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.     

The role of two f-box proteins, SLEEPY1 and SNEEZY, in Arabidopsis gibberellin signaling

The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCF(SLY1) (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.     

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。     

AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed‐plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed‐specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss‐of‐function atper1 mutants, atper1‐1 and atper1‐2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild‐type seeds. The suppressed primary seed dormancy of atper1‐1 was completely reduced by deletion of CYP707A genes. Furthermore, loss‐of‐function of AtPER1 cannot enhance the seed germination ratio of aba2‐1 or ga1‐t, suggesting that AtPER1‐enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild‐type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.     

Proteolysis-independent downregulation of DELLA repression in Arabidopsis by the gibberellin receptor GIBBERELLIN INSENSITIVE DWARF1

This article presents evidence that DELLA repression of gibberellin (GA) signaling is relieved both by proteolysis-dependent and -independent pathways in Arabidopsis thaliana. DELLA proteins are negative regulators of GA responses, including seed germination, stem elongation, and fertility. GA stimulates GA responses by causing DELLA repressor degradation via the ubiquitin-proteasome pathway. DELLA degradation requires GA biosynthesis, three functionally redundant GA receptors GIBBERELLIN INSENSITIVE DWARF1 (GID1a, b, and c), and the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase. The sly1 mutants accumulate more DELLA proteins but display less severe dwarf and germination phenotypes than the GA biosynthesis mutant ga1-3 or the gid1abc triple mutant. Interestingly, GID1 overexpression rescued the sly1 dwarf and infertility phenotypes without decreasing the accumulation of the DELLA protein REPRESSOR OF ga1-3. GID1 rescue of sly1 mutants was dependent on the level of GID1 protein, GA, and the presence of a functional DELLA motif. Since DELLA shows increasing interaction with GID1 with increasing GA levels, it appears that GA-bound GID1 can block DELLA repressor activity by direct protein-protein interaction with the DELLA domain. Thus, a SLY1-independent mechanism for GA signaling may function without DELLA degradation.     

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。     

A role for brassinosteroids in germination in Arabidopsis

This paper presents evidence that plant brassinosteroid (BR) hormones play a role in promoting germination. It has long been recognized that seed dormancy and germination are regulated by the plant hormones abscisic acid (ABA) and gibberellin (GA). These two hormones act antagonistically with each other. ABA induces seed dormancy in maturing embryos and inhibits germination of seeds. GA breaks seed dormancy and promotes germination. Severe mutations in GA biosynthetic genes in Arabidopsis, such as ga1-3, result in a requirement for GA application to germinate. Whereas previous work has shown that BRs play a critical role in controlling cell elongation, cell division, and skotomorphogenesis, no germination phenotypes have been reported in BR mutants. We show that BR rescues the germination phenotype of severe GA biosynthetic mutants and of the GA-insensitive mutant sleepy1. This result shows that BR stimulates germination and raises the possibility that BR is needed for normal germination. If true, we would expect to detect a germination phenotype in BR mutants. We found that BR mutants exhibit a germination phenotype in the presence of ABA. Germination of both the BR biosynthetic mutant det2-1 and the BR-insensitive mutant bri1-1 is more strongly inhibited by ABA than is germination of wild type. Thus, the BR signal is needed to overcome inhibition of germination by ABA. Taken together, these results point to a role for BRs in stimulating germination.     

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.     

Wheat seed proteins regulated by imbibition independent of dormancy status

Seed dormancy is an important trait in wheat (Trticum aestivum L.) and it can be released by germination-stimulating treatments such as after-ripening. Previously, we identified proteins specifically associated with after-ripening mediated developmental switches of wheat seeds from the state of dormancy to germination. Here, we report seed proteins that exhibited imbibition induced co-regulation in both dormant and after-ripened seeds of wheat, suggesting that the expression of these specific proteins/protein isoforms is not associated with the maintenance or release of seed dormancy in wheat.     

The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase

The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCF(SLY1)-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.     

The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.     

生长素调控种子的休眠与萌发

植物种子的休眠与萌发,是植物生长发育过程中的关键阶段,也是生命科学领域的研究热点。种子从休眠向萌发的转换是极为复杂的生物学过程,由外界环境因子、体内激素含量及信号传导和若干关键基因协同调控。大量研究表明,植物激素脱落酸(Abscisic acid, ABA)和赤霉素(Gibberellin acid, GA)是调控种子休眠水平,决定种子从休眠转向萌发的主要内源因子。ABA与GA在含量和信号传导两个层次上的精确平衡,确保了植物种子能以休眠状态在逆境中存活,并在适宜的时间启动萌发程序。生长素(Auxin)是经典植物激素之一,其对向性生长和组织分化等生物学过程的调控已有大量研究。但最近有研究证实,生长素对种子休眠有正向调控作用,这表明生长素是继ABA之后的第二个促进种子休眠的植物激素。本文在回顾生长素的发现历程、阐释生长素体内合成途径及信号传导通路的基础上,重点综述了生长素通过与ABA的协同作用调控种子休眠的分子机制,并对未来的研究热点进行了讨论和展望。     

羊草种子休眠机制及破除方法研究

羊草种子休眠程度深、发芽率低是限制栽培利用的重要因子.采用不同破除羊草种子休眠的方法,测定各处理对种子萌发的影响,以探索破除羊草种子休眠的有效途径.结果显示:(1)刺破种皮的裸种子较完整种子的萌发率、吸水速率、生活力分别由对照的6%、63%、0%显著增加到60%、86%、94%.(2)完整羊草种子分别用清水浸种1 d、30% NaOH浸种80 min、清水浸种1 d后用30% NaOH浸种60min其萌发率由6%分别显著提高到36%、60%、84%,而各浓度赤霉素处理完整种子其萌发率较对照均无显著变化. (3)采用清水浸种1 d后用30% NaOH处理60 min,再施加200 μg/g GA3综合处理,可使羊草完整种子的发芽率由6%提高到91%,接近其种子生活力94%.研究表明,羊草种子的稃与种皮不影响种子水分的吸收,但影响种子对GA3的吸收、不同程度地阻碍大分子物质的渗入、限制羊草种子内部萌发抑制物的渗出,从而引起种子休眠;分析认为稃和种皮以及种子内部萌发抑制物质是引起羊草种子休眠的主要原因.     

Seed after-ripening is a discrete developmental pathway associated with specific gene networks in Arabidopsis

After-ripening (AR) is a time and environment regulated process occurring in the dry seed, which determines the germination potential of seeds. Both metabolism and perception of the phytohormone abscisic acid (ABA) are important in the initiation and maintenance of dormancy. However, molecular mechanisms that regulate the capacity for dormancy or germination through AR are unknown. To understand the relationship between ABA and AR, we analysed genome expression in Arabidopsis thaliana mutants defective in seed ABA synthesis (aba1-1) or perception (abi1-1). Even though imbibed mutant seeds showed no dormancy, they exhibited changes in global gene expression resulting from dry AR that were comparable with changes occurring in wild-type (WT) seeds. Core gene sets were identified that were positively or negatively regulated by dry seed storage. Each set included a gene encoding repression or activation of ABA function (LPP2 and ABA1, respectively), thereby suggesting a mechanism through which dry AR may modulate subsequent germination potential in WT seeds. Application of exogenous ABA to after-ripened WT seeds did not reimpose characteristics of freshly harvested seeds on imbibed seed gene expression patterns. It was shown that secondary dormancy states reinstate AR status-specific gene expression patterns. A model is presented that separates the action of ABA in seed dormancy from AR and dry storage regulated gene expression. These results have major implications for the study of genetic mechanisms altered in seeds as a result of crop domestication into agriculture, and for seed behaviour during dormancy cycling in natural ecosystems.