Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase

A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.     

Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants

Summary. Serine acetyltransferase (SATase) and cysteine synthase (O-acetylserine (thiol)-lyase) (CSase) are committed in the final step of cysteine biosynthesis. Six cDNA clones encoding SATase have been isolated from several plants, e.g. watermelon, spinach, Chinese chive and Arabidopsis thaliana. Feedback-inhibition pattern and subcellular localization of plant SATases were evaluated. Two types of SATase that differ in their sensitivity to the feedback inhibition by l-cysteine were found in plants. In Arabidopsis, cytosolic SATase was inhibited by l-cysteine at a physiological concentration in an allosteric manner, but the plastidic and mitochondrial forms were not subjected to this feedback regulation. These results suggest that the regulation of cysteine biosynthesis through feedback inhibition may differ depending on the subcellular compartment. The allosteric domain responsible for l-cysteine inhibition was characterized, using several SATase mutants. The single change of amino acid residue, glycine-277 to cysteine, in the C-terminal region of watermelon SATase caused a significant decrease of the feedback-inhibition sensitivity of watermelon SATase. We made the transgenic Arabidopsis overexpressing point-mutated watermelon SATase gene whose product was not inhibited by l-cysteine. The contents of OAS, cysteine, and glutathione in transgenic Arabidopsis were significantly increased as compared to the wild-type Arabidopsis. Transgenic tobacco (Nicotiana tabacum) (F₁) plants with enhanced CSase activities both in the cytosol and in the chloroplasts were generated by cross-fertilization of two transgenic tobacco expressing either cytosolic CSase or chloroplastic CSase. Upon fumigation with 0.1 μL L⁻¹ sulfur dioxide, both the cysteine and glutathione contents in leaves of F₁ plants were increased significantly, but not in leaves of non-transformed control plants. These results indicated that both SATase and CSase play important roles in cysteine biosynthesis and its regulation in plants. Received November 27, 2001 Accepted December 21, 2001     

Differential expression of triplicate phosphoribosylanthranilate isomerase isogenes in the tryptophan biosynthetic pathway of Arabidopsis thaliana (L.) Heynh.

Phosphoribosylanthranilate isomerases (PAI) in the tryptophan biosynthetic pathway of Arabidopsis thaliana are encoded by a gene family. Expression patterns of each individual PAI isogene were investigated by analyzing expression of translation-fusions of promoter-β-glucuronidase (GUS) chimeras in transgenic plants. Quantification and histochemical staining of GUS activities expressed in PAI transgenic plants demonstrated that, first, expression of the three PAI isogenes was differentially regulated under normal growth conditions. Both PAI1 and PAI3 showed approximately 10-fold stronger expression than PAI2. Second, PAI isogenes differentially responded to environmental stresses such as ultraviolet irradiation and the abiotic elicitor silver nitrate. PAI2 displayed a stronger response to stresses than the other two PAI isogenes. Third, each individual PAI isogene was differentially expressed in a tissue- and cell-type-specific manner. Fourth, expression of PAI isogenes was coordinated to meet the requirement for normal growth and development of A. thaliana. Deletion of PAI1 is partially responsible for abnormal growth and development in the PAI deletion mutant trp6 as well as strong blue fluorescence in young leaves under ultraviolet irradiation. Received: 15 June 2000 / Accepted: 16 August 2000     

Molecular characterisation of a new mutant allele of the plastid phosphoglucomutase in Arabidopsis, and complementation of the mutant with the wild-type cDNA

Screening of transposon-associated mutants of Arabidopsis thaliana for altered starch metabolism resulted in the isolation of a mutant that did not accumulate starch in any tissue or at any developmental stage (starch-free mutant, stf1). Allelism tests with known mutants showed that stf1 represents a new mutant allele of the plastid isoform of the enzyme phosphoglucomutase (PGMp). The mutation was mapped to chromosome 5. An Arabidopsis EST that showed significant homology to the cytosolic isoform of phosphoglucomutase (PGM) from maize was able to complement the mutant phenotype. The Arabidopsis EST was transcribed and translated in vitro and the protein product was efficiently imported into isolated chloroplasts and processed to its mature form. The lack of starch biosynthesis in stf1 is accompanied by the accumulation of soluble sugars. The rate of CO₂ assimilation measured in individual leaves was substantially diminished only under conditions of high CO₂ and low O₂. Remarkably, stf1 exhibits an increase rather than a decrease in total leaf PGM activity, suggesting an induction of the cytosolic isoform(s) in the mutant. The substrate for PGM, glucose 6-phosphate, accumulated in stf1 during the day, resulting in 10-fold higher content than in the wild type at the end of the photoperiod. Received: 4 January 2000 / Accepted: 21 March 2000     

Overproduction of L-cysteine and L-cystine by expression of genes for feedback inhibition-insensitive serine acetyltransferase from Arabidopsis thaliana in Escherichia coli

Two cDNAs encoding feedback inhibition-insensitive serine acetyltransferases of Arabidopsis thaliana were expressed in the chromosomal serine acetyltransferase-deficient and L-cysteine non-utilizing Escherichia coli strain JM39-8. The transformants produced 1600 to 1700 mg l(-1) of L-cysteine and L-cystine from glucose. The amount of these amino acids produced per cell was 30 to 60% higher than that of an E. coli strain carrying mutant serine acetyltransferase less sensitive to feedback inhibition.     

Plastid transformation in Arabidopsis thaliana

Plastid transformation is reported in Arabidopsis thaliana following biolistic delivery of transforming DNA into leaf cells. Transforming plasmid pGS31A carries a spectinomycin resistance (aadA) gene flanked by plastid DNA sequences to target its insertion between trnV and the rps12/7 operon. Integration of aadA by two homologous recombination events via the flanking ptDNA sequences and selective amplification of the transplastomes on spectinomycin medium yielded resistant cell lines and regenerated plants in which the plastid genome copies have been uniformly altered. The efficiency of plastid transformation was low: 2 in 201 bombarded leaf samples. None of the 98 plants regenerated from the two lines were fertile. Received: 13 February 1998 / Revision received: 24 April 1998 / Accepted: 5 June 1998     

The essential role of the phosphorylated pathway of serine biosynthesis in Arabidopsis

In plants, 3 different pathways of serine biosynthesis have been described: the Glycolate pathway, which is associated with photorespiration, and 2 non-photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Serine Biosynthesis (PPSB) has been known since the 1950s, but has been studied relatively little, probably because it was considered of minor significance as compared with the Glycolate pathway. In the associated study¹, we described for the first time in plants the in vivo functional characterization of the PPSB, by targeting the phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Following a gain- and loss-of-function approach in Arabidopsis, we provided genetic and molecular evidence for the essential role of PSP1 for embryo and pollen development, and for proper root growth. A metabolomics study indicated that the PPSB affects glycolysis, the Krebs cycle, and the biosynthesis of several amino acids, which suggests that this pathway is an important link connecting metabolism and development. The mechanisms underlying the essential functions of PSP1 are discussed.     

Properties of serine：glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

The photorespiratory enzyme L-serine：glyoxylate amino- transferase （SGAT; EC 2.6.1.45） was purified from Arabidopsis thaliana leaves. The f＇mal enzyme was approximately 80 % pure as revealed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated by gel filtration chromato- graphy on Sephadex G-150 under non-denaturing conditions, mass spectrometry （matrix-assisted laser desorption/ ionization/time of flight technique） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum pH value was 9.2. The enzyme activity was inhibited by aminooxyacetate and β-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme＇s transaminating activity with L-alanine and glyoxylate as substrates was approximately 55 % of that observed with L-serine and glyoxylate. The lower Kmvalue （1.25 mM） for L-alanine, compared with that of other plant SGATs, and the kcat/Km（Ala） ratio being approxi- mately 2-fold higher than kcat/Km（Ser） suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant （Keq）, derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 mM for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1：1.8 for the native enzyme, whereas it was 1：7 for the recombinant SGAT. Native SGAT showed a much lower Km value for L-alanine compared to the recombinant enzyme.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

拟南芥白化突变体心口的基因定位与分析

EMS30是拟南芥经甲基磺酸乙酯（EMS）诱变得到的白化突变体。该突变体的叶绿体结构存在严重缺陷,同时伴随叶绿素缺失。遗传分析显示EMS30突变体的突变表型受隐性单基因控制。采用图位克隆的方法对EMS30突变基因进行定位的结果显示,该基因位于拟南芥第一条染色体的分子标记F21M12和F14N23之间的96kb区间内,该区间包含25个基因。通过生物信息学分析发现,该区间内有3个基因定位在叶绿体或与叶绿体发育相关。这些结果有助于该基因的克隆,为阐释叶绿体发育提供线索。     

Disposal of chloroplasts with abnormal function into the vacuole in Arabidopsis thaliana cotyledon cells

Summary. Autophagy is a process in which cell membrane rearrangement allows for the sequestration and degradation of part of the cytoplasm. Many protein components of the autophagic mechanism and their corresponding genes have been identified in yeast cells by molecular genetics, and this has enabled researchers to identify homologues of these genes in mammalian and plant systems. Autophagy is involved in the starvation response in which part of the cytoplasm is degraded in order to produce essential substrates to allow the cell to survive during extreme substrate-limiting conditions. However, autophagy may also be important as a quality control mechanism in normal cells. By screening Arabidopsis thaliana T-DNA insert mutants, we isolated an A. thaliana mutant that lacks the AtTIC40 gene and found that the cotyledon cells of this mutant contained undeveloped plastids. Moreover, many toluidine-stained particulate structures were found in the vacuoles of these mutant cells. The images from electron microscopy suggested that some of these particulate structures were partially degraded chloroplasts. Furthermore, oil bodies were found in the cotyledon cells of mutant and wild-type plants, which suggests that the mutant seedlings were not starved under the experimental conditions. These results may indicate that under nutrient-sufficient conditions, plant cells remove abnormal plastids by autophagy and that this mechanism is involved in the quality control of organelles.Present address: BioResource Center, Tsukuba Institute, Institute of Physical and Chemical Research (RIKEN), Tsukuba, Japan.Present address: Genomics Sciences Center, Yokohama Institute, Institute of Physical and Chemical Research (RIKEN), Yokohama, Japan.Correspondence and reprints: School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.     

拟南芥低磷胁迫反应分子机理研究的最新进展

本文综述了拟南芥低磷(Pi)胁迫反应分子机理的最新研究进展,重点介绍了低磷胁迫反应中的SUMOylation途径、转录因子在低磷反应中的功能、Pi平衡调节机制以及磷脂酶在Pi的循环利用过程中的作用,总结了已经鉴定的参与低磷胁迫反应的基因及其可能存在的相互关系。     

The Arabidopsis thaliana sulfiredoxin is a plastidic cysteine-sulfinic acid reductase involved in the photooxidative stress response

The 2-cysteine peroxiredoxins (2-Cys-Prxs) are antioxidants that reduce peroxides through a thiol-based mechanism. During catalysis, these ubiquitous enzymes are occasionally inactivated by the substrate-dependent oxidation of the catalytic cysteine to the sulfinic acid (-SO2H) form, and are reactivated by reduction by sulfiredoxin (Srx), an enzyme recently identified in yeast and in mammal cells. In plants, 2-Cys-Prxs constitute the most abundant Prxs and are located in chloroplasts. Here we have characterized the unique Srx gene in Arabidopsis thaliana (AtSrx) from a functional point of view, and analyzed the phenotype of two AtSrx knockout (AtSrx-) mutant lines. AtSrx is a chloroplastic enzyme displaying sulfinic acid reductase activity, as shown by the ability of the recombinant AtSrx to reduce the overoxidized 2-Cys-Prx form in vitro, and by the accumulation of the overoxidized Prx in mutant lines lacking Srx in vivo. Furthermore, AtSrx mutants exhibit an increased tolerance to photooxidative stress generated by high light combined with low temperature. These data establish that, as in yeast and in mammals, plant 2-Cys-Prxs are subject to substrate-mediated inactivation reversed by Srx, and suggest that the 2-Cys-Prx redox status and sulfiredoxin are parts of a signaling mechanism participating in plant responses to oxidative stress.     

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。     

The new understanding of Arabidopsis thaliana proteins associated with salinity

Abstract

Salt stress is a major abiotic stress limiting the productivity and the geographical distribution of many plant species. Arabidopsis thaliana is an excellent model with rich genetic resources for modern plant biology research. To comprehensively and representatively understand salt-response mechanisms in A. thaliana, we applied the first attempt to use the most data (252 of 10,469 reviewed A. thaliana protein) from public protein database for displaying the enriched protein domains, Kyoto Encyclopedia of Genes and Genomes pathways, molecular functions, and cell localizations involved in salt-response. The data were analyzed by Database for Annotation Visualization and Integrated Discovery. Our results indicated salt-response proteins cross-talked not only with drought and temperature stress as previously reported but also with further stresses such as bacterium, light, metal ion, radiation, and wounding stress. Multiple cellular localizations under salt stress indicated proteins were versatile. In addition, 27 proteins have the characteristics with response to multiple stresses and localization in multiple places. We called it the ‘space-stress’ double cross-talk effects, which indicated that A. thaliana proteins dealt with salt stress and other stresses in a reciprocal economical way. An enriched bioinformatics analysis of the large data could provide clues and basis for the development of salt-response potential biomarkers for plant growth and crop productivity.     

Molecular evolution of the histidine biosynthetic pathway

The available sequences of genes encoding the enzymes associated with histidine biosynthesis suggest that this is an ancient metabolic pathway that was assembled prior to the diversification of the Bacteria, Archaea, and Eucarya. Paralogous duplications, gene elongation, and fusion events involving different his genes have played a major role in shaping this biosynthetic route. Evidence that the hisA and the hisF genes and their homologues are the result of two successive duplication events that apparently took place before the separation of the three cellular lineages is extended. These two successive gene duplication events as well as the homology between the hisH genes and the sequences encoding the TrpG-type amidotransferases support the idea that during the early stages of metabolic evolution at least parts of the histidine biosynthetic pathway were mediated by enzymes of broader substrate specificities. Maximum likelihood trees calculated for the available sequences of genes encoding these enzymes have been obtained. Their topologies support the possibility of an evolutionary proximity of archaebacteria with low GC Gram-positive bacteria. This observation is consistent with those detected by other workers using the sequences of heat-shock proteins (HSP70), glutamine synthetases, glutamate dehydrogenases, and carbamoylphosphate synthetases.Abbreviations as amino acid - ORF open reading frame - bp base pair - kb 10³ bp - CarA carbamoyl phosphate synthetase (EC 6.3.5.5) - GAT glutamine amidotransferase - GuaA GMP synthetase (EC 6.3.4.1) - PabA 4-amino-4-deoxychorismate synthase (EC 4.1.3-) - PyrG GTP synthetase (EC 6.3.4.2) - AICAR 5-aminoimidazole-4-carboxamide-l--d ribofuranosyl 5-monophosphate - HAL l-histidinal - HOL l-histidinol - HP histidinol phosphate - IAP imidazole acetol-phosphate - IGP imidazole glycerol phosphate - PR phosphoribosyl - PRFAR N-[(5-phosphoribulosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide - 5-ProFAR N ¹-[(5-phosphoribosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide - PRPP phosphoribosyl-pyrophosphate - RFLP restriction fragment length polymorphism Correspondence to: R. Fani     

Histological Study of Seed Coat Development in Arabidopsis thaliana

Arabidopsis seed coat development using light and transmission electron microscopy revealed major morphological changes associated with the transition of the integuments into the mature seed coat. By the use of a metachromatic staining procedure, cytological events such as the production of phenolic compounds and acidic polysaccharides were followed. Immediately after fertilization, the cells of the inner epidermis of the inner integument became vacuolated and subsequently accumulated pigment within them. This pigment started to disappear from the cytoplasm at the torpedo stage of the embryo, as it became green. During the torpedo stage, mucilage began to accumulate in the cells of the external epidermis of the outer integument. Furthermore, starch grains accumulated against the central part of the inner periclinal wall of these cells, resulting in the formation of small pyramidal domes that persisted until seed maturity. At the maturation stage, when the embryo became dormant and colourless, a new pigment accumulation was observed in an amorphous layer derived from remnants of crushed integument layers. This second pigment layer was responsible for the brown seed colour. These results show that seed coat formation may proceed in a coordinated way with the developmental phases of embryogenesis. Received 25 May 1999/ Accepted in revised form 10 February 2000     

拟南芥abi5基因的分子克隆及其在原核细胞中的表达和纯化

拟南芥abi5基因编码了一个碱性亮氨酸拉链类转录因子,它在ABA信号转导过程中发挥着关键调控作用。本文以拟南芥为材料,通过RT-PCR扩增、克隆了包含abi5基因编码区的片段。核苷酸序列分析表明,所克隆的基因与NCBI数据库收录的abi5基因（GenBank登录号NM129185.3）有99.0%的一致性;氨基酸序列存在4个残基差异。所克隆的abi5基因被进一步亚克隆至pET-32a表达载体。序列测定核实构建正确的重组质粒（pET32a-ABI5）转化入大肠杆菌BL21 Star（DE3）中诱导表达。表达产物经Ni-NTA亲和层析柱分离纯化、SDS-PAGE分析和质谱鉴定。结果表明,重组abi5基因在大肠杆菌表达的较适宜条件为：异丙基-β-D-硫代半乳糖苷（IPTG）终浓度为0.3 mmol L-1、30℃下诱导4 h,可达到细菌裂解液上清蛋白的29.1%。经Ni-NTA亲和层析柱纯化后的ABI5融合蛋白在SDS-PAGE电泳分析时呈现一条蛋白带。该条带经串联质谱分析证明为重组ABI5融合蛋白。