Seed germination of GA-insensitive sleepy1 mutants does not require RGL2 protein disappearance in Arabidopsis

We explore the roles of gibberellin (GA) signaling genes SLEEPY1 (SLY1) and RGA-LIKE2 (RGL2) in regulation of seed germination in Arabidopsis thaliana, a plant in which the hormone GA is required for seed germination. Seed germination failure in the GA biosynthesis mutant ga1-3 is rescued by GA and by mutations in the DELLA gene RGL2, suggesting that RGL2 represses seed germination. RGL2 protein disappears before wild-type seed germination, consistent with the model that GA stimulates germination by causing the SCF(SLY1) E3 ubiquitin ligase complex to trigger ubiquitination and destruction of RGL2. Unlike ga1-3, the GA-insensitive sly1 mutants show variable seed dormancy. Seed lots with high seed dormancy after-ripened slowly, with stronger alleles requiring more time. We expected that if RGL2 negatively controls seed germination, sly1 mutant seeds that germinate well should accumulate lower RGL2 levels than those failing to germinate. Surprisingly, RGL2 accumulated at high levels even in after-ripened sly1 mutant seeds with 100% germination, suggesting that RGL2 disappearance is not a prerequisite for seed germination in the sly1 background. Without GA, several GA-induced genes show increased accumulation in sly1 seeds compared with ga1-3. It is possible that the RGL2 repressor of seed germination is inactivated by after-ripening of sly1 mutant seeds.     

CONSTITUTIVE PHOTOMORPHOGENIC 1 promotes seed germination by destabilizing RGA-LIKE 2 in Arabidopsis

Under favorable moisture, temperature, and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGA-LIKE 2 (RGL2), a major repressor of germination in Arabidopsis (Arabidopsis thaliana) seeds. Analysis of seed germination phenotypes of constitutive photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacted directly with RGL2, and in vivo this interaction was strongly enhanced by SUPPRESSOR OF PHYA-105 1. COP1 directly ubiquitinated RGL2 to promote its degradation. Moreover, GA stabilized COP1 with consequent RGL2 destabilization. By uncovering this COP1–RGL2 regulatory module, we reveal a mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

A master regulator of photomorphogenesis positively regulates germination in Arabidopsis seeds by directly ubiquitinating and promoting the degradation of a key repressor of seed germination.     

拟南芥突变体种子休眠与萌发的研究进展

种子的休眠和萌发是一个复杂的过程, 至今尚未能清楚阐明其调控机制。目前已从拟南芥突变体中鉴定了一些与种子萌发和休眠相关的基因, 有助于阐明种子休眠和萌发的分子机制。本文综述了拟南芥突变体种子休眠与萌发方面的研究进展。赤霉素是促进种子萌发的主要因素之一, RGL、SPY、GCR、SLY和GAR等基因的表达参与赤霉素对种子萌发的调控。脱落酸与种子休眠有关, ABI1、ABI2、ABI3、ABI4、ABI5、FUS3、LEC、MARD和CIPK等基因参与了脱落酸的调控过程。对3类乙烯反应的突变体 (ein、etr和ctr) 以及油菜素内酯突变体 (det和bri) 的研究表明乙烯和油菜素内酯是通过拮抗脱落酸而促进种子萌发的。光对种子萌发的调节, 是通过具有Ser/Thr蛋白激酶活性的光敏色素PhyA、PhyB、 PhyC、PhyD和PhyE, 以磷酸化/去磷酸化方式调节其它与萌发相关基因的表达。含氮化合物对种子萌发的促进, 可能是以一种依赖一氧化氮的方式解除种子休眠。     

Loss of function of four DELLA genes leads to light- and gibberellin-independent seed germination in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Arabidopsis severe gibberellin-deficient mutant ga1-3 does not germinate even when the optimal light and temperature conditions are provided. This fact suggests that (1) gibberellin (GA) is absolutely necessary for the germination of an intact seed and (2) the ga1-3 mutant can be used as a good system to identify factors that repress seed germination. In this report, using ga1-3 mutation as the genetic background, we confirm that RGL2, one member of the DELLA family, encodes the predominant repressor of seed germination in Arabidopsis and show that the other DELLA genes GAI,RGA and RGL1 enhance the function of RGL2. More importantly, we show that ga1-3 seeds lacking RGA, RGL1 and RGL2 or GAI, RGL1 and RGL2, confer GA-independent germination in the light but not in the darkness whilst ga1-3 seeds lacking GAI, RGA and RGL2 germinate both in the light and darkness. This suggests that the destabilization or inactivation of RGA and GAI is not only triggered by GA but also possibly by light. In addition, ga1-3 seeds lacking in all the aforementioned four DELLA genes have elongated epidermal cells and confer light-, cold- and GA-independent seed germination. Therefore, DELLA proteins likely act as integrators of environmental and endogenous cues to regulate seed germination.     

AtEXP2 Is Involved in Seed Germination and Abiotic Stress Response in Arabidopsis

Expansins are cell wall proteins that promote cell wall loosening by inducing pH-dependent cell wall extension and stress relaxation. Expansins are required in a series of physiological developmental processes in higher plants such as seed germination. Here we identified an Arabidopsis expansin gene AtEXPA2 that is exclusively expressed in germinating seeds and the mutant shows delayed germination, suggesting that AtEXP2 is involved in controlling seed germination. Exogenous GA application increased the expression level of AtEXP2 during seed germination, while ABA application had no effect on AtEXP2 expression. Furthermore, the analysis of DELLA mutants show that RGL1, RGL2, RGA, GAI are all involved in repressing AtEXP2 expression, and RGL1 plays the most dominant role in controlling AtEXP2 expression. In stress response, exp2 mutant shows higher sensitivity than wild type in seed germination, while overexpression lines of AtEXP2 are less sensitive to salt stress and osmotic stress, exhibiting enhanced tolerance to stress treatment. Collectively, our results suggest that AtEXP2 is involved in the GA-mediated seed germination and confers salt stress and osmotic stress tolerance in Arabidopsis.     

拟南芥突变体种子休眠与萌发的研究进展

种子的休眠和萌发是一个复杂的过程,至今尚未能清楚阐明其调控机制。目前已从拟南芥突变体中鉴定了一些与种子萌发和休眠相关的基因,有助于阐明种子休眠和萌发的分子机制。本文综述了拟南芥突变体种子休眠与萌发方面的研究进展。赤霉素是促进种子萌发的主要因素之一,RGL、SPY、GCR、SLY和GAR等基因的表达参与赤霉素对种子萌发的调控。脱落酸与种子休眠有关,ABI1、ABI2、ABI3、ABI4、ABI5、FUS3、LEC、MARD和CIPK等基因参与了脱落酸的调控过程。对3类乙烯反应的突变体（ein、etr和ctr）以及油菜素内酯突变体（det和bri）的研究表明乙烯和油菜素内酯是通过拮抗脱落酸而促进种子萌发的。光对种子萌发的调节,是通过具有Ser／Thr蛋白激酶活性的光敏色素PhyA、PhyB、PhyC、PhyD和PhyE,以磷酸化／去磷酸化方式调节其它与萌发相关基因的表达。含氮化合物对种子萌发的促进,可能是以一种依赖一氧化氮的方式解除种子休眠。     

DELLA Proteins and GA Signalling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin (GA) is a classical plant hormone involved in many aspects of plant growth and development. A family of five homologs called the DELLA proteins, comprised of GAI, RGA, RGL1, RGL2 and RGL3, were recently found to act as critical GA signal mediators in Arabidopsis. Reports have shown that GAI and RGA are coupled together to repress stem elongation growth whereas RGL2 is a major negative regulator of seed germination. GA down-regulates DELLA proteins through protein degradation likely via the proteasome pathway. The conserved and functionally important DELLA domain is responsible for protein stability in response to GA.     

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.     

Identification of the conserved serine/threonine residues important for gibberellin-sensitivity of Arabidopsis RGL2 protein

The DELLA proteins GAI, RGA, RGL1 and RGL2 in Arabidopsis are plant growth repressors, repressing diverse developmental processes. Studies have shown that gibberellin (GA) attenuates the repressive function of DELLA proteins by triggering their degradation via the proteasome pathway. However, it is not known if GA-induced protein degradation is the only pathway for regulating the bioactivity of DELLA proteins. We show here that tobacco BY2 cells represent a suitable system for studying GA signaling. RGL2 exists in a phosphorylated form in BY2 cells. RGL2 undergoes GA-induced degradation, and this process is blocked by proteasome inhibitors and serine/threonine phosphatase inhibitors; however, serine/threonine kinase inhibitors had no detectable effect, suggesting that dephosphorylation of serine/threonine is probably a prerequisite for degradation of RGL2 via the proteasome pathway. Site-directed substitution of all 17 conserved serine and threonine residues showed that six mutants (RGL2(S441D, RGL2(S542D), RGL2(T271E), RGL2(T319E), RGL2(T411E) and RGL2(T535E)) mimicking the status of constitutive phosphorylation are resistant to GA-induced degradation. This suggests that these sites are potential phosphorylation sites. A functional assay based on the expression of GA 20-oxidase revealed that RGL2(T271E) is probably a null mutant, RGL2(S441D), RGL2(S542D), RGL2(T319E) and RGL2(T411E) only retained about 4-17% of the activity of the wild type RGL2, whereas RGL2(T535E) retained about 66% of the activity of the wild type RGL2. However, expression of GA 20-oxidase in BY2 cells expressing these mutant proteins is still responsive to GA, suggesting that the stabilization of RGL2 protein is not the only pathway for regulating its bioactivity.