The use of different terms and definitions of seed dormancy have lead to confusion between ecologists and physiologists. Baskin & Baskin (2004) proposed that seed scientists state the class, level and type of dormancy based on their new classification system (see section II.3). This hierarchical system accurately reflects the ‘whole-seed’ ecologists’ view of the control of germination by dormancy and should be widely used in future work. At the molecular level, very little is known about MD, MPD, PY, PY + PD and deep PD. In contrast, recent physiological and molecular work has provided insight into mechanisms of nondeep PD, which are the focus of this section. These studies show that the intrinsic molecular mechanisms determining dormancy can have an embryo and/or a coat component (Hilhorst, 1995; Bewley, 1997a; Kucera et al., 2005). The terms ‘embryo’ and ‘coat dormancy’ will therefore be used here to distinguish between these two mechanisms, but it should be emphasized that they are not used as part of a classification system for ‘whole seeds’sensu Baskin & Baskin (2004). Embryo dormancy and coat dormancy are components of PD; their sum and interaction determine the degree of ‘whole-seed’ PD.
Embryo dormancy is characterized by a block that inhibits extension growth, and therefore excised embryos do not grow. Coat dormancy is characterized by a block that is conferred by the covering layers. Nondormant embryos excised from coat-dormant seeds will therefore extend and grow. ‘Coat’ is used in a loose sense and can be any embryo-covering structure, for example the testa, endosperm and/or pericarp, and terms such as ‘testa dormancy’ and ‘endosperm dormancy’ will be used to specify these. For example, mechanical resistance from combined testa and endosperm dormancy, which is greater than the embryo growth potential opposing it, appears to be the cause of nondeep PD in seed model systems such as A. thaliana and Solanaceae species (Hilhorst, 1995; Bewley, 1997b; Koornneef et al., 2002; Leubner-Metzger, 2003a).
1. Induction, maintenance and release of physiological dormancy by plant hormones and environmental signals
There is considerable evidence that ABA is an important positive regulator of both the induction of dormancy and the maintenance of the dormant state in imbibed seeds following shedding. As this evidence for ABA involvement has been described in detail in a very recent review (Kucera et al., 2005) and in several earlier reviews (Hilhorst, 1995; Bewley, 1997a; Li & Foley, 1997; Koornneef et al., 2002) only an update is provided here.
ABA deficiency during seed development is associated with absence of primary dormancy in the mature seed, whereas overexpression of ABA biosynthesis genes can increase seed ABA content and enhance seed dormancy or delay germination (e.g. Finkelstein et al., 2002
; Nambara & Marion-Poll, 2003
; Kushiro et al., 2004
ABA produced by the seed itself during seed development can impose a lasting dormancy, whereas maternal ABA or ABA application during seed development fails to induce lasting seed dormancy, but has other functions (Kucera et al., 2005
In A. thaliana
, the members of the AtNCED
gene family encode g-cis-epoxycarotenoid dioxygenases catalysing the key regulatory step in ABA biosynthesis (Lefebvre et al., 2006
). This work suggests that ABA synthesis in the embryo and that in the endosperm both contribute to the induction of seed dormancy.
High ABA contents are present in the imbibed seeds of the strongly dormant A. thaliana
ecotype Cape Verde Island (Cvi) and decrease as dormancy is lost (Ali-Rachedi et al., 2004
). A recent transcriptome analysis with this ecotype strongly supports the view that increased ABA biosynthesis is associated with the dormant state (Cadman et al., 2006
ABA biosynthesis during imbibition of dormant, but not nondormant, seeds has been demonstrated in the A. thaliana
ecotype Cvi (Ali-Rachedi et al., 2004
), as well as in other species including Nicotiana plumbaginifolia
(Grappin et al., 2000
), Helianthus annuus
(Le Page-Degivry & Garello, 1992
), and Hordeum vulgare
(Wang et al., 1995
). This de novo
ABA biosynthesis has been interpreted as a mechanism for dormancy maintenance.
Work with the strongly dormant A. thaliana ecotype Cvi shows that dormancy may depend on an intrinsic balance of GA and ABA biosynthesis and catabolism, which will determine the dominance of either of the hormones (Ali-Rachedi et al., 2004; Cadman et al., 2006). While PD release of Cvi seeds occurs effectively by after-ripening, stratification or inhibition of ABA biosynthesis, the addition of GA appears less effective. GA treatment of dormant Cvi seeds caused a transient increase in ABA concentration (Ali-Rachedi et al., 2004), suggesting that in dormant seeds a feedback mechanism exists that maintains a high ABA:GA ratio.
Thus, the net result of the dormant state is characterized by increased ABA biosynthesis and GA degradation. According to the revised hormone-balance hypothesis for seed dormancy proposed by Karssen and Laçka (1986), ABA and GA act at different times and sites during ‘seed life’. ABA induces dormancy during maturation, and GA plays a key role in dormancy release and in the promotion of germination. Newer evidence suggests that it is likely that this revision went too far. Experiments with sorghum (Sorghum bicolor) (Steinbach et al., 1997), and with ABA-deficient and -insensitive mutants of maize (Zea mays) (White & Rivin, 2000; White et al., 2000) demonstrated that GA and ABA can act at the same time on dormancy and germination. Inhibition of GA biosynthesis during seed development mimics the effects of exogenous ABA, for example in suppressing vivipary. It appears to be the ABA:GA ratio, and not the absolute hormone contents, that controls germination. Thus, it seems that GA directly antagonizes ABA signalling during dormancy induction of cereal grains. Experiments with other species are needed to determine whether this is a general phenomenon.
While dormancy maintenance also depends on high ABA:GA ratios, dormancy release involves a net shift to increased GA biosynthesis and ABA degradation resulting in low ABA:GA ratios (e.g. Ali-Rachedi et al., 2004; Cadman et al., 2006). This supports the proposal of Le Page-Degivry et al. (1996) that ABA is the primary hormone involved in any step during dormancy maintenance and release, and that GAs are present at sufficient concentrations to promote germination as soon as ABA biosynthesis is inhibited. There is further support from genetic work with Avena fatua (Fennimore & Foley, 1998) showing that GA itself, although its addition to the medium can cause germination of dormant seeds, is not involved in (embryo) dormancy loss, but in stimulating seed germination. Thus, dormancy release is characterized by the capacity for enhanced ABA degradation and increased GA biosynthesis, which is followed by GA promotion of seed germination.
We assume that these conclusions regarding the role of ABA and GA concentrations/synthesis in dormancy and germination are valid for the regulation of embryo dormancy. However, the emerging picture is incomplete without considering coat dormancy (see sections III.3 and III.4) and hormone sensitivities. The sensitivities for GA and ABA, their perception by receptors, their interconnected signalling chains, and their developmental regulation are of utmost importance for germination and dormancy (Kucera et al., 2005). In addition to hormone content and synthesis, the transition from the dormant to the nondormant state of many seeds is characterized by a decrease in ABA sensitivity and an increase in GA sensitivity (e.g. Le Page-Degivry et al., 1996; Corbineau et al., 2002; Koornneef et al., 2002; Leubner-Metzger, 2002; Ali-Rachedi et al., 2004; Chiwocha et al., 2005). The seed phenotypes of the A. thaliana ABA-insensitive (abi) response mutants abi1 to abi5 demonstrate that ABI1 to ABI5 are involved in seed dormancy and/or germination (Finkelstein, 2004; Kucera et al., 2005). Transcript expression of ABI1 to ABI5 is regulated in a complex manner during dormancy induction and release of A. thaliana ecotype Cvi (Cadman et al., 2006). An important finding of this study is that the dormant state is characterized by the transcription of genes with an overrepresentation of ABA-responsive elements (ABRE) in their promoters and of genes for transcription factors that bind to the ABRE. Such an overrepresentation of ABRE-containing genes is also evident in stored mRNAs of dry A. thaliana seeds (Nakabayashi et al., 2005). ABRE-binding transcription factors appear to be master regulators that mediate ABA responses in seeds, including the regulation of dormancy.
Among the genes that are induced in A. thaliana ecotype Landsberg erecta (Ler) and Columbia (Col) seeds during imbibition are many GA-responsive genes, but GA also causes down-regulation of many ABRE-containing genes (Yamaguchi & Kamiya, 2002; Ogawa et al., 2003; Yamauchi et al., 2004). Bioactive GAs accumulate in the embryo just before radicle protrusion, and light is one of the environmental factors that induces this GA biosynthesis, which occurs in two separate embryo tissues during germination: (1) the provascular tissue, where ent-copalyl diphosphate synthase 1 (AtCPS1) gene promoter activity is localized, has the early biosynthetic pathway, including the geranylgeranyl diphosphate cyclization reaction catalysed by CPS; (2) the cortex and endodermis of the root, where GA 3-oxidase 1 (AtGA3ox1) and AtGA3ox2 transcripts accumulate and AtGA3ox2 gene promoter activity is localized, have the late biosynthetic pathway, including the formation of bioactive GA by GA3ox. This physical separation of the early and late GA biosynthetic pathway implies that intercellular transport of an intermediate (probably ent-kaurene) is required for the production of bioactive GA by the embryo.
Cold and light responses are mediated, at least in part, by promoting GA biosynthesis via enhanced expression of AtGA3ox (Yamaguchi & Kamiya, 2002; Oh et al., 2004; Yamauchi et al., 2004; Liu et al., 2005b; Penfield et al., 2005). The Blue Micropylar End 3 (BME3) GATA zinc finger transcription factor is expressed in the radicle and seems to be involved as a positive regulator of seed germination and GA biosynthesis in response to cold stratification (Liu et al., 2005b). The recent model by Penfield et al. (2005) explains the control by these two environmental factors (cold and light) through the interaction of the basic helix-loop-helix (bHLH) transcription factors Spatula (SPT) and Phytochrome-Interacting-Factor-Like5 (PIL5). In the dark, SPT and PIL5 are both active as repressors of germination, while in ‘light plus cold’ their repressive activities are low. The regulation of PIL5 activity is controlled at the level of protein stability by light, which causes its repressive activity to decrease. In dark stratified seeds, SPT activity appears to be dependent on PIL5. Tsiantis (2006) speculates whether natural allelic variants of these transcription factors are responsible for determining some of the observed differences in dormancy behaviour in response to these environmental variables (temperature and light) between different ecotypes of the same species and indeed between species.
A number of GA-responsive genes are found to be differentially expressed when global transcript abundances are compared among seeds with different depths of dormancy and nondormant seeds of A. thaliana ecotype Cvi (Cadman et al., 2006). This study also suggests that there is active biosynthesis of GA precursors in all states: AtGA20ox1 transcripts are always present at high abundance, suggesting that biologically inactive GA9 and GA20 are always produced. There is also a high abundance of AtGA2ox1 transcripts present in all states, suggesting that any biologically active GAs formed (e.g. GA4 and GA1) are degraded rapidly. In seeds that require only light to germinate and those exposed to light, the transcript expression of AtGA3ox2 increases dramatically, presumably completing the final step of the biosynthesis of biologically active GA (e.g. GA4 from GA9, and GA1 from GA20). Therefore, a dynamic balance of biosynthesis and degradation of ABA and GA may exist that determines a state-specific equilibrium in the ABA:GA ratio (Cadman et al., 2006). High ABA signalling is associated with dormancy and high GA signalling with germination, while the transition between the two programmes is controlled by shifting the signalling between the two hormones.
Two functions for GA during seed germination have been proposed (reviewed in Kucera et al., 2005). First, GA increases the growth potential of the embryo and promotes germination. Secondly, GA is necessary to overcome the mechanical restraint conferred by the seed-covering layers by weakening of the tissues surrounding the radicle (see sections III.3 and III.4). Other plant hormones are involved in regulating gene expression during the induction, maintenance and release of PD (reviewed in Kucera et al., 2005). Further key information about the control of germination may come from the study of natural allelic variation at loci linked to dormancy and germination. Quantitative trait loci (QTL) mapping approaches for A. thaliana (Alonso-Blanco et al., 2003; Clerkx et al., 2004; Koornneef et al., 2004), Brassica oleracea (Bettey et al., 2000; Finch-Savage et al., 2005a) and cereals (Koornneef et al., 2002; Gu et al., 2004) are being used to identify germination and dormancy-related genes. Such QTL have been identified, but the cloning of the corresponding genes has not yet been reported.
2. Seed after-ripening: dormancy release and promotion of germination
After-ripening, i.e. a period of usually several months of dry storage at room temperature of freshly harvested, mature seeds, is a common method used to release dormancy (Bewley, 1997a; Probert, 2000; Leubner-Metzger, 2003; Kucera et al., 2005; Bair et al., 2006). Seed after-ripening can be characterized by: (1) a widening of the temperature range for germination; (2) a decrease in ABA concentration and sensitivity and an increase in GA sensitivity or loss of GA requirement (Fig. 2); (3) a loss of light requirement for germination in seeds that do not germinate in darkness; (4) an increase in seed sensitivity to light in seeds that do not germinate even with light; (5) a loss of the requirement for nitrate; (6) an increase of germination velocity.
The parameters that determine seed after-ripening are moisture and oil contents, seed-covering structures, and temperature (Manz et al., 2005 and references therein). After-ripening is prevented in very dry seeds; it requires seed moisture contents above a threshold value. This threshold moisture content is species-specific and lower in oilseeds compared with starchy seeds because they contain less bound water when equilibrated at any given relative humidity. After-ripening is also prevented during storage at very high air humidity (higher equilibrium moisture content). For several species, the conditions that generate optimal low-hydration values for after-ripening have been determined (e.g. Probert, 2000; Hay et al., 2003; Steadman et al., 2003; Leubner-Metzger, 2005; and references therein). The molecular mechanisms of after-ripening are not known. Nonenzymatic reactions that remove germination inhibitors, reactive oxygen species and antioxidants (Bailly, 2004), membrane alterations (Hallett & Bewley, 2002), and specific protein degradation via the proteasome (Skoda & Malek, 1992; Borghetti et al., 2002) have been proposed. Using cDNA-amplified fragment length polymorphism (cDNA-AFLP) gene expression analysis, Bove et al. (2005) provide evidence that Nicotiana seed after-ripening generates a developmental switch at the transcript level. This is in agreement with the A. thaliana Cvi transcriptome work (Cadman et al., 2006). In wild oat (Avena fatua), transcriptional regulation and post-transcriptional regulation are both important for the expression of dormancy-associated genes (Li & Foley, 1997).
Two recent publications provide evidence for gene expression in air-dry Nicotiana seeds during after-ripening (Bove et al., 2005; Leubner-Metzger, 2005). A rapid promotion of testa rupture of Nicotiana tabacum seeds occurred after c. 60 days of dry storage (Leubner-Metzger, 2005). This was associated with transient β-1,3-glucanase gene expression in the covering layer during tobacco after-ripening. Bove et al. (2005) found that at least eight specific mRNAs accumulated in air-dry, low-hydrated seeds of Nicotiana plumbaginifolia during after-ripening. Thus, while degradation of mRNAs and proteins for positive regulators of dormancy and for negative regulators of germination appears to be part of the molecular mechanisms of seed after-ripening, the possibility of de novo gene expression during seed after-ripening should also be considered.
4. Control of germination by the endosperm: endosperm weakening
The endosperm acts as a mechanical barrier to the germination of seeds in several angiosperm clades (Fig. 4). A decline in this mechanical resistance of the micropylar endosperm (the endosperm layer covering the radicle tip) appears to be a prerequisite for radicle protrusion during seed germination (for reviews, see Hilhorst, 1995; Bewley, 1997b; Leubner-Metzger, 2003; Sanchez & Mella, 2004; Kucera et al., 2005). This endosperm weakening (C↓ in Fig. 4) can be promoted by GA and, at least in part, inhibited by ABA (GA↓ and ABA↑ in Fig. 4). Solanaceae species such as tomato (Lycopersicon esculentum), tobacco (Nicotiana spp.), pepper (Capsicum annuum) and Datura have become model species for endosperm weakening. Although freshly harvested mature seeds of these species have nondeep PD, endosperm weakening has been studied using these seeds in the nondormant (e.g. after-ripened) or conditionally dormant state. Possibly as a consequence of this, in most of these cases, it has been proposed that the endosperm-weakening mechanism is part of the germination process of nondormant seeds and is not part of a dormancy release process per se (Baskin & Baskin, 2004). There are some exceptions to this in gymnosperm seeds, where weakening of the embryo-covering layer (megagametophyte) occurred during dormancy-breaking treatments, well separated from the germination process (see References in Baskin & Baskin, 2004).
In Fig. 6 we present unpublished work with ash (Fraxinus excelsior, Oleaceae; W. E. Finch-Savage, unpublished results) seeds that clearly shows that endosperm weakening can be part of the dormancy release process in angiosperms. The fruits of ash are deeply dormant and require prolonged warm followed by cold periods of stratification to break dormancy (optimally 16 wk warm, 16 wk cold; Nikolaeva, 1969; Finch-Savage & Clay, 1997). Warm stratification is required for the release of dormancy in the small but fully differentiated embryo and is associated with a decline in ABA concentration (Fig. 6a). Subsequent cold stratification is required for germination and is associated with an increase in GA concentration (Fig. 6c,d). If seeds are exposed to constant cold conditions after full stratification germination will continue slowly, but if seeds are exposed to alternating temperatures of 3 and 25°C germination will proceed more quickly. However, if the warm period exceeds the cold period in the 24-h cycle the seeds will not germinate and they become secondarily dormant. This implies that dormancy breaking continues during this regime, as only a limited proportion of seeds in the population germinate if transferred to 15°C. Within the dormant seed population there is a distribution of forces required to puncture the endosperm layer covering the radicle, which moves to lower puncture forces (Fig. 6f) during stratification, suggesting that these changes begin to occur while the seed is still dormant. Thus, the localized weakening of the enclosing tissues should be considered part of dormancy loss, rather than part of germination following dormancy loss in this species. Further evidence for this view is that dormancy is reinstated after stratification by constant warm conditions, but these same conditions do not prevent continued growth of the excised embryo. Based on current knowledge, it is not always possible to unambiguously assign endosperm weakening to either dormancy release or germination promotion. This apparent confusion is consistent with the proposal that a continuum appears to exist between dormancy and germination (Cohn, 1996). Thus, considering the fact that many of the molecular processes of endosperm weakening have been studied in more or less nondormant seeds, it seems reasonable to consider all the known molecular mechanisms of endosperm weakening as putative coat dormancy release mechanisms. The available evidence suggests that control of germination through GA-promoted endosperm weakening is a general phenomenon, associated with seeds that differ considerably in endosperm abundance. We propose, therefore, that at least some of the molecular mechanisms of endosperm weakening are widespread and constitute evolutionarily ancient traits.
Figure 6. Dormancy release during stratification in Fraxinus excelsior includes endosperm weakening that is mediated by gibberellins. The stratification treatment was 16 wk at 15°C followed by 16 wk at 3°C. Seeds were then transferred to dormancy breaking/germination conditions of 16 h at 3°C followed by 8 h at 25°C. (a–d) Original endogenous hormone data [abscisic acid (ABA) and gibberellins GA19, GA1 and GA3, respectively] sampled and measured by methods beccribed in Blake et al. (2002). Closed circles, endosperm; open circles, embryo. (b, c, d) The inactive precursor of GA1, GA19, accumulates in the endosperm during stratification, but significant GA1 concentrations do not occur for several weeks after the start of cold treatment when the ABA concentration is minimal. GA1 is not present in the embryo, but GA3 accumulates later in the embryo, coincident with radicle extension growth leading to germination. (e) Embryo growth within the seed. Initially the embryo will not grow when excised from the seed, but this physiological dormancy is progressively lost in the first half of the warm period as the endogenous ABA concentration (a) declines. The embryo will then grow to the full length of the endosperm cavity and will continue to grow if excised from the seed coat (e). However, germination of the whole seed will not reach completion without cold. If provided only with a cold treatment the embryo remains dormant and will not grow. (f) The distribution of forces in the population required to puncture the endosperm/seed coat layer which constrains the embryo (Finch-Savage & Clay, 1997). Radicle emergence was possible as the puncture force declined below 0.4 N. Solid dark line, dormant seed; dotted grey line, stratified seed; dotted dark line, hypothetical data following continued exposure to the dormancy breaking/germination conditions; the hatched area designates germination. Data in (e) and (f) are from Finch-Savage & Clay (1997). DW, dry weight.
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The germination of intact tomato seeds is inhibited by ABA, but surgical removal of the micropylar cap permits germination even in the presence of ABA (Liptay & Schopfer, 1983). Endosperm rupture has commonly been observed to be inhibited by ABA, and in the established model systems of the Asterid clade this occurs, at least in part, by inhibition of changes in the micropylar endosperm (e.g. Ni & Bradford, 1993; Leubner-Metzger, 2003a; da Silva et al., 2004). Puncture-force experiments investigating the effect of ABA on coffee (Coffea arabica) and tomato seeds (ABA↑ in Fig. 4) have shown that endosperm weakening is biphasic. The first phase is ABA-insensitive, and this is followed by the second phase which is inhibited by ABA. The ABA-inhibited second phase accounts for c. 53% of the total decrease in puncture force required for germination in coffee (da Silva et al., 2004), and c. 6% (Wu et al., 2000) or c. 24% (Toorop et al., 2000) in tomato. In coffee seeds, ABA controls germination by inhibiting both the embryo growth potential and the second step of endosperm weakening (da Silva et al., 2004).
Testa rupture and endosperm rupture are temporally separate events during the germination of many seeds of the Cestroideae subfamily of the Solanaceae, for example Nicotiana and Petunia (Fig. 2, Krock et al., 2002; Leubner-Metzger, 2003; Petruzzelli et al., 2003). These events are also mechanistically distinct processes, because the testa is dead and the endosperm is living tissue in these species. Testa rupture of tobacco occurs at predetermined breaking points and depends on water uptake and swelling of the embryo and the endosperm. It is associated with an additional increase in seed water content in the late part of phase II water uptake (Manz et al., 2005). In contrast, water uptake in dormant tobacco seeds is blocked before testa rupture and no additional phase II water uptake occurs (Mohapatra & Johnson, 1978). Following testa rupture, storage reserves in the micropylar endosperm cells are degraded and the radicle emerges through a hole in the endosperm which has a smooth outline. This hole always forms at the micropylar end of germinating tobacco seeds and results from tissue dissolution rather than increased growth potential of the emerging radicle (reviewed in Leubner-Metzger, 2003a).
Ikuma & Thiman (1963) in their ‘hatching hypothesis’ of seed biology suggested that ‘… the final step in the germination control process is the production of an enzyme whose action enables the tip of the radicle to penetrate through the coat’. In searching for this ‘hatching enzyme’, evidence has been uncovered for the contribution of various cell-wall-modifying proteins, including endo-β-1,4-mannanases and endo-β-1,3-glucanases (for reviews, see Hilhorst, 1995; Bewley, 1997b; Koornneef et al., 2002; Leubner-Metzger, 2003; Bailly, 2004; Kucera et al., 2005). Taken together, the current findings support the view that germination control by the seed-covering layers is achieved through the combined or successive actions of several cell-wall-modifying proteins. One intriguing issue arising from these studies is that there seem to be evolutionarily conserved molecular mechanisms as well as species-specific adaptations for endosperm weakening and/or coat dormancy release.
β-1,3-Glucanases are proposed to be involved in coat dormancy release, after-ripening and endosperm weakening (Fig. 2) and we use them here to illustrate how cell-wall-modifying proteins may act in the control of germination. These enzymes regulate symplastic trafficking, for example of cell-to-cell movement of GA, by controlling the strategically localized callose (β-1,3-glucan) deposition in the neck regions of plasmodesmata (Rinne et al., 2001; Leubner-Metzger, 2003). Increased callose deposition is associated with bud dormancy of trees, and release of bud dormancy by GA or chilling seems to involve callose degradation by β-1,3-glucanase. Expression of β-1,3-glucanase in the micropylar endosperm, its inhibition by ABA and the inhibition of endosperm rupture by ABA are widespread among the Solanaceae (Fig. 2, Leubner-Metzger, 2003; Petruzzelli et al., 2003). ABA inhibition of β-1,3-glucanase expression is also evident in perisperm weakening of Cucurbitaceae seeds (Welbaum et al., 1998; Yim & Bradford, 1998; Amritphale et al., 2005; Ramakrishna & Amrithhale, 2005). Proteomic analysis of A. thaliana showed that β-1,3-glucanases are glycosylphosphatidylinositol-anchored membrane proteins (GPI-APs; Elortza et al., 2003). GPI-APs are proposed to be involved as enzymes and receptors in cell adhesion, cell separation and differentiation processes. Karssen et al. (1989) proposed that the second step of tomato endosperm weakening resembles a cell separation process. β-1,3-Glucanase induction in the micropylar endosperm of tomato is associated with this second step (Toorop et al., 2000; Wu et al., 2000; Petruzzelli et al., 2003). Based on this, we speculate that β-1,3-glucanases facilitate endosperm rupture of seeds by breaking intercellular adhesion and causing cell separation.
The evolutionary trend towards cotyledon storage and seeds without endosperm at maturity is taken to the extreme in the Rosid clade (Fig. 4) and can be represented by the Brassicaceae. For example, the mature seeds of Raphanus and Brassica are without endosperm (Schopfer & Plachy, 1984; Schopfer et al., 2001), those of A. thaliana retain a single cell layer of endosperm (Pritchard et al., 2002; Liu et al., 2005a) and the mature seeds of Lepidium spp. have a thin endosperm layer (Nguyen et al., 2000; Müller et al., 2006). Endosperm weakening has recently been demonstrated in Brassicaceae seeds, indicating that the endosperm is also a constraint to germination in seeds of the Rosid clade (Müller et al., 2006). In this work, seeds of both A. thaliana and its much larger-seeded relative Lepidium sativum (garden cress) were studied. Both species belong to the Brassicoideae subfamily of the Brassicaceae (Hall et al., 2002; Koch et al., 2003) and are very similar in seed structure (FA2 type) and physiology. Testa rupture and endosperm rupture are separate events and only the latter is inhibited by ABA in both species (Liu et al., 2005a; Müller et al., 2006). Direct biomechanical measurement of the puncture force required to rupture the endosperm showed that the L. sativum micropylar endosperm weakened before radicle emergence (Müller et al., 2006). ABA delayed the onset and inhibited the rate of endosperm weakening in a dose-dependent manner. An early embryo signal which was required to induce endosperm weakening could be replaced by GA, and that weakening was found to be regulated by the GA:ABA ratio. These results suggest that the control of radicle protrusion in L. sativum and probably also A. thaliana seeds is mediated, at least in part, by endosperm weakening.