Over the last 20 years, the use of mutants, particularly those of Arabidopsis, has made a major contribution to our understanding of developmental processes in seeds, including dormancy and germination (Koornneef and Karssen, 1994; Bentsink and Koornneef, 2002; Koornneef et al., 2002). A vast range of seed phenotypes have been described and loci and genes that are associated with dormancy and germination have been identified (listed in Bentsink and Koornneef, 2002). However, we are still far from a comprehensive view of the regulation and mechanisms of dormancy and germination. Despite the dissimilarities among used protocols and often erroneous methodology, outlines are gradually becoming visible. Most studies have focused on hormonal signalling involved in the regulation of these processes, particularly abscisic acid (ABA) and gibberellins (GAs), and most progress has been made in this field (reviewed by Finkelstein et al., 2002; Olszewski et al., 2002). Here, we focus on the action of ABA.
ABA has long been implicated in the acquisition of dormancy during seed development. In many cases, the induction of primary dormancy is accompanied by a transient increase in ABA content, and ABA-deficient or ABA-insensitive mutants are characterized by the absence of dormancy. However, ABA is involved in the regulation of a number of developmental processes, including protein synthesis and the acquisition of desiccation tolerance. It has thus been suggested that dormancy is a developmental event and that ABA affects a multitude of developmental processes rather than directly inducing dormancy (Hilhorst, 1995). Genome-wide profiling of stored mRNA in Arabidopsis seeds has revealed that ~300 of the almost
500 most highly expressed genes in dry seeds (out of a total of 12,470 expressed genes) contained one or more ABA-responsive sequences (Nakabayashi et al., 2005). Genome-wide gene expression profiling by massively parallel signature sequencing (MPSS) yielded 1357 genes that were upregulated or downregulated by ABA in Arabidopsis seedlings (Hoth et al., 2002). In a gene-finding survey in existing databases, using the cis-regulatory ABA-responsive element (ABRE) and its coupling element (CE) to target at ABA-responsiveness, almost 2000 stress-inducible genes were found of diverse functional categories (Zhang et al., 2005). These studies have made clear that hundreds of genes are potentially controlled by ABA. In addition, evidence is accumulating that extensive cross-talk exists among signalling pathways of ABA and other hormones. For example, the etr1-2 mutant in Arabidopsis, which is impaired in downstream signalling from the ethylene receptor, displayed higher levels of ABA, GAs, cytokinins and auxins (Chiwocha et al., 2005).
Dormancy in Arabidopsis Cvi seeds is clearly correlated with ABA content. Dormancy-breaking treatments reduced ABA content, whereas conditions that maintained dormancy induced an increase (Ali-Rachedi et al., 2004). Thus, it is expected that expression of ABA-controlled genes will increase under conditions that are conducive to induction or maintenance of dormancy. This is indeed the case in seeds of Arabidopsis Cvi that were manipulated to go through several dormancy cycles by modulation of the temperature (Cadman et al., 2006). This study showed that dormant seeds were as highly transcriptionally active as non-dormant seeds. Dormancy could be characterized by the expression of 442 genes which is at least twofold higher when compared with the non-dormant state. ABREs were overrepresented in the dormant gene set. Among the genes, many were identified as stress-related, including those encoding for small heat shock proteins (sHSPs), superoxide dismutase (SOD) and peroxiredoxin, as well as late embryo abundant (LEA) proteins and others related to seed development and maturation. This suggests that acquisition of primary dormancy may be related to expression of the same set of dormancy genes. Higher expression of these genes was also observed under non-stressed conditions, at 20°C in the dark when non-dormant seeds acquired secondary dormancy (Cadman et al., 2006). Thus, it may be argued that any condition that induces an increase in ABA content will result in the expression of a similar set of ABA-controlled genes, apart from genes that may be specific for a certain response. In addition, it has been suggested that ABRE-mediated transcription may be affected by signals that do not alter ABA content (Nambara and Marion-Poll, 2003).
What is the role of ABA in a plant cell when so many genes are potentially controlled by the hormone and how is the broad response of the transcriptome of a cell or tissue to ABA fine-tuned to a more specific response, such as dormancy? It is hard to assume that the increase in expression of a majority of genes is simply redundant (e.g. when their translation is blocked). Yet, inhibition of transcription in imbibing Arabidopsis tt2-1 mutant seeds by a-amanitin did not inhibit germination sensu stricto but prevented further growth of the protruding radicle (Rajjou et al.,
2004). Thus, germination sensu stricto may only depend on preformed transcripts and not on de novo transcription. These results suggest that transcriptional activity during seed maturation may allow the subsequent stage of early germination, and transcriptional activity during germination may be in preparation for subsequent growth of the seedling.
Because of the large number of genes affected, it is clear that ABA signalling is not simply linear but consists of a complex signalling network. The cis-acting elements seem to play a crucial role and may be regarded as nodes within the network. The fast progress in transcriptome expression profiling has made it possible to identify various combinations of cis-acting elements involved in ABA-, stress-and other hormone responses. For example, among the dry seed transcriptome of Arabidopsis Cvi, the genes with the highest expression were more likely to contain multiple ABREs, a combination of ABRE with the CE, or a combination of ABRE with the seed-specific RY/Sph motif (RY: purine/pyrimidine repeat motif; Sph: a restriction enzyme site; Nakabayashi et al., 2005). Now, it is also possible to distinguish between combinations that are affected by ABA and those that are not.
Superposed upon the organization of cis-acting elements are the networks of transcription factors that interact with them. Thus, the combination of certain transcription factors with certain combinations of cis-acting elements may then determine the promoter activity of a much reduced number of genes compared with the potential number of genes under ABA control (e.g. as described for osmotic and cold stress responses) ( Yamaguchi-Shinozaki and Shinozaki, 2005). Furthermore, post-transcriptional and post-translational control mechanisms will be involved in further fine-tuning of the response.
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