Association of glutathione with flowering in Arabidopsis thaliana

In order to study the relationship between GSH and flowering, wild-type and late-flowering mutant, fca-1, of Arabidopsis thaliana were treated with L-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, under long-day conditions. BSO treatment of the fca-1 mutant starting at 17 d after imbibition promoted flowering. However, when the treatment was started at 12 d after imbibition, BSO treatment at 10(-4) M resulted in an inhibition of flowering. This inhibitory effect of BSO on flowering was abolished by GSH treatment at 10(-4) M, although GSH treatment at an increased concentration of 10(-3) M clearly delayed flowering. In contrast, BSO treatment of wild-type plants starting at 12 d after imbibition promoted flowering, whose effect was abolished by GSH application. In the fca-1 mutant, whose endogenous GSH levels were high, chilling treatment lowered the GSH levels and promoted flowering, as was the case in the BSO treatment. An A. thaliana mutant, cad2-1, which has a defect in GSH biosynthesis also exhibited late flowering. The late-flowering phenotype of this mutant tended to be strengthened by BSO and abolished by GSH treatment. These results suggest that flowering is associated with the rate of GSH biosynthesis and/or the levels of GSH in A. thaliana.     

The first exon coding region of cystathionine gamma-synthase gene is necessary and sufficient for downregulation of its own mRNA accumulation in transgenic Arabidopsis thaliana.

Expression of the gene for cystathionine gamma-synthase (CGS), which catalyzes the key step of methionine biosynthesis, is feedback regulated at the level of mRNA stability. The first exon polypeptide of CGS is suggested to be involved in this regulation and amino acid sequence alterations caused by mto1 mutations in that region lead to an overaccumulation of CGS mRNA [Chiba et al. (1999) Science 286: 1371-1374]. Transgenic Arabidopsis thaliana harboring chimeric constructs in which wild-type or mto1 mutant CGS exon 1 are fused in-frame to reporter genes and driven by the cauliflower mosaic virus 35S RNA promoter were constructed. Studies with these transgenic lines demonstrated that the coding region of CGS exon 1 is necessary and sufficient for downregulation of its own mRNA accumulation in response to methionine application and that this region acts in cis in this process.     

Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression

We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes. The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite. Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate. Arabidopsis plants expressing the E. coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type. However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants. When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone. This arsenic remediation strategy should be applicable to a wide variety of plant species.     

amp1 - a mutant with high cytokinin levels and altered embryonic pattern, faster vegetative growth, constitutive photomorphogenesis and precocious flowering

amp1 , a mutant of Arabidopsis thaliana has a phenotype altered in three different aspects of plant development; spatial pattern, photomorphogenetic growth, and initiation of flowering. While fewer than 0.1% of the seedlings of wild-type plants are non-dicot as many as 20% of the seedlings of the amp1 mutant are tricot or tetracot. The rate of leaf initiation is faster and vegetative phyllotaxy is altered in amp1 . When grown in the dark amp1 seedlings show morphogenetic properties similar to light-grown wild-type plants: they do not form an apical hook, have hypocotyls shorter than wild-type plants and form etiolated true leaves. amp1 mutant flowers significantly earlier than congenic Amp1 plants. The mutant has six times more cytokinin than wild-type suggesting that endogenous cytokinin levels might play an important role in mediating these different developmental processes. AMP1 might code for a negative regulator of cytokinin biosynthesis, or may be required for the degradation of cytokinin.     

Sinapic acid ester metabolism in wild type and a sinapoylglucose-accumulating mutant of arabidopsis.

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.     

植物螯合肽合酶基因AtPCS2的表达调控

植物螯合肽合酶（pcs）受重金属离子激活,并以还原型谷胱甘肽为底物合成植物螯合肽（PCs）,在植物和真菌的重金属解毒机制中起重要作用．拟南芥基因组中有两个编码PCS的基因AtPCS1和AtPCS2,但AtPCS1单基因功能缺失即可导致相应的突变体cad1—3对镉高度敏感,其体内也检测不到PCs;而体外表达分析表明,AtPCS2具有完全的PCs合酶活性,预示植物体内可能存在AtPCS2的负向调控机制．基于该推测,构建了CaMV35S启动子驱动的AtPCS2基因编码区与c—Myc抗原标签融合的过表达载体．结果表叽在cadl-3的MV35S／AtPCS2：cMyc的异位表达株系中,AtPCS2的mRNA和蛋白都保持较高的表达量．不仅如此,AtPCS2具有植物螯合肽合成能力,并完全互补了cad1-3突变体的镉敏感性状．AtPCS2和EYFP的融合蛋白在细胞质有明显表达,在细胞核也检测到一定信号．以上结果表明,AtPCS2在植物体内可能主要受转录水平调控,而且可能具有调节PCs合成以外的其他生化功能．     

AtMRD1 and AtMRU1, two novel genes with altered mRNA levels in the methionine over-accumulating mto1-1 mutant of Arabidopsis thaliana

The mto1-1 mutant of Arabidopsis thaliana over-accumulates soluble methionine (Met) up to 40-fold higher than that in its Col-0 wild type. In order to identify genes regulated by altered Met concentrations, microarray analysis of gene expression in young rosettes and developing siliques of the mto1-1 mutant were performed. Expression of selected genes was then examined in detail in three developmental stages of the mto1-1 mutant using a combination of Northern hybridisation analysis and real-time PCR. Eight genes were identified that had altered mRNA accumulation levels in the mto1-1 mutant compared to that in wild-type plants. Three of the genes have known roles in plant development unrelated to amino acid biosynthesis. One other gene up-regulated specifically in mto1-1 rosettes shared similarity with the embryo-specific protein 3 (ATS3). Two novel genes, referred to as AtMRD1 and AtMRU1, were also identified that were expressed in a developmental manner in wild-type Col-0 and do not share sequence similarity with genes of known function. AtMRD1 was strongly down-regulated in both rosette and young silique tissues of the mto1-1 mutant. AtMRU1 was up-regulated approximately 3-fold in young mto1-1 rosettes and exhibited a developmental response to the mto1-1 mutation.     

Cytosolic γ-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.     

Screening for Arabidopsis mutants affected in the Nii gene expression using the Gus reporter gene

A mutant screen was developed to isolate Arabidopsis thaliana mutants affected in the regulation of the nitrate assimilation pathway. A fusion between the tobacco Nii1 gene (that encodes a foliar nitrite reductase involved in nitrate assimilation) and the Gus reporter gene was introduced into A. thaliana , and shown to be properly regulated by nitrate. Moreover, β -glucuronidase (GUS) activity in the transgenic plants was essentially detected in the cotyledons and leaves, showing that the organ-specific expression of the tobacco Nii1 gene was retained in Arabidopsis . M2 plantlets derived from mutagenized seeds homozygous for the Nii-Gus fusion were screened by histochemical staining of whole plates for GUS activity after growth on nitrate or glutamine. About 250 progenies were screened, leading to the isolation of plants showing an enhanced or reduced staining compared to the control non-mutagenized plants. Several mutants were analyzed for the transmission of the phenotype to the M3 generation, as well as for levels of GUS or nitrite reductase activities or mRNA levels. A major problem encountered during the screening was the high background of false positives that reproducibly showed altered GUS histochemical staining compared to control plants and did not, however, display any changes in GUS activity levels. One interesting family of mutants was isolated that overexpressed GUS activity and Nii mRNA in the absence of nitrate. These mutants turned out to be cnx mutants impaired in the molybdenum cofactor biosynthesis that is necessary for nitrate reductase activity. These results may indicate that active nitrate reductase is necessary for a correct regulation of nitrate assimilation genes by nitrate.     

UGT87A2, an Arabidopsis glycosyltransferase, regulates flowering time via FLOWERING LOCUS C

? Family 1 glycosyltransferases comprise the greatest number of glycosyltransferases found in plants. The widespread occurrence and diversity of glycosides throughout the plant kingdom underscore the importance of these glycosyltransferases. ? Here, we describe the identification and characterization of a late-flowering Arabidopsis (Arabidopsis thaliana) mutant, in which a putative family 1 glycosyltransferase gene, UGT87A2, was disrupted. The role and possible mechanism of UGT87A2 in the regulation of flowering were analyzed by molecular, genetic and cellular approaches. ? The ugt87a2 mutant exhibited late flowering in both long and short days, and its flowering was promoted by vernalization and gibberellin. Furthermore, the mutant flowering phenotype was rescued by the wild-type UGT87A2 gene in complementation lines. Interestingly, the expression of the flowering repressor FLOWERING LOCUS C was increased substantially in the mutant, but decreased to the wild-type level in complementation lines, with corresponding changes in the expression levels of the floral integrators and floral meristem identity genes. The expression of UGT87A2 was developmentally regulated and its protein products were distributed in both cytoplasm and nucleus. ? Our findings imply that UGT87A2 regulates flowering time via the flowering repressor FLOWERING LOCUS C. These data highlight an important role for the family 1 glycosyltransferases in the regulation of plant flower development.     

Arabidopsis CLP1-SIMILAR PROTEIN3, an ortholog of human polyadenylation factor CLP1, functions in gametophyte, embryo, and postembryonic development

Polyadenylation factor CLP1 is essential for mRNA 3'-end processing in yeast and mammals. The Arabidopsis (Arabidopsis thaliana) CLP1-SIMILAR PROTEIN3 (CLPS3) is an ortholog of human hCLP1. CLPS3 was previously found to be a subunit in the affinity-purified PCFS4-TAP (tandem affinity purification) complex involved in the alternative polyadenylation of FCA and flowering time control in Arabidopsis. In this article, we further explored the components in the affinity-purified CLPS3-TAP complex, from which Arabidopsis cleavage and polyadenylation specificity factor (CPSF) subunits AtCPSF100 and AtCPSF160 were found. This result implies that CLPS3 may bridge CPSF to the PCFS4 complex. Characterization of the CLPS3 mutant revealed that CLPS3 was essential for embryo development and important for female gametophyte transmission. Overexpression of CLPS3-TAP fusion caused a range of postembryonic development abnormalities, including early flowering time, altered phyllotaxy, and abnormal numbers and shapes of flower organs. These phenotypes are associated with the altered gene expression levels of FCA, WUS, and CUC1. The decreased ratio of FCA-beta to FCA-gamma in the overexpression plants suggests that CLPS3 favored the usage of FCA regular poly(A) site over the alternative site. These observations indicate that Arabidopsis CLPS3 might be involved in the processing of pre-mRNAs encoded by a distinct subset of genes that are important in plant development.     

Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock-controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.     

Evidence for a direct link between glutathione biosynthesis and stress defense gene expression in Arabidopsis

The mutant regulator of APX2 1-1 (rax1-1) was identified in Arabidopsis thaliana that constitutively expressed normally photooxidative stress-inducible ASCORBATE PEROXIDASE2 (APX2) and had >/=50% lowered foliar glutathione levels. Mapping revealed that rax1-1 is an allele of gamma-GLUTAMYLCYSTEINE SYNTHETASE 1 (GSH1), which encodes chloroplastic gamma-glutamylcysteine synthetase, the controlling step of glutathione biosynthesis. By comparison of rax1-1 with the GSH1 mutant cadmium hypersensitive 2, the expression of 32 stress-responsive genes was shown to be responsive to changed glutathione metabolism. Under photo-oxidative stress conditions, the expression of a wider set of defense-related genes was altered in the mutants. In wild-type plants, glutathione metabolism may play a key role in determining the degree of expression of defense genes controlled by several signaling pathways both before and during stress. This control may reflect the physiological state of the plant at the time of the onset of an environmental challenge and suggests that changes in glutathione metabolism may be one means of integrating the function of several signaling pathways.