Seed Dormancy and Germination

Seed Dormancy and Germination
Author(s): Lenie Bentsink and Maarten Koornneef

Source: The Arabidopsis Book,
Published By: The American Society of Plant Biologists
DOI: http://dx.doi.org/10.1199/tab.0119
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The Arabidopsis Book
2008 American Society of Plant Biologists
First published on December 30, 2008: e0119. doi: 10.1199/tab.0119

This chapter is an updated version of a chapter originally published on April 4, 2002, doi:10.1199/tab.0050

Lenie Bentsink a,1 and Maarten Koornneef b ,c
a Department
of Molecular Plant Physiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Planck Institute for Plant Breeding Research, Carl-von-Linn-Weg 10, 50829 Cologne, Germany
c Laboratory of Genetics, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
1 Address correspondence to L.Bentsink@uu.nl
b Max
Seed dormancy allows seeds to overcome periods that are unfavourable for seedling established and is therefore important
for plant ecology and agriculture. Several processes are known to be involved in the induction of dormancy and in the switch
from the dormant to the germinating state. The role of plant hormones, the different tissues and genes involved, including
newly identified genes in dormancy and germination are described in this chapter, as well as the use transcriptome, proteome
and metabolome analyses to study these mechanistically not well understood processes.
INTRODUCTION
SEED DEVELOPMENT
The seed is an important stage in the higher plant life cycle

with respect to its survival as a species. It is the dispersal unit
of the plant, which is able to survive the period between seed
maturation and the establishment of the next generation as a
seedling after it has germinated. For this survival, the seed,
mainly in a dry state, is well equipped to sustain extended periods of unfavourable conditions. To optimise germination over
time, the seed enters a dormant state. Dormancy prevents
pre-harvest germination as well. Numerous studies have
been performed to better understand how germination is controlled by various environmental factors and applied chemicals. However, still very little is known about the process by
which the embryo emerges from the seed to complete germination and how embryo emergence is blocked in dormant
seeds (Bewley, 1997).
Arabidopsis possesses seed dormancy, as is the case for
many other plant species. This property is controlled by environmental factors such as light, temperature and time of seed
dry storage as well as by genetic factors. The use of genetics
and molecular genetics in Arabidopsis is starting to shed light on
some aspects of the mechanism of dormancy and germination
by the identification of mutants and genes that control these
processes. This review provides an overview of current knowledge of seed dormancy and germination in Arabidopsis based
mainly on the contribution that molecular genetics made to the
study of this process including a table with genes that are related to germination/dormancy. Several recent reviews (Finch
Savage and Leubner-Metzger 2006, Finkelstein et al., 2008,
Holdsworth et al., 2008a) describe more details of the molecular mechanism that were derived from the mainly molecular genetic studies combined with physiological experiments.
Seed development comprises two major phases: embryo development and seed maturation. Embryogenesis, which is a morphogenesis phase, starts with the formation of a single-cell zygote
and ends in the heart stage when all embryo structures have been
formed (Mayer et al., 1991). It is followed by a growth phase during which the embryo fills the seed sac (Goldberg et al., 1994). At
the end of the embryo growth phase, cell division in the embryo arrests (Raz et al., 2001). Hereafter, the seed, containing a full sized
embryo, undergoes maturation during which food reserves accumulate and dormancy and desiccation tolerance develops (Goldberg et al., 1994). Several detailed studies have been published on
the developmental (Baud et al. 2002), metabolic (Fait et al., 2006),
proteomic (Fu et al., 2005; Gallardo et al., 2001, 2002; Rajjou et
al., 2004 and Chibani et al., 2006 and gene expression (Girke et
al., 2000; Nakabayashi et al., 2005; Finch-Savage et al., 2007)
changes during seed development, in dry seeds and subsequent
germination.
Seed development has been extensively studied using mutants
defective in various aspects of the process. Mutants affected in
the morphogenesis phase result in lethality or have a seedling
phenotype (Mayer et al., 1991; Meinke, 1995). In seed germination
mutants, properties of germination and dormancy are affected
which sometimes are accompanied by pleiotropic effects that are
specific for maturation, such as desiccation intolerance (Goldberg
et al., 1994; Koornneef and Karssen, 1994).
SEED DORMANCY AND GERMINATION

Seed dormancy has been defined as the incapacity of a viable
seed to germinate under favorable conditions (Bewley, 1997;
2 of 18
Figure 1. Schematic presentation of processes controlling seed dormancy and germination in an Arabidopsis seed. The Arabidopsis seed is characterized
by the embryo with two cotyledons and a single cell layer endosperm. Germination promoting (green arrows) and inhibiting factors (red arrows) are indicated.
Finch-Savage and LeubnerMetzger, 2006). Dormancy in Arabidopsis should be described as physiologically non-deep, meaning that embryos released from surrounding structures grow
normally, and that dormancy is lost through moist chilling (stratification) or after-ripening (Baskin and Baskin, 2004). However, in
addition to the testa and endosperm layer surrounding the embryo, the growth potential of the embryo is also important to overcome the constraint of these structures and thereby affects the
dormancy state of a seed (Kucera et al., 2006).
Since dormancy is regulated at different developmental
phases, in interaction with environmental factors, it is difficult to
detect when the genetic and physiological differences are established. This difficulty arises because all dormancy assays are
based on seed germination, which is the result of the balance between the degree of dormancy and the capacity of the embryo to
overcome dormancy. Mechanistically one can distinguish factors
that influence dormancy and germination on the basis of their effect on germination, being either inhibiting or promoting. Mutants
that germinate better or faster can represent genes that promote
dormancy or those that repress germination. A further distinction
can be made by defining the timing and site of action of these factors (during maturation or during imbibition of the seeds, in the
embryo, the endosperm or in the testa). The interaction between
these factors and the large effect of the environment, both during
seed development and during imbibition, make seed dormancy a
very complex trait.
By definition, germination incorporates those events that commence with the uptake of water by the quiescent dry seed and terminates with the elongation of the embryonic axis (Bewley and
Black, 1994). Water uptake by a seed is triphasic; phase I rapid
initial uptake; phase II plateau phase and in phase III further in-
crease of water uptake, however, only when germination occurs

(Schopfer and Plachy, 1984; Bewley, 1997; Manz et al., 2005). The
first signs of germination are the resumption of essential
processes, including transcription, translation and DNA repair followed by cell-elongation and eventually at the time of radicle protrusion, resumption of cell division (Barroco et al., 2005;
Masubelele et al., 2005). Physically germination is a two-stage
process, where testa rupture is followed by endosperm rupture.
Following rupture of the micropylar endosperm by the emerging
radicle, germination is complete (Fig. 2; Karssen, 1976; Hepher
and Roberts 1985; Leubner-Metzger et al., 1995; Krock et al.,
2002; Petruzzelli et al., 2003; Leubner-Metzger, 2003; Liu et al.,
2005). Germination assays in Arabidopsis are often performed in
light on seeds freshly harvested or stored for a limited time (LonKloosterziel et al., 1996a). Other parameters are the germination
rate after different periods of cold treatment (Cutler et al., 1996)
and germination in darkness (Meng et al., 2008; Kim et al., 2008).
In addition to testing mature seeds, germination of immature
seeds, either excised from the silique or within fruits detached from
the plant, can be used to investigate genetic variation during the
early stages of seed development (Raz et al., 2001).
Since tissues from both maternal (testa) and zygotic origin
(embryo and endosperm) contribute to seed germination, genetic
analyses of seed dormancy have to take into account these different tissue origins. Maternal effects, in contrast to zygotic factors are maternally inherited and might be due to the genetic make
up of the testa surrounding the embryo, but can also be due to genetic differences related to factors that are transported into the
seed from the mother plant. Maternal inheritance can be deduced
from the germination of seeds obtained after reciprocal crosses,
where the parental genotypes are used both as female and as
3 of 18
Figure 2. Arabidopsis seed germination. A mature Arabidopsis seed can be either dormant or non-dormant. A dormant seed will not germinate when it
will be exposed to the right environmental conditions (light and water). Seed dormancy can be broken by dry storage or by a cold imbibition (stratification).
Seed germination in Arabidopsis is two-step: testa rupture followed by endosperm rupture.
male parent. The endosperm is the product of fertilization. However the genomic contribution of the female parent is twice that of
the male parent in this triploid tissue, which in some cases may
lead to differences in reciprocal crosses when dose effects are involved. The role of this single endosperm layer in dormancy and
the need to weaken this cell layer for germination has recently
been shown (Mller et al., 2006; Bethke et al., 2007a; for review
Holdsworth et al., 2008a).
Seed dormancy in Arabidopsis can be overcome by germination promoting factors such as after-ripening, light, cold treatment
(also called stratification). Furthermore applied chemicals such as
gibberellins and KNO3 (Derkx and Karssen, 1993a; Alboresi et al.
2005; Ali-Rachedi et al. 2004), NO (Bethke et al., 2007b) also have
germination promoting effects (Fig. 1). None of these environmental factors are an absolute requirement for germination because the need for one factor depends on the other factors, as
shown for the interaction between light and temperature by Cone
and Spruit (1983). This requirement for exogenous factors depends very much on the genotype. Dormancy, released by dry
after-ripening, or by exposure to cold or nitrate followed by light
exposure has been studied in the accession of the Cape Verde Islands (Cvi). It appeared that the sensitivity of seeds to cold, nitrate
and light was dependent upon the length of time that seeds had
been dry after-ripened. The seeds became first sensitive to nitrate,
then to cold and finally to light (Finch-Savage et al., 2007). Furthermore it has been shown that the rate of increase of sensitivity
to environmental signals was not fixed, seeds produced in different years had a different response. This is consistent with the fact
that the depth of dormancy is not only determined genetically, but
also by the ambient environment during seed formation (Donohue, 2005).
Non-dormant seeds that are exposed for some time to unfavourable germination conditions (imbibed seeds kept at relatively
high temperature in darkness for example) may enter a state of
dormancy again, which is called secondary dormancy (Cone and
Spruit, 1983; Derkx and Karssen 1993b; Hilhorst, 2007).
A challenge in dormancy and germination research is to identify the nature of the crucial regulator(s) that prevent(s) the onset
of germination (dormancy), that trigger(s) the germination process

and their mutual interaction. Furthermore, it is important to know
how the environmental factors such as light and cold affect the endogenous factors that control germination.
GENETIC VARIATION FOR GERMINATION CHARACTERS

Genetic variation can be induced by mutagenesis, but is also present among the many natural accessions (ecotypes) of Arabidopsis. Genetic variation for germination can be detected when
genotypes are compared in identical environments. This implies
that not only the conditions of the germination test must be identical, but also growth conditions during seed development and
storage conditions, including the time that the seeds are stored,
must be the same. Furthermore, the test conditions must be discriminative between genotypes.
Germination tests can be used efficiently for mutant screens
because of the large numbers that can be assayed. However, variability of the germination trait may lead to genetic misclassification of individual seeds and therefore to false-positives in mutant
screens. The Arabidopsis genotypes Landsberg erecta (Ler) and
Columbia (Col), which are mostly used in Arabidopsis research,
show only a low level of dormancy. This dormancy disappears after
approximately one month of after-ripening (van der Schaar et al.,
1997). Because of this relatively low level of dormancy it is impossible to saturate mutations in dormancy genes. However this
problem can be overcome by the use of more dormant accessions
(Koornneef et al., 2000).
The genetic variants described in this review have in common
that the seeds are viable, as shown by their ability to germinate
after special treatments, such as disruption of the seed coat or the
application of specific chemicals. Mutants that do not germinate
because they are lethal, including early ovule mutants (Schneitz,
1999) and many of the so-called embryo lethals (Meinke, 1995)
are not described in this review because they mainly have developmental defects that do not control specifically dormancy and
germination.
4 of 18
The analysis of natural variation

Arabidopsis is an annual plant for which large differences in dormancy can be found between accessions collected from nature
(Ratcliffe, 1976). Kugler (1951) performed the first genetic study of
differences in germination between Arabidopsis accessions. Differences in dormancy are amenable to genetic analysis in Arabidopsis as was shown by Quantitative Trait Loci (QTL) analysis for
seed germination/dormancy characteristics in several recombinant
inbred line (RIL) populations (van der Schaar et al., 1997; AlonsoBlanco et al., 2003 and Clerkx et al., 2004). These RIL populations have been made by crossing accessions that have different
levels of seed dormancy to the standard laboratory accession Ler.
In the analysis of van der Schaar et al. (1997), the Ler/Col RILs
were grown in three independent greenhouse experiments and
tested in different germination conditions. Despite little difference
in dormancy between the parental lines, altogether 14 QTL could
be identified, of which most had only small effects. Many of these
were only detected in specific germination conditions or in seeds
from one or two harvest dates, indicating the complexity of the genetic control of dormancy differences.
QTL analyses for seed dormancy that led to the identification
of loci with an effect on seed dormancy that could be confirmed
using Near Isogenic Lines (NILs) were described for the Ler/Cvi
RIL population (Alonso-Blanco et al., 2003). This QTL study identified loci affecting the after-ripening requirement, measured as
the number of days of seed dry storage required to reach 50%
germination (DSDS50). Thus, seven QTL were identified and
named Delay Of Germination (DOG) 17. To confirm and characterize these loci NILs carrying Cvi introgression fragments in a Ler
genetic background were constructed. The analysis of these lines
for germination confirmed four QTL (DOG1, DOG2, DOG3, and
DOG6). The same type of analyses in the Ler/Shahdara (Sha) RIL
population revealed four QTL. Three of them, DOG1, DOG3 and
DOG6 were at positions, identified before (Alonso-Blanco et al.,
2003) and most likely representing the same loci. In addition a
QTL on chromosome 2 was identified (Clerkx et al., 2004).
The first of these seed dormancy QTL, DOG1 a major QTL in
the Ler/Cvi population has been cloned (Bentsink et al. 2006). DOG1
is expressed during seed development and the transcript remains in
the mature dry and after-ripened seed but upon imbibition the transcript disappears. DOG1 is a member of a small gene family of unknown molecular function and has not been related with seed
dormancy before, indicating that natural variation can be valuable to
identify novel seed dormancy genes (Bentsink et al., 2006).
In total seven RIL populations derived form crosses with Ler
have presently been analysed, leading to the identification of
eleven DOG QTL (Bentsink et al., unpublished). This screen for
after-ripening requirement is reaching saturation as in newly
analysed populations the same QTL are being identified also in a
cross were Ler has not been involved (X. Huang, M. Koornneef
and K. Donohue unpublished data).
Analyses for seed germination in response to cold and dark
showed the feasibility of fine mapping and cloning of QTL responsible for cold-tolerant, dark germination (Meng et al., 2008).
The authors identified three major loci responsible for the variability of this trait in the Bay-0/Sha RIL population. One of these
QTL, CDG-1 (Cold-tolerant Dark Germination) was localized on
the same genomic region as the delay of germination loci DOG2
and DOG3 (Alonso-Blanco et al., 2003) and QTL that affect controlled deterioration and germination speed (Clerkx et al., 2004).
Another seed trait for which natural variation has been described is for mucilage release during imbibition. The Sha accession from central Asia carries a mutation in the MUM2 gene
(Macquet et al., 2007) resulting in the absence of mucilage.
Genes and factors affected in seed dormancy/germination
Genes and or factors that induce seed dormancy or inhibit

germination
The first important stage for dormancy induction is probably the
end of the morphogenetic program, when all tissues present in a
mature embryo have been formed and the embryo enters a phase
of growth arrest. ABA-INSENSITIVE3 (ABI3), FUSCA3 (FUS3)
and LEAFY COTYLEDON (LEC1 and LEC2) are four key regulators that play prominent roles in controlling mid- and late seed development (Meinke et al., 1994; Raz et al., 2001). ABI3, FUS3 and
LEC2 encode related plant-specific transcription factors containing
the conserved B3 DNA binding domain (Giraudat et al., 1992;
Luerssen et al., 1998; Stone et al., 2001), whereas LEC1 encodes
a HAP3 subunit of the CCAAT binding transcription factor (CBF,
also known as NF-Y (Lotan et al., 1998)). All four abi3, lec1, lec2
and fus3 mutants are severely affected in seed maturation and
share some common phenotypes, such as decreased dormancy
at maturation (Raz et al., 2001) and reduced expression of seed
storage proteins (Gutierrez et al., 2007). However, they also show
specific phenotypes, such as the absence of chlorophyll degradation in the dry seed (abi3), a reduced sensitivity to ABA (abi3 and,
to a lesser extent, lec1), the accumulation of anthocyanins (fus3,
lec1, and, to a lesser extent, lec2), an intolerance to desiccation
(abi3, fus3, and lec1), or defects in cotyledon identity (lec1, fus3,
and lec2) (Bumlein et al., 1994; Keith et al., 1994; Meinke et al.,
1994; Parcy et al., 1994; Parcy & Giraudat, 1997; Luerssen et al.,
1998; Vicient et al., 2000; Raz et al., 2001; Stone et al., 2001; Kroj
et al., 2003). It was recently shown that several of the fus3 phenotypes are due to pleiotropic effects caused by truncated gene
products of the mutant alleles. The direct effects of FUS3 are probably restricted to embryo-derived dormancy and determination of
cotyledon epidermis cell identity (Tiedemann et al., 2008).
The LEC1 gene is required for normal development during
early and late phases of embryogenesis and is sufficient to induce
embryonic development in vegetative cells (Lotan et al., 1998).
Loss of function of LEC1 leads to germination of excised embryos
at a similar stage (between 8-10 days after pollination) as lec2 and
fus3 mutants, but earlier during embryo development than found
for abi3 mutants (Raz et al., 2001). Ten HAP3 (AHAP3) subunits
have been identified in Arabidopsis, which can be divided into two
classes based on sequence identity in their central, conserved B
domain (Kwong et al., 2003). LEC1 and the closely related subunit,
LEC1-LIKE (L1L), constitute LEC1-type AHAP3 subunits, whereas
the remaining eight are designated non-LEC1-type. Similar to
LEC1, L1L is expressed primarily during seed development. However, suppression of L1L gene expression induced defects in embryo development that differed from those of lec1 mutants,
suggesting that LEC1 and L1L play different roles in embryogenesis (Kwong et al., 2003).
LEC2 directly controls a transcriptional program involved in the

maturation phase of seed development. Induction of LEC2 activity in seedlings causes rapid accumulation of RNAs normally present primarily during the maturation phase, including seed storage
and lipid-body proteins. Promoters of genes encoding these maturation RNAs all possess RY motifs (cis-elements bound by B3
domain transcription factors) (Braybrook et al., 2006). This provides strong evidence that these genes represent transcriptional
targets of LEC2. One of these genes is DOG1, the first seed dormancy gene accounting for variation occurring in natural populations that has been identified at the molecular level (Bentsink et al.,
2006).
It has been shown that ABI3, FUS3, LEC1 and LEC2 interact
as a network to control various aspects of seed maturation. LEC1
was shown to regulate the expression of both ABI3 and FUS3 (Kagaya et al., 2005), FUS3 and LEC2 have been shown to act in a
partially redundant manner to control gene expression of seed
specific proteins, and LEC2 was shown to locally regulate FUS3
expression in regions of the cotyledons (Kroj et al., 2003). The indication of redundant regulation within this group of genes was recently shown (To et al., 2006). By analyzing ABI3 and FUS3
expression in various single, double, and triple maturation mutants, multiple regulatory links among all four genes were identified. It was found that one of the major roles of LEC2 was to
up-regulate FUS3 and ABI3. The lec2 mutation leads to a dramatic
decrease in ABI3 and FUS3 expression, and most lec2 phenotypes can be rescued by ABI3 or FUS3 constitutive expression. In
addition, ABI3 and FUS3 were shown to positively regulate themselves and each other, thereby forming feedback loops essential
for their sustained and uniform expression in the embryo. Finally,
LEC1 also positively regulates ABI3 and FUS3 in the cotyledons
(To et al., 2006). Although multiple regulatory links were identified
amongst these four genes, molecular mechanisms underlying this
network, and the downstream targets of the network associated
with dormancy induction still require further investigation.
Apart from mutants that influence general seed maturation,
other mutants more specifically influence seed dormancy, i.e. mutants, which are altered in ABA biosynthesis or its mode of action.
ABA regulates various aspects of plant growth and development,
including seed dormancy. The absence of ABA-induced dormancy
allows seeds to germinate without gibberellins. Therefore, the selection of mutants that germinate in the presence of GA biosynthesis inhibitors, such as paclobutrazol and tetcyclacis, is an
effective way to isolate ABA biosynthesis mutants (Lon-Kloosterziel et al., 1996b). Reciprocal crosses between wild type and the
ABA deficient aba1 mutants showed that dormancy is controlled
by the ABA genotype of the embryo and not by that of the mother
plant. The latter is responsible for the relatively high ABA levels
found in seeds halfway through seed development (Karssen et al.,
1983). At this phase ABA may prevent precocious germination as
shown by the maternal ABA effects in the extreme aba abi3-1 double mutants (Koornneef et al., 1989). Key genes for ABA biosynthesis during seed development are NCED6 and NCED9, both
members of the 9-cis-epoxycarotenoid dioxygenese family (Lefebvre et al., 2006). In contrast to what was proposed by Karssen et
al (1983) it is now clearly shown that endogenous ABA is required
for the maintenance of seed dormancy. In wild type ABA levels decrease at the end of seed maturation and during imbibition due to
the activity of ABA catabolism genes belonging to the P450
5 of 18
CYP707A family (Okamoto et al., 2006), indicating that ABA levels can be modified at different phases of seed development and
germination with significant effects on germination. Furthermore,
the observation that inhibitors of ABA biosynthesis, such as norfluorazon, promote germination (Debeaujon and Koornneef, 2000)
indicated that the maintenance of dormancy in imbibed seeds is
an active process involving de novo ABA synthesis as was also
shown for dormant seed batches of the accession Cvi (Ali-Rachedi
et al., 2004).
ABA has a major effect on seed dormancy and therefore it can
be expected that defective ABA signalling also leads to changes
in germination characteristics. Substantial progress has been
made in the characterization of such ABA signal transduction pathways (Bonetta and McCourt, 1998; Leung and Giraudat, 1998).
Genetic screens to identify ABA signalling mutants were based
primarily on the inhibition of seed germination by applied ABA. The
ABA-insensitive (abi) mutants (Koornneef et al., 1984) and several others described thereafter (Rock, 2000 and Holdsworth et
al., 2008a) are able to germinate and grow in the presence of ABA
concentrations that are inhibitory to the wild type. It was expected
that such screens would yield ABA receptor and signal transduction mutants. However most of ABA receptor genes were identified
using reverse genetics in which screening for germination characteristics were performed that often showed no or small effects
on ABA sensitivity for germination and dormancy it self was often
not tested (reviewed in Holdsworth et al., 2008a). Forward screens
for mutants in which seed germination is prevented by low concentrations of ABA that ordinarily permit germination of the wildtype seed were first described by Cutler et al. (1996) resulting in
the era1 (enhanced response to ABA) to era3 mutants. Subsequently using similar screens identified many additional loci that
are involved in removal of sensitivity to ABA function, that when
mutated lead to ABA hypersensitivity of imbibed after-ripened or
moist-chilled seeds (Hugouvieux et al., 2001; Xiong et al., 2001;
Nishimura et al., 2004; Katagiri et al., 2005; Zhang et al., 2005;
Pandey et al., 2006; Saez et al., 2006; Yoine et al., 2006; Nishimura
et al., 2007). These loci encode diverse functions, including those
associated with RNA translation and metabolism, protein degradation pathways and phosphatase components of signalling pathways and transcription factors (Holdsworth et al., 2008a). As
judged from their effects on seed dormancy, these two sets of mutations also alter the regulation of seed germination by endogenous ABA. The abi3, abi4 and abi5 mutants exhibit reduced
expression of various seed maturation genes but only abi3 mutants are non-dormant, which coincides with desiccation intolerance (Nambara et al., 1992, Ooms et al., 1993, Bies et al., 1999,
Finkelstein et al., 2008) in strong alleles. Surprisingly no dormancy
or other seed maturation phenotype was observed in abi4 and
abi5 mutants (Finkelstein, 1994; Finkelstein et al., 2008), except
reduction of some seed maturation specific mRNAs (Finkelstein
and Lynch, 2000; Sderman, et al., 2000). This may indicate that
other genes are redundant in function to these seed specific transcription factors, which are members of the APETALA2 domain
(ABI4, Finkelstein et al., 1998; Sderman, et al., 2000) and basic
leucine zipper factor family (ABI5, Finkelstein and Lynch, 2000;
Lopez-Molina et al., 2001).
According to a recent report, ABA levels might be positively
regulated by DELLA protein through upregulation of XERICO expression (Zentella et al., 2007). XERICO over-expression, which
6 of 18
encodes an E3 ubiquitin ligase, leads to both an elevated level of

ABA and increased drought tolerance, although the mechanism
is still unknown (Ko et al., 2006).
A class of mutants that was directly selected on the basis of
reduced dormancy are the rdo1-rdo4 mutants (Lon-Kloosterziel
et al., 1996a; Peeters et al., 2002). The fact that all four mutants
show some mild pleiotropic effects in adult plants indicates that
the genes are not specific for dormancy/germination but affect
other processes as well. The RDO4 (renamed as HUB1) was
shown to encode a C3HC4 Ring finger protein involved in the monoubiquitination of histone H2B, revealing a role for chromatin
modelling in seed dormancy (Liu et al., 2007). The dag1-1 mutant
also displays reduced dormancy, but in contrast to the rdo mutants
the effect is determined by the maternal genotype. This is in agreement with the expression pattern of the DAG1 gene in the vascular tissue of the developing seed. DAG1, which encodes a DOF
transcription factor, may influence the import of compounds from
the mother plant into the seed (Papi et al., 2000). It is the first gene
identified as being specifically involved in maternal control of seed
germination. However, the germination phenotype of dag2, mutant in the related DAG2 gene, with a similar expression pattern as
DAG1 is opposite to that of dag1 seeds (Gualaberti et al., 2002)
showing increased dormancy. Additional mutants with a reduced
dormancy phenotype at other loci, including mutants with no obvious pleiotropic effect have been isolated (Bentsink et al., 2006
and M. Schwab and W. Soppe unpublished results), indicating the
complexity of the genetic regulation of seed dormancy. Instead of
selecting for mutants that germinate when the wild type is still dormant, Salaita et al. (2005) used germination speed at 10C as a
selection criterion when screening activation tagged lines of the
Col accession. Except for two tt mutants (see below) none of these
cold temperature germination (ctg) has been characterized molecularly.
Another group of mutants that shows reduced seed dormancy
are mutants with an altered seed coat or testa (Debeaujon et al.,
2000; reviewed in Debeaujon et al., 2007; Lepiniec et al., 2006).
The seed coat is a multifunctional organ that plays an important
role in embryo nutrition during seed development and in protection
against detrimental agents from the environment afterwards (Debeaujon et al., 2007). The seed coat is formed from two integuments of epidermal origin that surround the mature ovule. The
development of the seed coat from the ovule has been described
by Beeckman et al. (2000).
The seed coat together with the endosperm layer exerts a germination-restrictive action, either by being impermeable to water
and /or oxygen, by producing germination inhibiting compounds
or by its mechanical resistance to radicle protrusion. In Arabidopsis, phenolic compounds and their derivatives present in the inner
layer of the testa, called endothelium, affect seed coat properties
that influence germination as can be concluded from the reduced
dormancy phenotype of many testa mutants.
Seed coat mutants consist of two major groups. One group, affected in flavonoid pigmentation is represented by the transparent
testa (tt) and transparent testa glabra (ttg) mutants. Mutants identified are tt1 to tt15, ttg1 and ttg2 and banyuls (ban). The color of
the tt mutants ranges from yellow to pale brown (Debeaujon et al.,
2000). Ban mutants accumulate pink flavonoid pigments in the endothelium of immature seeds, but do not contain proanthocyanins
due to a mutation in the anthocyanin reductase (ANR) gene (Xie
et al., 2003) resulting in grayish-green, spotted mature seeds (Albert et al, 1997; Devic et al, 1999). The ttg1 mutant lacks mucilage
and trichomes and is affected in the morphology of the outer layer
of the seed coat as well as in pigment production. Many of the
seed coat mutants have now been cloned, for more details see
supplemental Table 1 online and Debeaujon et al. (2007).
The second group is represented by mutants affected in testa
structure. The aberrant testa shape (ats) mutant, mutated in the
KANADI 4 gene (McAbee et al., 2006) produces a single integument instead of the two integuments seen in wild-type ovules and
shows less dormancy.
Genes and or factors that decrease seed dormancy or promote

the germination potential of seeds
The germination of Arabidopsis seeds is under phytochrome-mediated photocontrol. It is therefore to be expected that phytochrome deficient mutants are affected in seed germination. The
complexity of the phytochrome system comes from the presence
of distinct types of phytochromes, for which five genes in the Arabidopsis genome encode different, but related, apoproteins (Sharrock and Quail, 1989). In addition different modes of action of
phytochrome, described as very-low-fluence response (VLFR),
low-fluence response (LFR) and high-irradiance response (HIR),
which have their own fluence dependency, can affect germination
(reviewed by Casal and Snchez, 1998). Mutants lacking phytochrome B (phyB) show a reduced sensitivity to red light, indicating that phyB has a primary role in seed germination. PhyA can
only induce germination after a prolonged imbibition of seeds (Shinomura et al., 1994). Detailed action spectra for seed germination
performed in wild type, phyA and phyB mutants revealed a typical
red/ far-red (R/FR) -reversible LFR mediated by phyB, whereas
the germination response mediated by phyA turned out to be a
VLFR with a 104 -fold higher sensitivity to light (Shinomura et al,
1996). The observation that also phyA phyB double mutants show
some light- dependent germination indicates the involvement of another R/FR-reversible photoreceptor system (Yang et al., 1995;
Poppe and Schfer, 1997) probably mediated by phyC, D, and/or E.
Although the main role of phytochrome is in light-induced stimulation of seed germination, a role in the onset of dormancy or the
setting of the light requirement is suggested by the experiments of
McCullough and Shropshire (1970) and Hayes and Klein (1974).
These authors showed that the R/FR ratio experienced by the
mother plant and therefore during seed maturation, affects the
subsequent germination behaviour of mature seeds. Munir et al.
(2001) showed that photoperiod conditions during seed formation
may also influence seed germination. However, this effect was
strongly genotype dependent. In addition, it appears that phytochrome mediated pathways are required to break cold-induced
dormancy (Donohue et al., 2007). Cool temperatures during seed
maturation induced seed dormancy which could not be overcome
in the hy2-1 (deficient in phytochrome chromophore, common to
all five phytochromes) mutant.
The plant hormone gibberellin plays an important role in promoting seed germination. GA-deficient mutants are unable to germinate without exogenous GAs (Koornneef and van der Veen,
1980; Mitchum et al., 2006). De novo biosynthesis of GAs is required during imbibition, as was concluded from the observation
that inhibitors of GA biosynthesis, such as paclobutrazol and tetcyclacis prevent germination (Karssen et al., 1989) unless ABA is
absent. As expected also GA signalling mutants such as the
sleepy1 (sly1) mutant, which was selected in a screen for suppressors of the ABA insensitive mutant abi1-1 (Steber et al., 1998)
and Della protein encoding genes such as RGL2 (Lee et al., 2002;
Bassel et al., 2004) have germination defective phenotypes. For
the GA receptors mutants (gid1a, gid1b, gid1c) triple mutants had
to be constructed to see this phenotype due to redundancy of the
function of these genes (Griffith et al., 2006, Iuchi et al., 2007, reviewed by Hirano et al., 2008). GAs can promote germination by
their ability to overcome germination constraints that exist in seeds
requiring after-ripening, light and cold. This led to the suggestion
that such environmental factors may induce GA biosynthesis during the early phases of germination. At present the changes of GA
content and the expression of GA biosynthesis and catabolism
genes during dormancy release and germination is well documented (Yamauchi et al., 2007).
The phytochrome (light) effect was supported by Yamaguchi et
al. (1998), who showed that one of two 3- hydroxylase enzymes,
encoded by the GA4H gene is induced in germinating seeds by
phytochrome. The mechanism of the GA signalling proteins and
the effect of light is now well established. Crucial in this are the
DELLA proteins which repress GA action, RGL2 (repressor of
GA1-3 like 2) is the major DELLA regulating seed germination.
These DELLA proteins are degraded by the 26S proteosome
therefore GA charged GID1 DELLA proteins interact with the F
box protein SLY1 needed for DELLA ubiquitination (Steber, 2007,
Finkelstein et al., 2008). An important signal transduction component of light induced germination is the bHLH transcription factor
PIF1 (Phytochrome Interacting Factor 1) also named PIL5 (Phytochrome-Interacting factor 3-Like 5) (Oh et al., 2004, 2006, Castillon et al., 2007), which can bind to Pfr that thereafter results in
proteosome degradation (Oh et al., 2006). This increases GA levels because GA biosynthesis genes such as GA3ox1 and GA3ox2
are repressed by PIF1/PIL5, whereas the GA catabolic GA2ox
gene and genes encoding DELLA proteins are activated (Oh et
al., 2007). These higher GA levels further lead to inactivation of
the DELLA proteins as described above. Somnus (SOM) which
encodes a nucleus-localized CCCH-type zinc finger protein is another gene acting downstream of PIL5 (Kim et al., 2008). The som
mutant germinates in darkness, independently of various light
regimes. Kim et al. (2008) showed that PIL5 activates the expression of SOM by binding directly to the SOM promoter. It is suggested that PIL5 regulates ABA and GA metabolic genes partly
through SOM.
Cold treatments were also found to stimulate GA biosynthesis
(Yamauchi et al., 2004) by inducing the GA3ox1 and GA3ox2
genes. This cold effect is mediated by a light stable bHLH transcription factor SPATULA (SPT), which suppresses the expression
of these genes (Penfield et al., 2005). In addition cold may increase the sensitivity of seeds to GAs because it also has an effect in GA deficient mutants (Debeaujon and Koornneef,
2000).The fact that often stratification is more effective than GA
treatment (Alonso-Blanco et al., 2003) suggests that also other
factors, promoting germination, are affected by stratification.
Brassinosteroids (BRs), are naturally occurring plant steroid
hormones found in a wide variety of plant species (Clouse and
Sasse, 1998) are also involved in the control of germination in Ara-
7 of 18
bidopsis. The BR signal leads to reduced sensitive to ABA and

thereby stimulates germination, although the normal germination
of BR deficient and BR signalling mutants indicates that there is no
absolute BR requirement for germination (Steber and McCourt,
2001). BRs could overcome the lack of germination of the sleepy1
mutant, probably by bypassing its GA requirement through a GA
independent mechanism (Steber and McCourt, 2001; Finkelstein
et al., 2008).
Mutants in ethylene signalling are also affected in their germination response. Ethylene is produced in trace amounts by almost
all higher plants and is involved in the control of growth and developmental processes that range from germination to senescence. Often seeds that respond to ethylene are light sensitive for
germination (Kepczynski and Kepczynska, 1997). Ethylene insensitive mutants such as etr and ein2 germinate less well or after
a longer period of after-ripening than wild type (Bleecker et al.,
1988; Beaudoin et al., 2000). The ein2 and etr mutants are hypersensitive to ABA (Beaudoin et al., 2000; Ghassemian et al., 2000),
which agrees with the observation that ein2 mutants were isolated
as abi1-1 suppressors. The ctr1 mutant, which is characterised by
a constitutive ethylene response, appeared among mutants selected as enhancers of the ABA insensitive mutant abi1-1 and ctr1
monogenic mutants are also slightly ABA resistant (Beaudoin et
al., 2000). These observations, in combination with the non-dormant phenotype of the ein2 abi3-4 double mutant indicated that
ethylene negatively regulates seed dormancy by inhibiting ABA
action (Beaudoin et al., 2000). However, Ghassemian et al., (2000)
and Chiwocha et al. (2005) showed that, in addition to signalling,
the slightly more dormant ethylene insensitive mutants, such as
ein2 and etr1-2 have higher ABA levels. The higher levels of germination promoting hormones indicate compensation effects. The
presence of cross talk between sugar signalling and ethylene was
suggested by the sugar insensitive phenotype of ctr1 (Gibson et
al., 2001) and the sugar hypersensitive phenotype of etr (Zhou et
al., 1998). Apparently ABA, ethylene and sugar signalling strongly
interact at the level of germination and early seedling growth.
Auxins are known to play important roles in embryogenesis.
However, its role in the regulation of germination and seedling establishment remained obscure (Kucera et al., 2006). Auxin alone
was not generally considered to be important in the control of seed
germination but cross-talk between auxin, ABA, GA and ethylene
was suggested to both affect germination and seedling establishment (Fu and Harberd, 2003; Ogawa et al., 2003; Chiwocha et al.,
2005, Carrera et al., 2008; Liu, PP et al., 2007 reviewed by
Holdsworth et al., 2008a). Analysis of the expression of the
DR5:GUS auxin reporter indicated that auxin accumulates during
embryogenesis and is present in the seed following imbibition. Expression was observed at the radicle tip prior to germination in
one study (Liu, PP et al., 2007) and throughout the embryo at the
end of embryogenesis in another study (Ni et al., 2001). Analysis
of transcriptome expression showed that RNA encoding auxin
transporters AUX1, PIN2, and PIN7 were highly up-regulated in
response to treatment of ga1 mutant seeds with GA (Ogawa et
al., 2003), and that both efflux and influx transporters are up-regulated in after-ripened compared to dormant seeds (Carrera et al.,
2008). This may indicate a role for these transporters in germination per-se, or with the establishment of the root apex and gravitropism following radicle emergence. Clearer genetic evidence of a
role for auxin in germination has been obtained from an analysis
8 of 18
of the regulation of Auxin Response Factor10 (ARF10) by microRNA (miRNA) miR160 (Liu, PP et al., 2007). miRNAs have
been shown to down-regulate target genes at the post-transcriptional level, and play crucial roles in a broad range of developmental processes (Dugas & Bartel, 2004). It was shown that
transgenic seeds expressing an miR160-resistant form of ARF10
(mARF10) were hypersensitive to germination inhibition by exogenous ABA, whereas ectopic expression of miR160 resulted in
reduced sensitivity to ABA (Liu, PP et al., 2007).
These results indicate a role of auxin in germination associated pathways, and suggest that interactions between auxin
and ABA signalling pathways may contribute to the germination
potential of seeds. An analysis of the function of key components of auxin signalling in relation to after-ripening, germination potential and vigour may reveal novel roles for auxin in
these processes.
Compounds that have been identified as being important stimulants of germination, mainly using farmacological tools are several nitrogen-containing compounds, including nitric oxide (NO)
gas (Bethke et al., 2006), nitrite (NO2-) and nitrate (NO3-) (Alboresi
et al., 2005; Bethke et al., 2007a for review). It is suggested
(Bethke et al., 2007a) that all N compounds affect germination via
conversion into NO. Enzymatic NO production occurs mainly via
nitrate reductase as by product of lipid catabolism or nitric oxide
synthase (Crawford and Guo, 2005). Non-enzymatic conversion
of nitrite to NO, has also been demonstrated and was suggested
to have special significance for seeds (Bethke et al., 2007a). The
observation by Alboresi et al. (2005) that the high nitrate levels
that accumulate in nitrate reductase deficient mutants lead to reduced dormancy implies that nitrate reductase is not essential either because NO is generated by this non-enzymatic pathway or
nitrate acts on its own.
An effect of NO is that it might function as an antioxidant
(Lamatinna et al., 2003). However, it is also reported that NO inhibits catalase leading to higher H2O2 and reactive oxygen species
(ROS), which has the opposite effect as antioxidants (Bethke et
al., 2007a). ROS are a by product of oxidation of stored seed
fatty acids. Thereby ROS are increased, which it self may alleviate
dormancy (Bailly et al., 2004). However, other and additional
mechanisms have also been suggested and include interaction
with ABA catabolism enzymes and light and GA signalling (Finkelstein et al., 2008, Bethke et al., 2007b)
THE GENETIC CONTROL OF SEED STORAGE

COMPOUNDS, SEED DETERIORATION AND EARLY
SEEDLING DEVELOPMENT
Seeds can survive for a long time without germinating, either when
stored in dry conditions or when buried in the soil. These seeds
form the seed bank waiting until environmental conditions become
favorable for germination. Despite the strong desiccation tolerance
of many seeds, storage also under dry conditions ultimately leads
to a loss of viability. It has been suggested that compounds such
as certain sugars and proteins, such as LEA (Late embryogenesis abundant) (Skriver and Mundy, 1990) and Heat shock proteins
(Hsps) (Wehmeyer and Vierling, 2000; Hong and Vierling, 2001),
that accumulate during the later stages of seed development, have
a desiccation protective role. However, mutants like abi5, which
show a strong reduction in some LEA proteins (Finkelstein and

Lynch, 2000) do not have obvious defects in seed storability. A
knock-out mutant of Hsp101 shows a loss of thermotolerance during seed germination (Hong and Vierling, 2001), but under optimal conditions germination seems unaffected, suggesting that at
least Hsp101 does not have a general function in the survival of
desiccated seeds. The expression of these heath shock proteins
are developmentally controlled by the seed specific heat stress
transcription factor HsfA9, which is regulated by the ABI3 protein
(Kotak et al., 2007).
The presence of genetic variation for storability is clear in desiccation intolerant genotypes such as abi3, lec1, fus3 and dog1
mutants (Meinke et al., 1994; Weber and Wobus, 1999; Bentsink
et al., 2006) and effects on testa mutants are reported as well (Debeaujon et al., 2000). QTL analysis in the Ler/Cvi and Ler/Sha RIL
lines (Bentsink et al., 2000; Clerkx et al., 2004) also revealed genetic variation for this trait, which was however not related to the
levels of raffinose series oligosaccharides segregating in the same
population.
For a seed it is important that after surviving a period in which
it could not germinate, it can grow into a vigorous seedling that
can compete with other seedlings. Factors that are important for
seedling establishment probably mainly have to do with the availability and mobilization of storage materials. The accumulation of
storage proteins and lipids is defective in seed maturation mutants
such as lec1, lec2 and fus3, which although they germinate on filter paper they cannot be transferred efficiently to soil from this substrate, in contrast to other seeds, and require establishment as
seedling on sugar supplemented media before they can be transplanted to soil. The wrinkled1 (wri1; Focks and Benning, 1998) mutant affects specifically seed storage compounds with a strong
reduction in seed oil content. In addition it shows reduced germination potential and reduced seedling establishment and responds
different than wild type to sugar and ABA signals (Cernac et al.,
2006). WRI1 encodes an AP2/EREB family transcription factor
(Cernac and Benning, 2004), which itself is regulated by LEC2
(Baud et al., 2007). WRI1 controls carbon metabolism in developing seeds and is a prerequisite for oil accumulation in seeds.
The mobilization of food reserves during germination and early
seedling growth require the activity of lipid and carbohydrate degrading enzymes. Storage lipid breakdown is a complex process
involving a number of steps for which many of the genes have
been identified in recent years and their effect of mutations in
these genes have been studied in mutants and double mutants
(reviewed in Penfield et al., 2007; Graham et al., 2008). Fatty acids
are stored in oil bodies as triacylglycerol (TAG), which are hydrolysed by lipases. The released fatty acids are passed to glyoxysomes (a peroxisome in which the glyoxylate cycle occurs) via
an ABC transporter encoded by the CTS (comatose) protein (Russell et al., 2000; Footitt et al., 2002). In here oxidation takes
place converting fatty acids, activated to acyl-CoA esters to acetyl
Co-A, which is subsequently converted to four-carbon sugars.
These sugars are then transported to the mitochondria from where
they are converted to malate and transported to the cytosol for
gluconeogenesis or used for respiration. Gene expression profiling
revealed that flavonoids were absent from cts and kat2-1 mutant
seeds, but accumulated in the presence of sucrose. This indicates
an essential role for oxidation in inducing flavonoid biosynthetic
genes. The same analyses revealed that CTS functions very late
in phase II of germination and that its function is required for the

expression of specific gene sets related to an important biochemical pathway associated with seedling establishment and survival
(Carrera et al., 2007).
Proteins that play a role in oxidation are LACS (Acyl-CoA
synthetase), ACX (Acyl-CoA oxidase), MFP2 (Multifunctional protein 2) and KAT2 (3-keto-acyl-CoA thiolase). The glyoxylate cycle
involve citrate synthase (CSY), isocitrate lyase (ICL), malate synthase (MLS) and phosphophoenolpyruvate carboxykinase (PCK1)
(reviewed by Penfield et al., 2007; Graham et al., 2008).
As expected from mutants that are deficient in storage lipids,
many mutants in these genes described above (or double mutants
when duplicated genes control the step) have defects in hypocotyl
elongation in darkness and seedling establishment that can be rescued by sugars (Eastmond et al., 2000; Cornah et al., 2004). Therefore, the glyoxylate cycle can be described as being non-essential
in Arabidopsis germination, although it does play an important
physiological role in seedling establishment. However a subset of
the lipid mobilisation single mutants (cts, kat2) and double (in case
of acx and csy) show strongly reduced germination, which explains
why cts mutants were isolated in a seed germination screen (Russell et al., 2000). All these mutants have in common that they act
in the glyoxysome and are involved in transport into (CTS) or, export from (CSY, Pracharoenwattana et al., 2005, Graham, 2007 for
review) or -oxidation in this organelle. This suggests that a product(s) of -oxidation apparently has a germination promoting function. The nature of this signal, which is not the absence of lipid
mobilisation as such, is not known. Penfield et al. (2007) mention
a number of possibilities, such as involvement of jasmonate biosynthesis or redox control. However, the direct involvement of these
factors is unlikely because other mutants in these factors do not
show a germination phenotype (Penfield et al., 2007).
An example of another gene controlling metabolism in developing and germinating seeds is plastidic pyruvate kinase (PKP1)
controlling the last step of glycolysis. Pkp1 mutants which are reduced in oil accumulation, contain less chlorophyll and tocopherol
and consequently a seedling establishment defect (Andre et al.,
2007; Baud et al., 2007).
Sugars arrest early seedling establishment. It has been shown
that this process is closely associated with plant hormone biosynthesis and signalling, in particular with that of ABA (for review see
Finkelstein and Gibson, 2001; Gazzarini and McCourt, 2001; Len
and Sheen, 2003; Rook et al., 2006; Dekkers and Smeekens,
2007; Rognoni et al., 2007). Mutants that were insensitive to the inhibiting effect of glucose and sucrose on early seedling development were isolated by several groups and identified ABA deficient
mutants (i.e. aba2/isi4/gin1/sis4 and aba3/gin5) and ABA insensitive4 (abi4/sun6/isi3/gin6/sis5) as sugar insensitive (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al.,
2001). The fact that ABI4 encodes an AP2 domain containing transcription factor that binds a CE1-like element present in many ABA
and sugar regulated promoters (Finkelstein et al., 1998; Niu et al.,
2002; Acevedo-Hernandez et al., 2005) linked sugar regulation to
ABA signalling. However there are many more examples of
processes and genes that are regulated by ABA and sugar. Recently Dekkers et al. (2008) showed that ABI3 is a key regulator of
glucose insensitive (GIN) signalling which is in agreement with
previous reports showing that ABI3 overexpression (either AtABI3
or CnABI3) in Arabidopsis results in a glucose oversensitive phe-
9 of 18
notype (Finkelstein et al., 2002; Zeng and Kermode, 2004). The

same authors showed that ABA and glucose signalling overlap to
a larger extend than originally thought (Dekkers et al., 2008). Recently it has been identified that DOG1 is involved as well in the
ABA mediated sugar signalling pathway in seedlings. This role is
specific for the GSQ5/DOG1 Cvi allele as glucose addition induced the expression of the GSQ5/DOG1 Cvi allele whereas the
Ler and Col allele did not respond (Teng et al., 2008).
OMIC ANALYSES RELATED TO DORMANCY AND

GERMINATION
Several recent studies have analysed the expression of the
genome in Arabidopsis seeds at both the transcriptome and proteome levels (reviewed by Holdsworth et al., 2008b). These studies have identified a major role for translation in germination and
dormancy release. Rajjou et al. (2004) reported that transcription
is not the restricting step in the completion of germination following imbibition of non-dormant seeds. Imbibition of seeds in the
presence of an excess of -amanitin (a specific inhibitor of DNAdependent RNA polymerase II) failed to inhibit germination completion, whereas the translational inhibitor cyclohexamide totally
inhibited germination. This indicates the importance of the in the
dry seeds stored mRNA that are used for translation in imbibed
seeds (Rajjou et al., 2004)
Several recent studies have shown that important transcript
and protein changes are happening in the dry seeds during storage and that these changes might be targeted to release of dormancy as seeds after-ripen. These studies indicate that both the
accumulation of specific proteins (Chibani et al., 2006) and gene
transcripts (Bove et al., 2005, Leubner-Metzger, 2005) can occur.
The dry state, as very clearly described by Holdsworth et al.
(2008b), are mature seeds containing 5-10% water depending on
the species. This residual water is not uniformly distributed in the
seed tissues indicating that some areas (hydrated pockets within
cells) may contain enough water to support gene expression
(Leubner-Metzger, 2005).
The transcriptome
Developmental phases during late embryonesis and subsequent
germination are characterized by spatial and temporal patterns of
gene expression (Nambara et al., 2000; Parcy et al., 1997). Several studies have been performed in order to understand
processes that occur in the dry seed, for example during afterripening. Carrera et al. (2008) performed a study in order to understand the relationship between ABA and after-ripening. It has
been shown that mutants defective in seed ABA synthesis (aba11) or perception (abi1-1) which do not show dormancy, exhibit
changes in global gene expression resulting from dry after-ripening that were comparable with changes occurring in wild type
seeds. Thus, the action of ABA has been separated from afterripening and dry storage regulated gene expression. The authors
also use their seed-specific gene ontology (GO) TAGGIT, which
have been described in an earlier paper (Carrera et al., 2007),
which facilitates the identification and visualisation of the germination signature.
10 of 18
Stored mRNA species in dry seeds are thought to be required

for late embryogenesis and seed germination. To differentiate
these classes Nakabayaski et al. (2005) analyzed expression profiles obtained from seeds at 6, 12 and 24 hours after imbibition. It
has been indicated that embryonic genes are largely down-regulated within 6 hours of imbibition, and that induction of metabolic
genes involved in germination started around 12 hours after imbibition. The same study revealed that upstream regions of genes
that are highly expressed in dry seeds over-represent ABREs
(ABA-responsive elements). The ABRE requires a second element
to form a functional ABA response complex. The coupling element
(CE) is such a motif and elicits a response to ABA when located
adjacent to an ABRE (ABRC; Shen and Ho., 1995). It was indeed
shown, that genes that contain both ABRE and CE are expressed
at higher levels in seeds. In addition to this also the RY/Sph motif
(known to regulate seed specific expression; Bumlein et al., 1992;
Hattori et al., 1992) showed enrichment in seeds when associated
with ABRE (Nakabayashi et al., 2005).
Penfield et al. (2006) established the relative roles of embryo
and endosperm in the control of seed germination and seedling
establishment. They showed that the endosperm responds to both
ABA and gibberellins, but that ABA in particular regulates nuclear
but not plastid-encoded photosynthetic gene expression in the embryo. They also showed that ABI4 expression is confined to the
embryo, accounting for the major differences in embryo response
to ABA, and defining a role for ABI4 as a repressor of lipid breakdown. Furthermore, ABI5 expression in the endosperm defines a
second region of altered ABA signalling in the micropylar endosperm cap. Finally, embryo and endosperm ABA signalling mutants demonstrate the spatial specificity of ABA action in seed
germination. It was concluded that the single cell endosperm plays
an active role in the regulation of seed germination in Arabidopsis
(Penfield et al., 2006).
Other studies have produced microarray data related to germination under different conditions. Experiments using GA-deficient seeds (Ogawa et al., 2003) have described temporal
transcript abundance changes in GA-deficient mutants seeds stimulated to germinate by GA treatment or in nongerminating waterimbibed controls. Dormancy cycling was studied in the Arabidopsis
accession Cvi in a range of dormant and after-ripening states. The
data support an ABA-GA hormone balance mechanism controlling cycling through dormant states that depends of synthetic and
catabolic pathways of both hormones (Cadman et al., 2006). The
same authors studied the effect of after-ripening, low temperature,
nitrate and light on gene expression during dormancy release
(Finch-Savage et al., 2007). Here they show that dormancy and
after-ripening gene sets respond in a quantitative way to specific
environmental signals. Recently, Bassel et al. (2008) have performed a chemical genetic approach in order to identify small-molecules that inhibit germination. The authors identified three
mechanistically distinct inhibitors which they used subsequently
to isolate a transcriptional signature that defines germinating
seeds. The data was compared to data published by Carrera et al.
(2007).
To help to visualize the data mentioned above a seed mRNA
expression browser was designed using the electronic Fluorescent Pictograph platform (eFP). This eFP is included as part of
the Bio-Array (BAR) online bioinformatics tools package
(www.bar.utoronto.ca). The seed eFP browser which is developed
and described by Bassel et al. (2008) visualizes experimental

gene expression data painted onto pictographic representations
of the tissues and conditions from which the RNA samples were
obtained. The TAGGIT GO (Carrera et al., 2007) has also been integrated into the eFP browser.
The proteome
Several proteome analyses have been performed to analyse protein abundance in Arabidopsis seeds in relation to dormancy and
germination using two-dimensional gels. Gallardo et al. (2001,
2002a) described protein abundance related to germination and
seed priming (pre-germination treatment which overcomes seed
dormancy) and the role of gibberellins in seed germination. In addition the importance of methionine biosynthesis for Arabidopsis
seed germination and seedling growth has been shown (Gallardo
et al., 2002b). Results indicated that methionine synthase and Sadenosylmethionine synthetase are fundamental components
controlling metabolism in the transition from a quiescent to a highly
active state during seed germination.
Recently a genome-scale proteomics analyses was published
were proteome dynamics are described for different plant organs
using mass spectrometry (Baerenfaller et al., 2008). This research
includes data on after-ripened dry seeds, in these seeds 13,901
distinct peptides were detected that represent 3,789 proteins. In
total 133 seed specific biomarkers have been identified. These biomarkers were enriched for specific functional classes that are: embryonic development ending in seed dormancy (GO:0009793),
sequestering of lipids (GO:0019915), lipid transport (GO:0006869),
seed dormancy (GO:0010162) and response to water
(GO:0009415). Although this paper only describes a protein survey in dry after-ripened seeds, the seed specific peptide map
shows that proteomics can be used as a routine scoring method
comparing different dormancy and germination states.
The metabolome
Fait et al. (2006) have analysed metabolic profiles using gas chromatography-mass spectrometry (GS-MS) on seeds throughout
development and germination. The authors conclude that seed
maturation was associated with a significant reduction of most
sugars, organic acids and amino acids, suggesting their incorporation into storage reserves. Furthermore, it appeared that the
transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids,
and shikimate-derived metabolites. In contrast to this, seed stratification was associated with a decrease in the content of several
of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly,
the levels of other metabolites significantly increased during stratification and were boosted further during germination sensu
stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and
germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as
determined by semi-quantitative RT-PCR and analysis of publicly

available microarray data. In conclusion, this study provides a
comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation
for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct
metabolic switches during stratification and early germination (Fait
et al., 2006).
CONCLUSIONS
Dormancy and germination are very complex traits under the control of a large number of genes. The number of mutants and genes
that are involved has increased rapidly in recent years. These studies confirmed the crucial role of the plant hormones GA and ABA.
However it also showed the involvement and complex interaction
with other plant hormones such as ethylene, brassinosteriods and
auxin. Furthermore novel players were detected such as the HUB
genes affecting histone modifications and genes such as DOG1
for which loss of function mutants show their importance but for
which the biochemical function is unknown. The use of transcriptomic and proteomic analysis refined the variation and similarities
of the pathways affected by dormancy and germination and provides a basis for studying the role of individual genes in these
pathways. In future studies apart from distinguishing dormancy
and germination promotion also the temporal and spatial components will be taken into account, as well as the specific roles and
sensing mechanisms of environmental factors both during seed
development, seed storage and seed germination. This together
should allow an in depth insight in this essential and intriguing biological system.
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Seed Dormancy and Germination

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Seed Dormancy and Germination

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Seed Dormancy and Germination

Author(s): Lenie Bentsink and Maarten Koornneef

The Arabidopsis Book

2008 American Society of Plant Biologists

First published on December 30, 2008: e0119. doi: 10.1199/tab.0119