Dormancy is imposed during the seed maturation phase
As stated in the first part of the Introduction, at maturity, dry, viable Arabidopsis seeds are dormant, i.e. they cannot germinate even under favorable for germination environmental conditions. In nature, dormancy is lost progressively after seed exposure to a number of environmental factors such as cold and moist or after-ripening (i.e. dry storage). Dormancy is established during the seed maturation phase and ABA plays a crucial role in promoting dormancy.
Dormancy is a property of a dry seed, but whether a mature wild type seed is dormant can be determined only after its imbibition. Seeds that are dormant will not germinate, while seeds that are after-ripened will germinate within 48 hours. Global gene expression studies comparing dormant and after-ripened seeds showed that dormant seeds upon imbibition have equally active gene expression programs as after-ripened seeds but with different expression patterns (Cadman et al., 2006; Finch-Savage et al., 2007). These results suggest that dormant seeds, upon imbibition, maintain a dormant state by actively repressing germination. I will briefly describe factors that contribute to this active maintenance of seed dormancy and, at the end, I will characterize factors that are known to alleviate dormancy: after-ripening and nitrate treatment.
A key role of de novo ABA synthesis on dormancy maintenance
Arabidopsis dormant seeds after imbibition maintain high ABA levels unlike non dormant or stratified seeds that decrease ABA levels and germinate (Ali-Rachedi et al., 2004).
Both dormant and after-ripened dry seeds have similarly high ABA levels, which decrease within the first 24 hours after imbibition (Ali-Rachedi et al., 2004). However, only seeds that are dormant increase their endogenous ABA levels, maintaining them high for several days (Ali-Rachedi et al., 2004). When dormant imbibed seeds are treated with an inhibitor of ABA synthesis they germinate suggesting that de novo ABA synthesis upon seed imbibition is necessary to prevent dormant seed germination. Dormancy breaking factors like stratification and NO3-
treatment (see below) prevented the increase of ABA levels in dormant seeds, allowing them to germinate (Ali-Rachedi et al., 2004; Matakiadis et al., 2009). Unsurprisingly, mutations in ABA biosynthetic or signaling genes lead to production of seeds that do not maintain the dormancy state.
Microarray mRNA expression analysis showed that, consistent with the key role of ABA in maintaining seed dormancy, dormant Cvi seeds accumulate high levels of transcripts related to abiotic stress (including those encoding for LEA and Em-like proteins) when compared to after-ripened seeds. A significant set of genes highly expressed in dormant seeds contained ABA-responsive elements (Cadman et al., 2006). Interestingly, genes associated with the assembly of translation machinery were expressed at lower levels in dormant seeds, while expression of these genes was high when seeds were after-ripened or when dormancy was broken by cold or nitrate treatment (Cadman et al., 2006; Finch-Savage et al., 2007). Similarly, dormant Cvi seeds had relatively low expression levels of genes associated with reserve mobilization and cell wall modification, while their expression was high in seeds where dormancy was broken by after-ripening, cold and nitate treatment (Cadman et al., 2006; Finch-Savage et al., 2007). Both studies analyzed transcriptomes of dormant or after-ripened seeds imbibed for 24 hours in the presence or absence of dormancy-breaking factors. However, it is hard to conclude about a potential role of differentially expressed genes. In fact, differences in gene expression might simply reflect the actual developmental state of a seed committed to germinate after dormancy breaking treatment.
The comparison of the transcriptome profiles of dormant dry Cvi seeds and dry Cvi seeds that went through a period of after-ripening did not reveal drastic changes in mRNA expression
suggesting that processes other than transcription break dormancy during dry seed storage (Finch-Savage et al., 2007).
The seed coat is necessary to maintain dormancy
The role of the seed coat on seed dormancy was demonstrated in the experiment in which the seed coat was removed from a dormant seed. Such a seed coat-less embryo was able to green and grow into a normal seedling, while the embryo that was not removed from the seed coat did not germinate even after several days. This showed that the seed coat is necessary to maintain dormancy of imbibed wild type seed. The seed coat is composed of two layers: the testa that is a dead tissue and the endosperm that is alive and biologically active. I will describe how those two layers might influence dormancy maintenance.
A role of testa in dormancy maintanance
The role of the testa in dormancy maintenance has been studied using testa pigmentation and/or structure mutants (Debeaujon et al., 2000). It was shown that various testa mutants are less dormant than wild type seeds. None of the testa mutants germinated when introduced into the ga1 background suggesting that they still require GA biosynthesis for proper germination (Debeaujon and Koornneef, 2000). However, contra to this possibility, few testa mutants in the ga1 background displayed higher sensitivity to exogenous GA application, light and chilling than ga1 mutant (Debeaujon and Koornneef, 2000). The mechanism by which pigmentation or the structure of the testa influences seed dormancy and GA responsiveness is not known. An important experiment addressing directly a role of the testa on seed dormancy maintenance was performed in the dormant C24 ecotype (Bethke et al., 2007). It was shown that testa removal was not sufficient to trigger dormant seed germination as long as the endosperm layer was covering the embryo (Bethke et al., 2007). Moreover, it cannot be excluded that mutations in genes involved in testa pigmentation or testa shape have some additional effect for example on endosperm formation or seed development. In summary, the contribution of the testa layer to dormancy maintenance, if any, is minor. This suggests that the major germination restrictive activity provided by the seed coat in dormant seeds resides in the endosperm (described below).
A role of endosperm on dormancy maintenance
As stated above, removal of the testa from imbibed, dormant wild type seeds was not sufficient to trigger their germination as long as the endosperm layer was covering the embryo (Bethke et al., 2007). These testa-less embryos could remain dormant and viable for over one month without germinating or greening. However, when the endosperm layer was damaged or removed, dormant C24 seeds germinated (Bethke et al., 2007). Moreover, endosperm layers isolated from dormant seeds were shown to respond to dormancy breaking factors like nitrogen-containing compounds or stratification. Indeed, stratification or NaN3 treatment led to a reduction in protein storage vacuole (PSV) number per endosperm cell, which is a marker of endosperm activity correlated with the ability of the seed to germinate (Bethke et al., 2007).
Thus, the endosperm has been proposed to be the primary determinant of Arabidopsis seed dormancy maintenance but the mechanism by which it exerts its germination inhibitory role is unknown.
Genetic determinants of dormancy
DOG1, the first dormancy-promoting gene isolated after QTL analysis
Different Arabidopsis accessions display various levels of seed dormancy. Ecotypes such as Cape Verde Island (Cvi) show high levels of dormancy, while the commonly used Ler ecotype shows low dormancy levels. This natural variation between Ler and Cvi ecotypes allowed the mapping of quantitative trait loci (QTL) conferring dormancy. Seven QTL were identified and named Delay Of Germination (DOG) 1-7 (Alonso-Blanco C, 2003) and four of them were confirmed in Near Isogenic Lines (NILs) carrying Cvi introgression fragments in the Ler genetic background (DOG1, DOG2, DOG3 and DOG6). Only DOG1 has been cloned so far (Bentsink et al., 2006). DOG1 encodes a protein whose function is difficult to predict based on its sequence.
DOG1 expression is seed specific; its mRNA starts to accumulate 9 days after pollination and reaches its highest level during the last phases of seed development. DOG1 transcripts are present in dormant as well as in after-ripened dry seeds. Upon imbibition, DOG1 mRNA rapidly
disappears in both dormant and after-ripened seeds (Bentsink et al., 2006) suggesting that DOG1 might function only in the establishment of dormancy during seed maturation while not directly participating in repressing germination upon imbibition of dormant seeds. DOG1 belongs to a small gene family that consists of four additional members (named DOG1-Like 1-4 (DOGL1-4));
however, only a mutation in DOG1, and not in any of DOGL genes, leads to the loss of dormancy (Bentsink et al., 2006). A DOG1 sequence comparison between eight Arabidopsis accessions showed various polymorphisms in the coding regions of DOG1 alleles; however, a correlation between the sequence and the dormancy level was not observed. The authors suggested that natural variation present between various ecotypes may result from differences in DOG1 cis-regulatory regions (Bentsink et al., 2006).
RDO, the role of histone ubiquitination in dormancy maintenance
The Ler ecotype shows a low level of dormancy since a four week (i.e. short) period of after-ripening is sufficient to break its dormancy. However, when freshly harvested Ler seeds can display dormancy, since they germinate poorly upon imbibition. This ecotype was used to identify 4 mutants of reduced dormancy (rdo1-4) (Leon-Kloosterziel K., 1996). RDO4 has been cloned and shown to encode a C3HC4 RING-motif containing protein involved in the monoubiquitination of histone H2B (thus, RDO4 was renamed HISTONE MONOUBIQUITINATION1, HUB1) (Liu et al., 2007b). Monoubiquitination of histone H2B is a prerequisite for histone H3 methylation at Lys-4 and -79 and is associated with actively transcribed genes. This suggests that HUB1 influences seed dormancy by ubiquitination of H2B that leads to changes in histone H3 methylation that in turn changes the expression of genes that might influence seed dormancy. HUB1 has a homologue in Arabidopsis genome (HUB2) and the hub2 mutant seeds also lack dormancy (Liu et al., 2007b). Cloning of other RDO genes might further elucidate the complexity of dormancy regulation.
Dormancy breaking factors
After-ripening
In the natural environment, dormancy is usually broken by after-ripening, i.e. dry storage of seeds for an extended period of time. The mechanisms by which such dry storage of seeds leads to dormancy breaking is unknown, since a very low content of water in dry seeds is believed to be not sufficient for biochemical reactions. However, it was shown that changes of gene expression can occur in dry seeds of Nicotiana tabacum suggesting that after-ripening involves specific molecular genetic mechanisms occurring in dry seeds (Leubner-Metzger, 2005). Alternatively, non-enzymatic reactions such as lipid and protein oxidation were proposed to break seed dormancy. Indeed, after ripening and dormancy release was associated with an enhancement in hydrogen peroxide and superoxide anion content in dry seeds (Oracz et al., 2007). Moreover, after-ripening was associated with increased oxidation of specific protein sets of sunflower embryos (Oracz et al., 2007). Thus, reactive oxygen species-dependent modifications of proteins is a proposed mechanism for dormancy alleviation during seed after-ripening.
Exposure to nitrate breaks seed dormancy
Dormancy can be broken when seeds are exposed to nitrogen-containing compounds like nitric oxide gas (NO), nitrite (NO2
-), nitrate (NO3
-), nitrogen dioxide, nitrogen cyanide and others. Even nitrate provided during seed development via the maternal plant leads to lower dormancy (Matakiadis et al., 2009). It was shown that breaking dormancy by nitrate treatment led to higher expression of the ABA catabolic enzyme CYP707A2 and that cyp707a2 dormant seeds did not respond to nitrate (Matakiadis et al., 2009). Thus, nitrate may promote CYP707A2 accumulation that in turn lowers endogenous ABA levels and leads to dormancy alleviation.
However, the expression pattern of the other ABA metabolic genes in response to nitrate needs to be analyzed to determine if the nitrate effect is specific to CYP707A2 expression. Furthermore, lower CYP707A2 expression in nitrate-treated seeds might be a consequence of their germination ability rather than the determinant of a dormancy state.
In summary, seed dormancy is a complex trait and “one of the least understood phenomena in seed biology” (Bentsink and Koornneef, 2009) and despite extended research the mechanism of dormancy imposition and breaking is unknown.