The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.     

DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus.

We have identified a mutation at the DAG locus of Antirrhinum majus which blocks the development of chloroplasts to give white leaves with green revertant sectors. The green areas contain normal chloroplasts whereas the white areas have small plastids that resemble proplastids. The cotyledons of dark-grown dag mutant seedlings have plastids which also resemble proplastids. The palisade cells in the white areas of dag mutant leaves also lack their characteristic columnar shape. The DAG locus was cloned by transposon tagging: DAG encodes a novel protein with a predicted Mr of 26k, which is targeted to the plastids. Cleavage of its predicted transit peptide gives a mature protein of Mr 20k. Screening of databases and analysis of Southern blots gave evidence that DAG belongs to a protein family with homology to several proteins of unknown function from plants. Expression of DAG is required for expression of nuclear genes affecting the chloroplasts, such as CAB and RBCS, and also for expression of the plastidial gene RPOB encoding the plastidial RNA polymerase beta subunit, indicating that it functions very early in chloroplast development.     

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.     

Tam3 produces a suppressible allele of the DAG locus of Antirrhinum majus similar to Mu-suppressible alleles of maize

Tam3 from Antirrhinum majus belongs to the Ac/Ds family of transposable elements. An allele of the DAG locus of Antirrhinum ( dag ::Tam3), which is required for chloroplast development and leaf palisade differentiation, has been generated by Tam3 insertion into the untranslated leader sequence of the gene. This allele gives rise to a cold-sensitive phenotype, where mutant tissue containing wild-type revertant somatic sectors is observed in the leaves of plants grown at 15°C, while leaves of plants grown at 25°C appear near wild-type. The temperature sensitivity of dag ::Tam3 results from expression of the DAG locus responding to the activity of the transposable element, the transposition of which is very sensitive to growing temperature. Genetic suppression of Tam3 transposition, using the STABILISER locus, also results in suppression of the dag mutant phenotype. dag ::Tam3 represents a Tam3-suppressible allele similar to those described for Mu transposons in maize. Suppression of the dag mutant phenotype in response to element inactivation appears to result from use of an alternative promoter at the 3' end of the Tam3 element. The production of suppressible alleles by an Ac-like element is discussed in relation to the mutagenic potential of plant transposons in producing complex genetic diversity.     

陆地棉SUPERMAN类似锌指蛋白基因的克隆与表达分析

锌指蛋白是生物体内数量最多的转录调控因子,它在动植物的生长发育中都起到十分重要的作用。SUPERMAN类锌指蛋白只含有1个锌指结构。我们根据这类蛋白的保守结构域设计简并引物,通过RT-PCR从棉花中获得了3个这个家族成员的EST,得到1个锌指蛋白基因的全长序列,该基因的编码区长744 bp,编码长248个氨基酸的多肽,其氨基酸序列与GenBank中登录的一个拟南芥RBE蛋白有40%的同源性。此基因被命名为GZFP。它含有保守的锌指结构并在多肽链的C-端具有富含亮氨酸的保守结构域,GZFP含有核定位信号并且没有内含子。GZFP基因在棉花花蕾、子房、花瓣和根中的表达量要高于木质部、韧皮部、叶片、纤维和种子。GZFP基因的表达量很低,在GenBank中没有任何和它同源的EST序列存在。对GZFP 5′侧翼区进行分析发现有数个花粉和根特异表达相关元件,4个与Dof蛋白作用的核心序列,4个与光诱导相关的元件。      

转基因PnDof30拟南芥非生物胁迫下的抗性分析

Dof(DNA-binding with one finger)转录因子是植物中特有的一类转录因子,是锌指蛋白家族中的一个具有众多成员的家族,氨基酸长度一般在200~400,含有非常保守的N端和较为多变的C端。已有研究表明,Dof转录因子家族在参与植物发育的多种生理途径和调节碳氮代谢、增加氮素的吸收与利用,提高植株抗逆能力中起着重要作用。为了探究小黑杨(Populus simonii×P.nigra)中Dof30基因的抗逆能力,本研究以转基因PnDof30拟南芥为研究对象,对干旱、盐和渗透胁迫后过表达PnDof30拟南芥株系L2和野生型拟南芥WT的生理指标进行比较。发现胁迫后拟南芥株系L2的种子萌发率、根长和鲜重等指标均高于WT;同时SOD、POD、脯氨酸含量高于WT,叶绿素和MDA含量下降;胁迫后L2中的PnDof30基因表达量显著提高。这些结果表明了PnDof30基因具有抗旱、耐盐和渗透胁迫的能力,对全面了解Dof转录因子的抗逆胁迫功能具有重要的意义。     

Functional analyses of the Dof domain, a zinc finger DNA-binding domain, in a pumpkin DNA-binding protein AOBP

AOBP, a DNA-binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA-binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA-binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA-binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.