番茄线粒体解偶联蛋白基因（LeUCP）沉默对番茄光合作用、呼吸作用及抗逆性的影响
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摘要
植物线粒体是进行呼吸作用为细胞提供能量的重要场所,其在感知外界逆境信号,调控植物抗逆防御反应中也扮演着重要的角色。植物线粒体对氧化胁迫的消除机制除存在交替氧化酶为末端氧化酶的交替电子传递链路径外,还可以通过线粒体解偶联蛋白UCPs来减弱活性氧的产生。研究发现,AtUCPl基因对低温、干旱等氧化胁迫抗性以及有效的光合作用均起着积极的作用。然而其对蔬菜作物的调节及在抗逆中的作用机制尚不清楚。因此,研究UCPs基因在调节植物光合、呼吸作用及在抗逆反应中的作用机制无疑具有非常重要的科学和现实意义。
本文以烟草脆裂病毒(TRV)和番茄为材料,选择八氢番茄红素去饱和酶基因(PDS)作为参照基因,构建了TRV-LePDS和TRV-LeUCP重组质粒,建立了LePDS和LeUCP基因VIGS体系,LePDS基因沉默植株的叶片出现典型的光漂白现象；LeUCP基因沉默植株其基因表达量显著降低。
利用TRV-LeUCP基因沉默植株,研究了番茄LeUCP基因对光合参数以及相关酶活性、荧光参数、电子流分配的影响,分析了LeUCP基因对线粒体呼吸、活性氧积累、交替氧化酶的影响。结合三羧酸循环中的顺乌头酸酶活性和柠檬酸含量及氧化还原状态以的变化,探明线粒体解偶联蛋白对Redox状态的影响。在PQ和高温逆境条件下,探明线粒体解偶联蛋白在氧化胁迫下是否起到保护作用。所得主要结果如下：
1、研究了番茄LeUCP基因沉默对光合作用的影响。结果表明,TRV病毒载体侵染未引起番茄植株生长表型差异；基因沉默植株叶片在不同光照强度下的光合速率均低于对照植株,Pn、4sat、Vc，max值降低,气孔导度Gs提高,但对Ci无明显影响。番茄LeUCP基因沉默植株Rubisco和FBPase酶活性受抑制,分别降低了23.3%和20.3%。
2、研究了番茄LeUCP基因沉默对叶绿素荧光参数的影响。LeUCP基因表达量的降低对Fv/Fm无明显影响,但降低了光化学猝灭系数(qP)和电子传递速率ETR,与Mock处理植株相比qP值降低了54%。提高了非光化学猝灭系数NPQ,并且明显降低了Je(PSII)和分配于Je(PCR)与Je(PCO)的比例以及NAD+/NADH比值。说明LeUCP基因编码的线粒体解偶联蛋白对有效的光合是必需的,可能通过影响卡尔文循环关键酶、重新分配PSII的电子流比例、降低电子传递速率和光呼吸而起作用。
3、研究了番茄LeUCP基因沉默对呼吸作用和Redox状态的影响。基因沉默植株线粒体总呼吸和交替呼吸下降明显,抑制AOXl基因家族的基因表达,其中对AOXla基因表达量影响显著。LeUCP基因表达量的降低影响还原态泛醌的含量和丙酮酸含量,分别较Mock下降了54.3%和29.1%,表明线粒体交替氧化酶和解偶联蛋白两者呈协同表达关系。三羧酸循环中的顺乌头酸酶活性和柠檬酸含量与Mock相比分别降低34.6%、14.4%。NADH含量增加且NAD+/NADH明显下降,表明线粒体解偶联蛋白对维持Redox状态起一定作用。
4、研究了PQ和高温氧化胁迫下解偶联蛋白基因的响应。与Mock和TRV组植株相比,LeUCP基因沉默诱导了植株叶片H2O2和O2.-的积累,并且在PQ和高温胁迫处理下,ROS积累显著增加,其中PQ处理后H2O2和O2.-分别较Mock增加44.67%和23%。PQ胁迫后造成了严重的氧化胁迫,Fv/Fm明显下降,MDA含量增加。CAT1、CAT2、SOD、cAPX、GR、POD基因表达量提高,但TRV-LeUCP+PQ仍然低于Mock+PQ与TRV+PQ。与Mock相比,PQ处理植株提高了AsA、GSH的含量,并且AsA/DHA、GSH+GSSG也有所增加,但TRV-LeUCP+PQ处理植株叶片AsA含量和GSH含量均低于Mock+PQ与TRV+PQ处理,进一步表明LeUCP对维持Redox信号平衡和增强氧化胁迫抗性具有重要作用。
Plant mitochondrion is the important place to provide energy for cell respiration. It plays an important role for plants in perceiving in stress signal, regulating the defense response. Not only plant mitochondrial alternative oxidase electron transport chain in the mechanism of oxidative stress, it also posses mitochondrial uncoupling proteins to decrease reactive oxygen species. Recent studies found that AtUCPl gene play an active role in abiotics stress tolerance to low temperature, drought and other oxidative stress and effective photosynthesis. However, the mechanism in the regulation function for the vegetable crops and its role in stress resistance mechanism are not clear. Therefore, it is great importance to investigate mechanism of plant UCP gene in the regulation of photosynthesis, respiration and the reaction in stress tolerance.
In this study, tobacco rattle virus (TRV) and tomato plants (Lycopersicon esculentum Mill. cv. Zongza 9) were used to construct the gene silencing vector carrying the fragment of LePDS and LeUCP gene.VIGS transform system of LePDS and LeUCP genes were established. The silencing of the reference gene LePDS appeared in photo-bleached young leaves; RT-PCR analysis showed that LeUCP gene was degraded significantly.
TRV-LeUCP gene silencing plants were used to study the effect mitochondria LeUCP genes of photosynthetic parameters and related enzymes activity, fluorescence, electron flow distribution. Mitochondria respiration, reactive oxygen species accumulation and alternative oxidase (AOX) were also analyzed. The effects on redox state of mitochondrial uncoupling protein were studied combination with aconitase citric acid content in the Krebs cycle activity and redox state changes. Under PQ and high temperature stress, the roles of mitochondrial uncoupling protein in oxidative stress were also investgate. The following are the major results:
1. The effects of tomato LeUCP gene silencing on photosynthesis were examined in the present study. No phenotypic differences were detected in the growth of tomato plants infected with TRV-LeUCP viral vector. The photosynthesis rate in the leaves of LeUCP gene silencing was lower than the control plants under different light intensities. Net Photosynthetic rate, Asat, Vc,max values decrease, stomatal conductance increase with LeUCP gene silence. However, no significant effects were observed on intercellular CO2 concentralion between different treatments. Activity of Rubisco and FBPase of tomato plants with LeUCP gene silencing was inhibited, it decreased by 23.3% and 20.3% respectively.
2. We have studied tomato LeUCP gene silencing on the chlorophyll fluorescence parameters. LeUCP gene expression reduce had no effect on Fv/Fm, but decreased photochemical quenching and electron transport rate. Compared with mock treatment plants qP values decrease by 54%. Meanwhile, non-photochemical quenching and Je (PSII), the distribution of Je (PCR) and Je (PCO) and the ratio of NAD+/NADH ratio were significantly reduced.The results demonstrated that LeUCP gene is necessary for effective photosynthesis. LeUCP gene may play a role through influencing Calvin cycle key enzymes activities, re-distributing of the proportion of PSII electron flow, reducing electron transfer and light respiration rate.
3. Mitochondrial LeUCP gene silencing significantly decreased respiration and alternative respiratory and inhibited of AOX1 gene family expression, especially AOXla gene expression. LeUCP gene silencing reduce content of ubiquinone and pyruvic acid, which decreased by 54.3% and 29.1% respectively compared with Mock and. It indicated that mitochondrial alternative oxidase and uncoupling protein expression appear synergistic. Compared with mock, aconitase activity and citric acid content decreased as 34.6%,14.4% respectively.NADH was increased and NAD+ /NADH decreased, it showed mitochondrial uncoupling protein played important role in redox state maintenance.
4. Compared with mock and TRV treatment, LeUCP gene silencing induced of H2O2 and O2·-accumulation on leaves. Meanwhile, ROS accumulation increased significantly under PQ and heat stress treatments, in which H2O2 and O2·- increase 44.67% and 23% after PQ treatment respectively compared with Mock. PQ treatment caused severe oxidative stress, Fv/Fm decreased significantly and MDA content increased. PQ treatment induced antioxidant genes such as CAT1, CAT2, SOD, cAPX, GR, POD, but the expression levels of TRV-LeUCP+PQ were still below Mock+PQ and TRV+PQ treatment. Compared with Mock, PQ treatment plants improved AsA, GSH content associated with AsA/DHA, GSH+GSSG increased. However, the AsA content and GSH content of TRV-LeUCP+PQ treatment leaves were lower than Mock+PQ and the TRV+PQ treatment. Therefore, it was further evidence that LeUCP gene had an important role in promoting stress resistance and redox signaling balance under oxidative stress.
本文以烟草脆裂病毒(TRV)和番茄为材料,选择八氢番茄红素去饱和酶基因(PDS)作为参照基因,构建了TRV-LePDS和TRV-LeUCP重组质粒,建立了LePDS和LeUCP基因VIGS体系,LePDS基因沉默植株的叶片出现典型的光漂白现象；LeUCP基因沉默植株其基因表达量显著降低。
利用TRV-LeUCP基因沉默植株,研究了番茄LeUCP基因对光合参数以及相关酶活性、荧光参数、电子流分配的影响,分析了LeUCP基因对线粒体呼吸、活性氧积累、交替氧化酶的影响。结合三羧酸循环中的顺乌头酸酶活性和柠檬酸含量及氧化还原状态以的变化,探明线粒体解偶联蛋白对Redox状态的影响。在PQ和高温逆境条件下,探明线粒体解偶联蛋白在氧化胁迫下是否起到保护作用。所得主要结果如下：
1、研究了番茄LeUCP基因沉默对光合作用的影响。结果表明,TRV病毒载体侵染未引起番茄植株生长表型差异；基因沉默植株叶片在不同光照强度下的光合速率均低于对照植株,Pn、4sat、Vc，max值降低,气孔导度Gs提高,但对Ci无明显影响。番茄LeUCP基因沉默植株Rubisco和FBPase酶活性受抑制,分别降低了23.3%和20.3%。
2、研究了番茄LeUCP基因沉默对叶绿素荧光参数的影响。LeUCP基因表达量的降低对Fv/Fm无明显影响,但降低了光化学猝灭系数(qP)和电子传递速率ETR,与Mock处理植株相比qP值降低了54%。提高了非光化学猝灭系数NPQ,并且明显降低了Je(PSII)和分配于Je(PCR)与Je(PCO)的比例以及NAD+/NADH比值。说明LeUCP基因编码的线粒体解偶联蛋白对有效的光合是必需的,可能通过影响卡尔文循环关键酶、重新分配PSII的电子流比例、降低电子传递速率和光呼吸而起作用。
3、研究了番茄LeUCP基因沉默对呼吸作用和Redox状态的影响。基因沉默植株线粒体总呼吸和交替呼吸下降明显,抑制AOXl基因家族的基因表达,其中对AOXla基因表达量影响显著。LeUCP基因表达量的降低影响还原态泛醌的含量和丙酮酸含量,分别较Mock下降了54.3%和29.1%,表明线粒体交替氧化酶和解偶联蛋白两者呈协同表达关系。三羧酸循环中的顺乌头酸酶活性和柠檬酸含量与Mock相比分别降低34.6%、14.4%。NADH含量增加且NAD+/NADH明显下降,表明线粒体解偶联蛋白对维持Redox状态起一定作用。
4、研究了PQ和高温氧化胁迫下解偶联蛋白基因的响应。与Mock和TRV组植株相比,LeUCP基因沉默诱导了植株叶片H2O2和O2.-的积累,并且在PQ和高温胁迫处理下,ROS积累显著增加,其中PQ处理后H2O2和O2.-分别较Mock增加44.67%和23%。PQ胁迫后造成了严重的氧化胁迫,Fv/Fm明显下降,MDA含量增加。CAT1、CAT2、SOD、cAPX、GR、POD基因表达量提高,但TRV-LeUCP+PQ仍然低于Mock+PQ与TRV+PQ。与Mock相比,PQ处理植株提高了AsA、GSH的含量,并且AsA/DHA、GSH+GSSG也有所增加,但TRV-LeUCP+PQ处理植株叶片AsA含量和GSH含量均低于Mock+PQ与TRV+PQ处理,进一步表明LeUCP对维持Redox信号平衡和增强氧化胁迫抗性具有重要作用。
Plant mitochondrion is the important place to provide energy for cell respiration. It plays an important role for plants in perceiving in stress signal, regulating the defense response. Not only plant mitochondrial alternative oxidase electron transport chain in the mechanism of oxidative stress, it also posses mitochondrial uncoupling proteins to decrease reactive oxygen species. Recent studies found that AtUCPl gene play an active role in abiotics stress tolerance to low temperature, drought and other oxidative stress and effective photosynthesis. However, the mechanism in the regulation function for the vegetable crops and its role in stress resistance mechanism are not clear. Therefore, it is great importance to investigate mechanism of plant UCP gene in the regulation of photosynthesis, respiration and the reaction in stress tolerance.
In this study, tobacco rattle virus (TRV) and tomato plants (Lycopersicon esculentum Mill. cv. Zongza 9) were used to construct the gene silencing vector carrying the fragment of LePDS and LeUCP gene.VIGS transform system of LePDS and LeUCP genes were established. The silencing of the reference gene LePDS appeared in photo-bleached young leaves; RT-PCR analysis showed that LeUCP gene was degraded significantly.
TRV-LeUCP gene silencing plants were used to study the effect mitochondria LeUCP genes of photosynthetic parameters and related enzymes activity, fluorescence, electron flow distribution. Mitochondria respiration, reactive oxygen species accumulation and alternative oxidase (AOX) were also analyzed. The effects on redox state of mitochondrial uncoupling protein were studied combination with aconitase citric acid content in the Krebs cycle activity and redox state changes. Under PQ and high temperature stress, the roles of mitochondrial uncoupling protein in oxidative stress were also investgate. The following are the major results:
1. The effects of tomato LeUCP gene silencing on photosynthesis were examined in the present study. No phenotypic differences were detected in the growth of tomato plants infected with TRV-LeUCP viral vector. The photosynthesis rate in the leaves of LeUCP gene silencing was lower than the control plants under different light intensities. Net Photosynthetic rate, Asat, Vc,max values decrease, stomatal conductance increase with LeUCP gene silence. However, no significant effects were observed on intercellular CO2 concentralion between different treatments. Activity of Rubisco and FBPase of tomato plants with LeUCP gene silencing was inhibited, it decreased by 23.3% and 20.3% respectively.
2. We have studied tomato LeUCP gene silencing on the chlorophyll fluorescence parameters. LeUCP gene expression reduce had no effect on Fv/Fm, but decreased photochemical quenching and electron transport rate. Compared with mock treatment plants qP values decrease by 54%. Meanwhile, non-photochemical quenching and Je (PSII), the distribution of Je (PCR) and Je (PCO) and the ratio of NAD+/NADH ratio were significantly reduced.The results demonstrated that LeUCP gene is necessary for effective photosynthesis. LeUCP gene may play a role through influencing Calvin cycle key enzymes activities, re-distributing of the proportion of PSII electron flow, reducing electron transfer and light respiration rate.
3. Mitochondrial LeUCP gene silencing significantly decreased respiration and alternative respiratory and inhibited of AOX1 gene family expression, especially AOXla gene expression. LeUCP gene silencing reduce content of ubiquinone and pyruvic acid, which decreased by 54.3% and 29.1% respectively compared with Mock and. It indicated that mitochondrial alternative oxidase and uncoupling protein expression appear synergistic. Compared with mock, aconitase activity and citric acid content decreased as 34.6%,14.4% respectively.NADH was increased and NAD+ /NADH decreased, it showed mitochondrial uncoupling protein played important role in redox state maintenance.
4. Compared with mock and TRV treatment, LeUCP gene silencing induced of H2O2 and O2·-accumulation on leaves. Meanwhile, ROS accumulation increased significantly under PQ and heat stress treatments, in which H2O2 and O2·- increase 44.67% and 23% after PQ treatment respectively compared with Mock. PQ treatment caused severe oxidative stress, Fv/Fm decreased significantly and MDA content increased. PQ treatment induced antioxidant genes such as CAT1, CAT2, SOD, cAPX, GR, POD, but the expression levels of TRV-LeUCP+PQ were still below Mock+PQ and TRV+PQ treatment. Compared with Mock, PQ treatment plants improved AsA, GSH content associated with AsA/DHA, GSH+GSSG increased. However, the AsA content and GSH content of TRV-LeUCP+PQ treatment leaves were lower than Mock+PQ and the TRV+PQ treatment. Therefore, it was further evidence that LeUCP gene had an important role in promoting stress resistance and redox signaling balance under oxidative stress.
引文
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Senthil-Kumar M, Govind G, Kang L, Mysore KS, Udayakumar M. Functional characterization of Nicotiana benthamiana homologs of peanut water deficit-induced genes by virus-induced gene silencing. Planta.2007,225:523-539.
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Suarez MH, Rodriguez ER, Romero CD. Analysis of organic acid content in cultivars of tomato harvested in Tenerife. European Food Research and Technology.2008, 226:423-435.
Sweetlove LJ, Lytovchenko A, Morgan M, Nunes-Nesi A, Taylor NL, Baxter CJ, Eickmeier I, Fernie AR. Mitochondrial uncoupling protein is required for efficient photosynthesis. Proceedings of the National Academy of Sciences.2006,103(51): 19587-19592
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Vercesi AE, Borecky J, Maia IG,Arruda P, Cuccovia IM, Chaimovich H. Plant uncou pling mitochondrial proteins. Annual Review Plant Biology.2006,57:383-404.
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Watanabe A, Nakazono M, Tsutsumi N, Hirai A. AtUCP2:a novel isoform of the mitochondrial uncoupling protein of Arabidopsis thaliana. Plant Cell Physiology. 1999,40:1160-1166.
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