转<italic>codA</italic> 基因提高番茄植株的耐热性

引用本文

李枝梅, 窦海鸥, 卫丹丹, 孟庆伟, 杨兴洪. 转codA 基因提高番茄植株的耐热性 . 作物学报, 2013, 39(11): 2046-2054[LI Zhi-Mei, DOU Hai-Ou, WEI Dan-Dan, MENG Qing-Wei, Tony Huihuang CHEN, YANG Xing-Hong, Tony Huihuang CHEN. codA Transgenic Tomato Plants Enhance Tolerance to High Temperature Stress . 作物学报, 2013, 39(11): 2046-2054] 复制到剪切板

Permissions

作物学报编辑部

转codA 基因提高番茄植株的耐热性

李枝梅¹, 窦海鸥¹, 卫丹丹¹, 孟庆伟¹, 杨兴洪^1,^*

¹山东农业大学生命科学学院 / 作物生物学国家重点实验室, 山东泰安 271018

²Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA

^*通讯作者(Corresponding author): 杨兴洪, E-mail:xhyang@sdau.edu.cn, Tel: 0538-8246167

收稿日期:2013-06-24

基金:本研究由国家重点基础研究发展计划(973计划)项目(2009CB118500), 国家高技术研究发展计划(863计划)项目(2012AA10A309), 国家自然科学基金项目(30970229)和教育部高校博士点基金(20103702110007)资助。

摘要

以野生型番茄(cv. Moneymaker)和转codA 番茄为材料, 用不同温度(25、30、35、40、45和50℃)分别处理2 h, 测定叶片净光合速率(P_n)、PSII最大光化学效率(F_v/F_m)、过氧化氢(H₂O₂)含量、丙二醛(MDA)含量、相对电导率(REC)和抗氧化酶活性等生理指标; 42℃高温处理0、3和6 h后, 检测热响应基因的表达量以及D1蛋白的含量, 研究高温胁迫对上述参数的影响, 探讨转codA 基因提高番茄叶片耐热性的机制。结果表明, 高温胁迫下, 转codA 基因番茄叶片P_n和F_v/F_m的抑制程度明显低于野生型, H₂O₂、MDA的积累量以及REC均低于野生型, 而且明显增强了过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)的活性。此外, 转codA 基因番茄叶片中抗氧化酶基因和热胁迫基因的表达水平均高于野生型, 而D1蛋白的降解水平低于野生型。转codA 基因番茄体内合成的甜菜碱提高了转基因番茄的耐热性, 这与提高和维持较高的抗氧化酶活性、促进热激响应基因的表达及减缓D1蛋白的降解等有关。

关键词: codA基因; 番茄; 高温胁迫; 耐热性; 甜菜碱

codA Transgenic Tomato Plants Enhance Tolerance to High Temperature Stress

LI Zhi-Mei¹, DOU Hai-Ou¹, WEI Dan-Dan¹, MENG Qing-Wei¹, Tony Huihuang CHEN², YANG Xing-Hong^1,^*, Tony Huihuang CHEN²

¹State Key Laboratory of Crop Biology / College of Life Sciences, Shandong Agricultural University, Tai’an 271018, China

²Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA

Abstract

The effects ofcodA gene on photosynthesis, activities of antioxidative enzymes, the expression of the heat response genes and the accumulation of D1 protein in tomato leaf under different temperature stresses were investigated to reveal the mechanism of thermotolerance incodA -transgenic tomato plants. The wild type (cv. Moneymaker) andcodA transgenic tomato plants were treated with 25, 30, 35, 40, 45, and 50℃ respectively for two hours, then net photosynthetic rate (P_n), the maximal efficiency of PSII photochemistry (F_v/F_m), hydrogen peroxide (H₂O₂) content, malondialdehyde (MDA) content, relative electric conductivity (REC) and activities of antioxidative enzymes were detected. After 42℃ heat stress for 0, 3, 6 hours, the expressions of antioxidant enzyme genes and heat stress genes and the accumulation of the D1 protein were also determined. The results demonstrated that under high temperature stress, the inhibition degree ofP_n andF_v/F_m incodA transgenic tomato plants was lower than that in wild type plants. The accumulation of H₂O₂, MDA and REC incodA transgenic tomato plants was less than that in wild type plants. AndcodA transgenic tomato plants also greatly enhanced the activities of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX). Moreover, the expression levels of antioxidant genes and heat response genes incodA transgenic tomato plants was higher than those in wild type plants and the degradation degree of D1 protein incodA transgenic tomato plants were lower than that in wild type plants. It indicated thatcodA transgenic tomato plants enhance thermotolerance via maintaining higher activities of antioxidant enzymes, accelerating the expression of heat response genes and reducing the degradation of D1 protein.

Keyword: codA gene; Tomato; High temperature stress; Thermotolerance; Glycinebetaine

Show Figures

引言

高温是影响植物生长发育的一个重要因素。高温能够影响植物光合作用、增加细胞膜的通透性、抑制蛋白质的合成进而抑制植株生长^[1]。番茄是保护地栽培的主要蔬菜之一, 对温度敏感, 温度过高会使其生长发育受阻。番茄植株生长发育的最适温度为昼温24~26℃, 夜温18℃左右, 30℃则过高, 到40℃就停止生长, 如果高温达到45℃时, 在短时间内茎叶就会出现日灼, 叶脉泛白, 以致坏死^[2]。甜菜碱作为一种渗透调节物质, 参与植物对许多环境胁迫的响应^[3]。 codA 基因是合成甜菜碱的重要基因, 可以催化胆碱直接合成甜菜碱。因此研究转 codA 基因对植物耐热性的影响, 探讨转 codA 基因番茄的耐热性机制有重要意义。有研究表明, 转 codA 基因番茄通过合成甜菜碱能够缓解低温胁迫对番茄幼苗生长发育以及开花的影响, 是因为在低温下甜菜碱能够维持一些抗氧化酶的活性, 降低细胞膜的膜脂过氧化作用, 减缓PSII最大光化学效率的降低程度^[4]。Kathuria等^[5]认为转 codA 基因水稻通过合成甜菜碱能够增加一些胁迫响应基因的表达来提高水稻植株的抗旱能力。另外一些研究发现甜菜碱能够缓解盐胁迫对卡尔文循环中一些酶的损伤, 从而提高转基因烟草的耐盐性^[6]。Yang等^[7,8]指出在高温胁迫下, 甜菜碱能够通过提高烟草的光合能力和PSII活性来增强其耐热性。Li等^[9]发现在高温胁迫下外源甜菜碱能够促进番茄种子的萌发和幼苗的生长。关于高温胁迫下甜菜碱增强番茄耐热性与热响应基因表达及D1蛋白关系的研究目前仍未见报道。本文拟研究高温胁迫下, 野生型和转 codA 基因番茄叶片的净光合速率、荧光参数、MDA含量、REC、抗氧化酶活性、热响应基因和D1蛋白的变化, 探讨甜菜碱提高番茄耐热性与热胁迫基因表达和D1蛋白积累间的关系。

1 材料与方法

1.1 试验材料与种植

以自交获得的第5代野生型番茄(cv . Moneymaker)和转 codA (胆碱氧化酶)基因番茄为材料, 根据转 codA 基因番茄株系表达量的差异, 选择3个株系, 分别是CL1、CL2和CL4, 其中转 codA 基因番茄以野生型番茄(cv . Moneymaker)为受体获得。转 codA 番茄由美国俄勒冈州立大学Tony Chen实验室提供, 该基因定位于叶绿体和细胞质^[10]。将番茄种子种于MS培养基中, 于25℃条件下生长15 d后移至内含蛭石的花盆(直径15 cm, 高10 cm)中, 用Hoagland营养液培养, 在昼夜温度为25/20℃、光周期为光照16 h/黑暗8 h、相对湿度为65%~70%、光强为300 μmol m^-2 s^-1的温室中培养。

1.2 材料处理

选取生长至八叶期长势一致的番茄植株在光照培养箱中进行高温处理。将在温室生长6周后的番茄植株置不同温度(25、30、35、40、45和50℃)的光照培养箱中分别处理2 h或者于42℃的培养箱中处理0、3和6 h, 培养箱光照保持在300 μmol m^-2 s^-1, 湿度保持在65%~70%。从每处理选用4~6株, 选取完全展开的从上向下第3片或第4片叶子测定相关生理指标。3次重复测定, 取平均值。

1.3 测定项目与方法

1.3.1 甜菜碱含量的测定参考Rhodes等^[11]的方法提取和测定甜菜碱

1.3.2 光合速率和气孔导度的测定采用CIRAS-2便携式光合系统(PP Systems, 英国)测定净光合速率( P_n)、气孔导度( G_s)。控制光强为800 μmol m^-2 s^-1, CO₂ 浓度为380 μL L^-1, 温度为25℃, 空气相对湿度65%~70%^[7]。

1.3.3 荧光参数的测定采用便携脉冲调制式荧光仪(FMS-2, Hansatech, 英国)测定^[12]。叶片暗适应30 min后测定其荧光参数, 其中, F_o为初始荧光, F_m为最大荧光, 光系统II最大光化学效率 F_v/ F_m= ( F_m- F_o)/ F_m。然后将叶片暴露在300 μmol m^-2 s^-1的光强下大约8 min后, 测定光适应下的稳态荧光( F_s), 然后施加8000 µmol m^-2 s^-1的强脉冲光, 测定光适应下的最大荧光( F_m'), 最后远红光照射3 s后测定光适应下的最小荧光( F_o')。实际光化学效率 Ф_PSII= ( F_m- F_o)/ F_m'。

1.3.4 过氧化氢(H₂O₂)含量的测定参照 Brennan和Frenkel^[13]的方法测定。

1.3.5 丙二醛(MDA)含量和相对电导率(REC)的测定

参照赵世杰等^[14]的硫代巴比妥酸(TBA)比色法测定MDA含量, 参照Clarke等^[15]的方法测定REC。

1.3.6 酶活性测定称取剪碎的番茄叶片0.5 g, 加入5 mL预冷的提取液(pH 7.8磷酸缓冲液, 含 0.1 mmol L^-1 EDTA, 1% PVP), 冰浴研磨, 于4℃下12 000× g 离心20 min, 上清液即为酶提取液。按Aebi^[16]的方法测定过氧化氢酶(CAT)活性, 按Giannopolitis和Ries^[17]的方法测定超氧化物歧化酶(SOD)活性, 按愈创木酚法^[18]测定过氧化物酶(POD)活性, 按Nakano和Asada^[19]的方法测定抗坏血酸过氧化物酶(APX)活性。

1.3.7 实时定量PCR分析利用TRIzol试剂盒提取总RNA, 取1 μg总RNA进行DNase I处理, 采用cDNA第一链合成试剂盒反转录为cDNA。

将反转录所得cDNA样品稀释至0.5~2.0 ng μL^-1, 作为实时定量PCR反应的模板, 参照SYBR Green Real time PCR Master Mix(QPK-201)说明书, 反应体系含2.5 μL cDNA, 9 μL SYBR Green, 0.5 μL正向/反向引物, 7.5 μL ddH₂O, 反应条件和步骤为预变性95˚C 30 s, 变性95˚C 10 s, 退火59˚C 30 s, 延伸72˚C 20 s, 变性到退火过程为43个循环, 以番茄 Actin 作为内参, 基因相对表达量由Bio-Rad CFX 96实时定量PCR仪器自带软件分析, 扩增曲线及样品Ct值由软件自动生成。引物由生工生物工程(上海)有限公司合成, 引物序列见表1。

表1 实时定量PCR所用引物 Table 1 Primers for the real-time PCR

1.3.8 D1蛋白的测定按文献[20-21], 取新鲜番茄叶片1 g, 在液氮中研磨成粉末, 加预冷的提取缓冲液(50 mol L^-1 Hepes, pH 7.8, 10 mol L^-1 NaCl, 2 mol L^-1 EDTA, 2 mol L^-1 MgCl₂和400 mol L^-1蔗糖), 经3层纱布过滤匀浆, 用提取缓冲液冲洗类囊体颗粒后在5000× g 离心10 min, 将所得类囊体膜最后悬浮于贮存缓冲液中, 于-80℃保存备用。采用赵世杰等^[22]的方法测定叶绿素含量。

D1蛋白的分离与测定: 采用SDS-PAGE(聚丙烯酰胺凝胶电泳)和Western blot(免疫印迹)法^[23]。分离胶15%, 浓缩胶5%, 均含6 mol m^-2 s^-1尿素。上样量以叶绿素含量为准, 保证每泳道一致的叶绿素含量^[24]。

在Western blot分析中, 一抗为叶绿体D1蛋白多克隆抗体, 二抗为碱性磷酶标记羊抗兔抗体IgG, 用半干法蛋白转移法转膜。

1.4 统计分析

采用Microsoft Excel 2003, Sigmaplot 10.0与SPSS 13.0软件的LSD法对数据进行方差分析和差异显著性检验。

2 结果与分析

2.1 转 codA 基因番茄叶片中甜菜碱的含量

在正常条件下, 转 codA 基因番茄在叶片中积累甜菜碱。如图1所示, 野生型番茄叶片中不能检测到甜菜碱, 而不同转基因番茄株系叶片中甜菜碱含量也不

	Figure Option View Download New Window
	图1 野生型和转 codA 基因番茄叶片中甜菜碱的含量相同, 本试验中所选用的CL1、CL2和CL4株系中甜菜碱的含量分别为2.79、2.35和2.22 µmol g^-1 FW。Fig. 1 Contents of glycinebetaine (GB) in leaves of wild type and codA transgenic tomato plants

2.2 不同温度处理对野生型和转 codA 番茄叶片光合速率、气孔导度和光化学效率的影响

当温度低于30℃时, 野生型和转基因株系的 P_n均呈增加趋势, 当温度超过30℃时, 随处理温度提高, 番茄叶片 P_n下降, 且野生型下降的幅度明显高于转基因株系( P<0.01)。45℃处理2 h后, 野生型番茄的 P_n比25℃时降低62%, 而转基因CL1、CL2和CL4株系分别降低34%、22%和40%。50℃处理2 h后, 野生型番茄的 P_n比25℃时降低80%, 而转基因株系分别降低59%、54%和64% (图2-A)。在高温处理下, 番茄各株系 G_s均下降, 且野生型下降幅度均高于各转基因株系(图2-B)。

F_v/ F_m是暗适应下PSII的最大光化学效率, 反映了PSII反应中心最大光能转化效率^[25]。当温度低于40℃时, 温度处理对番茄 F_v/ F_m没有显著影响, 而且野生型和转 codA 基因番茄的 F_v/ F_m没有显著差异。当处理温度高于40℃时, 野生型和转基因番茄叶片的 F_v/ F_m均下降, 且转基因番茄的下降幅度明显低于野生型( P<0.01)。45℃处理2 h后, 野生型番茄的 F_v/ F_m比25℃时降低22%, 而转基因CL1、CL2和CL4株系分别降低11%、10%和13%。50℃处理2 h后, 野生型番茄的 F_v/ F_m比25℃时降低96%, 而转基因株系分别降低56%、57%和58% (图2-C)。各个株系的 F_o与 F_v/ F_m呈相反的变化趋势。45℃高温胁迫2 h后, F_o均上升, 而野生型番茄叶片 F_o显著高于转基因株系( P<0.01)(图2-D)。

以上结果表明, 转 codA 基因番茄在高温胁迫下比野生型番茄能够维持较高的光合速率和PSII活性。

	Figure Option View Download New Window
	图2 不同温度处理2 h对野生型和转 codA 基因番茄叶片光合和荧光参数 P_n(A)、 G_s(B)、 F_v/ F_m(C)和 F_o(D)的影响Fig. 2 Effects of temperature treatments for two hours on P_n (A), G_s (B), F_v/ F_m (C), and F_o (D) in wild type and codA transgenic tomato leaves

2.3 不同温度对野生型和转 codA 番茄叶片过氧化氢(H₂O₂)含量的影响

转 codA 番茄在合成甜菜碱时有副产物H₂O₂产生^[4], 而在胁迫条件下细胞中活性氧的积累与细胞膜的氧化损伤有关^[26]。图3所示, 常温下, 转基因番茄叶片中H₂O₂含量比野生型高8%~10%。随着处理温度的升高, 野生型和转基因番茄H₂O₂含量均逐渐增加, 且前者明显高于后者( P<0.01), 50℃处理2 h后, 野生型番茄叶片中H₂O₂含量比25℃时增加87%, 而转基因CL1、CL2和CL4株系分别增加了43%、40%和44%。同野生型番茄相比, 高温胁迫下转 codA 基因番茄叶片中H₂O₂积累较少。

	Figure Option View Download New Window
	图3 不同温度处理2 h对野生型和转 codA 基因番茄叶片中H₂O₂含量的影响Fig. 3 Effects of temperature treatments for two hours on H₂O₂ content in wild type and codA transgenic tomato leaves

2.4 不同温度处理对野生型和转 codA 番茄叶片丙二醛(MDA)含量和相对电导率(REC)的影响

丙二醛(MDA)含量是膜脂过氧化的一个重要指标, 相对电导率(REC)是胁迫条件下膜稳定性的重要指标, 它们均反映了胁迫对细胞膜的伤害程度。如图4所示, 在25℃条件下, 野生型和转基因番茄叶片中的MDA含量没有显著差异, 30℃以上随处理温度的升高, 番茄叶片MDA含量增加, 野生型番茄MDA含量增加幅度较转基因番茄高。50℃胁迫2 h后, 野生型番茄叶片中MDA含量增加了87%, 而各转基因CL1、CL2和CL4株系分别增加了61%、50%和52% (图4-A)。番茄叶片相对电导率的变化趋势与MDA的变化趋势相似, 当温度高于35℃时, 番茄叶片的相对电导率明显升高, 而野生型番茄叶片REC增加幅度明显高于转基因株系( P<0.01)(图4-B)。以上结果表明, 在高温胁迫下, 转 codA 基因番茄叶片膜脂过氧化程度较轻, 电解质外渗较少, 高温胁迫下膜的伤害程度较野生型轻。

	Figure Option View Download New Window
	图4 不同温度处理2 h对野生型和转 codA 基因番茄叶片MDA含量和REC的影响Fig. 4 Effects of temperature treatments for two hours on MDA content and REC in wild type and codA transgenic tomato leaves

2.5 不同温度处理对野生型和转 codA 番茄叶片抗氧化酶活性的影响

在正常条件下, 转 codA 基因番茄叶片中CAT活性比野生型高16%~17%。随着处理温度的升高, 各株系CAT活性呈先上升后下降的趋势, 转基因番茄CAT活性始终显著高于野生型( P<0.01)(图5-A)。SOD和POD活性随温度升高各株系均呈先上升后下降的趋势, 当处理温度低于35℃时, 野生型和转基因株系之间SOD和POD活性无显著差异, 处理温度高于35℃时, 野生型番茄中SOD和POD活性均低于各转基因株系(图5-B~C)。番茄叶片中APX活性随温度升高呈逐渐上升的趋势, 野生型番茄APX活性低于转基因株系(图5-D)。以上结果表明, 同野生型番茄相比, 转 codA 基因番茄在高温胁迫下具有较高的抗氧化酶活性。

	Figure Option View Download New Window
	图5 不同温度处理2 h对野生型和转 codA 基因番茄叶片CAT、SOD、POD和APX活性的影响Fig. 5 Effects of temperature treatments for two hours on activities of CAT, SOD, POD, and APX in wild type and codA transgenic tomato leaves

2.6 高温胁迫对野生型和转 codA 番茄基因表达的影响

以番茄 Actin 基因为内参基因, 分别对高温胁迫下谷胱甘肽还原酶(GR)、脱氢抗坏血酸还原酶(MDHAR)、过氧化氢酶(CAT)、热激蛋白100 (HSP100)、热激蛋白70 (HSP70)以及小分子热激蛋白17.6 (HSP17.6)的基因进行实时定量PCR分析。在正常条件下, 野生型番茄叶片中 GR 的表达量高于转基因番茄叶片, 但是在42℃处理后, 转基因番茄叶片的表达量高于野生型番茄叶片(图6-A)。 MDHAR 在42℃处理3 h后, 转基因番茄叶片的表达量也明显高于野生型番茄叶片(图6-B)。 CAT1 在正常条件下, 转基因番茄的表达量比野生型明显高, 在42℃处理后, 野生型和转基因的表达量都大幅度地降低(图6-C)。 HSP100 、 HSP70 和 HSP17.6 基因在正常条件下表达量均很低, 但是在42℃处理3 h后, 野生型和转基因番茄的表达量均大幅度地增加, 在42℃处理6 h后, 野生型和转基因番茄的表达量又大幅降低, 在42℃高温处理之后, 转基因番茄的表达量始终高于野生型(图6-D~F)。这些结果表明, 在高温胁迫下, 抗氧化酶基因和热激蛋白基因在转 codA 基因番茄中的表达量高于野生型。

	Figure Option View Download New Window
	图6 42℃高温胁迫对野生型和转 codA 番茄基因( GR 、 MDHAR 、 CAT1 、 HSP100 、 HSP70 和 HSP17.6 )表达的影响Fig. 6 Expression of the genes ( GR , MDHAR , CAT1 , HSP100 , HSP70 , and HSP17.6 ) in wild type and codA transgenic tomato plants treated with 42℃

2.7 高温胁迫对野生型和转 codA 基因番茄叶片中D1蛋白的影响

如图7所示, 正常条件下, 野生型和转基因番茄叶片D1蛋白含量没有显著差别, 当42℃高温处理后含量均明显降低, 但转基因叶片中含量明显高于野生型番茄( P<0.01)。

3 讨论

本研究结果表明, 高温胁迫导致番茄叶片净光合速率、PSII最大光化学效率降低(图2)。高温胁迫下转基因番茄叶片维持了较高的光合速率, 而较高的光合速率与较高的气孔导度有关(图2-A~B)。前人研究表明较高浓度的H₂O₂能够诱导气孔关闭^[27,28]。甜菜碱的积累显著降低了高温胁迫下番茄叶片H₂O₂的积累(图3)。这可能是高温胁迫下转 codA 基因番茄维持较高气孔导度和光合速率的原因之一。另外, 前人对转基因烟草^[6]和芸薹属植物^[29]的研究认为, 在胁迫条件下, 甜菜碱能够通过保护光合机构增强光合能力。

	Figure Option View Download New Window
	图7 42℃高温胁迫对野生型和转 codA 基因番茄D1蛋白含量的影响Fig. 7 Effects of 42℃ treatment on D1 content in wild type and codA transgenic tomato plants

	Figure Option View Download New Window
	图8 42℃高温胁迫下野生型和转 codA 基因番茄D1蛋白的相对含量Fig. 8 Effects of 42℃ treatment on relative content of D1 protein in wild type and codA transgenic tomato plants

高温对PSII的伤害主要包括放氧复合体和光化学反应中心的构象变化^[30]。在本研究中, 高温胁迫下, F_v/ F_m的降低和 F_o的增高表明PSII反应中心受到了损伤。而转 codA 基因番茄植株中甜菜碱的积累缓解了 F_v/ F_m的降低和 F_o的增高(图2-C~D), 保护了PSII反应中心使其免受高温的伤害。Park等^[4]对冷胁迫下转 codA 基因番茄的研究和Kathuria等^[5]对干旱胁迫下转 codA 基因水稻的研究也得出了同样的结果; Allakhverdiev等^[31]的研究证实, 在高温下转 codA 基因蓝藻的放氧复合体和光反应中心的半失活温度均有所提高。而进一步研究表明, 高温对PSII的抑制作用主要是通过干扰D1蛋白的合成而抑制PSII的修复, 而不是直接通过光损伤来损伤PSII的^[32]。本研究结果表明, 高温胁迫严重抑制了D1蛋白的合成, 转 codA 基因番茄体内合成的甜菜碱有效缓解了高温胁迫对D1蛋白合成的抑制作用(图7), 减轻了高温胁迫对PSII的损伤(图2)。Allakhverdiev等^[31]通过对转 codA 蓝藻的研究表明甜菜碱能够通过促进D1蛋白的合成而保护PSII反应中心。

在光合细胞的电子传递过程中会产生活性氧, 而高温对PSII的损伤主要是由活性氧的积累造成的^[33]。前人研究证实, H₂O₂在胁迫中起着双重作用, 即浓度较低时, 作为一种信号物质, 而浓度超过一定限度时作为一种毒害物质^[4]。在正常条件下, 转基因番茄比野生型积累的H₂O₂高出16%, 当冷处理后, 转基因番茄仍维持正常条件下的H₂O₂含量, 而野生型比正常条件下的H₂O₂含量增高了0.5倍^[4]。本研究发现, 在正常条件下, 野生型番茄叶片中H₂O₂含量比转基因番茄低8%~10%, 高温胁迫之后, 野生型番茄叶片中H₂O₂含量明显高于转基因番茄(图3)。这是由于正常条件下转基因番茄产生甜菜碱的过程中有副产物H₂O₂产生, 而在高温胁迫下, 体内甜菜碱能够减轻活性氧的积累。

甜菜碱可通过提高抗氧化酶的活性清除ROS^[8]。CAT是通过将H₂O₂分解为H₂O和O₂而直接清除H₂O₂的酶, 在正常条件下, CAT酶的活性在转基因番茄叶片中始终高于野生型, 可能是转基因番茄叶片中较高的H₂O₂含量诱导所致^[10]。高温胁迫下, CAT、SOD、POD和APX的活性在转基因番茄叶片中均高于野生型(图5)。在强光胁迫下, 转 codA 基因拟南芥的CAT、APX和GR的活性均明显高于野生型^[34]。研究表明在高温胁迫下, 转基因番茄体内甜菜碱的积累提高了抗氧化酶的活性, 从而减少了活性氧的积累。

Kathuria等^[5]对转 codA 基因水稻转录组分析发现, 与野生型相比, 转基因水稻中有165个基因上调表达, 这些基因与胁迫响应、跨膜转运、激素信号、植物生长发育以及细胞代谢等过程有关。Park等^[35]通过cDNA芯片技术也证明, 与野生型相比, 转 codA 基因番茄花芽中30个基因的表达增强, 这些基因的表达与细胞分裂有关。本研究结果表明, 在高温胁迫下, 转 codA 基因番茄叶片的 CAT1 、 GR 、 MDHAR 、 HSP100 、 HSP70 和 HSP17.6 基因的表达均高于野生型(图6)。Li等^[9]在转 codA 基因番茄种子中研究发现, 热激蛋白的转录水平和HSP70的积累量均高于野生型种子, 推测甜菜碱可能通过某种机制上调抗氧化酶基因和热激蛋白等基因的表达以提高耐热性。

胆碱在胆碱氧化酶的作用下合成甜菜碱和副产物H₂O₂, 因此, 转基因番茄叶片中H₂O₂的含量高于野生型(图3), 有研究表明, 低浓度的H₂O₂可以通过诱导抗氧化酶基因 CAT 的表达来对氧化胁迫起到保护作用^[36]。本实验也表明, 转基因番茄叶片中较高的H₂O₂诱导了正常条件下较高的CAT活性(图5-A), 而且, 在正常条件下, 转基因番茄叶片中 CAT1 基因的表达量也明显高于野生型番茄(图6-C)。低浓度的H₂O₂可作为信号分子激活一系列抗氧化保护机制^[37,38]。推测在转 codA 番茄叶片中, H₂O₂与甜菜碱共同增强番茄的抗热性, 但对于H₂O₂在热胁迫条件下增强抗热性的机制还有待更深入研究。

4 结论

转 codA 基因缓解了高温胁迫对番茄叶片气孔导度的影响, 维持了较高的光合速率。较高气孔导度的维持与转 codA 基因能够抑制高温胁迫下番茄体内活性氧的产生有关; 转 codA 基因提高了高温胁迫下抗氧化酶活性, 降低了膜脂过氧化程度, 维持了膜的稳定性, 促进了D1蛋白的修复循环, 减轻了活性氧对PSII的损伤, 提高了PSII的耐热性; 高温胁迫下, 转 codA 基因番茄提高了抗氧化酶基因和热胁迫基因的表达以应对高温胁迫。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

参考文献

View Option

[1]	Finaka A, Cuendet A F, Maathuis F J H, Saidi Y, Goloubinoff P. Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance. Plant Cell, 2012, 24: 3333-3348 [本文引用:1] [JCR: 8.987]
[2]	Wang D-M(王冬梅), Xu X-Y(许向阳), Li J-F(李景富). The research progress of heat resistance in tomato. China Veg(中国蔬菜), 2003, (2): 58-60 (in Chinese) [本文引用:1]
[3]	Chen T H H, Murata N. Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ, 2011, 34: 1-20 [本文引用:1] [JCR: 5.215]
[4]	Park E J, Jeknić Z, Sakamoto A, DeNoma J, Yuwansiri R, Murata N, Chen T H H. Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. Plant J, 2004, 40: 474-487 [本文引用:5] [JCR: 6.16]
[5]	Kathuria H, Giri J, Nataraja K N, Murata N, Udayakumar M, Tyagi A K. Glycinebetaine-induced water-stress tolerance in codA-expressing transgenic indica rice is associated with up-regulation of several stress responsive genes. Plant Biotechnol J, 2009, 7: 512-526 [本文引用:3] [JCR: 5.442]
[6]	Yang X H, Liang Z, Wen X G, Lu C M. Genetic engineering of the biosynthesis of glycinebetaine leads to increased tolerance of photosynthesis to salt stress in transgenic tobacco plants. Plant Mol Biol, 2008, 66: 73-86 [本文引用:2] [JCR: 4.15]
[7]	Yang X H, Liang Z, Lu C M. Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants. Plant Physiol, 2005, 138: 2299-2309 [本文引用:2] [JCR: 6.535]
[8]	Yang X H, Wen X G, Gong H M, Lu Q T, Yang Z P, Tang Y L, Liang Z, Lu C M. Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants. Planta, 2007, 225: 719-733 [本文引用:2] [JCR: 3.0]
[9]	Li S F, Li F, Wang J W, Zhang W, Meng Q W, Chen T H H, Murata N, Yang X H. Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings. Plant Cell Environ, 2011, 34: 1931-1943 [本文引用:2] [JCR: 5.215]
[10]	Park E J, JekniĆ Z, Pino M T, Murata N, Chen T H H. Glycinebetaine accumulation is more effective in chloroplasts than in the cytosol for protecting transgenic tomato plants against abiotic stress. Plant Cell Environ, 2007, 30: 994-1005 [本文引用:2] [JCR: 5.215]
[11]	Rhodes D, Rich P J, Brunk D G, Ju G C, Rhodes J C, Pauly M H, Hansen L A. Development of two isogenic sweet corn hybrids differing for glycinebetaine content. Plant Physiol, 1989, 91: 1112-1121 [本文引用:1] [JCR: 6.535]
[12]	Kooten O, Snel J F H. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res, 1990, 25: 147-150 [本文引用:1] [JCR: 3.243]
[13]	Brennan T, Frenkel C. Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol, 1977, 59: 411-416 [本文引用:1] [JCR: 6.535]
[14]	Zhao S-J(赵世杰), Xu C-C(许长成), Zou Q(邹琦), Meng Q-W(孟庆伟). Improvements of method for measurement of malondialdehvde in plant tissues. Plant Physiol Commun(植物生理学通讯), 1991, 30(3): 207-210 (in Chinese) [本文引用:1] [CJCR: 0.833]
[15]	Clarke S M, Mur L A J, Wood J E, Scott I M. Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana. Plant J, 2004, 38: 432-447 [本文引用:1] [JCR: 6.16]
[16]	Aebi H. Catalase in vitro. Method Enzymol, 1984, 105: 121-126 [本文引用:1] [JCR: 2.042]
[17]	Giannopolitis G N, Reis S K. Superoxide dismutase I. occurrence in higher plants. Plant Physiol, 1977, 59: 309-315 [本文引用:1] [JCR: 6.535]
[18]	Cakmak I, Marschner H. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol, 1992, 98: 1222-1227 [本文引用:1] [JCR: 6.535]
[19]	Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate peroxidase in spinach chloroplasts. Plant Cell Physiol, 1981, 22: 867-880 [本文引用:1] [JCR: 4.702]
[20]	Zhang L X, Paakkarinen V, Van Wijk K J, Aro E M. Cotranslational assembly of the D1 protein into photosystem II. J Biol Chem, 1999, 274: 16062-16067 [本文引用:1] [JCR: 4.773]
[21]	Zhang Q Y, Wang L Y, Kong F Y, Deng Y S, Li B, Meng Q W. Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation. Physiol Plant, 2012, 146: 363-373 [本文引用:1] [JCR: 6.535]
[22]	Zhao S-J(赵世杰), Zou Q(邹琦), Zheng G-S(郑国生). The study of the method of modulation determination of chloroplast pigment in tobacco. Acta Tab Sin (中国烟草), 1993, 3: 17-19 (in Chinese with English abstract) [本文引用:1]
[23]	Callahan F E, Ghirardi M L, Sopory S K, Mehta A M, Edelman M, Mattoo A K. A novel metabolic from of the 32kDa-D1 protein in the grana-localized reaction center of photosystem II. J Biol Chem, 1990, 265: 15357-15360 [本文引用:1] [JCR: 4.773]
[24]	Guo J-W(郭军伟), Wei H-M(魏慧敏), Wu S-F(吴守锋), Du L-F(杜林方). Effects of low temperature on the distribution of excitation energy in photosystem and the phosphorylation of thylakoid membrane proteins in rice. Acta Biophys Sin(生物物理学报), 2006, 22(3): 197-202 (in Chinese) [本文引用:1] [JCR: 1.376] [CJCR: 0.65]
[25]	Chen J-M(陈建明), Yu X-P(俞晓平), Cheng J-A(程家安). The application of chlorophyll fluorescence kinetics in the study of physiological responses of plants to environmental stresses. Acta Agric Zhejiangensis(浙江农业学报), 2006, 18(1): 51-55 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.7981]
[26]	Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Plant Biol, 2004, 55: 373-399 [本文引用:1] [JCR: 25.962]
[27]	McAinsh M R, Clayton H, Mansfield T A, Hetherington A M. Changes in stomatal behavior and guard cell cytosolic free calcium inresponse to oxidative stress. Plant Physiol, 1996, 111: 1031-1042 [本文引用:1] [JCR: 6.535]
[28]	Lee S, Choi H, Suh S, Doo I S, Oh K Y, Choi E J, Schroeder Taylor A T, Low P S, Lee Y. Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis. Plant Physiol, 1999, 121: 147-152 [本文引用:1] [JCR: 6.535]
[29]	Wang Q, Xu W, Xue Q, Su W. Transgenic Brassica chinensis plants expressing a bacterial codA gene exhibit enhanced tole- rance to extreme temperature and high salinity. J Zhejiang Univ-Sci B, 2010, 11: 851-861 [本文引用:1] [JCR: 1.099] [CJCR: 0.7148]
[30]	Heber U, Bukhov N G, Shuvalov V A, Kobayashi Y, Lange O L. Protection of the photosynthetic apparatus against damage by excessive illumination in homoiohydric leaves and poikilohydric mosses and lichens. J Exp Bot, 2001, 52: 1999-2006 [本文引用:1] [JCR: 5.364]
[31]	Allakhverdiev S, Los D, Mohanty P, Nishiyama Y, Murata N. Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition. Biochim Biophys Acta, 2007, 1767: 1363-1371 [本文引用:2]
[32]	Ohnishi N, Murata N. Glycinebetaine counteracts the inhibitory effects of salt stress on the degradation and synthesis of D1 protein during photoinhibition in Synechococcus sp. PCC 7942. Plant Physiol, 2006, 141: 758-765 [本文引用:1] [JCR: 6.535]
[33]	Guo Y P, Zhou H F, Zhang L C. Photosynthetic characteristics and protective mechanisms against photooxidation during high temperature stress in two citrus species. Sci Hortic, 2006, 108: 260-267 [本文引用:1] [JCR: 1.527]
[34]	Alia, Kondo Y, Sakamoto A, Nonaka H, Hayashi H, Saradhi P P, Chen T H H, Murata N. Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase. Plant Mol Biol, 1999, 40: 279-288 [本文引用:1] [JCR: 4.15]
[35]	Park E J, Jeknic Z, Chen T H H, Murata N. The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato. Plant Biotechnol J, 2007, 5: 422-430 [本文引用:1] [JCR: 5.442]
[36]	Prasad T K, Anderson M D, Martin B A, Stewart C R. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell, 1994, 6: 65-74 [本文引用:1] [JCR: 8.987]
[37]	Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci, 2002, 7: 405-410 [本文引用:1] [JCR: 11.047]
[38]	Miller G, Shulaev V, Mittler R. Reactive oxygen signaling and abiotic stress. Physiol Plant, 2008, 133: 481-489 [本文引用:1] [JCR: 6.535]

2012

8.987

0.0

Plant Cell. 2012, 24(8):3333-48

Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Finka A, Cuendet AF, Maathuis FJ, Saidi Y, Goloubinoff P

Department of Plant Molecular Biology, University of Lausane, CH-1015 Lausane, Switzerland.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.

PMID: 22904147

... 高温能够影响植物光合作用、增加细胞膜的通透性、抑制蛋白质的合成进而抑制植株生长^[1] ...

2003

0.0

... 番茄植株生长发育的最适温度为昼温24~26℃, 夜温18℃左右, 30℃则过高, 到40℃就停止生长, 如果高温达到45℃时, 在短时间内茎叶就会出现日灼, 叶脉泛白, 以致坏死^[2] ...

2011

5.215

0.0

... 甜菜碱作为一种渗透调节物质, 参与植物对许多环境胁迫的响应^[3] ...

2004

6.16

0.0

Plant J.. 2004, 40(4):474-87

Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage.

Park EJ, Jeknić Z, Sakamoto A, DeNoma J, Yuwansiri R, Murata N, Chen TH

Department of Horticulture, ALS 4017, Oregon State University, Corvallis, OR 97331, USA.

Tomato (Lycopersicon esculentum Mill.) plants, which normally do not accumulate glycinebetaine (GB), are susceptible to chilling stress. Exposure to temperatures below 10 degrees C causes various injuries and greatly decreases fruit set in most cultivars. We have transformed tomato (cv. Moneymaker) with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase to catalyze the conversion of choline to GB. These transgenic plants express codA and synthesize choline oxidase, while accumulating GB in their leaves and reproductive organs up to 0.3 and 1.2 micromol g(-1) fresh weight (FW), respectively. Their chloroplasts contain up to 86% of total leaf GB. Over various developmental phases, from seed germination to fruit production, these GB-accumulating plants are more tolerant of chilling stress than their wild-type counterparts. During reproduction, they yield, on average, 10-30% more fruit following chilling stress. Endogenous GB contents as low as 0.1 micromol g(-1) FW are apparently sufficient to confer high levels of tolerance in tomato plants, as achieved via transformation with the codA gene. Exogenous application of either GB or H2O2 improves both chilling and oxidative tolerance concomitant with enhanced catalase activity. These moderately increased levels of H2O2 in codA transgenic plants, as a byproduct of choline oxidase-catalyzed GB synthesis, might activate the H2O2-inducible protective mechanism, resulting in improved chilling and oxidative tolerances in GB-accumulating codA transgenic plants. Thus, introducing the biosynthetic pathway of GB into tomato through metabolic engineering is an effective strategy for improving chilling tolerance.

PMID: 15500464

... 有研究表明, 转codA 基因番茄通过合成甜菜碱能够缓解低温胁迫对番茄幼苗生长发育以及开花的影响, 是因为在低温下甜菜碱能够维持一些抗氧化酶的活性, 降低细胞膜的膜脂过氧化作用, 减缓PSII最大光化学效率的降低程度^[4] ...

... 3 不同温度对野生型和转codA 番茄叶片过氧化氢(H₂O₂)含量的影响转codA 番茄在合成甜菜碱时有副产物H₂O₂产生^[4], 而在胁迫条件下细胞中活性氧的积累与细胞膜的氧化损伤有关^[26] ...

... Park等^[4]对冷胁迫下转codA 基因番茄的研究和Kathuria等^[5]对干旱胁迫下转codA 基因水稻的研究也得出了同样的结果 ...

... 前人研究证实, H₂O₂在胁迫中起着双重作用, 即浓度较低时, 作为一种信号物质, 而浓度超过一定限度时作为一种毒害物质^[4] ...

... 5倍^[4] ...

2009

5.442

0.0

Plant Biotechnol. J.. 2009, 7(6):512-26 DOI:10.1111/j.1467-7652.2009.00420.x

Glycinebetaine-induced water-stress tolerance in codA-expressing transgenic indica rice is associated with up-regulation of several stress responsive genes.

Kathuria H, Giri J, Nataraja KN, Murata N, Udayakumar M, Tyagi AK

Department of Plant Molecular Biology, Interdisciplinary Centre for Plant Genomics, University of Delhi South Campus, New Delhi, India.

Rice (Oryza sativa L.), a non-accumulator of glycinebetaine (GB), is highly susceptible to abiotic stress. Transgenic rice with chloroplast-targeted choline oxidase encoded by the codA gene from Arthrobacter globiformis has been evaluated for inheritance of transgene up to R5 generation and water-stress tolerance. During seedling, vegetative and reproductive stages, transgenic plants could maintain higher activity of photosystem II and they show better physiological performance, for example, enhanced detoxification of reactive oxygen species compared to wild-type plants under water-stress. Survival rate and agronomic performance of transgenic plants is also better than wild-type following prolonged water-stress. Choline oxidase converts choline into GB and H2O2 in a single step. It is possible that H2O2/GB might activate stress response pathways and prepare transgenic plants to mitigate stress. To check this possibility, microarray-based transcriptome analysis of transgenic rice has been done. It unravelled altered expression of many genes involved in stress responses, signal transduction, gene regulation, hormone signalling and cellular metabolism. Overall, 165 genes show more than two-fold up-regulation at P-value< 0.01 in transgenic rice. Out of these, at least 50 genes are known to be involved in plant stress response. Exogenous application of H2O2 or GB to wild-type plants also induces such genes. Our data show that metabolic engineering for GB is a promising strategy for introducing stress tolerance in crop plants and which could be imparted, in part, by H2O2- and/or GB-induced stress response genes.

PMID: 19490479

... Kathuria等^[5]认为转codA 基因水稻通过合成甜菜碱能够增加一些胁迫响应基因的表达来提高水稻植株的抗旱能力 ...

... Park等^[4]对冷胁迫下转codA 基因番茄的研究和Kathuria等^[5]对干旱胁迫下转codA 基因水稻的研究也得出了同样的结果 ...

... Kathuria等^[5]对转codA 基因水稻转录组分析发现, 与野生型相比, 转基因水稻中有165个基因上调表达, 这些基因与胁迫响应、跨膜转运、激素信号、植物生长发育以及细胞代谢等过程有关 ...

2008

4.15

0.0

Plant Mol Biol. 2008, 66(1-2):73 - 86 DOI:10.1007/s11103-007-9253-9

Genetic engineering of the biosynthesis of glycinebetaine leads to increased tolerance of photosynthesis to salt stress in transgenic tobacco plants

Xinghong Yang(12) Zheng Liang(1) Xiaogang Wen(1) Congming Lu(13)

1.Institute of Botany, Chinese Academy of Sciences Photosynthesis Research Center, Key Laboratory of Photosynthesis and Environmental Molecular Physiology Beijing 100093 China
2.Shandong Agricultural University College of Life Science, Key Laboratory of Crop Biology of Shandong Province Taian 211018 China
3.National Plant Gene Center Beijing 100093 China

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to synthesis glycinebetaine (GB) in chloroplasts was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea L.). The genetic engineering resulted in enhanced tolerance of growth of young seedlings to salt stress. This increased tolerance was not due to improved water status, since there were no significant differences in accumulation of sodium and chloride, leaf water potential, and relative water content between wild type and transgenic plants under salt stress. Salt stress resulted in a decrease in CO₂ assimilation and such a decrease was much greater in wild type plants than in transgenic plants. Though salt stress showed no damage to PSII, there were a decrease in the maximal PSII electron transport rate in vivo and an increase in non-photochemical quenching (NPQ) and these changes were greater in wild type plants than in transgenic plants. In addition, salt stress inhibited the activities of ribulose 1,5-bisphosphate carboxylase/oxygenase, chloroplastic fructose-1,6-bisphosphatase, fructose-1,6-bisphosphate aldolase, and phosphoribulokinase and such a decrease was also greater in wild type plants than in transgenic plants, suggesting that GB protects these enzymes against salt stress. However, there were no significant changes in the activities of phosphoglycerate kinase, triose phosphate isomerase, ribulose-5-phosphate isomerase, transketolase, and sedoheptulose-1,7-bisphosphatase in both wild type and transgenic plants. The results in this study suggest that enhanced tolerance of CO₂ assimilation to salt stress may be one of physiological bases for increased tolerance of growth of transgenic plants to salt stress.

... 另外一些研究发现甜菜碱能够缓解盐胁迫对卡尔文循环中一些酶的损伤, 从而提高转基因烟草的耐盐性^[6] ...

... 另外, 前人对转基因烟草^[6]和芸薹属植物^[29]的研究认为, 在胁迫条件下, 甜菜碱能够通过保护光合机构增强光合能力 ...

2005

6.535

0.0

Plant Physiol.. 2005, 138(4):2299-309

Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants.

Yang X, Liang Z, Lu C

Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Photosynthesis Research Center, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China.

Genetically engineered tobacco (Nicotiana tabacum) with the ability to synthesis glycinebetaine was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea). The genetic engineering enabled the plants to accumulate glycinebetaine mainly in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO2 assimilation of transgenic plants was significantly more tolerant to high temperatures than that of wild-type plants. The analyses of chlorophyll fluorescence and the activation of Rubisco indicated that the enhancement of photosynthesis to high temperatures was not related to the function of photosystem II but to the Rubisco activase-mediated activation of Rubisco. Western-blotting analyses showed that high temperature stress led to the association of Rubisco activase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than in transgenic plants. The results in this study suggest that under high temperature stress, glycinebetaine maintains the activation of Rubisco by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fractions and thus enhances the tolerance of CO2 assimilation to high temperature stress. The results seem to suggest that engineering of the biosynthesis of glycinebetaine by transformation with the BADH gene might be an effective method for enhancing high temperature tolerance of plants.

PMID: 16024688

... Yang等^[7,8]指出在高温胁迫下, 甜菜碱能够通过提高烟草的光合能力和PSII活性来增强其耐热性 ...

... 控制光强为800 μmol m^-2 s^-1, CO₂ 浓度为380 μL L^-1, 温度为25℃, 空气相对湿度65%~70%^[7] ...

2007

3.0

0.0

Planta. 2007, 225(3):719 - 733 DOI:10.1007/s00425-006-0380-3

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants

Xinghong Yang(12) Xiaogang Wen(1) Hongmei Gong(1) Qingtao Lu(1) Zhipan Yang(1) Yunlai Tang(1) Zheng Liang(1) Congming Lu(1)

1.Chinese Academy of Sciences Photosynthesis Research Center, Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany Beijing 100093 People’s Republic of China
2.Shandong Agricultural University College of Life Science, Key Laboratory of Crop Biology of Shandong Taian 217017 People’s Republic of China

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25–50°C) for 4 h in the dark and under growth light intensity (300 μmol m⁻² s⁻¹). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H₂O₂, O₂⁻ and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.

... Yang等^[7,8]指出在高温胁迫下, 甜菜碱能够通过提高烟草的光合能力和PSII活性来增强其耐热性 ...

... 甜菜碱可通过提高抗氧化酶的活性清除ROS^[8] ...

2011

5.215

0.0

... Li等^[9]发现在高温胁迫下外源甜菜碱能够促进番茄种子的萌发和幼苗的生长 ...

... Li等^[9]在转codA 基因番茄种子中研究发现, 热激蛋白的转录水平和HSP70的积累量均高于野生型种子, 推测甜菜碱可能通过某种机制上调抗氧化酶基因和热激蛋白等基因的表达以提高耐热性 ...

2007

5.215

0.0

Plant Cell Environ.. 2007, 30(8):994-1005

Glycinebetaine accumulation is more effective in chloroplasts than in the cytosol for protecting transgenic tomato plants against abiotic stress.

Park EJ, Jeknić Z, Pino MT, Murata N, Chen TH

Department of Horticulture, ALS 4017, Oregon State University, Corvallis, OR 97331, USA.

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants were transformed with a gene for choline oxidase (codA) from Arthrobacter globiformis. The gene product (CODA) was targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). These three transgenic plant types accumulated different amounts and proportions of glycinebetaine (GB) in their chloroplasts and cytosol. Targeting CODA to either the cytosol or both compartments simultaneously increased total GB content by five- to sixfold over that measured from the chloroplast targeted lines. Accumulation of GB in codA transgenic plants was tissue dependent, with the highest levels being recorded in reproductive organs. Despite accumulating, the lowest amounts of GB, Chl-codA plants exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses. This suggests that chloroplastic GB is more effective than cytosolic GB in protecting plant cells against chilling, high salt and oxidative stresses. Chloroplastic GB levels were positively correlated with the degree of oxidative stress tolerance conferred, whereas cytosolic GB showed no such a correlation. Thus, an increase in total GB content does not necessarily lead to enhanced stress tolerance, but additional accumulation of chloroplastic GB is likely to further raise the level of stress tolerance beyond what we have observed.

PMID: 17617827

... 转codA 番茄由美国俄勒冈州立大学Tony Chen实验室提供, 该基因定位于叶绿体和细胞质^[10] ...

... CAT是通过将H₂O₂分解为H₂O和O₂而直接清除H₂O₂的酶, 在正常条件下, CAT酶的活性在转基因番茄叶片中始终高于野生型, 可能是转基因番茄叶片中较高的H₂O₂含量诱导所致^[10] ...

1989

6.535

0.0

Plant Physiol.. 1989, 91(3):1112-21

Development of two isogenic sweet corn hybrids differing for glycinebetaine content.

Rhodes D, Rich PJ, Brunk DG, Ju GC, Rhodes JC, Pauly MH, Hansen LA

Center for Plant Environmental Stress Physiology, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907.

A hybrid of sweet corn, Zea mays L. (;1720'; Rogers Brothers Seed Co.), was found to be comprised of glycinebetaine-positive and glycinebetaine-deficient individuals in a 1:1 mixture. This phenomenon was traced to segregation for a single, nuclear, dominant gene determining leaf glycinebetaine content within the female inbred parent of this hybrid. Selection for homozygous recessive (glycinebetaine-deficient) and homozygous dominant (glycinebetaine-positive) genotypes of the female inbred parent enabled production of two isogenic versions of hybrid ;1720' differing with respect to a single copy of the dominant allele, by mating these female parent selections with the common homozygous recessive (glycinebetaine-deficient) male parent. These two isogenic hybrids are shown to differ by a factor of 300- to 400-fold in glycinebetaine titer of young expanding leaves of salinized plants, but exhibit no striking differences in the levels of free amino acids or the level of N-methylnicotinic acid (nicotinic acid betaine; trigonelline). The only significant difference between the two hybrids in terms of amino acid composition was found to be in the level of alanine under nonsalinized conditions. The betaine-deficient hybrid exhibited a 14% lower alanine level than the betaine-positive hybrid. Betaine deficiency was not associated with altered stress-induced accumulation of amino acids such as proline, serine, and asparagine plus aspartate, attesting to the high specificity of the genetic difference between these isogenic hybrids with respect to betaine accumulation. This germplasm offers unique opportunities to test whether a single dominant allele determining stress-induced betaine accumulation capacity influences stress resistance in maize.

PMID: 16667120

... 1 甜菜碱含量的测定参考Rhodes等^[11]的方法提取和测定甜菜碱 ...

1990

3.243

0.0

... 3 荧光参数的测定采用便携脉冲调制式荧光仪(FMS-2, Hansatech, 英国)测定^[12] ...

1977

6.535

0.0

Plant Physiol.. 1977, 59(3):411-6

Involvement of hydrogen peroxide in the regulation of senescence in pear.

Brennan T, Frenkel C

Department of Botany and Department of Horticulture and Forestry, Rutgers University, New Brunswick, New Jersey 08903.

Endogenous peroxide levels in pear fruit (Pyrus communis) were measured using a titanium assay method, and were found to increase during senescence in both Bartlett and Bosc varieties. Application of glycolic acid or xanthine, serving as substrates for the formation of H(2)O(2), increased the peroxide content of the tissue and accelerated the onset of ripening, as measured by increased softening and ethylene evolution. Application of ethylene also induced increased peroxide levels. Ripening processes were similarly promoted when peroxides were conserved by inhibiting the activity of catalase with hydroxylamine or potassium cyanide. By comparison, the inhibition of glycolate oxidase with alphahydroxy-2-pyridinemethanesulfonic acid decreased the peroxide content of the tissue and delayed the onset of ripening. These results indicate that the onset of ripening correlates with the peroxide content of fruit tissues as occurring under normal conditions or as influenced by the treatments. Hydrogen peroxide may be involved in oxidative processes required in the initiation and the promotion of ripening.

PMID: 16659863

... 4 过氧化氢(H₂O₂)含量的测定参照 Brennan和Frenkel^[13]的方法测定 ...

1991

0.0

0.833

... 参照赵世杰等^[14]的硫代巴比妥酸(TBA)比色法测定MDA含量, 参照Clarke等^[15]的方法测定REC ...

2004

6.16

0.0

... 参照赵世杰等^[14]的硫代巴比妥酸(TBA)比色法测定MDA含量, 参照Clarke等^[15]的方法测定REC ...

1984

2.042

0.0

... 按Aebi^[16]的方法测定过氧化氢酶(CAT)活性, 按Giannopolitis和Ries^[17]的方法测定超氧化物歧化酶(SOD)活性, 按愈创木酚法^[18]测定过氧化物酶(POD)活性, 按Nakano和Asada^[19]的方法测定抗坏血酸过氧化物酶(APX)活性 ...

1977

6.535

0.0

Plant Physiol.. 1977, 59(2):309-14

Superoxide dismutases: I. Occurrence in higher plants.

Giannopolitis CN, Ries SK

Department of Horticulture, Michigan State University, East Lansing, Michigan 48824.

Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium. The enzyme is present in the shoots, roots, and seeds of the three species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated a total of 10 distinct forms of the enzyme. Corn contained seven of these forms and oats three. Peas contained one of the corn and two of the oat enzymes. Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme. Some of the leaf superoxide dismutases were found primarily in mitochondria or chloroplasts. Peroxidases at high concentrations interfere with the assay. In test tube assays of crude extracts from seedlings, the interference was negligible. On gels, however, peroxidases may account for two of the 10 superoxide dismutase forms.

PMID: 16659839

1992

6.535

0.0

Plant Physiol.. 1992, 98(4):1222-7

Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves.

Cakmak I, Marschner H

Department of Soil Science and Plant Nutrition, University of Cukurova, Adana, Turkey.

The influence of varied Mg supply (10-1000 micromolar) and light intensity (100-580 microeinsteins per square meter per second) on the concentrations of ascorbate (AsA) and nonprotein SH-compounds and the activities of superoxide dismutase (SOD; EC 1.15.11) and the H(2)O(2) scavenging enzymes, AsA peroxidase (EC 1.11.1.7), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were studied in bean (Phaseolus vulgaris L.) leaves over a 13-day period. The concentrations of AsA and SH-compounds and the activities of SOD and H(2)O(2) scavenging enzymes increased with light intensity, in particular in Mg-deficient leaves. Over the 12-day period of growth for a given light intensity, the concentrations of AsA and SH-compounds and the activities of these enzymes remained more or less constant in Mg-sufficient leaves. In contrast, in Mg-deficient leaves, a progressive increase was recorded, particularly in concentrations of AsA and activities of AsA peroxidase and glutathione reductase, whereas the activities of guaiacol peroxidase and catalase were only slightly enhanced. Partial shading of Mg-deficient leaf blades for 4 days prevented chlorosis, and the activities of the O(2) (.-) and H(2)O(2) scavenging enzymes remained at a low level. The results demonstrate the role of both light intensity and Mg nutritional status on the regulation of O(2) (.-) and H(2)O(2) scavenging enzymes in chloroplasts.

PMID: 16668779

1981

4.702

0.0

1999

4.773

0.0

J. Biol. Chem.. 1999, 274(23):16062-7

Co-translational assembly of the D1 protein into photosystem II.

Zhang L, Paakkarinen V, van Wijk KJ, Aro EM

Department of Biology, University of Turku, FIN-20520 Turku, Finland.

Assembly of multi-subunit membrane protein complexes is poorly understood. In this study, we present direct evidence that the D1 protein, a multiple membrane spanning protein, assembles co-translationally into the large membrane-bound complex, photosystem II. During pulse-chase studies in intact chloroplasts, incorporation of the D1 protein occurred without transient accumulation of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa. These N-terminal D1 intermediates could be co-immunoprecipitated with antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a co-translational assembly of the D1 protein into photosystem II was obtained by analyzing ribosome nascent chain complexes liberated from the thylakoid membrane after a short pulse labeling. Radiolabeled D1 intermediates could be immunoprecipitated under nondenaturing conditions with antisera raised against the D1 and D2 protein as well as CP47. However, when the ribosome pellets were solubilized with SDS, the interaction of these intermediates with CP47 was completely lost, but strong interaction of a 25-kDa D1 intermediate with the D2 protein still remained. Taken together, our results indicate that during the repair of photosystem II, the assembly of the newly synthesized D1 protein into photosystem II occurs co-translationally involving direct interaction of the nascent D1 chains with the D2 protein.

PMID: 10347157

2012

6.535

0.0

Physiol Plant. 2012, 146(3):363-73 DOI:10.1111/j.1399-3054.2012.01645.x

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation.

Zhang QY, Wang LY, Kong FY, Deng YS, Li B, Meng QW

College of Life Science, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, Shandong 271018, PR China.

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non-photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress-induced photoinhibition and photooxidation, antisense-mediated suppression of zeaxanthin epoxidase transgenic plants and the wild-type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H(2)O(2)), superoxide anion radical (O(2)(•-)) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress-induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.

PMID: 22578286

1993

0.0

... 采用赵世杰等^[22]的方法测定叶绿素含量 ...

1990

4.773

0.0

J. Biol. Chem.. 1990, 265(26):15357-60

A novel metabolic form of the 32 kDa-D1 protein in the grana-localized reaction center of photosystem II.

Callahan FE, Ghirardi ML, Sopory SK, Mehta AM, Edelman M, Mattoo AK

Plant Molecular Biology Laboratory, United States Department of Agriculture-ARS, Beltsville Agricultural Research Center (West), Maryland 20705.

Two forms of the 32 kDa-D1 reaction center protein of photosystem II (PSII), having slightly different mobilities on denaturing polyacrylamide gels, have been resolved in Spirodela oligorrhiza, Glycine max L., Gossypium hirsutum L., Triticum aestivum L., and Zea mays L. The protein band with faster mobility is identified as the 32 kDa-D1 protein, and the less mobile band as a novel form, designated 32*. The two forms are structurally similar based on immunological and partial proteolytic tests. 32* is associated exclusively with the grana and is present in the PSII reaction center. Temporally, 32* appears several hours after the translocation of newly synthesized and processed 32 kDa-D1 protein from the stroma lamellae to the grana. Formation of the 32* is strictly light-dependent under physiological light intensities and correlates with a reciprocal loss of the 32-kDa form. Light induced formation of 32* is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea but is not coupled to linear electron transport.

PMID: 2203777

... D1蛋白的分离与测定: 采用SDS-PAGE(聚丙烯酰胺凝胶电泳)和Western blot(免疫印迹)法^[23] ...

2006

1.376

0.65

... 上样量以叶绿素含量为准, 保证每泳道一致的叶绿素含量^[24] ...

2006

0.0

0.7981

Acta Agric Zhejiangensis. 2006, (1):51 - 55

The application of chlorophyll fluorescence kinetics in the study of physiological responses of plants to environmental stresses

叶绿素荧光动力学及其在植物抗逆生理研究中的应用

CHENJian-ming^[1];YUXiao-ping^[1];CHENGJia-an^[2]

陈建明^[1];俞晓平^[1];程家安^[2]

概述了叶绿素荧光动力学的基本原理和测量方法,以及近年来叶绿素荧光动力学在植物抗逆生理研究中的应用,包括植物对光抑制、低温、热、水分和盐碱、营养、病原菌侵染等各种环境因子胁迫的反应.

... F_v/F_m是暗适应下PSII的最大光化学效率, 反映了PSII反应中心最大光能转化效率^[25] ...

2004

25.962

0.0

Annu Rev Plant Biol. 2004, 55:373-99

Reactive oxygen species: metabolism, oxidative stress, and signal transduction.

Apel K, Hirt H

Institute of Plant Sciences, Swiss Federal Institute of Technology, Universitatstr. 2, 8092 Zurich, Switzerland.

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of aerobic metabolism. Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Whereas plants are surfeited with mechanisms to combat increased ROS levels during abiotic stress conditions, in other circumstances plants appear to purposefully generate ROS as signaling molecules to control various processes including pathogen defense, programmed cell death, and stomatal behavior. This review describes the mechanisms of ROS generation and removal in plants during development and under biotic and abiotic stress conditions. New insights into the complexity and roles that ROS play in plants have come from genetic analyses of ROS detoxifying and signaling mutants. Considering recent ROS-induced genome-wide expression analyses, the possible functions and mechanisms for ROS sensing and signaling in plants are compared with those in animals and yeast.

PMID: 15377225

1996

6.535

0.0

Plant Physiol.. 1996, 111(4):1031-1042

Changes in Stomatal Behavior and Guard Cell Cytosolic Free Calcium in Response to Oxidative Stress.

McAinsh MR, Clayton H, Mansfield TA, Hetherington AM

Institute of Environmental and Biological Sciences, Division of Biological Sciences, Lancaster University, Lancaster, United Kingdom LA1 4YQ.

We have investigated the cellular basis for the effects of oxidative stress on stomatal behavior using stomatal bioassay and ratio photometric techniques. Two oxidative treatments were employed in this study: (a) methyl viologen, which generates superoxide radicals, and (b) H2O2. Both methyl viologen and H2O2 inhibited stomatal opening and promoted stomatal closure. At concentrations [less than or equal to]10-5 M, the effects of methyl viologen and H2O2 on stomatal behavior were reversible and were abolished by 2 mM EGTA or 10 [mu]M verapamil. In addition, at 10-5 M, i.e. the maximum concentration at which the effects of the treatments were prevented by EGTA or verapamil, methyl viologen and H2O2 caused an increase in guard cell cytosolic free Ca2+ ([Ca2+]i), which was abolished in the presence of EGTA. Therefore, at low concentrations of methyl viologen and H2O2, removal of extracellular Ca2+ prevented both the oxidative stress-induced changes in stomatal aperture and the associated increases in [Ca2+]i. This suggests that in this concentration range the effects of the treatments are Ca2+-dependent and are mediated by changes in [Ca2+]i. In contrast, at concentrations of methyl viologan and H2O2 > 10-5 M, EGTA and verapamil had no effect. However, in this concentration range the effects of the treatments were irreversible and correlated with a marked reduction in membrane integrity and guard cell viability. This suggests that at high concentrations the effects of methyl viologen and H2O2 may be due to changes in membrane integrity. The implications of oxidative stress-induced increases in [Ca2+]i and the possible disruption of guard-cell Ca2+ homeostasis are discussed in relation to the processes of Ca2+-based signal transduction in stomatal guard cells and the control of stomatal aperture.

PMID: 12226345

... 前人研究表明较高浓度的H₂O₂能够诱导气孔关闭^[27,28] ...

1999

6.535

0.0

Plant Physiol.. 1999, 121(1):147-52

Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis.

Lee S, Choi H, Suh S, Doo IS, Oh KY, Choi EJ, Schroeder Taylor AT, Low PS, Lee Y

Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea.

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (beta-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) and Commelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca(2+) and H(2)O(2) mediate the elicitor-induced decrease of stomatal apertures. We show that the H(2)O(2) that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H(2)O(2) production, thus interfering with the continuous invasion of pathogens through the stomatal pores.

PMID: 10482669

... 前人研究表明较高浓度的H₂O₂能够诱导气孔关闭^[27,28] ...

2010

1.099

0.7148

J Zhejiang Univ-sc B. 2010, 11(11):851 - 861 DOI:10.1631/jzus.B1000137

TransgenicBrassica chinensis plants expressing a bacterialcodA gene exhibit enhanced tolerance to extreme temperature and high salinity

Qing-bin Wang(13) Wen Xu(2) Qing-zhong Xue(1) Wei-ai Su(2)

1.Department of Agronomy, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, 310029 China
2.Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Science, Shanghai, 200032 China
3.School of Resource and Environment Management, Guizhou College of Finance and Economics, Guiyang, 550004 China

TransgenicBrassica compestris L. spp.chinensis plants expressing a choline oxidase (codA) gene fromArthrobacter globiformis were obtained throughAgrobacterium tumefaciens-mediated transformation. In the transgenic plants,codA gene expression and its product transportation to chloroplasts were detected by the enzyme-linked immunosorbent assay (ELISA) examination, immunogold localization, and¹H-nuclear magnetic resonance (¹H-NMR). Stress tolerance was evaluated in the T₃ plants under extreme temperature and salinity conditions. The plants of transgenic line 1 (L1) showed significantly higher net photosynthetic rate (P_n) andP_n recovery rate under high (45 °C, 4 h) and low temperature (1 °C, 48 h) treatments, and higher photosynthetic rate under high salinity conditions (100, 200, and 300 mmol/L NaCl, respectively) than the wild-type plants. The enhanced tolerance to high temperature and high salinity stresses in transgenic plants is associated with the accumulation of betaine, which is not found in the wild-type plants. Our results indicate that the introduction ofcodA gene fromArthrobacter globiformis intoBrassica compestris L. spp.chinensis could be a potential strategy for improving the plant tolerance to multiple stresses.

... 另外, 前人对转基因烟草^[6]和芸薹属植物^[29]的研究认为, 在胁迫条件下, 甜菜碱能够通过保护光合机构增强光合能力 ...

2001

5.364

0.0

... 高温对PSII的伤害主要包括放氧复合体和光化学反应中心的构象变化^[30] ...

2007

0.0

Biochim. Biophys. Acta. 2007, 1767(12):1363-71

Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition.

Allakhverdiev SI, Los DA, Mohanty P, Nishiyama Y, Murata N

National Institute for Basic Biology, Okazaki 444-8585, Japan.

Transformation with the bacterial gene codA for choline oxidase allows Synechococcus sp. PCC 7942 cells to accumulate glycinebetaine when choline is supplemented exogenously. First, we observed two types of protective effect of glycinebetaine against heat-induced inactivation of photosystem II (PSII) in darkness; the codA transgene shifted the temperature range of inactivation of the oxygen-evolving complex from 40-52 degrees C (with half inactivation at 46 degrees C) to 46-60 degrees C (with half inactivation at 54 degrees C) and that of the photochemical reaction center from 44-55 degrees C (with half inactivation at 51 degrees C) to 52-63 degrees C (with half inactivation at 58 degrees C). However, in light, PSII was more sensitive to heat stress; when moderate heat stress, such as 40 degrees C, was combined with light stress, PSII was rapidly inactivated, although these stresses, when applied separately, did not inactivate either the oxygen-evolving complex or the photochemical reaction center. Further our studies demonstrated that the moderate heat stress inhibited the repair of PSII during photoinhibition at the site of synthesis de novo of the D1 protein but did not accelerate the photodamage directly. The codA transgene and, thus, the accumulation of glycinebetaine alleviated such an inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein. We propose a hypothetical scheme for the cyanobacterial photosynthesis that moderate heat stress inhibits the translation machinery and glycinebetaine protects it against the heat-induced inactivation.

PMID: 17991419

... Allakhverdiev等^[31]的研究证实, 在高温下转codA 基因蓝藻的放氧复合体和光反应中心的半失活温度均有所提高 ...

... Allakhverdiev等^[31]通过对转codA 蓝藻的研究表明甜菜碱能够通过促进D1蛋白的合成而保护PSII反应中心 ...

2006

6.535

0.0

Plant Physiol.. 2006, 141(2):758-65

Glycinebetaine counteracts the inhibitory effects of salt stress on the degradation and synthesis of D1 protein during photoinhibition in Synechococcus sp. PCC 7942.

Ohnishi N, Murata N

National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan.

Glycinebetaine (hereafter referred to as betaine) is a compatible solute that accumulates in certain plants and microorganisms in response to various types of stress. We demonstrated previously that when the cyanobacterium Synechococcus sp. PCC 7942 (hereafter Synechococcus) is transformed with the codA gene for choline oxidase, it can synthesize betaine from exogenously supplied choline, exhibiting enhanced tolerance to salt and cold stress. In this study, we examined the effects of salt stress and betaine synthesis on the photoinhibition of photosystem II (PSII). Salt stress due to 220 mm NaCl enhanced photoinhibition of PSII and betaine protected PSII against photoinhibition under these conditions. However, neither salt stress nor betaine synthesis affected photodamage to PSII. By contrast, salt stress inhibited repair of photodamaged PSII and betaine reversed this inhibitory effect of salt stress. Pulse-chase-labeling experiments revealed that salt stress inhibited degradation of D1 protein in photodamaged PSII and de novo synthesis of D1. By contrast, betaine protected the machinery required for degradation and synthesis of D1 under salt stress. Neither salt stress nor betaine affected levels of psbA transcripts. These observations suggest that betaine counteracts the inhibitory effects of salt stress, with resultant accelerated repair of photodamaged PSII.

PMID: 16632587

... 而进一步研究表明, 高温对PSII的抑制作用主要是通过干扰D1蛋白的合成而抑制PSII的修复, 而不是直接通过光损伤来损伤PSII的^[32] ...

2006

1.527

0.0

... 在光合细胞的电子传递过程中会产生活性氧, 而高温对PSII的损伤主要是由活性氧的积累造成的^[33] ...

1999

4.15

0.0

Plant Mol. Biol.. 1999, 40(2):279-88

Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase.

Alia , Kondo Y, Sakamoto A, Nonaka H, Hayashi H, Saradhi PP, Chen TH, Murata N

Department of Regulation Biology, National Institute for Basic Biology, Okazaki, Japan.

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis. This gene encodes choline oxidase, an enzyme that converts choline to glycinebetaine. The photosynthetic activity, monitored in terms of chlorophyll fluorescence, of transformed plants was more tolerant to light stress than that of wild-type plants. This enhanced tolerance to light stress was caused by acceleration of the recovery of the photosystem II (PS II) complex from the photo-inactivated state. The transformed plants synthesized glycinebetaine, but no changes were detected in the relative levels of membrane lipids or in the relative levels of fatty acids in the various membrane lipids. Transformation with the codA gene increased levels of H2O2, a by-product of the reaction catalyzed by choline oxidase, by only 50% to 100% under stress or non-stress conditions. The activity of ascorbate peroxidase and, to a lesser extent, that of catalase in transformed plants were significantly higher than in the wild-type plants. These observations suggest that H2O2 produced by choline oxidase in the transformed plants might have stimulated the expression of H2O2 scavenging enzymes, with resultant maintenance of the level of H2O2 within a certain limited range. It appears that glycinebetaine produced in vivo, but not changes in membrane lipids or in the level of H2O2, protected the PS II complex in transformed plants from damage due to light stress.

PMID: 10412906

... 在强光胁迫下, 转codA 基因拟南芥的CAT、APX和GR的活性均明显高于野生型^[34] ...

2007

5.442

0.0

... Park等^[35]通过cDNA芯片技术也证明, 与野生型相比, 转codA 基因番茄花芽中30个基因的表达增强, 这些基因的表达与细胞分裂有关 ...

1994

8.987

0.0

... 胆碱在胆碱氧化酶的作用下合成甜菜碱和副产物H₂O₂, 因此, 转基因番茄叶片中H₂O₂的含量高于野生型(图3), 有研究表明, 低浓度的H₂O₂可以通过诱导抗氧化酶基因CAT 的表达来对氧化胁迫起到保护作用^[36] ...

2002

11.047

0.0

Trends Plant Sci.. 2002, 7(9):405-10

Oxidative stress, antioxidants and stress tolerance.

Mittler R

Dept of Botany, Plant Sciences Institute, 353 Bessey Hall, Iowa State University, Ames, IA 50011, USA. rmittler@iastate.edu

Traditionally, reactive oxygen intermediates (ROIs) were considered to be toxic by-products of aerobic metabolism, which were disposed of using antioxidants. However, in recent years, it has become apparent that plants actively produce ROIs as signaling molecules to control processes such as programmed cell death, abiotic stress responses, pathogen defense and systemic signaling. Recent advances including microarray studies and the development of mutants with altered ROI-scavenging mechanisms provide new insights into how the steady-state level of ROIs are controlled in cells. In addition, key steps of the signal transduction pathway that senses ROIs in plants have been identified. These raise several intriguing questions about the relationships between ROI signaling, ROI stress and the production and scavenging of ROIs in the different cellular compartments.

PMID: 12234732

... 低浓度的H₂O₂可作为信号分子激活一系列抗氧化保护机制^[37,38] ...

2008

6.535

0.0

Physiol Plant. 2008, 133(3):481-9 DOI:10.1111/j.1399-3054.2008.01090.x

Reactive oxygen signaling and abiotic stress.

Miller G, Shulaev V, Mittler R

Department of Biochemistry and Molecular Biology, University of Nevada, MS200, Reno, NV 89557, USA.

Reactive oxygen species (ROS) play a dual role in plant biology acting on the one hand as important signal transduction molecules and on the other as toxic by-products of aerobic metabolism that accumulate in cells during different stress conditions. Because of their toxicity as well as their important signaling role, the level of ROS in cells is tightly controlled by a vast network of genes termed the 'ROS gene network'. Using mutants deficient in key ROS-scavenging enzymes, we have defined a signaling pathway that is activated in cells in response to ROS accumulation. Interestingly, many of the key players in this pathway, including different zinc finger proteins and WRKY transcription factors, are also central regulators of abiotic stress responses involved in temperature, salinity and osmotic stresses. Here, we describe our recent findings and discuss how ROS integrate different signals originating from different cellular compartments during abiotic stress.

PMID: 18346071

... 低浓度的H₂O₂可作为信号分子激活一系列抗氧化保护机制^[37,38] ...