Gibberellins are phytohormones regulating growth and development throughout a plant’s life cycle; they are essential in processes such as stem elongation, floral development and seed germination
[1–4]. The importance of gibberellins in these processes is most obvious in gibberellin-deficient mutants; GIBBERELLIC ACID REQUIRING 1 (GA1) encodes for the ent-kaurene synthetase A of the gibberellin biosynthetic pathway, ga1 plants are therefore unable to synthesise gibberellins. These mutants are extremely dwarfed, male-sterile, and most importantly their seeds fail to germinate without exogenous gibberellin
Significant progress has been made in recent years in elucidating the gibberellin signalling pathway
[7, 8]. The gibberellin signal is perceived by the soluble receptors GIBBERELLIN INSENSITIVE DWARF 1 (OsGID1 or OsGID1-like)
[9, 10]. Arabidopsis contains three GID1-like genes, GID1a, GID1b and GID1c. The gibberellin-GID1 interaction triggers the degradation of DELLA proteins, the major negative regulators of gibberellin signalling, via the 26S proteasome pathway
[12–15]. The gibberellin-specific F-box proteins OsGID2 and SLEEPY1 (AtSLY1) mediate this degradation
DELLA proteins are a subfamily of the GRAS family of transcriptional regulators
, named after their highly conserved N-terminal DELLA motif, which mediates gibberellin-responsiveness
[21–23]. In Arabidopsis, there are five DELLA proteins: GIBBERELLIN INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA), RGA-like1 (RGL1), RGL2 and RGL3
[13, 24–26]. Extensive genetic studies using various combinations of DELLA knock-out mutations have elucidated overlapping as well as distinct functions of each protein in repressing plant growth and development: RGA and GAI are the main repressors of stem elongation, whereas floral development is regulated by a combination of RGA, RGL1 and RGL2, and RGL2 is the key DELLA protein repressing seed germination
However, the events downstream of DELLAs in the gibberellin-mediated regulation of growth and development are less well understood. Until recently, it was not clear how DELLA proteins repress gibberellin-mediated gene expression. Although they have been classified as transcriptional regulators, they do not have conserved DNA binding domains. A major break-through in understanding the molecular mechanism of DELLA-action was achieved when the direct interaction of DELLA proteins with PHYTOCHROME INTERACTING FACTOR 3 (PIF3) and PIF4 was shown
[32, 33]. Since then, the main mode of DELLAs in regulating transcription is thought to occur via the sequestering of transcription factors. Recently, the interaction in yeast with more members of the basic helix-loop-helix (bHLH) subfamily 15, namely PIF3-like 5 (PIL5), PIL2 and SPATULA (SPT) was shown; the authors thus hypothesised that DELLAs could interact with all members of this subfamily
. This further corroborates the conclusion that the main molecular mechanism of DELLA function is their interaction with transcription factors, which leads to the formation of inactive complexes
. This model has been further revised to show that DELLA proteins are also able to activate transcription by sequestration of inhibitors
Here, we focus on gibberellin-mediated regulation of germination, in particular the molecular mechanism by which the DELLA protein RGL2 suppresses germination. Germination is a complex process of three phases, each being tightly regulated at various levels. Phase I describes the intake of water, during which the seed imbibes, whereas in phase II metabolic processes are re-initiated (also called germination sensu stricto), and in phase III the radicle emerges
[39, 40]. Seed germination is regulated by the balance of the two phytohormones abscisic acid and gibberellins, which inhibit and promote germination, respectively. Gibberellins function in late phase II of germination
, a phase which is physiologically characterised mainly by post-imbibitional cell elongation in embryonic radicle and hypocotyl as well as endosperm weakening
. Gibberellins are therefore key players in the actual commitment of seeds to germination.
RGL2 is the major DELLA protein involved in repressing germination
[25, 31]. It performs this function, at least partly, through increasing abscisic acid biosynthesis as well as activities of ABI5 and ABI3
[43, 44]. This has been further elucidated by Lee et al., showing that RGL2 regulates abscisic acid release in the endosperm to control embryo growth. In fact, RGL2 has been identified as one of the genes to be involved in the regulation of seed germination at the phase II to phase III transition
. Despite all these findings, it remains unclear exactly how RGL2 suppresses this complex process of germination. Therefore, elucidation of the molecular mechanism of RGL2 function would not only allow us to gain a deeper understanding of gibberellin-mediated germination, but also enable us to manipulate details of germination, for example to prevent pre-harvest sprouting in crops, at the same time ensure full and synchronous germination upon sowing.
In this study, we show that RGL2 function causes seeds to enter a state of dormancy. Microarray analysis showed that RGL2 up-regulates several genes associated with dormant states of seeds. Enforcing dormancy is partly achieved by RGL2 directly inhibiting transcription of cell wall-modulating genes ALPHA EXPANSIN3 (EXPA3) and EXPA8, by binding to their promoters, perhaps as a complex with as yet unidentified transcription factors. Thus, RGL2 directly affects cell growth. These data suggest that RGL2 inhibits seed germination both directly and indirectly.