or size of
the plant. In BPM2 and 5 clones, the usual age
to start inflorescence formation is three to four
months, but the start of inflorescence formation
can sometimes be delayed until the age of six
to seven months. There is also much variation
in the number of inflorescence formation
during the two to three first months after the
start of flowering. During this period the
abortion of inflorescences is frequent and
inflorescence formation can also cease until it
starts again at the age of about eight months
and at this stage the inflorescence formation is
more permanent and the abortion is not so
evident (unpublished observations).
The functions of genes are often studied by
expressing them ectopically in the sense or in
antisense orientation using a general promoter.
The CaMV 35S promoter is widely used for
this purpose in several plant species. Generally
it is considered to be tissue-unspecific,
although some variation in expression levels
in various tissues has been detected (Benfey
et al., 1989). Testing the CaMV 35S promoter
in birch leaves has shown that its activity is of
the same order of magnitude than that of the
promoters of ubiquitin or actin (KeinonenMettälä et al. 1998). However, more detailed
examination of its specificity and activity in
birch inflorescence tissues was necessary in
order to evaluate its suitability to express
ectopically the MADS box genes isolated by
our group.
The quantitative measurement of GUS
activity (MUG) in different parts of plants
showed that all parts studied had some activity,
but the inflorescences, buds and roots had the
highest activities. In addition, nine (eight)
different transgenic lines (3 lines of BPM2, 4
lines of BPM5 and 2 lines of JR1/4) were
analysed for GUS activity and it was found that
great variation existed between different lines
cultured in vitro. The cultivation of the lines
was continued and the same lines were
analysed again ten months after the first
analysis. Interestingly, the expression levels
had dropped dramatically to a minor fraction
of the levels measured in the previous assay.
Therefore, a significant silencing of transgenes
had apparently occurred in these transgenic
birch lines. This is interesting because in
tobacco several of our transgenic lines have
maintained their phenotypes several years
without any indication of decreased activity of
transferred genes. The same GUS construct that
was used in birch has also retained its activity
over six years in tobacco. The silencing seems
to depend on inverted or duplicated sequences
which are a consequence of several copies of
the transferred gene (De Buck et al., 2001;
Kumar and Fladung, 2001). It is possible that
transgenes in birch are silenced more readily
than those in tobacco or Arabidopsis.
4.2. BpMADS1 and BpMADS6
4.2.1. Isolation of MADS-box genes (II, III)
In order to rationally manipulate flowering in
birch, some knowledge on genes involved in
this process in birch was necessary, although
the flowering in Arabidopsis has been studied
in some detail. The isolation of genes would
also give tools necessary for biotechnical
applications. Among the genes regulating
flowering in model plants the MADS box genes
19
are the best characterised. For this reason, I
have isolated two MADS box genes from birch.
The cDNA clone of BpMADS1 was first
isolated using RT-PCR with degenerative
primers. The corresponding full-length cDNA
clone was then isolated by screening a cDNA
library using the PCR clone as the probe (III).
A genomic clone was isolated by screening of
the genomic library with the cDNA as the probe
and then a 3.1 kb fragment upstream to the
cDNA clone was isolated and sequenced (III).
This fragment is hereafter called the promoter
of BpMADS1.
For the isolation of BpMADS6, the same
cDNA library as that used for BpMADS1 was
screened with an AG fragment (Yanofsky et al.,
1990) as a heterologous probe.
As the first characterisation, the isolated
genes were sequenced and the sequences were
used in phylogenetic analyses (II). According
to most analyses, BpMADS1 and BpMADS6
are most similar to the Arabidopsis genes SEP3
and AG, respectively. However, in Arabidopsis
there are two genes very similar to SEP3 (SEP1
and 2) and three genes similar to AG (SHP1
and 2 and AGL11), and therefore the
identification of the nearest homologues for the
birch genes is not quite unequivocal on a
sequence basis alone.
later. The expression continued in the
developing stamens. In axillary buds, where
female inflorescences develop, the expression
of BpMADS1 was first detected in cells from
which female flowers (pistil) apparently
develop. The strongest expression of
BpMADS1 was detected in the proximal and
central parts of the pistil. The expression
continued in the developing pistil and later
could be detected in the integuments and the
embryo sac. No expression was detected in the
tepals, scales or axis. We do not know whether
BpMADS1 is also expressed in female
inflorescence meristems. The terminal buds of
birches often give rise to male inflorescences,
whereas the lateral buds producing female
inflorescences are difficult to screen and
identify at very early stages of development.
For this reason, we could not find any good
specimens for this phase.
The expression of BpMADS6 in male
inflorescences was first detected in cells from
which stamen formation apparently starts later
and continues in developing anthers and
filaments. In female inflorescences, the
expression of BpMADS6 was detected in the
distal parts of the pistil in contrast to the
expression of BpMADS1. Later the expression
of BpMADS6 was also detected in the
integuments and the embryo sac. No expression
was detected in the tepals, scales or axis.
4.2.2. Expression (II, III)
The localisation of the expression of
BpMADS1 and 6 was first carried out using
the RNA gel blot analysis (II and III). Both
genes were expressed in both male and female
inflorescences but not in any vegetative tissues.
At early developmental phases, the expression
in the inflorescences was weak. The strongest
expression was in the inflorescences of late
developmental phases and it also remained
high during seed development in female
catkins.
The expression was further localised using
in situ hybridisation (II). In terminal buds,
where male inflorescences develop, the
expression of BpMADS1 was first detected at
the earliest stage when the inflorescence
meristems could be identified. However, this
expression lasted only for a short time. Next,
the expression was detected in the cells from
which flowers (or stamens) apparently develop
4.2.3. Effects of overexpression and
antisense constructs (II)
The localisation of gene activity by in situ
hybridisation to certain tissues and cells tells
something about gene function. Much more
information on the exact functions can be
obtained by over-expressing the studied genes
and especially by suppressing them, for
example, by using antisense technique or
mutagenesis. Therefore, both BpMADS1 and
6 were overexpressed with the CaMV 35S
promoter in Arabidopsis, tobacco and birch.
In addition, both cDNAs were also expressed
in antisense orientation in birch.
In tobacco, the BpMADS1 sense construct
resulted in accelerated flowering but did not
have any other effects, for example, the
structure of the flowers did not change.
However, in Arabidopsis the BpMADS1 sense
20
construct caused reduction in the numbers of
floral organs and/or whorls, for example,
sometimes a complete lack of sepals and petals.
In addition, it resulted in mixed organ identities
with the characteristics of the three outer
whorls changed towards those of the inner
whorls, e.g. sepals with white areas or stigmatic
tissues, filament-like petals, and stamens with
ovules.
In birch, the effect of the BpMADS1
constructs was usually not strong. We could
detect an altered phenotype in 11 of the 56
kanamycin-resistant transgenic lines obtained.
In the phenotype obtained with the BpMADS1
antisense construct (eight lines), the developing
male inflorescences first looked normal but
after having attained the size of about 8 mm
they started to produce leaves instead of
stamens. Because the scales (which are
modified bracts) were still present in these
flowers, the leaves could not originate from
the scales. Later these “inflorescences” died
or continued their development as branches.
This indicates that BpMADS1 is necessary for
the formation of stamens. We have not tested
the effect of these constructs on the
development of the female inflorescence,
because this would require an overwintering
treatment. Surprisingly, the effects with sense
construct (three lines) were similar to those of
antisense construct, which suggests that the
sense construct has caused co-suppression,
similarly to the sense construct of FBP2 in
petunia (Angenent et al., 1994). This has been,
however, difficult to prove because the
constructs are not effective enough to cause
constant phenotypes. We could not detect any
effect of the overexpression of BpMADS1 on
the flowering time in birch, but a weak effect
cannot be excluded, because it might have been
obscured by large variation in the flowering
time of control plants in these experiments.
Similarly to the effect of the BpMADS1
sense construct, the BpMADS6 sense construct
resulted in accelerated flowering in tobacco.
In addition, it had an effect on sepals and petals
(paper II, figure 6). In a weak phenotype, the
size of the tube and the limbs was reduced and
the edges of petal tips were strongly curved
outwards. In a strong phenotype, the sepals
formed a tube resembling the carpel and had
some stigma-like tissue at the edges. When an
AG homologue, BAG1 from Brassica, was
overexpressed in tobacco, a similar effect was
observed (Mandel et al., 1992).
In Arabidopsis, the effect of the
overexpression of BpMADS6 was dramatic
(paper II, figure 6). All of the 19 kanamycinresistant plants obtained flowered extremely
early, reaching the height of only 0.5 cm in
extreme cases. Even the cotyledons were
narrow and grooved as were the very small
rosette leaves formed thereafter. The flowers
lacked petals and the sepals had stigmatic tissue
or ovule-like structures.
In birch, only two lines (one sense and one
antisense line) out of 47 lines obtained with
BpMADS6 constructs had an altered
phenotype. In several male inflorescences of
these lines, the flowers were undetermined with
tepals instead of stamens and, in extreme cases,
similarly to the phenotypes obtained with the
BpMADS1 constructs, some “leafy”
inflorescences were formed.
4.2.4. Analysis of the BpMADS1 promoter
(III)
In order to find out whether the isolated
promoter region of BpMADS1 has all necessary
elements for flower-specific expression, the
BpMADS1::GUS construct was generated and
transferred into Arabidopsis and tobacco and
analysed with histochemical staining. In both
species, the first signs of expression were
detected early in inflorescence development.
In tobacco, the expression was detected in
shoot apex at an early stage of inflorescence
development. The staining was strong in young
flower buds and later it was strongest in the
basal part of the flower, especially in the inner
parts of the receptacle. In Arabidopsis, the
expression was detected in the apical bud
before the elongation of the inflorescence.
Later, expression was detected in the flower
buds, pedicel, receptacle, filament, anther,
carpel (especially in stigma) and sepals. No
vegetative expression was detected except
some occasional staining of very small areas
in the tips of some young rosette or upper
leaves.
The BpMADS1::GUS construct was also
transferred into the early-flowering birch
21
clones BPM2 and 5. In some transgenic lines
some staining could be detected in male
flowers, but generally this staining was quite
weak (unpublished results). One problem with
birch was its endogenous activity for the
staining substrate, which might be caused, at
least sometimes, by pathogenic fungi .
4.3. Prevention of flowering using the
BpMADS1::BARNASE construct (III, IV)
Several characters of BpMADS1 and its
promoter supported the suitability of the
promoter for a cytotoxin construct for the
prevention of flower formation. The BARNASE
gene (Hartley, 1988) was selected for a
cytotoxin gene. For the construct designed to
prevent flowering, the signal sequence was
removed from BARNASE. BARSTAR, the gene
for a strong inhibitor protein to BARNASE
(Hartley, 1989), was included in the construct
to protect the bacteria (E. coli and
Agrobacterium) during cloning and gene
transfer. Because BARSTAR is in the same
cistron under the same promoter as BARNASE,
but after BARNASE, it is not expressed in
plants.
The BpMADS1::BARNASE construct was
first tested in Arabidopsis and tobacco (III).
In both species the construct had an effect on
flower formation in most transgenic lines. In
tobacco, the BpMADS1::BARNASE construct
prevented most effectively the stamen and
carpel formation leading to sterile flowers
consisting of only sepals and petals even in the
weakest phenotypes. In stronger phenotypes,
the petals were also lacking. In the strongest
phenotypes, no inflorescences were formed. In
these lines, the growth of the main shoot
stopped and a few sepal-like leaves formed at
its apex. Later we have obtained some
transgenic lines in which the growth continued
without any signs of inflorescence formation
(unpublished results) (Fig. 4).
Interestingly, in the lines which produced
small inflorescences with sterile flowers, the
mass was increased at the end of the growth
period, the dry weights of these sterile
tobaccoes being 140-200% of those of the
controls. The initial growth of the control and
sterile plants was similar until inflorescence
formation. However, in wild type control
Figure 4. Transgenic tobacco containing the
BpMADS1::BARNASE construct (right). The shoot
growth continues without the formation of the inflorescence. The non-transgenic control plant (left).
plants, the vegetative growth ceased during
flower development and seed ripening, in
contrast to the plants with sterile flowers, in
which usually one or two axillary shoots
formed soon after the main shoot had stopped
its growth. This was repeated when the new
branches formed new inflorescences with
sterile flowers. In some new non-flowering
transgenic lines, we have later observed some
reduction of growth and in one line the height
of plants was about half of that in controls at
the time when controls started to flower
(unpublished results). This suggests that in
some cases the promoter may also function in
the vegetative parts of the plant.
In Arabidopsis, the prevention of flower/
inflorescence formation resulted in a
considerably increased number of leaves in
long days (III). Some transgenic plants never
formed real inflorescence shoots, but in most
transgenic lines there was some inflorescence
22
shoot formation. However, no flowers were
formed in many cases. Instead of flowers, a
rosette of leaves formed at the ends of the
shoots and then a new inflorescence shoot
started to grow in the middle of this “rosette”.
The inflorescence shoots without flowers were
usually shorter (in extreme cases below 0.5 cm)
and at the same time thicker than the shoots in
the non-transgenic wild type. This indicates
that the construct could not totally prevent
inflorescence formation, but reduced their
length either by preventing flower formation
or by inhibiting inflorescence meristem
development. In the case of weak effect,
flowers without stamens and carpels formed.
In short days the effect was similar.
Interestingly, in one plant grown in short-day
conditions, the petals, stamens and carpels did
not develop properly, and the number of
flowers was much increased (Fig. 5)
(unpublished results). This suggests that some
signals coming out of developing flowers may
be needed for the regulation of inflorescence
meristems negatively.
In the early-flowering birch, 45 out of 81
transgenic lines transformed with the
BpMADS1::BARNASE construct had
phenotypic effects (IV). Only seven lines were
non-flowering without considerable
disturbances in the initial vegetative growth.
The plant development in these seven nonflowering lines was normal until inflorescence
development. Similarly to the control plants,
their leaves turned dark green at the onset of
flowering but no inflorescences were formed.
Sometimes some malformed brownish
structures, instead of inflorescences, were
formed and some small inflorescences without
stamens were formed in some lines. These
inflorescences without stamens died and
aborted soon. However, they occasionally
continued their development as branches.
When the control plants were producing
inflorescences, the non-flowering plants
sometimes formed dichotomic branches or
bundles of branches at their shoot apexes. The
new branches formed were also shorter and
their number was reduced in comparison to the
controls.
A great number of transgenic lines showed
severe defects even before the flowering of the
Figure
5.
The
introduction
of
the
BpMADS1::BARNASE construct into Arabidopsis
resulted in an inflorescence with high number of flowers, when grown in short days.
controls. The common features were bushy and
reduced growth due to extensive branching,
small leaves and brown, necrotic areas in the
leaves. Some of these lines produced
inflorescences but 12 of them were nonflowering.
After two of the non-flowering lines with
normal initial growth had been subjected to a
wintering treatment in a preliminary
experiment, no female inflorescences emerged
in one of them. In the other line, some female
inflorescences emerged but in them the pistils
were lacking or they were severely malformed
and without stigmata.
5. DISCUSSION
5.1. Advantages and disadvantages of the
early-flowering birches
Originally the early-flowering birches were
bred for conventional breeding (Stern, 1961),
but we have shown that they are especially
useful tools both in the basic molecular
research on flower development and in the
studying of applications based on the genetic
23
manipulation of flowering (II, IV).
The reduction of flowering time from 5-10
years to 3-6 months represents a major
advantage as does the fact that our clones form
inflorescences when only 50-100 cm high
allowing the cultivation of relatively large
numbers of plants until flowering in a relatively
small space (30-50 plants/m 2 when
inflorescence development starts). In addition,
our clones BPM2 and 5 give higher
transformation frequences than any of the
ordinary birch clones tested thus far (Keinonen,
1999).
Unfortunately, the early-flowering birch
clones also have some disadvantages. Some
original trees bred by Stern bore only
generative buds (Stern, 1961) but none of the
seedlings we obtained were such early
flowering (I). Although the BPM2 and 5 clones
were not as extremely early flowering, they still
have some abnormalities, for example, in the
positions where the inflorescences form.
Further, these two clones seem to behave in
slightly different ways, for example, they prefer
different growth media and they show
differences in the preferred positions of the first
inflorescences (I). Because the reason for their
early flowering is not yet known and because
there seem to be differences in the regulation
of inflorescence formation, two different clones
were used for the testing of gene constructs (I,
II, IV). Because the BPM2 and 5 clones are
not as extremely early flowering as the original
trees bred by Stern, they may actually be more
similar to ordinary birches and therefore more
suitable tools for studies on transgene effects
on flower development.
The structure of the inflorescences in the
early-flowering clones seems to be quite
normal. This suggests that most differences in
comparison to ordinary birch clones are in the
induction of inflorescence formation. The
downward curving of the leaves at the onset of
flowering suggests a hormonal imbalance (I),
which may be connected to the regulation of
flowering time and to the atypical formation
of male inflorescences in the axils of leaves,
as well as to good regeneration properties, too.
On the other hand, the reason for early
flowering can be either an abnormal expression
of a gene/genes accelerating flowering/flower
formation or suppression of a gene/genes
preventing flowering or a combination of both
alternatives. Whatever the reasons are, they
cause the observed abnormalities. One of those
abnormalities is that not only the terminal buds
give rise to male inflorescences they are also
produced by axillary buds, which Stern (1961)
also observed in the original trees. Although
an abnormality, it enables a continuous
formation of male inflorescences. The
sometimes abundant formation of
inflorescences may be an advantage when
constructs geared to prevent flowering are
tested, because it accelerates the revelation of
possible failures or problems in the functioning
of the constructs (IV). In normally flowering
birches, the revealed problems would perhaps
not be as severe. A clear disadvantage of the
early-flowering clones is that, because of the
differences in flowering, the results obtained
with them cannot be directly applied to normal
birches and they must be interpreted with some
caution. The clarification of the reason for early
flowering would greatly improve the usefulness
of these clones.
The large variation of the BPM clones in the
flowering time and inflorescence formation
suggest great sensitivity for various
environmental factors in the induction of
flowering. This is a disadvantage when
studying the effects of various gene constructs
on the flowering time. There is much variation
in the size at which these birches start
inflorescence formation as well as in the
abundance of inflorescence formation. Because
the inflorescence formation of these birches is
continuous, they can first form only some
inflorescences and later abundantly or vice
versa. Because the inflorescence formation is
also dependent on environmental effects, the
formation of inflorescences varies much when
grown in the greenhouse. Most likely, the
flowering would be much more homogenous
if the cultivation could be done in completely
controlled growth conditions in phytotrons. It
can also cease for a while.
The parents of these early-flowering birch
clones suffered from some fertility problems
(Stern, 1961), which explains the poor
germination percentage of the open-pollinated
seeds (I). This partial sterility suggests some
24
defects in floral structure or physiology. The
germination of the seeds of the BPM2 and 5
clones have not been tested but it is possible
that they behave in the same way. This was not
a problem when the constructs geared to
prevent flower formation were tested (IV).
However, when constructs geared to accelerate
flowering are tested, it would also be important
to test their effect on fertility because early
flowering with sterile flowers would have no
use in breeding. Fortunately, the acceleration
of flowering can also be tested in normallyflowering genotypes.
Together, the advantages, in spite of the
disadvantages mentioned, make the earlyflowering birch clones BPM2 and 5 very much
better that ordinary birches for studies
concerning the effects of transgenes on
inflorescence and flower development.
also expressed in the inflorescence meristems.
In addition, in Sinapis, a close relative to
Arabidopsis, the SEP3 homologue SaMADS
D is expressed in the shoot apex but just below
the actual inflorescence meristem (Bonhomme
et al., 1997). In petunia, FBP2 is not expressed
in the inflorescence meristem. However, two
other genes, FBP5 and pMADS12, which are
closely related to FBP2, are both expressed in
the inflorescence meristem (Ferrario et al.,
2003).
Differentiation of the perianth into sepals
and petals is most pronounced in eudicots
(Endress, 2001). However, the morphological
distinction between sepals and petals is less
simple than it may seem from consideration of
typical cases and sometimes sepals and petals
cannot unambiguously be defined by their
structures and functions. Therefore, it has been
hoped that genetic studies would help in this
problem. In many plant species studied, the
genes of the SEP3 group are expressed in
carpels, stamens and petals with little or no
expression in sepals (Angenent et al., 1994;
Pnueli et al., 1994; Mandel and Yanofsky,
1998). Lack of BpMADS1 expression in tepals
suggests that these might be homologous to
sepals. Lack of the expression of the birch
homologues of AP3 and PI from tepals (S.
Vepsäläinen, T. Savola and T. Sopanen,
unpublished results) is in harmony with this
suggestion.
The use of overexpression in the elucidation
of the functions of MADS box genes has been
criticised because of difficulties in
understanding and explaining the phenotypes
obtained. Because the MADS box genes are
quite similar to each other, high levels resulting
from their overexpression may result in the
binding of the gene products to regulatory
elements, to which they normally do not bind,
or to the formation of complexes, which
normally do not form. This may cause effects
that they do not normally have. However, in
the case of redundant genes, overexpression is
often a better choice than the use of suppression
of a single gene. Moreover, over-expression
can also be used in other species and,
importantly, it can also lead to practical
applications, for example, by resulting in
earlier flowering. An additional feature of using
5.2. BpMADS1
When BpMADS1 was isolated from birch, the
central role of the three SEPALLATA genes in
flower development was not known although
the results obtained with similar genes in
petunia (Angenent et al., 1994) and tomato
(Pnueli et al., 1994) gave some indications that
they would have some function in the
development of floral organs. The roles of the
SEP genes were difficult to find because of the
high redundancy of SEP genes. In petunia,
FBP2 and 5 also function redundantly (Ferrario
et al., 2003). However, in Gerbera hybrida,
the suppression of CRCD1, a gene similar to
SEP genes, resulted in homeotic changes of
sterile staminoides into petals (Kotilainen et
al., 2000), which suggests that in Gerbera the
redundancy in this gene group is not so high.
Southern hybridisation indicates that in birch
there is no other gene very similar to
BpMADS1, which suggests that in birch there
may be only one gene belonging to this group.
One of the interesting questions of genes in
the SEP3 group is their possible expression in
inflorescence meristems. BpMADS1 differs
from SEP3 in being expressed in the
inflorescence meristem (II). On the other hand,
BpMADS1 resembles in that respect the
monocot genes AOM in Asparagus officinalis
(Caporali et al., 2000) and DOMADS1 in
Dendrobium (Yu and Goh, 2000), which are
25
heterologous plants is the possible avoidance
of the homology dependent co-suppression.
In tobacco, the early flowering of transgenic
plants overexpressing BpMADS1 showed that
BpMADS1 can induce the development of
inflorescence meristems. The early expression
of BpMADS1 in the inflorescence meristem is
consistent with its ability to accelerate
flowering. The results with BpMADS1 are
similar to the results obtained by the overexpression of BpMADS1 homologues,
OsMADS1 (Chung et al., 1994), NsMADS2 and
3 (Jang et al., 1999), SEP3 (Pelaz et al., 2001)
or FBP2 (Ferrario et al., 2003). However, many
genes accelerate flowering when expressed
ectopically although there is no late flowering
mutants for them (Koornneef et al., 1998). This
indicates that the function of these genes may
be redundant or that they may be involved in
other related processes and that the early
flowering is just an artefact.
Perhaps the most confusing of all
phenotypes obtained in this study was obtained
with the sense construct of BpMADS1 in
Arabidopsis. In Arabidopsis, the overexpression of BpMADS1 showed that
BpMADS1 is able to regulate the development
of the floral organs (II). The main two
tendencies obtained with phenotypes were the
conversion of sepals, petals and stamens into
organs which had features of organs from the
whorls inner to them and the reduction of the
number of organs in the three outer whorls.
Most of the differences in phenotypes were
apparently due to the strength of the
phenotypes. The variation in the reduction in
the number of organs and/or whorls together
with mixed organ identity may result from the
construct having a variable, moderate effect on
the three outer whorls. In addition, the
construct seems to affect different whorls and
organs quite independently and this results in
variability on phenotypes. The expression data
obtained in Arabidopsis and tobacco contained
many phenotypes (not shown) that were
intermediates to the phenotypes presented in
the paper (II).
In birch, the formation of leaves instead of
stamens in plants with the BpMADS1 antisense
construct suggests that the gene is needed for
flower development and, in male flowers,
especially for stamen development (II). The
change of stamens into leaves also takes place
in the sep triple mutants (Pelaz et al., 2000).
This suggests that the function of BpMADS1
is similar to the function of the SEP genes. The
function of BpMADS1 in female inflorescence
development was not studied by the antisense
suppression because the transgenic lines
obtained showed altered phenotypes only
occasionally and female inflorescences emerge
first after a lengthy and space requiring overwintering treatment in the phytotrone and in
the cold room. The use of the RNAi technique
is probably more suitable for this purpose and
will be used in the near future.
The isolation of the BpMADS1 promoter
was necessary in order to make the BARNASE
construct for the prevention of flowering. In
addition, the isolation and analysis of the
BpMADS1 promoter region would be
important for the elucidation of gene regulation
in detail. Until now the sequences of only some
promoters in the SEP3 group are known, for
example, that of DOMADS1 in Dendrobium
(Yu et al., 2002) and SEP3 (The Arabidopsis
Genome Initiative, 2000). Although the cDNAs
and putative amino acid sequences of these
genes are highly similar (78 % identity between
BpMADS1 and SEP3 at amino acid level), the
promoter regions are almost completely
different from each other (unpublished results)
and do not allow an easy recognition of
important areas.
A simultaneous analysis of two or more
promoters, which are different but have quite
similar specificities, may help in the
identification of the regions necessary for
expression and for tissue-specificity. The
determination of the important regions for the
promoter function is useful for practical
reasons. The identification of tissue-specific
elements/regions is necessary for the designed
changes in the manipulation of promoters in
cases when no natural promoter with perfect
properties can be found.
When the promoter of BpMADS1 was
analysed in Arabidopsis and tobacco using a
deletion series with GUS, the shortest
fragments of the promoter did not have any
activity, intermediate length fragments showed
some non-specific activity and only the longest
26
(3.1 kb) fragment was flower/inflorescencespecific (III, P. Rinne, J. Lemmetyinen and T.
Sopanen, unpublished results). This indicates
that in the BpMADS1 promoter there are some
important regulatory elements in the region
between -1.6 kb and -3.1 kb. None of the
constructs made with the 2.5 kb long SEP3
promoter fragment were specific to flowers,
and all of them were expressed in leaves (P.
Rinne, J. Lemmetyinen and T. Sopanen,
unpublished results). This indicates that in the
SEP3 promoter there are some regulatory
elements outside the isolated promoter
fragment.
induction of flowering or in the determination
of the inflorescence meristems in these species.
It is quite interesting that the overexpression
of AGAMOUS homologues has such a strong
effect on the flowering time because, so far,
they have not been found to have any function
on the induction of inflorescence meristem.
AGAMOUS homologues are apparently able
to have an effect on many processes but are
harnessed only to flower development. It is also
possible that the proteins encoded by
AGAMOUS homologues form non-functional
complexes with some proteins inhibiting
flowering and in this way eliminate their
inhibitory effect and induce flowering.
5.3. BpMADS6
AG was among the first plant MADS box genes
isolated and characterized (Yanofsky et al.,
1990). Since then, many genes similar to AG
have been isolated in a variety of species. Most
of these genes, including BpMADS6, are
expressed flower-specifically in stamens and
carpels. Interestingly, the poplar genes PTAG1
and 2 (Brunner et al., 2000) and the apple gene
MdMADS15 (van der Linden et al., 2002) are,
however, expressed vegetatively, too. In the
curly leaf mutant, AG is also expressed
vegetatively (Goodrich et al., 1997). Vegetative
expression is not, however, characteristic for
the AG genes in trees, because BpMADS6 in
birch (II), DAL2 in Picea abies (Tandre et al.,
1998) and SAG1 in Picea mariana (Rutledge
et al., 1998) are not expressed vegetatively.
The function of BpMADS6 seemed to be
quite similar to the functions of its homologues
according to overexpression and suppression
studies in Arabidopsis, tobacco and birch and
expression studies in birch. All the results
suggest that BpMADS6 is needed for the
formation of stamens and pistil. In tobacco, the
overexpression of BpMADS6 accelerated the
flowering and had an effect on the development
of sepals and petals as expected from an AG
homologue. Interestingly, in Arabidopsis the
effect of BpMADS6 was similar but much
stronger than in tobacco. Although the effect
on the flowering time was quite similar with
BpMADS1 and BpMADS6 in tobacco, in
Arabidopsis the plants overexpressing
BpMADS6 flowered very early. This might be
a consequence of different mechanisms in the
5.4. Suitability of BpMADS1 and BpMADS6
for the prevention of flowering
Several reasons advocated the use of the
promoter of BpMADS1 for the BARNASE
construct. The expression of BpMADS1 was
inflorescence specific (II, III), which made the
BpMADS1 promoter potentially suitable for the
BARNASE construct. BpMADS1 was
expressed quite early (II, III) and therefore the
BpMADS1::BARNASE construct was
supposed to prevent the flower/inflorescence
development at an early stage. The homologues
of BpMADS1 were mainly expressed in the
three inner whorls (petals, stamens and carpels)
(see e.g. Theissen et al., 1996) but some of
them were also expressed in the outmost whorl
(sepals) (Bonhomme et al., 1997). This
suggested that the BpMADS1::BARNASE
construct introduced into a plant would prevent
the formation of at least petals, stamens and
carpels. In Arabidopsis and tobacco, the
BpMADS1::GUS construct was active, as
expected, in flowers from the early phases to
the late seed development phases (III,
unpublished results). The long duration of the
expression is a beneficial feature because it
ensures that the BpMADS1::BARNASE
construct has enough time to prevent flower
formation.
The effect of the BpMADS1::BARNASE
construct in tobacco and Arabidopsis was as
expected. The best effectivity in the prevention
of stamen and carpel formation together with
the prevention of inflorescence formation is
consistent with the results obtained with the
27
BpMADS1::GUS construct (III) and with in
situ hybridisations in birch (II). The results also
showed that the prevention of flowering can
result in increased growth, too.
Surprisingly,
in
birch
the
BpMADS1::BARNASE construct had an
adverse effect on the vegetative growth of
many transgenic lines (IV). Although the
results with northern and in situ analyses in
ordinary birch indicate that BpMADS1 is not
expressed in vegetative tissues, it is still
possible that some weak expression exists.
Because the expression of BpMADS1 in BPM2
and 5 is not known, it is possible that the
construct is expressed precociously in them and
might be one of the factors leading to early
flowering. Similarly it is possible that the
isolated promoter region does not have all the
elements that are needed for flower-specific
expression. However, because we have
obtained some non-flowering transgenic lines
without the reduction of growth before
flowering, these alternatives are not likely.
Alternatively, the reduction in vegetative
growth can be the result of uncompleted
transfer of the construct to the plant or of the
flanking sequence in the site of genome
integration. As mentioned above, the reduction
in promoter length results in vegetative
expression (unpublished results). In case of
several copies of genes, one truncated gene
would be enough to cause some vegetative
disturbances.
Although potato is a close relative to
tobacco, the BpMADS1::BARNASE
construct did not function in potato as well as
in tobacco (J. Lemmetyinen and T. Sopanen,
unpublished results). Although we obtained
some lines, in which the flower formation was
prevented, there was some reduction in the
vegetative growth in many lines. Because
both
BpMADS1::BARNASE
and
BpMADS1::GUS constructs functioned well
in as far relatives as Arabidopsis and tobacco,
it is quite likely that the BpMADS1 promoter
also functions well in many other species. At
least, BpMADS1 promoter functions in the
flowers of Dianthus, which was shown by the
transient expression of BpMADS1::GUS (K.
Nissinen, J. Lemmetyinen and T. Sopanen,
unpublished results).
BARNASE has also been used for preventing
seed germination in tobacco (Kuvshinov et al.,
2001). In this method, the flowers and
inflorescences develop normally, but the
sulfhydryl endopeptidase (SH-EP) promoter in
the gene construct guides the expression of
BARNASE to the embryos and seedlings thus
preventing their development. The induced
plant production via seeds and seedlings was
possible by using the gene encoding the
inhibitor protein BARSTAR, which was
regulated with the heat shock (HS) promoter.
This system is good for plants, in which the
flower and seed productions are needed, e.g.
in cereals. However, with plants, from which
only vegetative parts are utilised, the
prevention of the flower formation might be
more beneficial because it increases
productivity, at least in some cases (III).
Recently, an interesting way to solve the
problem caused by the non-specificity of the
promoter was developed (Burgess et al., 2002).
In this system, two different promoters are used
and each promoter expresses only part of the
functional BARNASE protein. These two parts
combine in the cell and form a functional
protein. In this system, only cells where both
promoters are active are destroyed. Another
hypothetical solution to prevent the
disturbances caused by the BARNASE (because
the leakage of the promoter) is to use
BARSTAR. The BARSTAR gene could be
expressed by using a constant and ubiquitous
promoter, which is weak enough to allow the
desired ablation to take place but strong enough
to prevent the undesired vegetative
disturbances. However, it may be difficult to
set the right level. One solution would be to
find a promoter to BARSTAR, which leads the
expression to vegetative tissues but not to
inflorescences.
One possibility to eliminate the disturbances
caused by the low vegetative expression of
BARNASE is to use another gene. The flowerspecific use of a gene, for example, which only
prevents cell division, is possibly less harmful
for vegetative growth.
Because we are not sure that the promoter
of BpMADS1 as such is the most suitable
promoter in the prevention of flowering, we
are also testing some other alternatives. For
28
example, the use of the promoters of
BpMADS2, 5 and 8 has given good preliminary
results in tobacco and Arabidopsis (M.
Lännenpää and T. Sopanen, unpublished
results).
The use of the antisense constructs of
BpMADS1 and BpMADS6 (II) suggested that
the efficient downregulation of either of these
genes might lead to complete sterility, which
is also evident in the light of mutations in other
plants (Pelaz et al., 2000). Although it seems
that the antisense technique is not able to
suppress strongly enough the gene expression,
the use of the RNAi technique is likely to give
better results. Antisense and RNAi techniques
have the advantage that vegetative effects are
very unlikely. The RNAi technique seems to
function very well in many different organisms
(e.g. Chuang and Meyerowitz, 2000) and,
therefore, is likely to function well also in birch
although it has not yet been tested.
to get early flowering transgenic lines might
be co-suppression, which is a probable reason
for some detected phenotypes of the transgenic
lines obtained (II). It is also possible that these
early-flowering birch clones already express
one or both of these genes precociously.
Therefore, with a normally-flowering birch
clone, the effects of these constructs may be
quite different.
Previously, we have shown that the birch
homologues of AP1 and FUL (BpMADS3-5)
accelerate flowering very strongly in tobacco
(Elo et al., 2001). With these genes the
acceleration is more efficient than with
BpMADS1 or 6 (II). Similarly, in birch,
BpMADS3 and 4 are more efficient and, in
extreme cases, the ectopic expression of
BpMADS4 resulted in inflorescence formation
when the plants were still a few cm high
(unpublished results, see Lemmetyinen and
Sopanen, in press; Sopanen and Elo, 2002).
At least in these transgenic lines, the
overexpression of genes accelerating flowering
does not lead to co-suppression. The
overexpression of BpMADS3 resulted in either
accelerated or delayed flowering (J.
Lemmetyinen and T. Sopanen, unpublished
results), which suggests that the same gene
construct can lead either to overexpression or
to co-suppression.
5.5. Suitability of BpMADS1 and BpMADS6
for the acceleration of flowering
In addition to the usefulness of the BpMADS1
promoter in the prevention of flowering, we
have found that its cDNA, as well as the cDNA
of BpMADS6 can be used to accelerate
flowering (II). The results with tobacco show
that the resulting transgenic plants are fertile.
In tobacco, the BpMADS1 sense construct did
not have any effect on the flower structure. In
contrast, the BpMADS6 sense construct had a
moderate effect on the flower structure. The
fertility of transgenic tobacco plants
overexpressing BpMADS1 and 6 suggests that
these genes might be suitable for practical
purposes. However, the very small size of all
Arabidopsis plants overexpressing BpMADS6
shows that applicability depends on the
species. Although the overexpression of, for
example, many MADS box genes or the
suppression of some other genes are able to
accelerate flowering, BpMADS1 and 6 may
have some applications for moderate
acceleration in some cases.
In birch, the effects of BpMADS1 and 6
genes on the flowering time have not been
evident and need further testing because of
large variation in our early-flowering birch
clones. One possible reason for our inability
6. GENERAL CONCLUSIONS
1. Early-flowering birch clones are useful in
the study of flower development in birch. They
help especially in the testing of the effect of
transgenes on flower formation. Although
early-flowering birches have many features
suitable for a research tool, they still have some
peculiarities in flowering. Therefore, caution
is necessary in the interpretation of results
obtained and possible practical applications
have to be tested with ordinary birches as well.
2. BpMADS1 and BpMADS6 are important
genes in the regulation of flower development.
Largely they are similar to their homologues
in other plants and they seem to function in
the same way. However, BpMADS1 is
expressed in the inflorescence meristem, in
29
contrast to its homologue SEP3, and it is
therefore possible that it also has some function
in the inflorescence development.
Special thanks are for Ilkka Porali for his
help and interest especially in any kind of
challenging technical problems (which have
inspired many captivating coffee table
discussions), Kaija Keinonen for her good
advices about any kind of problems, Mika
Lännenpää for excellent and self-denying
gardening and Pia Järvinen for her good,
definite views. I also wish to thank my
coauthors, Annakaisa Elo, Minna Hassinen,
Hannu Mäkelä and Tuija Pennanen and all
current and previous members of the group,
especially Helena Kaija, Katri Nissinen, Paula
Rinne, Liisa Tikka, Marja-Leena Turunen and
Saila Vepsäläinen. It has been a pleasure to
work with them.
I am also very grateful to Riitta Pietarinen
and Hannele Hakulinen for their help in
laboratory, Pekka Piironen for his help in
gardening, Professor Teemu Teeri for
providing the vector used in constructing sense
and antisense constructs of BpMADS1 and 6,
Dr. R.W. Hartley for providing the pMT1002
plasmid containing the BARNASE/BARSTAR
genes, Professor Jaakko Kangasjärvi for
providing the genomic library of silver birch,
Leena Ryynänen for telling us about the earlyflowering birches and Marga Margelin for
checking the language of the thesis. I also wish
to thank Tuula Konsti, Mervi Kinnunen, Eija
Ristola, Matti Savinainen, Matti Hallikainen,
Heikki Loikkanen and Kari Määttä for their
help in many practical problems. I also wish
to thank Pertti Nenonen for his help in cutting
down birches in order to help us to collect the
inflorescences.
Finally, I thank my wife, Päivi, my
daughters, Emilia and Milla, and my parents
for their patience and support during this
process.
3. The tests with the BpMADS1::BARNASE
construct proved that inflorescence formation
can be prevented in trees as well as in other
dicots using tissue-specific ablation. In tobacco
and Arabidopsis, the results showed that the
prevention of inflorescence/flower formation
can result in increased growth and productivity.
In birch, the defects in vegetative growth
detected in many transgenic lines need more
consideration and therefore the prevention
inflorescence formation needs to be further
studied before its practical applications are
ready for general use. However, the studies
with the BpMADS1::BARNASE gene
construct are an important step towards the
controlled containment of transgenes.
4. The overexpression of BpMADS1 and
BpMADS6 resulted in moderately accelerated
flowering in tobacco. Therefore, it is possible
these genes might be used for accelerated
flowering in several other species, as well.
ACKNOWLEDGEMENTS
This study was carried out at the Department
of Biology, University of Joensuu. I thank
Professor Heikki Hyvärinen and Dr. Markku
Kirsi, the heads of the Department of Biology,
for their support and encouragement and for
providing excellent facilities. The study was
funded by the Academy of Finland, TEKES
(as a part of Finnish Biodiversity Programme,
FIBRE), Faculty of Science, University of
Joensuu, the Graduate School of Biology and
Biotechnology of Forest Trees and later by the
Graduate School of Forest Sciences.
I wish to express my sincere gratitude to my
supervisor Professor Tuomas Sopanen for
providing an interesting project as well as for
his patience and encouraging, wise advices
during my work. I also thank Professor Markku
Keinänen for being as supervisor at the final
stage of the thesis and Docent Yrjö Helariutta
and Professor Ove Nilsson who kindly
reviewed the manuscript.
REFERENCES
Alvarez-Buylla ER, Liljegren SJ, Pelaz S, Gold SE,
Burgeff C, Ditta GS, Vergara-Silva F, and Yanofsky
MF. 2000. MADS-box gene evolution beyond
flowers: expression in pollen, endosperm, guard cells,
roots and trichomes. Plant Journal 24:457-466.
Angenent GC, Franken J, Busscher M, Weiss D, and van
Tunen AJ. 1994. Co-suppression of the petunia
homeotic gene fbp2 affects the identity of the
30
generative meristem. Plant Journal 5:33-44.
Araki T. 2001. Transition from vegetative to reproductive
phase. Current Opinion in Plant Biology 4:63-68.
Atkinson MD. 1992. Betula pendula Roth (B. verrucosa
Ehrh.) and B. pubescens Ehrh. Journal of Ecology
80:837-870.
Aubert D, Chen L, Moon Y-H, Martin D, Castle LA, Yang
C-H, and Sung ZR. 2001. EMF1, a novel protein
involved in the control of shoot architecture and
flowering in Arabidopsis. Plant Cell 13:1865-1875.
Battey NH and Tooke F. 2002. Molecular control and
variation in the floral transition. Current Opinion in
Plant Biology 5:62-68.
Benfey PN, Ren L, and Chua N-H. 1989. The CaMV
35S enhancer contains at least two domains which
can confer different developmental and tissuespecific expression patterns. EMBO Journal 8:21952202.
Bhalerao R, Nilsson O, and Sandberg G. 2003. Out of
the woods: forest biotechnology enters the genomic
era. Current Opinion in Biotechnology 14:206-213.
Blázquez MA. 2000. Flower development pathways.
Journal of Cell Science 113:3547-3548.
Blázquez MA, Soowal LN, Lee I, and Weigel D. 1997.
LEAFY expression and flower initiation in
Arabidopsis. Development 124:3835-3844.
Blázquez MA, Green R, Nilsson O, Sussman MR, and
Weigel D. 1998. Gibberellins promote flowering of
Arabidopsis by activating the LEAFY promoter. Plant
Cell 10:791-800.
Bonhomme F, Sommer H, Bernier G, and Jacqmard A.
1997. Characterization of SaMADS D from Sinapis
alba suggests a dual function of the gene: in
inflorescence development and floral organogenesis.
Plant Molecular Biology 34:573-582.
Bowman JL. 1994. Arabidopsis: an atlas of morphology
and development. New York: Springer-Verlag New
York, inc.
Bowman JL, Smyth DR, and Meyerowitz EM. 1989.
Genes directing flower development in Arabidopsis.
Plant Cell 1:37-52.
Bowman JL, Smyth DR, and Meyerowitz EM. 1991.
Genetic interactions among floral homeotic genes of
Arabidopsis. Development 112:1-20.
Brunner AM, Mohamed R, Meilan R, Sheppard LA,
Rottmann WH, and Strauss SH. 1998. Genetic
engineering of sexual sterility in shade trees. Journal
of Arboriculture 24:263-273.
Brunner AM, Rottmann WH, Sheppard LA, Krutovskii
K, DiFazio SP, Leonardi S, and Strauss SH. 2000.
Structure and expression of duplicate AGAMOUS
ortholoques in poplar. Plant Molecular Biology
44:619-634.
Burgess DG, Ralston EJ, Hanson WG, Heckert M, Ho
M, Jenq T, Palys JM, Tang K, and Gutterson N. 2002.
A novel, two-component system for cell lethality and
its use in engineering nuclear male-sterility in plants.
Plant Journal 31:113-125.
Caporali E, Spada A, Losa A, and Marziani G. 2000. The
MADS box gene AOM1 is expressed in reproductive
meristems and flowers of the dioecious species
Asparagus officinalis. Sexual Plant Reproduction
13:151-156.
Chuang C-F and Meyerowitz EM. 2000. Specific and
heritable genetic interference by double-stranded
RNA in Arabidopsis thaliana. Proceedings of the
National Academy of Science of the United States of
America 97:4985-4990.
Chung Y-Y, Kim S-R, Finkel D, Yanofsky MF, and An
G. 1994. Early flowering and reduced apical
dominance result from ectopic expression of rice
MADS box gene. Plant Molecular Biology 26:657665.
Clark JR. 1983. Age-related changes in trees. Journal of
Arboriculture 9:201-205.
Coen ES and Meyerowitz EM. 1991. The war of the
whorls: genetic interactions controlling flower
development. Nature 353:31-37.
Coen ES, Romero JM, Doyle S, Elliot R, Murphy G, and
Carpenter R. 1990. floricaula: A homeotic gene
required for flower development in Antirrhinum
majus. Cell 63:1311-1322.
Collins AJ, and Campbell MM. 2001. Eucalyptus genes
implicated in the control of maturation and flowering.
IUFRO/Molecular Biology of Forest Trees. 22-27 July
2001, Columbia River Gorge, United States of
America.
De Buck S, Van Montagu M, and Depicker A. 2001.
Transgene silencing of invertedly repeated transgenes
is released upon deletion of one of the transgenes
involved. Plant Molecular Biology 46:433-445.
Decroocq V, Zhu X, Kauffman M, Kyozuka J, Peacock
WJ, Dennis ES, and Llewellyn DJ.1999. A TM3-like
MADS-box gene from Eucalyptus expressed in both
vegetative and reproductive tissues. Gene 228:155160.
Dong Y-H, Yao J-L, Atkinson RG, Putterill JJ, Morris
BA, and Gardner RC. 2000. MDH1: an apple
homeobox gene belonging to the BEL1 family. Plant
Molecular Biology 42:623-633.
Dye SJ, Brunner AM, Skinner JS, and Strauss SH. 2001.
Isolation of TERMINAL FLOWER 1 homologs from
Populus and expression analyses over a seasonal cycle
and continuous maturation gradient. IUFRO/
Molecular Biology of Forest Trees. 22-27 July 2001,
Columbia River Gorge, United States of America.
Egea-Cortines M, Saedler H, and Sommer H. 1999.
Ternary complex formation between the MADS-box
proteins SQUAMOSA, DEFICIENS and GLOBOSA
is involved in the control of floral architecture in
Antirrhinum majus. EMBO Journal 18:5370-5379.
Elo A, Lemmetyinen J, Turunen M-L, Tikka L, and
Sopanen T. 2001. Three MADS-box genes similar to
APETALA1 and FRUITFULL from silver birch
(Betula pendula). Physiologia Plantarum 112:95-103.
Endress PK. 2001. The flowers in extant basal
angiosperms and inferences on ancestral flowers.
International Journal of Plant Sciences 162:11111140.
Ferrándiz C, Gu Q, Martienssen R, and Yanofsky MF.
2000. Redundant regulation of meristem identity and
plant architecture by FRUITFULL, APETALA1 and
CAULIFLOWER. Development 127:725-734.
Ferrario S, Immink RGH, Shchennikova A, BusscherLange J, and Angenent GC. 2003. The MADS box
gene FBP2 is required for SEPALLATA function in
31
petunia. Plant Cell 15:914-925.
Finnegan EJ, Genger RK, Kovac K, Peacock WJ, and
Dennis ES. 1998. DNA methylation and the
promotion of flowering by vernalization. Proceedings
of the National Academy of Science of the United
States of America 95:5824-5829.
Frohlich MW and Parker DS. 2000. The mostly male
theory of flower evolutionary origins: from genes to
fossils. Systematic Botany 25:155-170.
Fukui M, Futamura N, Mukai Y, Wang Y, Nagao A, and
Shinohara K. 2001. Ancestral MADS box genes in
sugi, Cryptomeria japonica D. Don (Taxodiaceae),
homologous to the B function genes in angiosperms.
Plant and Cell Physiology 42: 566-575.
Goldman MHS, Goldberg RB, and Mariani C. 1994.
Female sterile tobacco plants are produced by stigmaspecific cell ablation. EMBO Journal 13:2976-2984.
Goodrich J, Puangsomlee P, Martin M, Long D,
Meyerowitz EM, and Coupland G. 1997. A polycombgroup gene regulates homeotic gene expression in
Arabidopsis. Nature 386:44-51.
Gustafson-Brown C, Savidge B, and Yanofsky MF. 1994.
Regulation of the Arabidopsis floral homeotic gene
APETALA1. Cell 76:131-143.
Hagqvist R and Hahl J. 1998. Rauduskoivun
siemenviljelysten jalostushyöty Etelä- ja KeskiSuomessa. Summary in English: Genetic gain
provided by seed orchards of silver birch in Southern
and Central Finland. Reports from the Foundation
for Forest Tree Breeding 13:1-30.
Hannon GJ. 2002. RNA interference. Nature 418:244251.
Hartley RW. 1988. Barnase and barstar. Expression of
its cloned inhibitor permits expression of a cloned
ribonuclease. Journal of Molecular Biology 202:913915.
Hartley RW. 1989. Barnase and barstar: two small
proteins to fold and fit together. Trends in
Biochemical Sciences 14:450-454.
Hempel FD, Weigel D, Mandel MA, Ditta G, Zambryski
PC, Feldman LJ, and Yanofsky MF. 1997. Floral
determination and expression of floral regulatory
genes in Arabidopsis. Development 124:3845-3853.
Holopainen L and Pirttilä V. 1978. Kukittamishuoneen
varusteet ja käyttö. Summary in English: The
equipment in and use of the flower induction hall.
The Foundation for Forest Tree Breeding in Finland
Information 1/1978:1-4.
Honma T and Goto K. 2001. Complexes of MADS-box
proteins are sufficient to convert leaves into floral
organs. Nature 409:525-529.
Huijser P, Klein J, Lönnig W-E, Meijer H, Saedler H,
and Sommer H. 1992. Bracteomania, an inflorescence
anomaly, is caused by the loss of function of the
MADS-box gene squamosa in Antirrhinum majus.
EMBO Journal 11:1239-1249.
Jack T. 2001. Relearning our ABCs: new twists on an
old model. Trends in Plant Science 6:310-316.
Jang S, Hong MY, Chung YY, and An G. 1999. Ectopic
expression of tobacco MADS genes modulates
flowering time and plant architecture. Molecules and
Cells 9:576-586.
Järvinen P, Lemmetyinen J, Savolainen O, and Sopanen
T. 2003. DNA sequence variation in B p M A D S 2
gene in two populations of Betula pendula. Molecular
Ecology 12:369-384.
Jefferson RA, Kavanagh TA, and Bevan MW. 1987. GUS
fusions: -glucuronidase as a sensitive and versatile
gene fusion marker in higher plants. EMBO Journal
6:3901-3907.
Johanson U, West J, Lister C, Michaels S, Amasino R,
and Dean C. 2000. Molecular analysis of FRIGIDA,
a major determinat of natural variation in Arabidopsis
flowering time. Science 290:344-347.
Keinonen K. 1999. Towards genetic manipulation of
silver birch (Betula pendula) and Scots pine (Pinus
sylvestris). Ph.D. Dissertation, University of Joensuu,
Finland.
Keinonen-Mettälä K, Pappinen A, and von Weissenberg
K. 1998. Comparisons of the efficiency of some
promoters in silver birch (Betula pendula). Plant Cell
Reports 17:356-361.
Kieffer M and Davies B. 2001. Developmental
programmes in floral organ formation. Seminars in
Cell & Developmental Biology 12:373-380.
Klopfenstein NB, Chun YW, Kim M-S, and Ahuja MR.
(Eds) 1997. Micropropagation, genetic engineering,
and molecular biology of Populus. General Technical
Report RM-GTR-297. Fort Collins, CO:US.
Department of Agriculture, Forest Service, Rocky
Mountain Forest and Range Experiment Station.
Koltunow AM, Truettner J, Cox KH, Wallroth M, and
Goldberg RB. 1990. Different temporal and spatial
gene expression patterns occur during anther
development. Plant Cell 2:1201-1224.
Koornneef M, Alonso-Blanco C, Peeters AJM, and Soppe
W. 1998. Genetic control of flowering time in
Arabidopsis. Annual Review of Plant Physiology and
Plant Molecular Biology 49:345-370.
Kotilainen M, Elomaa P, Uimari A, Albert VA, Yu D,
and Teeri TH. 2000. GRCD1, an AGL2-like MADS
box gene, participates in the C function during stamen
development in Gerbera hybrida. Plant Cell 12:18931902.
Krizek BA, Prost V, and Macias A. 2000.
AINTEGUMENTA promotes petal identity and acts
as a negative regulator of AGAMOUS. Plant Cell
12:1357-1366.
Kumar S and Fladung M. 2001. Gene stability in
transgenic aspen (Populus). II. Molecular
characterization of variable expression of transgene
in wild and hybrid aspen. Planta 213:731-740.
Kuvshinov V, Koivu K, Kanerva A, and Pehu E. 2001.
Molecular control of transgene escape from
genetically modified plants. Plant Science 160:517522.
Kyozuka J, Harcourt R, Peacock WJ, and Dennis ES.
1997. Eucalyptus has functional equivalents of the
Arabidopsis AP1 gene. Plant Molecular Biology
35:573-584.
Lännenpää M, Jänönen I, Hölttä-Vuori M, Gardemeister
M, Porali I, and Sopanen T. A new SBP-box gene
BpSPL1 in silver birch (Betula pendula). Physiologia
Plantarum, in press.
Lee H, Suh S-S, Park E, Cho E, Ahn JH, Kim S-G, Lee
JS, Kwon YM, and Lee I. 2000. The AGAMOUS-
32
LIKE 20 MADS domain protein integrates floral
inductive pathways in Arabidopsis. Genes &
Development 14:2366-2376.
Lemmetyinen J and Sopanen T. Modification of flowering
in forest trees. In: Kumar S and Fladung M, (Eds).
Molecular Genetics and Breeding of Forest Trees. The
Haworth Press, Inc. In press.
Levy YY and Dean C. 1998. The transition to flowering.
Plant Cell 10:1973-1989.
Liljegren SJ, Gustafson-Brown C, Pinyopich A, Ditta GS,
and Yanofsky MF. 1999. Interactions among
APETALA1, LEAFY, and TERMINAL FLOWER1
specify meristem fate. Plant Cell 11:1007-1018.
Liu J-J, and Podila GP. 1997. Characterization of a
MADS box gene (Y09611) from immature female
cone of red pine (PGR97-032). Plant Physiology
113:665.
Liu J, Huang Y, Ding B, and Tauer CG. 1999. cDNA
cloning and expression of a sweetgum gene that
shows homology with Arabidopsis AGAMOUS. Plant
Science 142:73-82.
Liu Z, Franks RG, and Klink VP. 2000. Regulation of
gynoecium marginal tissue formation by LEUNIG
and AINTEGUMENTA. Plant Cell 12:1879-1891.
Lloyd GB and McCown BH. 1980. Commercially
feasible micropropagation of mountain laurel (Kalmia
latifolia) by use of shoot-tip culture. Proc. Inter. Plant
Propagators Soc. 30:421-437.
Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F,
Simon R, and Weigel D. 2001. A molecular link
between stem cell regulation and floral patterning in
Arabidopsis. Cell 105:793-803.
Longman KA and Wareing PF. 1959. Early induction of
flowering in birch seedlings. Nature 84:2037-2038.
Mandel MA and Yanofsky MF. 1995. A gene triggering
flower formation in Arabidopsis. Nature 377:522524.
Mandel MA and Yanofsky MF. 1998. The Arabidopsis
AGL9 MADS box gene is expressed in young flower
primordia. Sexual Plant Reproduction 11:22-28.
Mandel MA, Bowman JL, Kempin SA, Ma H,
Meyerowitz EM, and Yanofsky MF. 1992.
Manipulation of flower structure in transgenic
tobacco. Cell 71:133-143.
Mandel MA, Gustafson-Brown C, Savidge B, and
Yanofsky MF. 1992. Molecular characterization of
the Arabidopsis floral homeotic gene APETALA1.
Nature 360:273-277.
Mazzella MA, Cerdán PD, Staneloni RJ, and Casal JJ.
2001. Hierarchical coupling of phytochromes and
cryptochromes reconciles stability and light
modulation of Arabidopsis development.
Development 128:2291-2299.
Meilan R. 1997. Floral induction in woody angiosperms.
New Forests 14:179-202.
Mellerowicz EJ, Horgan K, Walden A, Coker A, and
Walter C. 1998. PRFLL - a Pinus radiata homologue
of FLORICAULA and LEAFY is expressed in buds
containing vegetative shoot and undifferentiated male
cone primordia. Planta 206:619-629.
Michaels SD and Amasino RM. 1999. FLOWERING
LOCUS C encodes a novel MADS domain protein
that acts as a repressor of flowering. Plant Cell
11:949-956.
Mizukami Y and Ma H. 1992. Ectopic expression of the
floral homeotic gene AGAMOUS in transgenic
Arabidopsis plants alters floral organ identity. Cell
71:119-131.
Mizukami Y and Ma H. 1997. Determination of
Arabidopsis floral meristem identity by AGAMOUS.
Plant Cell 9:393-408.
Moon Y-H, Chen L, Pan RL, Chang H-S, Zhu T, Maffeo
DM, and Sung ZR. 2003. EMF genes maintain
vegetative development by repressing the flower
program in Arabidopsis. Plant Cell 15:681-693.
Mouradov A, Glassick T, Hamdorf B, Murphy L, Fowler
B, Marla S, and Teasdale RD. 1998a. NEEDLY, a
Pinus radiata ortholog of FLORICAULA/LEAFY
genes, expressed in both reproductive and vegetative
meristems. Proceedings of the National Academy of
Science of the United States of America 95: 65376542.
Mouradov A, Glassick TV, Hamdorf BA, Murphy LC,
Marla SS, Yang Y, and Teasdale RD. 1998b. Family
of MADS-box genes expressed early in male and
female reproductive structures of Monterey pine.
Plant Physiology 117:55-61.
Mouradov A, Hamdorf B, Teasdale RD, Kim JT, Winter
K-U, and Theissen G. 1999. A DEF/GLO-like MADSbox gene from a gymnosperm: Pinus radiata contains
an ortholog of angiosperm B class floral homeotic
genes. Developmental Genetetics 25: 245-252.
Mouradov A, Cremer F, and Coupland G. 2002. Control
of flowering time: Interacting pathways as a basis
for diversity. Plant Cell Supplement 2002:S111-S130.
Müller BM, Saedler H, and Zachgo S. 2001. The MADSbox gene DEFH28 from Antirrhinum is involved in
the regulation of floral meristem identity and fruit
development. Plant Journal 28:169-179.
Murashige T and Skoog F. 1962. A revised medium for
rapid growth and bio assays with tobacco tissue
cultures. Physiologia Plantarum 15:473-497.
Nilsson O, Lee I, Blázquez MA, and Weigel D. 1998a.
Flowering-time genes modulate the response to
LEAFY activity. Genetics 150:403-410.
Nilsson O, Wu E, Wolfe DS, and Weigel D. 1998b.
Genetic ablation of flowers in transgenic Arabidopsis.
Plant Journal 15:799-804.
Parenicová L, de Folter S, Kieffer M, Horner DS, Favalli
C, Busscher J, Cook HE, Imgram RM, Kater MM,
Davies B, Angenent GC, and Colombo L. 2003.
Molecular and phylogenetic analyses of the complete
MADS-box transcription factor family in
Arabidopsis: New openings to the MADS world.
Plant Cell 15:1538-1551.
Pautot V, Dockx J, Hamant O, Kronenberger J, Grandjean
O, Jublot D, and Traas J. 2001. KNAT2: Evidence
for a link between knotted-like genes and carpel
development. Plant Cell 13:1719-1734.
Pelaz S, Ditta GS, Baumann E, Wisman E, and Yanofsky
MF. 2000. B and C floral organ identity functions
require SEPALLATA MADS-box genes. Nature
405:200-203.
Pelaz S, Gustafson-Brown C, Kohalmi SE, Crosby WL,
and Yanofsky MF. 2001. APETALA1 and
SEPALLATA3 interact to promote flower
33
development. Plant Journal 26:385-394.
PeZa L, Martín-Trillo M, Juárez J, Pina JA, Navarro L,
and Martínez-Zapater JM. 2001. Constitutive
expression of Arabidopsis LEAFY of APETALA1
genes in citrus reduces their generation time. Nature
Biotechnology 19:263-267.
Pnueli L, Hareven D, Broday L, Hurwitz C, and Lifschitz
E. 1994. The TM5 MADS box gene mediates organ
differentiation in the three inner whorls of tomato
flowers. Plant Cell 6:175-186.
Ratcliffe OJ, Amaya I, Vincent CA, Rothstein S,
Carpenter R, Coen ES, and Bradley DJ. 1998. A
common mechanism controls the life cycle and
architecture of plants. Development 125:1609-1615.
Ratcliffe OJ, Kumimoto RW, Wong BJ, and Riechmann
JL. 2003. Analysis of the Arabidopsis MADS
AFFECTING FLOWERING gene family: MAF2
prevents vernalization by short periods of gold. Plant
Cell 15:1159-1169.
Reeves PH, Murtas G, Dash S, and Coupland G. 2002.
early in short days 4, a mutation in Arabidopsis that
causes early flowering and reduces the mRNA
abundance of the floral repressor FLC. Development
129:5349-5361.
Riechmann JL and Meyerowitz EM. 1997. MADS
domain proteins in plant development. Biological
Chemistry 378:1079-1101.
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C-Z,
Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha
RR, Creelman R, Pilgrim M, Broun P, Zhang JZ,
Ghandehari D, Sherman BK, and Yu G-L. 2000.
Arabidopsis transcription factors: Genome-wide
comparative analysis among eukaryotes. Science
290:2105-2110.
Rigola D, PP ME, Fabrizio C, MP G, and Sari-Gorla M.
1998. CaMADS1, a MADS box gene expressed in
the carpel of hazelnut. Plant Molecular Biology
38:1147-1160.
Rottmann WH, Meilan R, Sheppard LA, Brunner AM,
Skinner JS, Ma C, Cheng S, Jouanin L, Pilate G, and
Strauss SH. 2000. Diverse effects of overexpression
of LEAFY and PTLF, a poplar (Populus) homolog of
LEAFY /FLORICAULA, in transgenic poplar and
Arabidopsis. Plant Journal 22:235-245.
Rutledge R, Regan S, Nicolas O, Fobert P, Côté C,
Bosnich W, Kauffeldt C, Sunohara G, Séguin A, and
Stewart D. 1998. Characterization of an AGAMOUS
homologue from the conifer black spruce (Picea
mariana) that produces floral homeotic conversions
when expressed in Arabidopsis. Plant Journal 15:625634.
Ryynänen L and Ryynänen M. 1986. Propagation of adult
curly-birch succeeds with tissue culture. Silva
Fennica 20:139-147.
Saedler H, Becker A, Winter K-U, Kirchner C, and
Theissen G. 2001. MADS-box genes are involved in
floral development and evolution. Acta Biochimica
Polonica 48:351-358.
Saitou N and Nei M. 1987. The Neighbor-Joining
method: A new method for reconstructing
phylogenetic trees. Molecular Biology and Evolution
4:406-425.
Samach A and Coupland G. 2000. Time measurement
and the control of flowering in plants. BioEssays
22:38-47.
Samach A, Onouchi H, Gold SE, Ditta GS, SchwarzSommer Z, Yanofsky MF, and Coupland G. 2000.
Distinct roles of CONSTANS target genes in
reproductive development of Arabidopsis. Science
288:1613-1616.
Savidge B, Rounsley SD, and Yanofsky MF. 1995.
Temporal relationship between the transcription of
two Arabidopsis MADS box genes and the floral
organ identity genes. Plant Cell 7:721-733.
Schwarz-Sommer Z, Huijser P, Nacken W, Saedler H,
and Sommer H. 1990. Genetic control of flower
development by homeotic genes in Antirrhinum
majus. Science 250:931-936.
Shannon S and Meeks-Wagner DR. 1993. Genetic
interactions that regulate inflorescence development
in Arabidopsis. Plant Cell 5:639-655.
Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA,
Peacock WJ, and Dennis ES. 1999. The FLF MADS
box gene: A repressor of flowering in Arabidopsis
regulated by vernalization and methylation. Plant Cell
11:445-458.
Sheldon CC, Finnegan EJ, Rouse DT, Tadege M, Bagnall
DJ, Helliwell CA, Peacock WJ, and Dennis ES. 2000.
The control of flowering by vernalization. Current
Opinion in Plant Biology 3:418-422.
Sheppard LA, Brunner AM, Krutovskii KV, Rottmann
WH, Skinner JS, Vollmer SS, and Strauss SH. 2000.
A DEFICIENS homolog from the dioecious tree black
cottonwood is expressed in female and male floral
meristems of the two-whorled, unisexual flowers.
Plant Physiology 124:627-639.
Shore P and Sharrocks AD. 1995. The MADS-box family
of transcription factors. European Journal of
Biochemistry 229:1-13.
Simola LK. 1985. Propagation of plantlets from leaf
callus of Betula pendula F. purpurea. Scientia
Horticulturae 26:77-85.
Simpson GG, Gendall AR, and Dean C. 1999. When to
switch to flowering. Annual Review of Cell and
Developmental Biology 99:519-550.
Skinner JS, Meilan R, Brunner AM, and Strauss SH.
2000. Options for genetic engineering of floral
sterility in forest trees. In: Jain SM and Minocha SC
(Eds). Molecular Biology of Woody Plants.
Dordrecht, The Netherlands: Kluwer Academic
Publishers. p 135-153.
Sopanen T and Elo A. 2002. Prevention and acceleration
of flowering in birch and other plants. Flowering
Newsletter 33:23-31.
Southerton SG, Strauss SH, Olive MR, Harcourt RL,
Decroocq V, Zhu X, Llewellyn DJ, Peacock WJ, and
Dennis ES. 1998a. Eucalyptus has a functional
equivalent of the Arabidopsis floral meristem identity
gene LEAFY. Plant Molecular Biology 37:897-910.
Southerton SG, Marshall H, Mouradov A, and Teasdale
RD. 1998b. Eucalypt MADS-box genes expressed in
developing flowers. Plant Physiology 118:365-372.
Southerton SG, Uren T, Yang Y, Watson J, Furbank B,
Dennis L, Peacock J, and Moran G. 2001 Flowering
control in eucalypts. IUFRO/Molecular Biology of
Forest Trees. 22-27 July 2001, Columbia River Gorge,
34
United States of America.
Stern K. 1961. Über den Erfolg einer über drei
Generationen geführten Auslese auf frühes Blühen
bei Betula verrucosa. Silvae genetica 10:48-51.
Strauss SH, Rottmann WH, Brunner AM, and Sheppard
LA. 1995. Genetic engineering of reproductive
sterility in forest trees. Molecular Breeding 1:5-26.
Suárez-López P, Wheatley K, Robson F, Onouchi H,
Valverde F, and Coupland G. 2001. CONSTANS
mediates between the circadian clock and the control
of flowering in Arabidopsis. Nature 410:1116-1120.
Sundström J. 2001. Evolution of genetic mechanisms
regulating reproductive development in plants.
Characterisation of MADS-box genes active during
cone development in Norway spruce. Acta Univ. Ups.,
Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 612. 44
pp. Uppsala.
Sundström J, Carlsbecker A, Svensson ME, Svenson M,
Johanson U, Theissen G, and Engström P. 1999.
MADS-box genes active in developing pollen cones
of Norway spruce (Picea abies) are homologous to
the B-class floral homeotic genes in angiosperms.
Developmental Genetics 25:253-266.
Sung S-K, and An G. 1997. Molecular cloning and
characterization of a MADS-box cDNA clone of the
Fuji apple. Plant and Cell Physiology 38: 484-489.
Sung S-K, Yu G-H, and An G. 1999. Characterization of
MdMADS2, a member of the SQUAMOSA subfamily
of genes, in apple. Plant Physiology 120:969-978.
Sung S-K, Yu G-H, Nam J, Jeong D-H, and An G. 2000.
Developmentally regulated expression of two MADSbox genes, MdMADS3 and MdMADS4, in the
morphogenesis of flower buds and fruits in apple.
Planta 210:519-528.
Sung ZR, Chen L, Moon Y-H, and Lertpiriyapong K.
2003. Mechanisms of floral repression in
Arabidopsis. Current Opinion in Plant Biology 6:2935.
Tandre K, Albert VA, SundDs A, and Engström P. 1995.
Conifer homologues to genes that control floral
development in angiosperms. Plant Molecular
Biology 27:69-78.
Tandre K, Svenson M, Svensson ME, and Engström P.
1998. Conservation of gene structure and activity in
the regulation of reproductive organ development of
conifers and angiosperms. Plant Journal 15:615-623.
Telfer A and Poethig RS. 1998. HASTY: a gene that
regulates the timing of shoot maturation in
Arabidopsis thaliana. Development 125:1889-1898.
Telfer A, Bollman KM, and Poethig RS. 1997. Phase
change and the regulation of trichome distribution
in Arabidopsis thaliana. Development 124:645-654.
The Arabidopsis Genome Initiative. 2000. Analysis of
the genome sequence of the flowering plant
Arabidopsis thaliana. Nature 408:796-815.
Theissen G. 2001. Development of floral organ identity:
stories from the MADS house. Current Opinion in
Plant Biology 4:75-85.
Theissen G, Kim JT, and Saedler H. 1996. Classification
and phylogeny of the MADS-box multigene family
suggest defined roles of MADS-box gene subfamilies
in the morphological evolution of eukaryotes. Journal
of Molecular Evolution 43:484-516.
Theissen G, Becker A, Di Rosa A, Kanno A, Kim JT,
Münster T, Winter K-U, and Saedler H. 2000. A short
history of MADS-box genes in plants. Plant
Molecular Biology 42:115-149.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,
and Higgins DG. 1997. The CLUSTAL_X windows
interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic
Acids Research 25:4876-4882.
Thorsness MK, Kandasamy MK, Nasrallah ME, and
Nasrallah JB. 1993. Genetic ablation of floral cells
in Arabidopsis. Plant Cell 5:253-261.
Vahala T, Oxelman B, and von Arnold S. 2001. Two
APETALA2-like genes of Picea abies are
differentially expressed during development. Journal
of Experimental Botany 52:1111-1115.
van der Linden CG, Vosman B, and Smulders MJM. 2002.
Cloning and characterization of four apple MADS
box genes isolated from vegetative tissue. Journal of
Experimental Botany 53:1025-1036.
Vancanneyt G, Schmidt R, O’Connor-Sanchez A,
Willmitzer L, and Rocha-Sosa M. 1990. Construction
of an intron-containing marker gene: Splicing of the
intron in transgenic plants and its use in monitoring
early events in Agrobacterium-mediated plant
transformation. Molecular and General and Genetics
220:245-250.
Viherä-Aarnio A and Ryynänen L. 1995. Growth, crown
structure and seed production of birch seedlings,
grafts and micropropagated plants. Silva Fennica
29:3-12.
Vrtala S, Hirtenlehner K, Susani M, Akdis M, Kussebi
F, Akdis CA, Blaser K, Hufnagl P, Binder BR, Politou
A, Pastore A, Vangelista L, Sperr WR, Semper H,
Valent P, Ebner C, Kraft D, and Valenta R. 2001.
Genetic engineering of a hypoallergenic trimer of the
major birch pollen allergen Bet v 1. FASEB Journal
15:2045-2047.
Wagner D, Sablowski RWM, and Meyerowitz EM. 1999.
Transcriptional activation of APETALA1 by LEAFY.
Science 285:582-584.
Walden AR, Wang DY, Walter C, and Gardner RC. 1998.
A large family of TM3 MADS-box cDNAs in Pinus
radiata includes two members with deletions of the
conserved K domain. Plant Science 138:167-176.
Waterhouse PM, Graham MW, and Wang M-B. 1998.
Virus resistance and gene silencing in plants can be
induced by simultaneous expression of sense and
antisense RNA. Proceedings of the National Academy
of Science of the United States of America 95:1395913964.
Weigel D and Nilsson O. 1995. A developmental switch
sufficient for flower initiation in diverse plants.
Nature 377:495-500.
Weigel D, Alvarez J, Smyth DR, Yanofsky MF, and
Meyerowitz EM. 1992. LEAFY controls floral
meristem identity in Arabidopsis. Cell 69:843-859.
Wesley SV, Helliwell CA, Smith NA, Wang M, Rouse
DT, Liu Q, Gooding PS, Singh SP, Abbott D,
Stoutjesdijk PA, Robinson SP, Gleeve AP, Green AG,
and Waterhouse PM. 2001. Construct design for
efficient, effective and high-throughput gene
35
silencing in plants. Plant Journal 27:581-590.
West AG, Shore P, and Sharrocks AD. 1997. DNA binding
by MADS-box transcription factors: a molecular
mechanism for differential DNA bending. Molecular
and Cellular Biology 17:2876-2887.
Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann
KA, and Meyerowitz EM. 1990. The protein encoded
by the Arabidopsis homeotic gene agamous
resembles transcription factors. Nature 346:35-39.
Yao J-L, Dong Y-H, Kvarnheden A, and Morris B.1999.
Seven MADS-box genes in apple are expressed in
different parts of the fruit. Journal of the American
Society for Horticultural Science 124:8-13.
Yao J-L, Dong Y-H, and Morris BAM. 2001.
Parthenocarpic apple fruit production conferred by
transposon insertion mutations in a MADS-box
transcription factor. Proceedings of the National
Academy of Science of the United States of America
98: 1306-1311.
Yoshida N, Yanai Y, Chen L, Kato Y, Hiratsuka J, Miwa
T, Sung ZR, and Takahashi S. 2001. EMBRYONIC
FLOWER2, a novel polycomb group protein
homolog, mediates shoot development and flowering
in Arabidopsis. Plant Cell 13:2471-2481.
Yu H and Goh CJ. 2000. Identification and
characterization of three orchid MADS-box genes of
the AP1/AGL9 subfamily during floral transition.
Plant Physiology 123:1325-1336.
Yu H, Xu Y, Tan EL, and Kumar PP. 2002. AGAMOUSLIKE 24, a dosage-dependent mediator of the
flowering signals. Proceedings of the National
Academy of Science of the United States of America
99:16336-16341.
Yu H, Yang SH, and Goh CJ. 2002. Spatial and temporal
expression of the orchid floral homeotic gene
DOMADS1 is mediated by its upstream regulatory
regions. Plant Molecular Biology 49:225-237.
Zhang H and van Nocker S. 2002. The VERNALIZATION
INDEPENDENCE 4 gene encodes a novel regulator
of FLOWERING LOCUS C. Plant Journal 31:663673.
Zuo J, Niu Q-W, Mrller SG, and Chua N-H. 2001.
Chemical-regulated, site-specific DNA excision in
transgenic plants. Nature Biotechnology 19:157-161.
36