亚高温强光胁迫下番茄幼苗光抑制及光保护机制研究

发布时间：2018-01-10 05:00

本文关键词：亚高温强光胁迫下番茄幼苗光抑制及光保护机制研究　出处：《沈阳农业大学》2016年博士论文　论文类型：学位论文

【摘要】：设施蔬菜栽培是我国现代农业生产重要组成部分,番茄(Solanum lycopersicum L.)作为我国北方地区温室栽培的主要蔬菜作物之一,在设施越夏栽培过程中常遭受高温和强光的双重胁迫,导致植株产量和果实品质下降,严重制约设施蔬菜产业发展。因此,研究高温强光对设施蔬菜的影响,特别是东北地区亚高温强光对设施番茄光合作用的影响,进而加深人们对光合作用规律的认知并因地、因时制定相关防控措施,以缓解逆境障碍,从而保证高产、优质栽培,显得尤为重要。本文以番茄品种“辽园多丽”为试材,研究了亚高温强光诱导番茄幼苗叶片光合作用及光系统抑制的机理及D1蛋白周转、叶黄素循环、环式电子传递、线性电子传递等途径在亚高温强光胁迫下番茄叶片中的光保护作用机制,主要研究结果如下：1.明确了亚高温强光胁迫对番茄叶片光合作用的影响。研究表明亚高温强光导致植株叶片光合速率下降,非气孔因素是主要限制因素。研究结果显示,与对照(CK,25℃, 500 μmol·m~(-2)·s~(-1))植株相比,亚高温强光胁迫(HH,35℃,1000μmol·m~(-2)·s~(-1)引起番茄叶片净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)及气孔限制值(Ls)大幅下降,而胞间CO_2浓度(Ci)大幅升高,此外,Rubisco酶活性及其大小亚基(rbcL和rbcS)基因相对表达量显著低于对照,说明非气孔因素起主导作用。2.明确了亚高温强光胁迫对光系统Ⅱ (PS Ⅱ)的影响。研究发现,亚高温强光胁迫导致番茄叶片PSⅡ最大光化学效率(Fv/Fm)、PS Ⅱ天线转化效率(Fv'/Fm')、PS Ⅱ潜在活性(Fv/Fo)、PSⅡ光下实际光化学效率[Y(Ⅱ)]、 PS Ⅱ电子传递速率[ETR(Ⅱ)]显著下降,与此同时PS Ⅱ光化学效率[Y(NO)]和PS Ⅱ非光化学效率[Y(NPQ)]显著升高,而反映PS Ⅱ反应中心失活状态的最大荧光(Fm)和初始荧光(Fo)及反映PS Ⅱ反应中心开放程度的光化学淬灭系数(qP)也显著降低,这说明亚高温强光胁迫引起PS Ⅱ反应中心关闭并发生不可逆失活,进而引起PSⅡ光化学效率的降低,从而导致PSⅡ发生光抑制和光破坏。而且亚高温强光处理导致的PSⅡ光抑制和光破坏的程度随着胁迫时间的增加而加重。3.明确了亚高温强光胁迫对光系统I(PS Ⅰ)的影响。研究表明,亚高温强光处理后,番茄叶片PS Ⅰ光下实际光化学效率[Y(Ⅰ)]、PSI电子传递速率[ETR(Ⅰ)]极显著下降,说明PS Ⅰ受到光抑制。由于Y(Ⅰ)下降是PS Ⅰ受体侧量子效率[Y(NA)]显著下降和PS Ⅰ供体侧量子效率[Y(ND)]显著上升引起的,我们推测过剩光能引起PS Ⅱ电子传递受阻并在PS Ⅰ供体侧积累,从而导致PS Ⅰ供体侧光抑制；同时Rubisco酶活性下降导致电子在PS Ⅰ受体侧积累,进而发生PS Ⅰ受体侧光抑制。4.明确了亚高温强光胁迫对活性氧(ROS)代谢的影响。研究结果发现,亚高温强光胁迫引起番茄叶片中丙二醛(MDA)和H_2O_2含量显著上升,可溶性蛋白(Sp)和游离脯氨酸(Pro)含量显著下降,而细胞相对电导率(K)和细胞膜受损程度(α)升高,说明亚高温强光导致番茄体内ROS大量积累,严重破坏细胞膜并导致膜内物质外流。此外我们还发现亚高温强光处理导致番茄叶片超氧化物歧化酶(SOD)和过氧化物酶(POD)活性严重下降,(Cu/Zn)SOD、GR基因相对表达量显著降低,而过氧化氢酶(CAT)活性提高,(Mn)SOD、APX基因相对表达量显著升高,说明逆境诱导ROS清除系统清除ROS,但亚高温强光胁迫严重抑制了相关抗氧化酶活性,造成ROS不能及时清除并在植株体内大量积累,进而导致ROS代谢失衡并破坏光合作用和光合系统。5.明确了亚高温强光胁迫下D1蛋白周转和叶黄素循环对番茄叶片光合作用和保卫细胞的影响。研究结果表明,与对照植株相比,硫酸链霉素(SM)和二硫苏糖醇(DTT)处理导致番茄叶片Pn、表观量子效率(AQY)降低,与此同时气孔数目、保卫细胞和气孔的宽、面积降低,而保卫细胞和气孔长、长宽比增加,说明D1蛋白周转或叶黄素循环被破坏诱导气孔关闭和气孔数量减少,减弱了植株对光的利用能力,光合作用受抑制。6.明确了亚高温强光胁迫下D1蛋白周转和叶黄素循环对番茄叶片光抑制的影响。结果显示,SM和DTT处理引起Fv/Fm、Fv/Fo、Y(Ⅱ)、qP极显著降低,在转录和翻译水平上分别导致psbA基因下调表达和D1蛋白含量降低,说明D1蛋白循环和叶黄素循环被破坏引起PS Ⅱ核心蛋白在分子和蛋白水平上受损,进而造成D1蛋白的净损失,最终导致PS Ⅱ反应中心被破坏,发生严重光抑制。此外,我们还发现SM和DTT处理导致叶片总叶绿素含量显著降低及叶绿素a (Cha)和叶绿素b (Chb)含量比(Cha/Chb)升高,说明D1蛋白循环和叶黄素循环被破坏提高了捕光色素复合体对氧化胁迫的敏感性,导致叶片天线色素被破坏。7.明确了亚高温强光胁迫下D1蛋白周转和叶黄素循环对番茄叶片ROS的影响。采用荧光染料DHE和DCFH-DA分别对番茄叶片保卫细胞中的02·-和H202进行染色观察,并结合其活体染色观察发现亚高温强光胁迫导致保卫细胞和叶片中ROS大量积累,SM和DTT处理大大加深了保卫细胞中ROS的荧光强度和叶片染色深度；此外,抗氧化酶活性如CAT、POD和SOD,及总抗氧化能力(T-AOC)也被强烈抑制,说明D1蛋白循环和叶黄素循环被破坏诱导植株抗氧化能力降低,ROS不能清除,保卫细胞中ROS含量增加,进而导致气孔关闭。8.明确了亚高温强光胁迫下环式电子传递(CEF)和线性电子传递(LEF)对番茄叶片能量分配的影响。结果显示,甲基紫精(MV)和敌草隆(DCMU)处理导致番茄叶片分配于PS Ⅱ的光能(β)、PS Ⅱ天线热耗散能(D)急剧上升,而分配于PS Ⅰ的光能(α)、PS Ⅱ光化学反应能(P)、有活性PS Ⅱ反应中心过剩能(E)急剧下降,说明CEF和LEF被抑制诱导能量过剩及激发能分配失衡,而大量分配于PSⅡ的激发能导致反应中心被破坏而不能进行光化学反应。9.明确了亚高温强光胁迫下CEF和LEF对光抑制的影响。研究结果显示,MV和DCMU处理造成Fv/Fm、Fv'/Fm'、qP极大降低,PSⅡ激发压(1-qP)极大升高,说明CEF和LEF被抑制造成PS Ⅱ反应中心失活,甚至被降解且无法恢复,可见CEF和LEF对亚高温强光逆境下番茄叶片PS Ⅰ和PSⅡ反应中心的光保护作用极为重要。10.明确了亚高温强光胁迫下CEF和LEF对跨膜质子动力势(pmf)的影响。我们研究发现,MV和DCMU处理显著降低了番茄叶片的pmf及其组分跨膜质子梯度(△pH)和跨膜电势(△Ψ)。同时测定叶片P515信号发现MV和DCMU处理后,番茄叶片暗适应后P515信号衰减快速下降,叶片预照光后则缓慢下降,说明类囊体膜完整性被破坏及ATP-ase活性被严重抑制。
[Abstract]:Vegetable cultivation in China is an important part of modern agricultural production, the tomato (Solanum lycopersicum L.) as one of the most important vegetable crops in Greenhouse in North China in summer, facilities often suffer from high temperature and strong light stress during the process of cultivation, resulting in a decline in plant yield and fruit quality, facilities seriously restrict the development of vegetable industry. Therefore, effects of high temperature and strong light on the vegetable, especially the influence of the Northeast Asia high temperature and strong light of tomato photosynthesis, and thus deepen the cognition of the law of photosynthesis and because, when formulating the relevant prevention and control measures, to alleviate the stress disorder, so as to ensure the high yield, high quality cultivation, is particularly important. This paper in tomato liaoyuanduoli as experimental material, studied the effects of high temperature and strong light induced inhibition and the mechanism of D1 protein in leaves of tomato seedlings photosynthesis and photosystem turnover, leaf The flavin ring cycle, electron transfer mechanism, light protective effect of tomato leaves under the linear electron transport pathway in the sub high temperature and strong light stress, the main results are as follows: 1. to understand the effects of high temperature and strong light stress on the photosynthesis of tomato leaves. The results show that the sub high temperature and strong light caused leaf photosynthetic rate decreased, the non stomatal factors are the main limiting factor. The results of the study showed that compared with the control (CK, 25 C, 500 mol - m~ (-2) - s~ (-1)) were compared, sub high temperature and strong light stress (HH, 35 C, 1000 mol - m~ (-2) - s~ (-1) caused by the net photosynthetic rate of tomato (Pn), stomatal conductance (Gs), transpiration rate (Tr) and stomatal limitation (Ls) decreased significantly, while the intercellular CO_2 concentration (Ci) significantly increased, in addition, enzyme activity and size of Rubisco subunits (rbcL and rbcS) gene expression was significantly lower than that of light, that non stomatal factors play the leading role of high clear.2. Wen Qiangguang stress on photosystem II (PS II). The study found that the sub high temperature and strong light stress caused tomato the maximum photochemical efficiency of PS (Fv/Fm), PS II antenna conversion efficiency (Fv'/Fm'), PS (Fv/Fo), potential activity of PS II light actual photochemical efficiency of [Y (II)], PS II the electron transfer rate [ETR (II)] decreased significantly at the same time, PS II photochemical efficiency of [Y (NO)] and PS II non photochemical efficiency of [Y (NPQ)] was significantly increased, which reflects the maximum fluorescence of PS II reaction center inactivated state (Fm) and initial fluorescence (Fo) and the photochemical quenching of PS II reaction the center of the open degree of quenching coefficient (qP) were significantly decreased, indicating that the sub high temperature and strong light stress induced by PS II reaction center is closed and the occurrence of irreversible inactivation, and cause a decrease in the photochemical efficiency of PS II PS II, resulting in photoinhibition and photodamage. And sub high temperature and strong light induced by PS II light The degree of inhibition and light damage increased with the increase of stress time and increase the.3. to understand the effects of high light stress on photosystem I (PS 1) effect. The study shows that the sub high temperature and strong light treatment, tomato leaves PS I light the actual photochemical efficiency of [Y (I)], PSI electron transfer rate [ETR (I)] decreased significantly, indicating PS I by photoinhibition. Because Y is PS I (I) decreased the acceptor side of the quantum efficiency of [Y (NA)] was significantly decreased and PS of the donor side of the quantum efficiency of [Y (ND)] significantly rise, we speculate that the excess energy caused by PS II electron transfer was blocked and in PS I donor the side accumulation, which leads to the donor side of PS I photoinhibition; while Rubisco activity declined to electron accumulation in PS receptor and PS receptor side, side light inhibition of.4. to understand the effects of high temperature and strong light stress on active oxygen metabolism (ROS). The results of the study showed that the sub high temperature and strong light stress Cause the malondialdehyde (MDA) in tomato leaves increased significantly and the content of H_2O_2, soluble protein (Sp) and free proline (Pro) content decreased and cell relative conductivity (K) and cell membrane damage (alpha) increased, the high temperature and high light led to a substantial accumulation of ROS in tomato, serious damage to the cell membrane and lead to membrane within the material flow. We also found that the sub high temperature and strong light treatment resulted in tomato leaf superoxide dismutase (SOD) and peroxidase (POD) activity declined seriously, (Cu/Zn) SOD, the relative expression of GR gene was significantly decreased, while catalase (CAT) activity increased, SOD (Mn), the relative expression of APX gene a significant increase in the amount, indicating the stress induced ROS scavenging system ROS removal, but the sub high temperature and strong light stress severely inhibited the antioxidant activity, caused by ROS could not be removed in time and accumulation in plants, leading to ROS damage and metabolic imbalance Photosynthesis and photosynthetic system.5. clear D1 protein turnover and xanthophyll cycle effects on Photosynthesis of tomato leaves and guard cell sub high temperature and strong light stress. The results show that, compared with the control plants, streptomycin sulfate (SM) and two dithiothreitol (DTT) treatment resulted in tomato leaves Pn, apparent quantum efficiency (AQY at the same time) decreased, the number of stomata, guard cells and stomatal width, area reduced, and guard cells and stomatal length, length width ratio increased, D1 protein turnover or xanthophyll cycle destruction induced stomatal closure and decrease the number of pore, weaken the plant capacity of using light, photosynthesis inhibited.6. clear D1 protein the turnover of xanthophyll cycle and the effect of photoinhibition on tomato leaves of high temperature and strong light stress. The results showed that SM and DTT caused Fv/Fm, Fv/Fo, Y (II), qP decreased significantly at the level of transcription and translation respectively. Lead to down-regulation of psbA gene and D1 protein expression decreased, D1 protein cycle and the xanthophyll cycle was destroyed by PS II core protein damage at the molecular and protein levels, resulting in a net loss of D1 protein, resulting in PS II reaction center was destroyed, the occurrence of serious photoinhibition. In addition, we also found that the chlorophyll SM and and DTT caused the total leaf chlorophyll content was significantly reduced a (Cha) and chlorophyll b (Chb) content ratio (Cha/Chb) increased, indicating that D1 protein cycle and xanthophyll cycle destruction enhances the light harvesting complex sensitivity to oxidative stress, resulting in the destruction of blade antenna pigment.7. clear D1 protein turnover and xanthophyll cycle effects on tomato leaf ROS sub high temperature and strong light stress. By using the fluorescent dye DHE and DCFH-DA respectively on tomato leaves in guard cells of 02 - and H202 staining, and combined with the in vivo staining Color observation sub high temperature and strong light stress in guard cell and leaf ROS accumulation, SM and DTT greatly enhance the fluorescence intensity of ROS in guard cells and leaves the dyeing depth; in addition, the activity of antioxidant enzymes such as CAT, POD and SOD, and the total antioxidant capacity (T-AOC) was also strongly inhibited D1 protein cycle xanthophyll cycle and destruction induced reduction in plant antioxidant capacity, ROS can not be removed, ROS content in guard cells increased, leading to stomatal closure.8. clear sub under high temperature and high light stress cyclic electron transport (CEF) and linear electron transfer (LEF) effect on Tomato Leaf energy distribution. The results showed that the methyl viologen (MV) and diuron (DCMU) treatment resulted in light distribution in tomato leaves of PS (beta), PS II antenna heat dissipation (D) increased sharply, and the light distribution in PS I (alpha), PS II (P), photochemical reaction can active PS II Should the center excess energy (E) fell sharply, indicating that CEF and LEF were induced by inhibition of excess energy and excitation energy distribution imbalance, and a lot of excitation can lead to the distribution of PS II reaction center was destroyed and can not clear the photochemical reactions of.9. CEF and LEF on light suppression effects of sub high temperature and strong light stress results. Show that MV and DCMU treatment caused Fv/Fm, Fv'/Fm', qP greatly reduced, PS II excitation pressure (1-qP) increased greatly, indicating that CEF and LEF were inhibited by PS II reaction center inactivation, even being degraded and can not be recovered, the protective effect of CEF and LEF in visible light to strong light and high temperature adversity in tomato leaves under PS 1 and PS II reaction center is very important for.10. CEF and LEF to clear the transmembrane proton motive force of high temperature and strong light stress (PMF) effect. We found that MV and DCMU significantly reduced PMF and tomato leaves the transmembrane proton gradient (pH) And the membrane potential (lpli). Simultaneous determination of leaf P515 signal MV and found that DCMU treated tomato leaves after dark adaptation of P515 signal intensity decreased rapidly, pre illumination of leaves decreased slowly, indicating the destruction of ATP-ase and the activity of thylakoid membrane integrity was severely inhibited.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S641.2

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