Auxiliary Photosynthetic Functions of Arabidopsis thaliana—Studies in vitro and in vivo

An improved cultivation system for Arabidopsis thaliana was developed, allowing advanced biochemical studies in vitro and in vivo of this important model plant. Highly functional Arabidopsis thylakoids were isolated and used to study both basic and regulatory photosynthetic functions with the aim to create a platform for the characterization of mutants deficient in auxiliary proteins. Light-induced proteolytic degradation of the D1 protein could be followed and shown to be a subsequent event to photoinactivation of electron transport. The phosphorylation and dephosphorylation of thylakoid proteins resembled that seen in spinach leaves although phospho-CP43 revealed an unusual regulatory behavior.     

Changes of Photosystem Ⅱ Electron Transport in the ChlorophyⅡ-deficient Oilseed Rape Mutant Studied by ChlorophyⅡ Fluorescence and Thermoluminescence

The photosystem Ⅱ （PSII） complex of photosynthetic membranes comprises a number of chlorophyll-binding proteins that are important to the electron flow. Here we report that the chlorophyll b-deficient mutant has decreased the amount of light-harvesting complexes with an increased amount of some core polypeptldes of PSII, including CP43 and CP47. By means of chlorophyll fluorescence and thermolumlnescence, we found that the ratio of Fv/Fm, qP and electron transport rate in the chlorophyll b-deficient mutant was higher compared to the wild type. In the chlorophyll lPdeflclent mutant, the decay of the primary electron acceptor quinones （QA-） reoxidation was decreased, measured by the fluorescence. Furthermore, the thermoluminescence studies in the chlorophyll bdeficient mutant showed that the B band （S2/S3QB-） decreased slightly and shifted up towards higher temperatures. In the presence of dlchlorophenyl-dlmethylurea, which is inhibited in the electron flow to the second electron acceptor quinines （QB） at the PSll acceptor side, the maximum of the Q band （S2QA-） was decreased slightly and shifted down to lower temperatures, compared to the wild type. Thus, the electron flow within PSll of the chlorophyⅡ b-deficient mutant was down-regulated and characterized by faster oxidation of the primary electron acceptor quinine QA-via forward electron flow and slower reduction of the oxidation S states.     

The Arabidopsis szl1 Mutant Reveals a Critical Role of β-Carotene in Photosystem I Photoprotection

Carotenes and their oxygenated derivatives, the xanthophylls, are structural determinants in both photosystems (PS) I and II. They bind and stabilize photosynthetic complexes, increase the light-harvesting capacity of chlorophyll-binding proteins, and have a major role in chloroplast photoprotection. Localization of carotenoid species within each PS is highly conserved: Core complexes bind carotenes, whereas peripheral light-harvesting systems bind xanthophylls. The specific functional role of each xanthophyll species has been recently described by genetic dissection, however the in vivo role of carotenes has not been similarly defined. Here, we have analyzed the function of carotenes in photosynthesis and photoprotection, distinct from that of xanthophylls, by characterizing the suppressor of zeaxanthin-less (szl) mutant of Arabidopsis (Arabidopsis thaliana) which, due to the decreased activity of the lycopene-β-cyclase, shows a lower carotene content than wild-type plants. When grown at room temperature, mutant plants showed a lower content in PSI light-harvesting complex I complex than the wild type, and a reduced capacity for chlorophyll fluorescence quenching, the rapidly reversible component of nonphotochemical quenching. When exposed to high light at chilling temperature, szl1 plants showed stronger photoxidation than wild-type plants. Both PSI and PSII from szl1 were similarly depleted in carotenes and yet PSI activity was more sensitive to light stress than PSII as shown by the stronger photoinhibition of PSI and increased rate of singlet oxygen release from isolated PSI light-harvesting complex I complexes of szl1 compared with the wild type. We conclude that carotene depletion in the core complexes impairs photoprotection of both PS under high light at chilling temperature, with PSI being far more affected than PSII.     

An Integrative Analysis of the Effects of Auxin on Jasmonic Acid Biosynthesis in Arabidopsis thaliana

Auxin and jasmonic acid （JA） are two plant phytohormones that both participate in the regulation of many developmental processes. Jasmonic acid also plays important roles in plant stress response reactions. Although extensive investigations have been undertaken to study the biological functions of auxin and JA, little attention has been paid to the cross-talk between their regulated pathways. In the few available reports examining the effects of auxin on the expression of JA or JA-responsive genes, both synergetic and antagonistic results have been found. To further investigate the relationship between auxin and JA, we adopted an integrative method that combines microarray expression data with pathway information to study the behavior of the JA biosynthesis pathway under auxin treatment. Our results showed an overall downregulation of genes involved in JA biosynthesis, providing the first report of a relationship between auxin and the JA synthesis pathway in Arabidopsis seedlings.     

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.     

Photosynthetic Properties of Photosystem II in Arabidopsis thaliana lpa1 Mutant

In a previous study, we characterized a high chlorophyll fluorescence lpa1 mutant of Arabidopsis thaliana, in which approximately 20% photosystem (PS) II protein is accumulated. In the present study, analysis of fluorescence decay kinetics and thermoluminescence profiles demonstrated that the electron transfer reaction on either the donor or acceptor side of PSII remained largely unaffected in the lpa1 mutant. In the mutant, maximal photochemical efficiency (Fv/Fm, where Fm is the maximum fluorescence yield and Fv is variable fluorescence) decreased with increasing light intensity and remained almost unchanged in wild-type plants under different light conditions. The Fv/Fm values also increased when mutant plants were transferred from standard growth light to low light conditions. Analysis of PSII protein accumulation further confirmed that the amount of PSII reaction center protein is correlated with changes in Fv/Fm in lpa1 plants. Thus, the assembled PSII in the mutant was functional and also showed increased photosensitivity compared with wild-type plants.(Author for correspondence. Tel: +86 (0)10 6283 6256; Fax: +86 (0)10 8259 9384; E-mail: zhanglixin@ibcas.ac.cn)     

The thylakoid protease Deg1 is involved in photosystem‐II assembly in Arabidopsis thaliana

DegP proteases have been shown to possess both chaperone and protease activities. The proteolytic activities of chloroplast DegP‐like proteases have been well documented. However, whether chloroplast Deg proteases also have chaperone activities has remained unknown. Here we show that chloroplast Deg1 also has chaperone activities, like its Escherichia coli ortholog DegP. Transgenic plants with reduced levels of Deg1 accumulated normal levels of different subunits of the major photosynthetic protein complexes, but their levels of photosystem‐II (PSII) dimers and supercomplexes were reduced. In vivo pulse‐chase protein labeling experiments showed that the assembly of newly synthesized proteins into PSII dimers and supercomplexes was impaired, although the synthesis rate of chloroplast proteins was unaffected in the transgenic lines. Protein overlay assays provided direct evidence that Deg1 interacts with the PSII reaction center protein D2. These results suggest that Deg1 assists the assembly of the PSII complex, probably through interaction with the PSII reaction center D2 protein.     

油菜素内酯不敏感型的拟南芥突变型筛选

自1979年Grove等首次从油菜（＆．wM－。。WL·）花粉中分离出油菜素内酯（brassinolide,BR）以来,人们已在该激素的生理反应和对植物生长发育等方面进行了许多研究（Kalinich等1985,Mandava1988,吴登如和赵硫橘1993）。但由于这类激素在10－'mol／L浓度水平就能诱导大豆、水稻等多种植物细胞的生长和分裂（Sasse1991）,而且在植物体内含量极低,因此用传统的方法研究它的作用方式非常困难。目前,利用激素突变体来研究激素代谢及其分子机制已有不少成功的例子,如生长素（Keily和Bradford1986,Lincoln等1990）、赤霉素（Singh…     

Knockdown of the Arabidopsis thaliana chloroplast protein disulfide isomerase 6 results in reduced levels of photoinhibition and increased D1 synthesis in high light

A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild‐type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non‐photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild‐type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light‐regulated manner.     

拟南芥雄性不育突变体ms1142的遗传定位与功能分析

经EMS诱变野生型拟南芥(Arabidopsis thaliana)群体筛选得到一株雄性不育突变体ms1142, 突变体的果荚短小, 不含种子。细胞学观察和扫描电镜结果表明, 突变体花药发育过程中, 花药中小孢子外壁异常、破裂, 最后没有花粉形成。遗传分析表明, 该突变体为隐性单核基因突变所致; 利用图位克隆的方法将MS1142基因定位于第1条染色体的BAC克隆F16P17上44 kb区间内, 目前尚未见该区间内有雄性不育基因的报道。以上结果结合生物信息学分析表明, MS1142是一个新的调控花药发育的关键基因。该工作为花药发育关键基因MS1142的克隆及功能分析奠定了基础。     

拟南芥prr5突变体对ABA的响应

以拟南芥为材料,统计PRRs （pseudo-response regulators）突变体 prr5及其野生型经ABA处理后的萌发率、根长和NaCl处理后的萌发率,并采用实时定量PCR方法,对不同浓度ABA处理的拟南芥幼苗中的PRR5基因表达进行分析.结果表明：prr5突变体对ABA弱敏感,其种子萌发率比野生型显著或极显著增高,主根比野生型长,且PRR5基因表达受ABA抑制.同时,NaCl处理后,prr5的萌发率比野生型极显著增高.因此,推测prr5可能为ABA信号通路相关基因.     

拟南芥叶绿体分裂突变体pdl37的基因鉴定与分析

pd137是经甲基磺酸乙脂（ethyl methane sulphonate, EMS）诱变并通过筛选得到的一个拟南芥叶绿体分裂突变体。该突变体的叶绿体表型与野生型相比有很大差异： 叶绿体面积显著增大, 细胞中叶绿体数量明显减少。遗传分析显示pd137的突变表型受隐性单基因控制。本研究通过遗传作图将该突变基因粗定位于拟南芥2号染色体的分子标记CH2-13.70和CH2-16.0区间内。该区间内已知的与叶绿体分裂相关的基因只有FtsZ2-1。对FtsZ2-1基因的测序结果显示pd137突变体的FtsZ2-1基因第505位碱基发生了无义突变, 使蛋白质翻译提前终止。该突变还严重影响了FtsZ2-1基因的mRNA水平。转基因互补实验进一步验证了该突变体表型是由于FtsZ2-1基因突变引起。本项工作为研究叶绿体分裂的机制提供了新材料和一些有用的线索。