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Characterization of a sodium-regulated glutaminase from cyanobacterium Synechocystis sp. PCC 6803

Science china(Life Sciences) 2008年第12期51卷 1066-1075页

作者： zhou jie1, zhou junxia1,{2., yan chengshi1 & huang fang1 1 key laboratory of photosynthesis and environmental molecular physiology, institute of botany, chinese academy of Sciences, beijing 100093, china 2.Graduate School of chinese academy of Sciences, beijing 100039, china key laboratory of {2. and environmental molecular physiology Institute of Botany Chinese Academy of Sciences Beijing China Graduate School of chinese academy of Sciences Beijing China

Glutaminase is widely distributed among microorganisms and mammals with important functions. Lit-tle is known regarding the biochemical properties and functions of the deamidating enzyme glutami-nase in cyanobacteria. In this study a putative glutaminase encoded by gene slr2.79 in Synechocystis sp. PCC 6803 was investigated. The slr2.79 was expressed as histidine-tagged fusion protein in Es-cherichia coli. The purified protein possessed glutaminase activity, validating the functional assign-ment of the genomic annotation. The apparent Km value of the recombinant protein for glutamine was 2..6 ± 0.9 mmol/L, which was comparable to that for some of other microbial glutaminases. Analysis of the purified protein revealed a two-fold increase in catalytic activity in the presence of 1 mol/L Na+. Moreover, the Km value was decreased to 12.2.± 1.9 mmol/L in the presence of Na+. These data demon-strate that the recombinant protein Slr2.79 is a glutaminase which is regulated by Na+ through in-creasing its affinity for substrate glutamine. The slr2.79 gene was successfully disrupted in Synecho-cystis by targeted mutagenesis and the △slr2.79 mutant strain was analyzed. No differences in cell growth and oxygen evolution rate were observed between △slr2.79 and the wild type under standard growth conditions, demonstrating slr2.79 is not essential in Synechocystis. Under high salt stress condition, however, △slr2.79 cells grew 1.2.-fold faster than wild-type cells. Moreover, the photosyn-thetic oxygen evolution rate of △slr2.79 cells was higher than that of the wild-type. To further charac-terize this phenotype, a number of salt stress-related genes were analyzed by semi-quantitative RT-PCR. Expression of gdhB and prc was enhanced and expression of desD and guaA was repressed in △slr2.79 compared to the wild type. In addition, expression of two key enzymes of ammonium assimi-lation in cyanobacteria, glutamine synthetase (GS) and glutamate synthase (GOGAT) was examined by semi-quant

关键词： cyanobacteria Synechocystis putative glutaminase enzyme activity mutagenesis salt tolerance

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Proteomic screening of high-pH-stress proteins in cyanobacterium Synechocystis sp. Strain PCC 6803

Proteomic screening of high-pH-stress proteins in cyanobacte...

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中国植物学会七十五周年年会

作者： Lifang Zhang~1 haomeng {2.~1 Jia Hu~2.Suxia Cui~2.fang huang~(1*) (1.key laboratory of photosynthesis and environmental molecular physiology,institute of {12.,chinese academy of Sciences,beijing 100093,china 2.College of Life Sciences,Capital Normal University,beijing 100037,china)

Cyanobacteria are ancient and the largest group of prokaryotes as judged by their widespread habitats,frequent abundance and morphological ***,the molecular adaptation mechanisms underlying in cyanobacterial cells is ...

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Characterization of a FeMo cofactor-deficient MoFe protein from a nifE-deleted strain (DJ35) of Azotobacter vinelandii

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Science Bulletin 2005年第20期 51-56页

作者： ZHAO Ying1,2, BIAN Shaomin1,2, ZHANG Chunxi3, zhou Huina1,2, WANG huangping1,4, ZHAO Jianfeng1 & huang Jufu1 1. key laboratory of photosynthesis and environmental molecular physiology, institute of botany, chinese academy of Sciences, Bei- jing 100093, china 2. Graduate School, chinese academy of Sciences, beijing 100039, china 3. key laboratory of Photochemistry, institute of Chemistry, chinese academy of Sciences, beijing 100080, china 4. Bioengineering College, Fujian Normal University, Fuzhou 360007, china

A MoFe protein （?nifE Av1） with a purity of80% was purified from a nifE-deleted mutant of Azotobac- ter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP （OP Av1）, ?nifE Av1 had the same subunits composition, and had immune reaction with anti- body to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis （PAGE） was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ?nifE Av1 both apparently decreased. When complemented with OP Fe protein, ?nifE Av1 had no C2.2.reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism （CD） spectrum of ?nifE Av1 at450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ?nifE Av1 puri- fied from DJ35 was a FeMoco-deficient but P-cluster-con- taining MoFe protein.

关键词： Azotobacter vinelandii ?nifE Av1 FeMoco P-cluster EPR.

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Effects of cerium on growth and physiological mechanism in plants under enhanced ultraviolet-B radiation

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Journal of environmental Sciences 2006年第1期18卷 125-129页

作者： LIANG Chan-iuan huang Xiao-hua TAO Wen-yi zhou Qing The key laboratory of Industrial Biotechnology Ministry of Education Southern Yangtze University Wuxi 214036 China School of Biotechnology Southern Yangtze University Wuxi 214036 China College of Chemistry and environmental Science Nanjing Normal University Nanjing 210097 China

Effect of cerium （Ce^3＋） on the growth, photosynthesis and antioxidant enzyme system in rape seedlings （Brassica juncea L.） exposed to two levels of UV-B radiation （T1： 0.15 W/m^2.and T2.0.35 W/m^2. was studied by hydroponics under laboratory conditions. After 5 d of UV-B treatment, the aboveground growth indices were obviously decreased by 13.2.-44. 1%（T1） and 2..4%-49.3% （T2., compared to CK, and except active absorption area of roots, the belowground indices by 14.1%-35.6%（T1） and 2..3%-42.6% （T2.. For Ce＋UV-B treatments, the aboveground and belowground growth indices were decreased respectively by 4.1%-2..6%, 5.2. -2..3%（Ce＋T1） and 10.8%-2..4%, 7.0%-2..8%（Ce＋T2., lower than those of UV-B treatments. The decrease of growth indices appeared to be the result of changes of physiological processes. Two levels of UV-B radiation induced the decrease in chlorophyll content, net photosynthesis rate, transpiration rate, stomatal conductance and water use efficiency by 11.2.-2..9%（T1） and 2..9%- 56.9%（T2., whereas increase in membrane permeability and activities of antioxidant enzymes including superoxide dismutase（SOD）, catalase （CAT） and peroxidase （POD） by 6.9%, 2..8%, 2..5%, 9.5%（T1） and 36.6%, 12..3%, 103.5%, 2.8.9%（T2., respectively. The reduction of the photosynthetic parameters in Ce＋UV-B treatments was lessened to 3.2.-13.8%（Ce＋T1） and 4.9%-2..6%（Ce＋T2., and the increase of membrane permeability and activities of antioxidant enzymes except POD in the same treatments were lessened to 2.4%, 8.4%, 6.6%（Ce＋T1） and 30.1%, 116.7%, 75.4%（Ce＋T2.. These results indicate that the regulative effect of Ce on photosynthesis and antioxidant enzymatic function is the ecophysiological basis of alleviating the suppression of UV-B radiation on growth of seedlings. Furthermore, the protective effect of Ce on seedlings exposed to TI level of UV-B radiation is superior to T2.level.

关键词： cerium(Ce^3+) UV-B radiation rape growth photosynthesis antioxidant enzyme system

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AtECB1/MRL7, a Thioredoxin-Like Fold Protein with Disulfide Reductase Activity, Regulates Chloroplast Gene Expression and Chloroplast Biogenesis in Arabidopsis thaliana

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molecular Plant 2014年第1期7卷 206-217页

作者： Qing-Bo Yu Qian Ma Meng-Meng Kong Tuan-Tuan Zhao Xiao-Lei Zhang Que zhou Chao huang Kang Chong Zhong-Nan {2. Biology Department Life and Environmental College Shanghai Normal University Shanghai 200234 China National laboratory of Plant molecular Genetics Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences Shanghai 200032 China Research Center for molecular and Developmental Biology Key Laboratory of Photosynthesis and Environmental Molecular Physiology Institute of Botany the Chinese Academy of Sciences 100093 Beijing China

Plastid-encoded RNA polymerase （PEP） is closely associated with numerous factors to form PEP complex for plastid gene expression and chloroplast development. However, it is not clear how PEP complex are regulated in chloroplast. Here, one thioredoxin-like fold protein, Arabidopsis early chloroplast biogenesis 1 （AtECB1）, an allele of MRL7, was identified to regulate PEP function and chloroplast biogenesis. The knockout lines for AtECB1 displayed albino phenotype and impaired chloroplast development. The transcripts of PEP-dependent plastid genes were barely detected, suggesting that the PEP activity is almost lost in atecbl-1. Although AtECB1 was not identified in PEP complex, a yeast two-hybrid assay and pull-down experiments demonstrated that it can interact with Trx Z and FSD3, two intrinsic subunits of PEP complex, respectively. This indicates that AtECB1 may play a regulatory role for PEP-dependent plastid gene expression through these two subunits. AtECB1 contains a βαβαββα structure in the thioredoxin-like fold domain and lacks the typical C-X-X-C active site motif. Insulin assay demonstrated that AtECB1 harbors disulfide reductase activity in vitro using the purified recombinant AtECB1 protein. This showed that this thioredoxin-like fold protein, AtECB1 also has the thioredoxin activity. AtECB1 may play a role in thioredoxin signaling to regulate plastid gene expression and chloroplast development.

关键词： Arabidopsis Thioredoxin disulfide reductase activity chloroplast transcription chloroplast biogenesis.

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Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

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Journal of Integrative Plant Biology 2005年第11期47卷 1364-1371页

作者： Hui-Na zhou Ying ZHAO Shao-Min BIAN Jian-Feng ZHAO Fei REN huang-Ping WANG Ju-Fu huang key laboratory of {2. and environmental molecular physiology Institute of Botany the Chinese Academy of Sciences Beijing 100093 China Graduate School of the chinese academy of Sciences Beijing 100039 China china Agricultural University Beijing 100094 China Fujian Normal University Fuzhou 350007 China

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52. and Sephacryl S-2.0 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-2.0 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.

关键词： Azotobacter vinelandii chaperonin GroEL glucose-6-phosphate isomerase nitrogenase Av 1.

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Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

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Journal of Integrative Plant Biology 2006年第5期48卷 567-572页

作者： huang-Ping Wang Ying Zhao Shao-Min Bian Hui-Na zhou Zhi-Gang Zhang Jin-Mao Zhu Ju-Fu huang Bioengineering College Fujian Normal University Fuzhou 350007 China key laboratory of {2. and environmental molecular physiology Institute of Botany the Chinese Academy of Sciences Beijing 100093 China College of Life Sciences Northwest University Xi＇an 710069 China

nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52. Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2., AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-2. column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512.m = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2. these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2. and Na2.oO4, or K2.rO4, or KMnO4. But In the absence of MgATP or Av2. the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that：（I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.

关键词： activation in vitro by reconstituent solutions Azotobacter vinelandii mutant MoFe protein nifB^-,△nifE and △nifZ mutants.

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Characterization of a FeMo cofactor-deficient MoFe protein from a nifE-deleted strain(DJ35)of Azotobacter vinelandii

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chinese Science Bulletin 2005年第20期50卷 2305-2310页

作者： ZHAO Ying BIAN Shaomin ZHANG Chunxi zhou Huina WANG huangping ZHAO Jianfeng huang Jufu key laboratory of {2. and environmental MolecuIar physiology Institute of Botany Chinese Academy of Sciences Beijing 100093 China Graduate School Chinese Academy of Sciences Beijing 100039 China key laboratory of Photochemistry Institute of Chemistry Chinese Academy of Sciences Beijing 100080 China Bioengineering College Fujian Normal University Fuzhou 360007 China

A MoFe protein (ΔnifE Av1) with a purity of ～80% was purified from a nifE-deleted mutant of Azotobacter vinelandii DJ35. Compared with MoFe protein purified from wild-type strain OP (OP Av1), ΔnifE Av1 had the same subunits composition, and had immune reaction with antibody to OP Av1, but its relative mobility in anaerobic native polyacrylamide gel electrophoresis (PAGE) was a little larger than that of OP Av1. Metal analysis showed that Mo and Fe contents of ΔnifE Av1 both apparently decreased. When complemented with OP Fe protein, ΔnifE Av1 had no C2.2.reduction activity, but it could be in vitro activated by FeMoco extracted from OP Av1. The circular dichroism (CD) spectrum of ΔnifE Av1 at ~450 nm was similar to that of OP Av1, while the EPR signal at g≈3.7 was absolutely silent, and the signal intensities at g≈4.3 and 2.0 decreased by 75% and 50%, respectively. The results indicated that ΔnifE Av1 purified from DJ35 was a FeMoco-deficient but P-cluster-con- taining MoFe protein.

关键词： MoFe蛋白质固氮细菌纯净度 ERP 厌氧性

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Characterization of a nitro-genase CrFe protein from a mutant UW3 of Azotobacter vinelandii grown on a Cr-containing medium

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chinese Science Bulletin 2006年第14期51卷 1729-1735页

作者： ZHANG Zhigang ZHAO Ying ZHANG Chunxi BIAN Shaomin zhou Huina WANG huangping YIN Hong huang Jufu College of Life Sciences Northwest University Xi'an 710069 China key laboratory of {2. and environmental molecular physiology Institute of Botany Chinese Academy of Sciences Beijing100093 China laboratory of Photochemistry Institute of Chemistry Chinese Academy of Sciences Beijing 100080 China Bioengineering College Fujian Normal University Fuzhou 350007 China

通过 DEAE-52.的列上的缺氧的层析， Q-Sepharose andSeph 压克力 S-2.0， CrFe 蛋白质准备从 UW3 被获得， Azotobactervinelandii 的异种，它在包含 Cr 媒介上成长了很好。与 MoFe 蛋白质(OP Av1 ) 相比 fromwild 类型紧张 OP，在准... 详细信息

通过 DEAE-52.的列上的缺氧的层析， Q-Sepharose andSeph 压克力 S-2.0， CrFe 蛋白质准备从 UW3 被获得， Azotobactervinelandii 的异种，它在包含 Cr 媒介上成长了很好。与 MoFe 蛋白质(OP Av1 ) 相比 fromwild 类型紧张 OP，在准备的约 50% 蛋白质有类似的子单元作文并且与 OP Av1 的抗体有有免疫力的反应。准备有 C_2._2. H~+-andN_2.的 -40 百分比(在 H~+ 减小活动在之间由差别表示了在 Ar 下面并且在 N_2.下面) 到那些的 OP Av1 和 OP Av1 的类似的电子对的减小活动。并且金属分析证明准备包含了 Fe， Cr 和瞬间。，在在约 450 nm 的准备的圆形的二色性(CD ) 光谱类似于 OP Av1 的出现在一样的 g 价值的 threeEPR 信号的相对紧张(g 约 4.3， 3.7 和 2.0 ) 是不同的。EPR-basedcalculated 结果显示出那(1 ) 到 Cr 的瞬间并且 Fe 到的比率(Cr+Mo ) 分别地， CrFe 蛋白质准备， 0.41 和 15 正在显示在准备，到 MoFe 蛋白质的比率包含 ofCr 蛋白质是大约 2.5；(2.) 到 Fe 并且到准备的 Cr 的活动的比率分别地接近了活动的比率到 Fe 并且到 OP Av1 的瞬间；(3 ) EPR 的比率在到 CrFe 蛋白质的 Cr 的值大约到瞬间的约 83% 那些， 0% 和约 40% 的上述三 g 表明紧张。OP Av1，分别地。结果进一步显示 CrFe 蛋白质力量是一个新 ni-trogenase 部件我有在余因子由 Mn MoFe 蛋白质包括 metallocluster 到那些而不是瞬间的简单代替的类似的功能和结构的蛋白质。

关键词：固氮菌突变异种 CrFe蛋白质循环二色性电子顺磁共振

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The decoupling between gas exchange and water potential of Cinnamomum camphora seedlings during drought recovery and its relation to ABA accumulation in leaves

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Journal of Plant Ecology 2020年第6期13卷 683-692页

作者： Honglang Duan Defu Wang Xiaohua Wei Guomin huang Houbao Fan Shuangxi {2. Jianping Wu Wenfei Liu David TTissue Songze Wan institute for Forest Resources&Environment of Guizhou Key Laboratory of Forest Cultivation in Plateau Mountain of Guizhou ProvinceCollege of ForestryGuizhou UniversityGuiyang 550025China Jiangxi Provincial key laboratory for Restoration of Degraded Ecosystems&Watershed Ecohydrology Nanchang Institute of TechnologyNanchang 330099China Department of Earth Environmental and Geographic SciencesUniversity of British Columbia OkanaganKelownaBC V1V 1V7Canada The New Zealand institute for Plant and Food Research Ltd. Hawke’s BayNew Zealand laboratory of Ecology and Evolutionary Biology Yunnan UniversityKunming 650500China Hawkesbury institute for the Environment Hawkesbury CampusWestern Sydney UniversityLocked Bag 1797PenrithNSW 2751Australia College of Forestry Jiangxi Agricultural UniversityNanchang 330045China

Aims Drought stress and the degree of drought severity are predicted to rise under highly variable patterns of precipitation due to climate change,while the capacity of trees to cope with drought recovery through physiological and biochemical adjustment remains *** aimed to examine the coupling of physiology and biochemistry in trees during drought and the following *** Potted seedlings of Cinnamomum camphora were grown under well watered conditions prior to the experimental drought stress,which was initiated by withholding *** were rewatered following attainment of two drought severities:mild drought(stomatal closure)and moderate drought(ψxylem=−1.5 MPa).We measured leaf-level water potential,gas exchange(photosynthesis and stomatal conductance),abscisic acid(ABA),proline and non-structural carbohydrates(NSCs)concentrations in seedlings of *** during drought and a 4-day *** Findings We found that drought severity largely determined physiological and biochemical responses and affected the rate of *** closure occurred at the mild drought stress,accompanied with ABA accumulation in leaves and decline in water potential,while leaf proline accumulation and variable NSC were evident at the moderate drought *** severe drought stress led to delayed recovery of gas exchange,but it did not have significant effect on water potential *** relationships of water potential and gas exchange differed during drought stress and post-drought *** was tight coupling between water potential and gas exchange during drought,but not during rewatering due to high ABA accumulation in leaves,thereby delaying recovery of stomatal *** results demonstrate that ABA could be an important factor in delaying the recovery of stomatal conductance following rewatering and after water potential recovery of ***,greater drought severity had significant impacts on the rate of recove

关键词： drought recovery gas exchange ABA NSC proline

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