Thioredoxin targets in Arabidopsis roots

The importance of redox‐regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol‐disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y‐type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy‐two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid‐dependent biosynthesis of isoprenoids as a new redox‐mediated process. The redox‐regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia‐lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT‐reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin

Thioredoxin (TRX) is a small multifunctional protein with a disulfide active site involved in redox regulation. To gain insight into the numerous proteins able to interact with thioredoxin in Arabidopsis thaliana, we have compared three different proteomic procedures. In the two first approaches targets present in a mixture of soluble leaf proteins were reduced by the cytosolic TRX h3, then the new thiols were labeled either with radioactive iodoacetamide allowing specific detection (first method) or with a biotinylated thiol-specific compound allowing selective retention on an avidin column (second method). The third method involved a chromatography on a mutated TRX h3 column, which is able to covalently trap potential targets. All together, the three approaches enabled us to propose 73 proteins as being TRX-linked, and involved in various processes. Methods 1 and 3 were not only efficient with respectively 47 and 41 potential targets, but also complementary as only 26% of the targets were identified by both procedures. The second method with only 12 proteins was less efficient. However, this approach, as well as the first one when coupled with differential labeling of the cysteine residues, could be more informative about the cysteines involved in the thiol-disulfide interchange.     

Comparative proteomic analysis of Arabidopsis thaliana roots between wild type and its salt-tolerant mutant

Two-dimensional electrophoresis (2-DE) showed the variation expression of Arabidopsis thaliana root proteins between wild type and its salt-tolerant mutant obtained from cobalt-60 γ ray radiation. Forty-six differential root protein spots were reproducibly presented on 2-DE maps, and 29 spots were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MS). Fifteen protein spots corresponding to 10 proteins, and 14 protein spots corresponding to 9 proteins were constitutively up-regulated and down-regulated in the salt-tolerant mutant root. Bioinformatic analysis indicated that those differential proteins might be involved in the regulation of redox homeostasis, nucleotide metabolism, signal transduction, stress response and defense, carbohydrate metabolism, and cell wall metabolism. Peroxidase 22 might be a versatile enzyme and might play dual roles in both cell wall metabolism and regulation of redox homeostasis. Our work provides not only new insights into salt-responsive proteins in root, but also the potential salt-tolerant targets for further dissection of molecular mechanism adapted by plants during salt stress.     

Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach

A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 μM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

蔗糖调节拟南芥花青素的生物合成

为了探讨糖在花青素合成过程中的调节作用,采用蔗糖和其代谢糖（葡萄糖 和果糖）组合处理拟南芥幼苗.实验结果表明,60 mmol/L蔗糖处理显著提高拟南芥 幼苗的花青素、还原糖含量,并上调花青素合成相关基因（CHS, FLS-1, DFR, LDOX, BANYULS）的转录,对叶绿素含量和UGT78D2基因的转录无影响;20 mmol/L 葡萄糖+20 mmol/L果糖处理,对花青素、叶绿素和还原糖的含量无影响,对花青素 合成相关基因转录影响不一;20 mmol/L蔗糖+20 mmol/L葡萄糖+20 mmol/L果糖处 理后,花青素和还原糖含量介于前两个处理之间,也上调花青素合成相关基因的转 录;但和蔗糖处理组相比,上调UGT78D2基因转录,下调FLS-1基因转录.在不同处 理组之间,花青素含量变化和还原糖含量变化趋势相同,有可能糖在调节花青素 合成的同时也调节还原糖含量.因此,蔗糖既可以通过蔗糖特异信号途径,也可以 和其代谢糖通过其他途径共同调节拟南芥花青素的生物合成.     

Islands of co-expressed neighbouring genes in Arabidopsis thaliana suggest higher-order chromosome domains

Biochemical and cytogenetic experiments have led to the hypothesis that eukaryotic chromatin is organized into a series of distinct domains that are functionally independent. Two expectations of this hypothesis are: (i) adjacent genes are more frequently co-expressed than is expected by chance; and (ii) co-expressed neighbouring genes are often functionally related. Here we report that over 10% of Arabidopsis thaliana genes are within large, co-expressed chromosomal regions. Two per cent (497/22,520) of genes are highly co-expressed (r > 0.7), about five times the number expected by chance. These genes fall into 226 groups distributed across the genome, and each group typically contains two to three genes. Among the highly co-expressed groups, 40% (91/226) have genes with high amino acid sequence similarity. Nonetheless, duplicate genes alone do not explain the observed levels of co-expression. Co-expressed, non-homologous genes are transcribed in parallel, share functions, and lie close together more frequently than expected. Our results show that the A. thaliana genome contains domains of gene expression. Small domains have highly co-expressed genes that often share functional and sequence similarity and are probably co-regulated by nearby regulatory sequences. Genes within large, significantly correlated groups are typically co-regulated at a low level, suggesting the presence of large chromosomal domains.     

拟南芥抗盐突变体的RAPD分析

以筛选得到可以稳定遗传的抗盐单基因突变体2^#和15^#以及野生型拟南芥为材料进行RAPD分析,150条引物中有3条引物在突变体之间扩增出多态性,不仅证明了DNA水平突变的发生,而且表明它们之间遗传背景相似,是一系列抗盐性不同的近似等位基因系。1条引物在突变体的扩增产物比在野生型的扩增产物多出一个大小约为1200bp的片段,初步认为该片段与抗盐的主效基因有关。     

拟南芥未知功能基因At4g22890 编码蛋白的叶绿体定位

蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890 编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208 bp 的DNA 片段, 与绿色荧光蛋白(GFP) 基因构建重组表达载体pMON530-cTP-GFP, 经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察, GFP 荧光仅在叶绿体中观察到, 表明所克隆的DNA 序列编码的多肽能够将At4g22890 编码蛋白质引导进入叶绿体, 由此推测该蛋白质为叶绿体蛋白质。     

A proteomic approach to identification of transmembrane proteins and membrane-anchored proteins of Arabidopsis thaliana by peptide sequencing.

A proteomic approach was developed for the identification of membrane-bound proteins of Arabidopsis thaliana. A subcellular fraction enriched in vacuolar membranes was prepared from 4-week-old plants and was washed with various agents to remove peripheral membrane proteins and contaminating soluble proteins. The remaining membrane-bound proteins were then subjected to proteomic analysis. Given that these proteins were resolved poorly by standard two-dimensional gel electrophoresis, we subjected them instead to SDS-polyacrylamide gel electrophoresis and to protein digestion within gel slices with lysylendopeptidase. The resulting peptides were separated by reverse-phase high-performance liquid chromatography and subjected to Edman sequencing. From the 163 peptide peaks analyzed, 69 peptide sequences were obtained, 64 of which were informative. The proteins corresponding to these peptide sequences were identified as belonging to 42 families, including two subfamilies, by comparison with the protein sequences predicted from annotation of the A. thaliana genome. A total of 34 proteins was identified definitively with protein-specific peptide sequences. Transmembrane proteins detected in the membrane fraction included transporters, channels, receptors, and unknown molecules, whereas the remaining proteins, categorized as membrane-anchored proteins, included small GTPases, GTPase binding proteins, heat shock protein 70-like proteins, ribosomal proteins, and unknown proteins. These membrane-anchored proteins are likely attached to membranes by hydrophobic anchor molecules or through tight association with other membrane-bound proteins. This proteomic approach has thus proved effective for the identification of membrane-bound proteins.     

光对拟南芥微管蛋白基因表达的负调节作用

白光处理后拟南芥白化苗中的微管蛋白基因mRNA都有不同程度降低。白化苗中TUBI的mRNA量很高,连续用白光处理由化苗2－6h,TUB1mRNA降低。分析拟南芥幼苗根、下胚轴中的RNA发现根中TUBI基因转录水平不受白光影响。具子叶的下胚轴中TUB1基因转录水平受白光抑制。白先对TUB1基因表达的负调节具有组织特异性。     

A critical role for disproportionating enzyme in starch breakdown is revealed by a knock-out mutation in Arabidopsis

Disproportionating enzyme (D-enzyme) is a plastidial alpha-1,4-glucanotransferase but its role in starch metabolism is unclear. Using a reverse genetics approach we have isolated a mutant of Arabidopsis thaliana in which the gene encoding this enzyme (DPE1) is disrupted by a T-DNA insertion. While D-enzyme activity is eliminated in the homozygous dpe1-1 mutant, changes in activities of other enzymes of starch metabolism are relatively small. During the diurnal cycle, the amount of leaf starch is higher in dpe1-1 than in wild type and the amylose to amylopectin ratio is increased, but amylopectin structure is unaltered. The amounts of starch synthesised and degraded are lower in dpe1-1 than in wild type. However, the lower amount of starch synthesised and the higher proportion of amylose are both eliminated when plants are completely de-starched by a period of prolonged darkness prior to the light period. During starch degradation, a large accumulation of malto-oligosaccharides occurs in dpe1-1 but not in wild type. These data show that D-enzyme is required for malto-oligosaccharide metabolism during starch degradation. The slower rate of starch degradation in dpe1-1 suggests that malto-oligosaccharides affect an enzyme that attacks the starch granule, or that D-enzyme itself can act directly on starch. The effects on starch synthesis and composition in dpe1-1 under normal diurnal conditions are probably a consequence of metabolism at the start of the light period, of the high levels of malto-oligosaccharides generated during the dark period. We conclude that the primary function of D-enzyme is in starch degradation.     

模式植物拟南芥开花时间分子调控研究进展

植物从营养生长到生殖生长的转变是开花发育的关键,在合适的时间开花对植物的生长和繁衍极为重要,植物开花时间的调控对农业生产发展意义重大。植物开花是由遗传因子和环境因子协同调节的一个复杂过程。近年来,对不同植物开花调控的研究,特别是对模式植物拟南芥（Arabidopsis thaliana（L.） Heynh.）的开花调控研究取得了显著进展,已探明开花时间分子调控的6条主要途径分别是光周期途径、春化途径、自主途径、温度途径、赤霉素途径和年龄途径。各遗传调控途径既相互独立又相互联系,构成一个复杂的开花调控网络。本文综述了模式植物拟南芥开花时间调控分子机制相关研究的最新进展,并对未来的研究进行了展望。     

Jasmonate biosynthesis in Arabidopsis thaliana--enzymes, products, regulation

Among the plant hormones jasmonic acid and related derivatives are known to mediate stress responses and several developmental processes. Biosynthesis, regulation, and metabolism of jasmonic acid in Arabidopsis thaliana are reviewed, including properties of mutants of jasmonate biosynthesis. The individual signalling properties of several jasmonates are described.     

我多组学关联分析揭示表观等位基因在拟南芥环境适应性进化中的作用及机制

表观等位基因一般是指仅由DNA甲基化差异引起的表达量不同的等位基因,对植物形态结构和各种生理过程具有重要影响。但自然条件下环境因素对植物表观等位基因的影响还不清楚,同时表观等位基因在植物环境适应性进化中的作用和机制还亟待探究。为了在全基组水平鉴定拟南芥(Arabidopsis thaliana)中与特定环境因素相关的表观等位基因,并分析它们参与拟南芥环境适应性进化的可能机制,本研究利用623株拟南芥生态型的转录组、甲基化组和种源地气候数据进行多组学关联分析,并同时进行了蛋白互作网络和基因富集分析。以春季和夏季降水量为例,本研究最终鉴定到5个基因(AGL36、AT2G34100、AT4G09360、LSU4和AT5G56910)可能具有相应的表观等位基因,基因内部或附近特定区域不同甲基化水平对它们的表达可能具有调控作用。其中与种子发育有关的印记基因AGL36首次被发现可能作为表观等位基因参与拟南芥环境适应性进化,其他4个基因均与生物胁迫响应有关。自然条件下降水量能影响当地病虫害的严重程度,而DNA甲基化能通过影响这4个免疫基因的表达来影响拟南芥免疫能力。在长期演化过程中有利于个体适应当地降水模式的表观等位基因受到正向选择,这可能是这些表观等位基因参与拟南芥降水适应性进化的潜在机制。通过蛋白互作网络、GO功能分析和KEGG通路分析,本研究还首次发现LSU4可能与LSU基因家族其他成员一样参与硫代谢网络,并通过影响硫代葡萄糖苷代谢参与拟南芥生物胁迫响应。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。