紫外线强烈诱导的谷胱甘肽转移酶基因的功能鉴定

植物谷胱甘肽转移酶（glutathione S-transferases,GSTs)基因家族在逆境反应和植物生长发育过程中都起着非常重要的作用。为了阐明GST在紫外辐射下是否对植物有保护作用,以紫外强烈诱导表达的GST、cDNA为探针,筛选拟南芥cDNA文库,获得了这种GST的全长cDNA;利用此cDNA构建植物表达载体,并通过农杆菌介导法转化拟南芬,使其在拟南芥中得到大量表达;通过对转基因植株的紫外辐射耐性分析,证实了该GST的过量表达可明显增强拟南芥对紫外辐射损伤作用的耐受性。     

Identifying functional residues in Arabidopsis thaliana zeta class glutathione S-transferase through screening inactive point mutants

The functional residues of z-class glutathione S-transferase were identified by screening inactive point mutants from a random mutagenesis library. First, a random mutant library was constructed using error-prone polymerase chain reaction, and then candidate inactive mutants were screened by a high-throughput colorimetric assay. Twenty-five mutants were obtained, and 12 that formed inclusion bodies were discarded. The remaining 13 mutants that expressed soluble protein were used for accurate quantification of enzymatic activity and sequencing. The mutants W15R, C19Y, R22H/K83E, P61S, S73P, S109P, and Q112R were found to have activity lower than 1% of the wild-type and were considered as “inactive mutants”, whereas the mutants K83E, Q102R, and L147F still have a large fraction of the activity and were thus considered as “partially inactivated mutants”. Molecular modeling experiments disclosed that mutations resulting in inactivation of the enzyme were found in or near the binding pocket, whereas mutations resulting in partial inactivation were distant from both substrates. The role of the residue Ser73 in the enzyme was verified by site-directed mutagenesis. The result suggested that screening inactive point mutants from a random mutagenesis library is an efficient way of identifying functional residues in enzymes.     

Evolution of the Catalytic Activity of Arabidopsis thaliana Glutathione Transferase Zeta Class-1 by Saturation Mutagenesis

Saturation mutagenesis was performed on three non-catalytic residues, Asn71, Leu108, and Gly177, in and near the active site of Arabidopsis thaliana GSTZ-1 (AtGSTZ-1). Forty unique mutants with more than 10% activity increases, were obtained. Of these, 12 resulted in large activity improvement and were purified for further characterization. Remarkably, 11 of them contained mutations at Leu108, suggesting that Leu108 plays an important role in dichloroacetic acid-dechlorinating (DCA-DC) activity. Kinetic analysis revealed that multiple mutations at these residues increased k _cat/K _m toward DCA and GSH by as much as 2.2- and 5.8-fold, respectively. Since the catalytic residues were not involved in mutagenesis, the activity enhancements were presumably due to structural change in the active site rather than to a change in catalysis. Our results also suggest that the specific shape of the active site in AtGSTZ-1 is essential to its unique DCA-DC activity.     

Characterisation of a zeta class glutathione transferase from Arabidopsis thaliana with a putative role in tyrosine catabolism

A glutathione transferase (GST) similar to zeta GSTs in animals and fungi has been cloned from Arabidopsis thaliana using RT-PCR. The Arabidopsis zeta GST (AtGSTZ1) was expressed in Escherichia coli as his-tagged polypeptides, which associated together to form the 50-kDa AtGSTZ1-1 homodimer. Following purification, AtGSTZ1-1 was assayed for a range of activities and compared with other purified recombinant plant GSTs from the phi, tau, and theta classes. AtGSTZ1-1 differed from the other GSTs in showing no glutathione conjugating activity toward xenobiotics and no glutathione peroxidase activity toward organic hydroperoxides. Uniquely among the plant GSTs, AtGSTZ1-1 showed activity as a maleylacetone isomerase (MAI). This glutathione-dependent reaction is analogous to the cis-trans isomerization of maleylacetoacetate to fumarylacetoacetate, which occurs in the course of tyrosine catabolism to acetoacetate and fumarate. Thus, rather than functioning as a conventional GST, AtGSTZ1-1 appears to be involved in tyrosine degradation. In addition to the MAI activity, the AtGSTZ1-1 also catalyzed the glutathione-dependent dehalogenation of dichloroacetic acid to glyoxylic acid. This latter activity was used to demonstrate the presence of functional AtGSTZ1-1 inplanta.     

Oxidative Stimulation of Glutathione Synthesis in Arabidopsis thaliana Suspension Cultures

A system based on Arabidopsis thaliana suspension cultures was established for the analysis of glutathione (GSH) synthesis in the presence of hydrogen peroxide. Mild oxidative stress was induced by use of the catalase inhibitor, aminotriazole, and its development was monitored by measurement of the oxidative inactivation of aconitase. Addition of 2 mM aminotriazole resulted in a 25% decrease in activity of aconitase over 4 h. During the subsequent 10 h, no further decrease in aconitase activity was measured despite a sustained inhibition of catalase. In combination with our failure to detect significant increases in the level of lipid peroxidation, another marker indicative of oxidative injury, these data suggest that although hydrogen peroxide initially leaked into the cytosol, its accumulation was limited by a cytosolic catalase-independent mechanism. A 4-fold increase in the level of GSH, which was almost exclusively in the reduced form, was observed under the same treatment. To determine to what extent this increase in reduced GSH played a role in limiting the accumulation of hydrogen peroxide in the cytosol, we inhibited GSH synthesis with buthionine sulfoximine (BSO), a specific inhibitor of [gamma]-glutamylcysteine synthetase. No significant oxidative injury was detected as a result of treatment with 50 [mu]M BSO alone, and furthermore, this treatment had no effect on cell viability, However, addition of 2 mM aminotriazole to cells preincubated with 50 [mu]M BSO for 15 h led to a rapid loss of aconitase activity (75% in 4 h), and significant accumulation of products of lipid peroxidation. Within 72 h, cell viability was lost completely. After removal of BSO from the growth medium, GSH levels recovered to normal over a period of 20 h. Addition of 2 mM aminotriazole to cells at different time points during this recovery period demonstrated a strong correlation between the level of reduced GSH and the degree of protection against oxidative injury. These data strongly suggest that the induction of GSH synthesis by an oxidative stimulus plays a crucial role in determining the susceptibility of cells to oxidative stress.     

Saving Insertional Recessive Lethal Mutants of Arabidopsis thaliana

A group of 13 recessive lethal mutants was selected on the basis of the collection of Arabidopsis thaliana transgenic plants with insertions of T-DNA vector plasmid pLD3 or pPCVRN4, which was produced by agrobacterial transformation of germinating seeds. The use of media containing exogenous hormones made it possible to compensate the lethal effect, identify phenotypes, and characterize six lines of recessive lethal germlings using genetic and molecular-genetic methods.     

Establishing gene function by mutagenesis in Arabidopsis thaliana

The nuclear genome of Arabidopsis thaliana was sequenced to near completion a few years ago, and ahead lies the challenge of understanding its meaning and discerning its potential. How many genes are there? What are they? What do they do? Computer algorithms combined with genome array technologies have proven efficient in addressing the first two questions as shown in a recent report ( Yamada et al., 2003 ). However, assessing the function of every gene in every cell will require years of careful analyses of the phenotypes caused by mutations in each gene. Current progress in generating large numbers of molecular markers and near‐saturation insertion mutant collections has immensely facilitated functional genomics studies in Arabidopsis. In this review, we focus on how gene function can be revealed through the analysis of mutants by either forward or reverse genetics. These mutants generally fall into two distinct classes. The first class typically includes point mutations or small deletions derived from chemical or fast neutron mutagenesis whereas the second class includes insertions of transferred‐DNA or transposon elements. We describe the current methods that are used to identify the gene corresponding to these mutations, which can then be used as a probe to further dissect its function.     

Characterization of Adaptation in Phototropism of Arabidopsis thaliana

Phototropic curvature has been measured for etiolated Arabidopsis thaliana seedlings with and without a preirradiation. A bilateral preirradiation with 450-nm light at a fluence greater than about 0.1 micromole per square meter causes a rapid desensitization to a subsequent 450-nanometer unilateral irradiation at 0.5 micromole per square meter. Following a refractory period, the capacity to respond phototropically recovers to the predesensitization level, and the response is then enhanced. The length of the refractory period is between 10 and 20 minutes. Both the time needed for recovery and the extent of enhancement increase with increasing fluence of the bilateral preirradiation. Based on the relative spectral sensitivities of desensitization and enhancement, these responses can be separated. Desensitization is induced by blue light but not by red light. Enhancement, however, is induced by both blue and red light. Thus, enhancement can be induced without desensitization but not vice versa. Both desensitization and enhancement affect only the magnitude of the response and do not affect the fluence threshold.     

Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana

Glucosinolates are secondary metabolites involved in pathogen and insect defense of cruciferous plants. Although seeds and vegetative tissue often have very different glucosinolate profiles, few genetic factors that determine seed glucosinolate accumulation have been identified. An HPLC-based screen of 5500 mutagenized Arabidopsis thaliana lines produced 33 glucosinolate mutants, of which 21 have seed-specific changes. Five of these mutant lines, representing three genetic loci, are compromised in the biosynthesis of benzoyloxyglucosinolates, which are only found in seeds and young seedlings of A. thaliana. Genetic mapping and analysis of T-DNA insertions in candidate genes identified BZO1 (At1g65880), which encodes an enzyme with benzoyl-CoA ligase activity, as being required for the accumulation of benzoyloxyglucosinolates. Long-chain aliphatic glucosinolates are elevated in bzo1 mutants, suggesting substrate competition for the common short-chain aliphatic glucosinolate precursors. Whereas bzo1 mutations have seed-specific effects on benzoyloxyglucosinolate accumulation, the relative abundance of 3-benzoyloxypropyl- and 4-benzoyloxybutylglucosinolates depends on the maternal genotype.     

拟南芥谷胱甘肽S-转移酶Zeta类进化酶的获得及其特性分析

拟南芥谷胱甘肽S-转移酶Zeta类(AtGSTZ)是一种与细胞代谢和环境净化密切相关的多功能酶.应用易错PCR和多轮DNA洗牌技术构建了AtGSTZ随机突变文库;再利用pH指示剂颜色改变法对突变文库进行筛选,获得了9个二氯乙酸脱氯活性提高的突变子.其中,NN23含25个氨基酸突变,比活力提高120%,NN20含24个氨基酸突变,比活力提高102%,EC1含2个氨基酸突变,比活力提高47%,其他6个为单点突变,比活力分别提高9%～60%.酶学分析显示,所有进化酶对底物二氯乙酸的催化效率和对谷胱甘肽的亲和力以及个别进化酶的复性能力都得到不同程度的提高,但热稳定性均没有明显改善.同时,对一系列与AtGSTZ空间折叠及催化活性相关位点进行了讨论.     

Characterization of a CENP-C homolog in Arabidopsis thaliana

Centromere protein C (CENP-C) is a component of the kinetochore essential for correct segregation of sister chromatids in mammals. In Arabidopsis thaliana, a single-copy gene encoding a protein homologous to CENP-C has been found by homology in the whole-genome sequence. To investigate the CENP-C homolog (AtCENP-C), we cloned cDNAs by RT-PCR and determined its full-length coding sequence. Antibodies against the synthetic peptide for the C-terminal residues of AtCENP-C detected a polypeptide in Arabidopsis cell extracts on western blots. Immunofluorescence labeling with the antibodies and fluorescence in situ hybridization demonstrated clearly that AtCENP-C is present at the centromeric regions throughout the cell cycle.     

Characterization of two Arabidopsis thaliana glutathione S-transferases

Glutathione S-transferases (GST) are multifunctional proteins encoded by a large gene family, divided on the basis of sequence identity into phi, tau, theta, zeta and lambda classes. The phi and tau classes are present only in plants. GSTs appear to be ubiquitous in plants and are involved in herbicide detoxification and stress response, but little is known about the precise role of GSTs in normal plant physiology and during biotic and abiotic stress response. Two cDNAs representing the two plant classes tau and phi, AtGSTF9 and AtGSTU26, were expressed in vitro and the corresponding proteins were analysed. Both GSTs were able to catalyse a glutathione conjugation to 1-chloro-2,4-dinitrobenzene (CDNB), but they were inactive as transferases towards p-nitrobenzylchloride (pNBC). AtGSTF9 showed activity towards benzyl isothiocyanate (BITC) and an activity as glutathione peroxidase with cumene hydroperoxide (CumHPO). AtGSTU26 was not active as glutathione peroxidase and towards BITC. RT-PCR analysis was used to evaluate the expression of the two genes in response to treatment with herbicides and safeners, chemicals, low and high temperature. Our results reveal that AtGSTU26 is induced by the chloroacetanilide herbicides alachlor and metolachlor and the safener benoxacor, and after exposure to low temperatures. In contrast, AtGSTF9 seems not to be influenced by the treatments employed.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

Isolation and initial characterization of virescent mutants of Arabidopsis thaliana

In higher plants, development of the chloroplasts must be coordinated with development of the leaf. In order to study the signals that synchronize these two developmental processes, we have isolated virescent (delayed in greening) mutants of Arabidopsis thaliana. Two such mutants that have pale-green young leaves which gradually green more fully during leaf maturation have been partially characterized. The two, vir1 and vir2, are due to separate nuclear recessive mutations. The pale leaves of vir1 and vir2 both had reduced 77°K fluorescence emission at 730–734 nm relative to that at 686–687 nm, indicating a reduction in the relative amount of LHC I compared to WT. As leaves greened, the amount of LHC I increased to near wildtype levels. The shift in the fluorescence emission peak from 730 nm to 734 nm, characteristic of maturing LHC I, was seen for vir1, but not vir2, suggesting that vir1 is a regulatory mutant while vir2 may be defective in a specific aspect(s) of LHC I function.Abbreviations D dark - EMS ethyl methanesulfonate - er erecta - gl1 glabrous1 - L light - LHC I light harvesting complex of Photosystem I - LHC II light harvesting complex of Photosystem II - M2 second generation of mutagenized seed - M3 third generation of mutagenized seed - vir virescent - WT wildtype     

Characterization of Arabidopsis thaliana telomeres isolated in yeast.

In an effort to learn more about the genomic organization of chromosomal termini in plants we employed a functional complementation strategy to isolate Arabidopsis thaliana telomeres in the yeast, Saccharomyces cerevisiae. Eight yeast episomes carrying A. thaliana telomeric sequences were obtained. The plant sequences carried on two episomes, YpAtT1 and YpAtT7, were characterized in detail. The telomeric origins of YpAtT1 and YpAtT7 insert DNAs were confirmed by demonstrating that corresponding genomic sequences are preferentially degraded during exonucleolytic digestion. The isolated telomeric restriction fragments contain G-rich repeat arrays characteristic of A. thaliana telomeres, as well as subterminal telomere-associated sequences (TASs). DNA sequence analysis revealed the presence of variant telomeric repeats at the centromere-proximal border of the terminal block of telomere repeats. The TAS flanking the telomeric G-rich repeat in YpAtT7 corresponds to a repetitive element present at other A. thaliana telomeres, while more proximal sequences are unique to one telomere. The YpAtT1 TAS is unique in the Landsberg strain of A. thaliana from which the clone originated; however, the Landsberg TAS cross-hybridizes weakly to a second telomere in the strain Columbia. Restriction analysis with cytosine methylation-sensitive endonucleases indicated that both TASs are highly methylated in the genome.     

Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana

The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.     

Characterization of sphingolipid long-chain base kinase in Arabidopsis thaliana

Sphingolipid long-chain base (LCB) kinase catalyses the phosphorylation of sphingolipid LCB to form LCB 1-phosphate. Based on sequence identity to a murine sphingosine kinase (murine SPHK1a), we isolated and characterized a LCB kinase-like cDNA in Arabidopsis thaliana. The deduced amino acid sequence of the homologous cDNA shows several regions that are highly conserved in LCB kinases from mouse, yeast, human and Caenorhabditis elegans. These regions are not similar to those of other known kinase families. For a functional identification, the homologous cDNA from A. thaliana was expressed in Escherichia coli, and LCB kinase activity was measured. The recombinant AtLcbk1 protein was found to utilize ATP and sphinganine. These results indicate the first identification of a gene coding for a LCB kinase in plants.     

Characterization of DUF724 gene family in Arabidopsis thaliana

Eighteen genes that encode the proteins with highly conserved Domain of Unknown Function 724 (DUF724) and Agenet domains were identified in plant taxa but not in animals and fungi. They are actively expressed in many different plant tissues, implying that they may play important roles in plants. Here we report the characterization of their structural organizations, expression patterns and protein–protein interactions. In Arabidopsis, the DUF724 genes were expressed in roots, leaves, shoot apical meristems, anthers and pollen grains. At least seven of the ten Arabidopsis DUF724 proteins (AtDuf1 to AtDuf10) were localized in nucleus. Three of them (AtDuf3, AtDuf5 and AtDuf7) may form homodimers or homopolymers, but did not interact with other members of the same family. Together with the significant similarity between DUF724 proteins and FMRP in the fundamental and characteristic molecular architecture, the results implies the DUF724 gene family may be involved in the polar growth of plant cells via transportation of RNAs.