毛竹茎秆伸长过程中赤霉素生物合成、降解和信号转导关键基因的鉴定及表达分析

赤霉素(Gibberellin)是一类非常重要的植物激素,在高等植物生命活动的整个周期都起着重要的调控作用。从毛竹Phyllostachys edulis基因组中共鉴定出23个赤霉素途径基因,包括赤霉素生物合成相关的8个GA20ox和1个GA3ox基因、降解相关的8个GA2ox基因、参与赤霉素感知的2个GID1基因以及信号转导的2个GID2基因和2个DELLA基因。拟南芥、水稻和毛竹的系统进化树和保守基序分析显示赤霉素的合成代谢与信号转导在这些物种中是高度保守的。利用外源赤霉素处理毛竹种子和幼苗,发现赤霉素能显著提高种子的萌发率和幼苗的茎秆伸长,并且有着最佳的作用浓度。在GA3处理后,毛竹体内赤霉素生物合成基因GA20ox和GA3ox表达量均下调而降解活性赤霉素的GA2ox基因表达量上调;赤霉素受体GID1和正调控基因GID2的转录水平显著提高而负调控基因DELLA的表达受到抑制。这些基因在竹笋茎秆的不同形态学位置表达差异明显,大部分赤霉素生物合成与降解的相关基因GA20ox、GA3ox和GA2ox以及赤霉素受体GID1和正调控基因GID2都在竹笋的形态学上端大量表达,而赤霉素信号转导的阻遏基因DELLA在笋体形态学底端大量积累而顶端基本不表达。     

Evolutionary conservation of plant gibberellin signalling pathway components

Background:  

Gibberellins (GA) are plant hormones that can regulate germination, elongation growth, and sex determination. They ubiquitously occur in seed plants. The discovery of gibberellin receptors, together with advances in understanding the function of key components of GA signalling in Arabidopsis and rice, reveal a fairly short GA signal transduction route. The pathway essentially consists of GID1 gibberellin receptors that interact with F-box proteins, which in turn regulate degradation of downstream DELLA proteins, suppressors of GA-controlled responses.     

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.     

Protein domain-domain interactions and requirements for the negative regulation of Arabidopsis CDC48/p97 by the plant ubiquitin regulatory X (UBX) domain-containing protein, PUX1

CDC48/p97 is an essential AAA-ATPase chaperone that functions in numerous diverse cellular activities through its interaction with specific adapter proteins. The ubiquitin regulatory X (UBX)-containing protein, PUX1, functions to regulate the hexameric structure and ATPase activity of AtCDC48. To characterize the biochemical mechanism of PUX1 action on AtCDC48, we have defined domains of both PUX1 and AtCDC48 that are critical for interaction and oligomer disassembly. Binding of PUX1 to AtCDC48 was mediated through a region containing both the UBX domain and the immediate C-terminal flanking amino acids (UBX-C). Like other UBX domains, the primary binding site for the UBX-C of PUX1 is the N(a) domain of AtCDC48. Alternative plant PUX protein UBX domains also bind AtCDC48 through the N terminus but were found not to be able to substitute for the action imparted by the UBX-C of PUX1 in hexamer disassembly, suggesting unique features for the UBX-C of PUX1. We propose that the PUX1 UBX-C domain modulates a second binding site on AtCDC48 required for the N-terminal domain of PUX1 to interact with and promote dissociation of the AtCDC48 hexamer. Utilizing Atcdc48 ATP hydrolysis and binding mutants, we demonstrate that PUX1 binding was not affected but that hexamer disassembly was significantly influenced by the ATP status of AtCDC48. ATPase activity in both the D1 and the D2 domains was critical for PUX1-mediated AtCDC48 hexamer disassembly. Together these results provide new mechanistic insight into how the hexameric status and ATPase activity of AtCDC48 are modulated.     

Chemical screening and development of novel gibberellin mimics

Gibberellin (GA) plays versatile roles in the regulation of plant growth and development and therefore is widely used as a regulator in agriculture. We performed a chemical library screening and identified a chemical, named 67D, as a stimulator of seed germination that was suppressed by paclobutrazol (PAC), a GA biosynthesis inhibitor. In vitro binding assays indicated that 67D binds to the GID1 receptor. Further studies on the structure–activity relationship identified a chemical, named chemical 6, that strongly promoted seed germination suppressed by PAC. Chemical 6 was further confirmed to promote the degradation of RGA (for repressor of ga1-3), a DELLA protein, and suppress the expression levels of GA3ox1 in the same manner as GA does. 67D and its analogs are supposed to be agonists of GID1 and are expected to be utilized in agriculture and basic research as an alternative to GA.     

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.     

Understanding gibberellic acid signaling--are we there yet?

The phytohormone gibberellic acid (GA) controls important aspects of plant growth such as seed germination, elongation growth, and flowering. The key components of the GA signaling pathway have been identified over the past 10 years. The current view is that GA binds to a soluble GID1 receptor, which interacts with the DELLA repressor proteins in a GA-dependent manner and thereby induces DELLA protein degradation via the E3 ubiquitin ligase SCF(GID2/SLY1). GA-dependent growth responses can generally be correlated with and be explained by changes in DELLA repressor abundance, where the DELLA repressor exerts a growth restraint that is relieved upon its degradation. However, it is obvious that other mechanisms must exist that control the activity of this pathway. This review discusses recent advances in the understanding of GA signaling, of its homeostasis, and of its cross-talk with other signaling pathways.     

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins regulate gibberellin signaling and photomorphogenesis in Arabidopsis

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. In Arabidopsis (Arabidopsis thaliana), cryptochrome 1 (CRY1) mediates blue light-induced photomorphogenesis, which is characterized by reduced hypocotyl elongation and enhanced anthocyanin production, whereas gibberellin (GA) signaling mediated by the GA receptor GA-INSENSITIVE DWARF1 (GID1) and DELLA proteins promotes hypocotyl elongation and inhibits anthocyanin accumulation. Whether CRY1 control of photomorphogenesis involves regulation of GA signaling is largely unknown. Here, we show that CRY1 signaling involves the inhibition of GA signaling through repression of GA-induced degradation of DELLA proteins. CRY1 physically interacts with DELLA proteins in a blue light-dependent manner, leading to their dissociation from SLEEPY1 (SLY1) and the inhibition of their ubiquitination. Moreover, CRY1 interacts directly with GID1 in a blue light-dependent but GA-independent manner, leading to the inhibition of the interaction between GID1 with DELLA proteins. These findings suggest that CRY1 controls photomorphogenesis through inhibition of GA-induced degradation of DELLA proteins and GA signaling, which is mediated by CRY1 inhibition of the interactions of DELLA proteins with GID1 and SCF^SLY1, respectively.

Blue light-dependent interactions of CRY1 with GID1 and DELLA proteins inhibit gibberellin (GA)-induced degradation of DELLA proteins to regulate GA signaling and photomorphogenesis.     

Plant UBX domain-containing protein 1, PUX1, regulates the oligomeric structure and activity of arabidopsis CDC48

p97/CDC48 is a highly abundant hexameric AAA-ATPase that functions as a molecular chaperone in numerous diverse cellular activities. We have identified an Arabidopsis UBX domain-containing protein, PUX1, which functions to regulate the oligomeric structure of the Arabidopsis homolog of p97/CDC48, AtCDC48, as well as mammalian p97. PUX1 is a soluble protein that co-fractionates with non-hexameric AtCDC48 and physically interacts with AtCDC48 in vivo. Binding of PUX1 to AtCDC48 is mediated through the UBX-containing C-terminal domain. However, disassembly of the chaperone is dependent upon the N-terminal domain of PUX1. These findings provide evidence that the assembly and disassembly of the hexameric p97/CDC48 complex is a dynamic process. This new unexpected level of regulation for p97/CDC48 was demonstrated to be critical in vivo as pux1 loss-of-function mutants display accelerated growth relative to wild-type plants. These results suggest a role for AtCDC48 and PUX1 in regulating plant growth.     

Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.     

Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant

The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.     

Overexpression of a GmGBP1 ortholog of soybean enhances the responses to flowering, stem elongation and heat tolerance in transgenic tobaccos

Soybean is a typical short-day crop, and its photoperiodic and gibberellin (GA) responses for the control of flowering are critical to seed yield. The GmGBP1 mRNA abundance in leaves was dramatically increased in short-days (SDs) compared to that in long-days in which it was consistently low at all time points from 0 to 6 days (days after transfer to SDs). GmGBP1 was highly expressed in leaves and exhibited a circadian rhythm in SDs. Ectopic overexpression of GmGBP1 in tobaccos caused photoperiod-insensitive early flowering by increasing NtCO mRNA levels. GmGBP1 mRNA abundance was also increased by GAs. Transgenic GmGBP1 overexpressing (-ox) tobacco plants exhibited increased GA signaling-related phenotypes including flowering and plant height promotion. Furthermore, the hypocotyl elongation, early-flowering and longer internode phenotypes were largely accelerated by GA₃ application in the GmGBP1-ox tobacco seedlings. Being consistent, overexpression of GmGBP1 resulted in significantly enhanced GA signaling (evidenced suppressed expression of NtGA20ox) both with and without GA treatments. GmGBP1 was a positive regulator of both photoperiod and GA-mediated flowering responses. In addition, GmGBP1-ox tobaccos were hypersensitive to ABA, salt and osmotic stresses during seed germination. Heat-inducible GmGBP1 also enhanced thermotolerance in transgenic GmGBP1-ox tobaccos during seed germination and growth. GmGBP1 protein was localized in the nucleus. Analyses of a series of 5′-deletions of the GmGBP1 promoter suggested that several cis-acting elements, including P-BOX, TCA-motif and three HSE elements necessary to induce gene expression by GA, salicic acid and heat stress, were specifically localized in the GmGBP1 promoter region.     

The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis

Gibberellic acid (GA) promotes seed germination, elongation growth, and flowering time in plants. GA responses are repressed by DELLA proteins, which contain an N-terminal DELLA domain essential for GA-dependent proteasomal degradation of DELLA repressors. Mutations of or within the DELLA domain of DELLA repressors have been described for species including Arabidopsis thaliana, wheat (Triticum aestivum), maize (Zea mays), and barley (Hordeum vulgare), and we show that these mutations confer GA insensitivity when introduced into the Arabidopsis GA INSENSITIVE (GAI) DELLA repressor. We also demonstrate that Arabidopsis mutants lacking the three GA INSENSITIVE DWARF1 (GID1) GA receptor genes are GA insensitive with respect to GA-promoted growth responses, GA-promoted DELLA repressor degradation, and GA-regulated gene expression. Our genetic interaction studies indicate that GAI and its close homolog REPRESSOR OF ga1-3 are the major growth repressors in a GA receptor mutant background. We further demonstrate that the GA insensitivity of the GAI DELLA domain mutants is explained in all cases by the inability of the mutant proteins to interact with the GID1A GA receptor. Since we found that the GAI DELLA domain alone can mediate GA-dependent GID1A interactions, we propose that the DELLA domain functions as a receiver domain for activated GA receptors.     

植物赤霉素生物合成和信号传导的分子生物学

赤霉素 (GAs)在植物的种子萌发、茎的伸长和花的发育等许多方面起着非常重要的作用。最近几年 ,对GA生物合成及其信号传导途径相关基因的研究取得了惊人的进展。这些进展促进了对其生物合成及其信号传导途径的认识。GA生物合成相关基因的表达受到多种内源和外源因子的调控 ,其中研究较多的是发育阶段、激素水平和光信号等内源及环境因子的调控。GA信号传导通常处于抑制状态 ,GA信号通过去抑制作用激活该传导途径而促进GA刺激植物生长和发育。     

赤霉素作用机理的分子基础与调控模式研究进展

赤霉素(gibberellins或gibberellic acid, GA)作为植物生长的必需激素之一, 调控植物生长发育的各个方面, 如: 种子萌发, 下胚轴的伸长, 叶片的生长和植物开花时间等。近年来随着植物功能基因组学的进一步发展, 有关赤霉素生物合成及其调控, 赤霉素信号转导途径, 以及赤霉素与其他激素和环境因子的互作等领域的研究取得了较大的进展。本文综述了赤霉素生物合成的生物学途径及其调控研究; GA信号转导通道的研究进展, 特别是DELLA蛋白阻遏植物生长发育的分子机理和GA解除阻遏作用(derepress)的分子模型; GA受体研究的新进展; 探讨GA与其它激素之间的相互作用, 以及植物在应答环境过程中的作用。     

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.     

植物赤霉素生物合成和信号传导的分子生物学

赤霉素(GAs)在植物的种子萌发、茎的伸长和花的发育等许多方面起着非常重要的作用。最近几年,对GA生物合成及其信号传导途径相关基因的研究取得了惊人的进展。这些进展促进了对其生物合成及其信号传导途径的认识。GA生物合成相关基因的表达受到多种内源和外源因子的调控, 其中研究较多的是发育阶段、激素水平和光信号等内源及环境因子的调控。GA信号传导通常处于抑制状态, GA信号通过去抑制作用激活该传导途径而促进GA刺激植物生长和发育。