【作者】 田风霞；

【导师】 王玮；

【作者基本信息】 山东农业大学 ， 植物学， 2013， 博士

【摘要】 干旱是作物生长过程中经常遇到的逆境胁迫之一。小麦（Triticum aestivum L.）是世界性的粮食作物，干旱胁迫严重影响其生长和产量。研究小麦的抗旱生理及其分子机制，培育抗旱型小麦品种，对于保障小麦高产和稳产具有重大意义。衰老是一个受基因调控的程序性过程，是植物发育过程中的一个重要阶段。在衰老的过程中，植物细胞在结构、功能、新陈代谢以及基因表达等方面都发生着一系列的变化，其中比较明显的变化就是叶绿素的降解和光合作用的降低。小麦在灌浆后期易遭受干旱、高温等逆境的影响导致早衰并且降低籽粒的品质和产量。在衰老后期小麦滞绿突变体叶绿素基本不降解或降解缓慢，延缓小麦在灌浆后期的衰老，对于提高小麦产量具有一定的潜能。我们通过甲基磺酸乙酯（EMS）诱变，获得了一个明显延缓衰老的小麦滞绿突变体，命名为tasg1。本文以小麦滞绿突变体tasg1及其野生型（WT）核生2号为材料，设计大田与室内实验两部分进行研究，旨在探讨滞绿突变体tasg1的抗旱生理基础及分子机制，为滞绿突变体在小麦育种中的应用提供理论依据。主要结论如下：（1）tasg1是一个功能型滞绿突变体1）正常生长条件下，生长发育前期tasg1和野生型的表型基本一致，没有明显的区别；进入灌浆后期，二者叶片颜色开始出现差异。当野生型小麦已经表现明显的衰老症状即叶片发黄时，突变体tasg1叶片仍保持绿色，衰老延缓，表现出明显的滞绿表型。2）叶绿素（Chl）a的含量在衰老的起始阶段，tasg1和WT之间没有明显差异。但是从开花后的第22天到第30天，tasg1的Chl a的含量高于野生型，特别是经过干旱胁迫处理以后。Chl b的含量也表现出相似的差异。旗叶净光合速率同叶绿素含量变化趋势基本一致。花后30天，与野生型相比tasg1旗叶维持较高光合速率，表明突变体tasg1具有较长的叶片光合功能期。突变体tasg1和WT旗叶气孔导度（Gs）、蒸腾速率（E）、细胞间隙CO2浓度（Ci）在开花灌浆期的变化趋势和Pn的变化趋势基本一致。3）在对照田（CK，正常灌溉）和旱田（DS，控制灌溉和遮雨）tasg1的产量比野生型高9.5%和7.0%。尽管每行穗数是tasg1中高于野生型，但是不显著，其中每穗粒数增加对产量的贡献相对较大。结果表明滞绿突变体tasg1在灌浆后期维持较高的叶绿素含量能够增加产量。说明tasg1的滞绿特性具有一定的生产利用价值。（2）大田条件下tasg1与WT开花灌浆期叶细胞类囊体结构1）正常情况下，小麦叶片叶绿体紧贴着细胞壁单行排列，呈椭圆状，类囊体片层紧密排列成典型的基粒和类囊体基质，tasg1的叶绿体和类囊体超微结构稍微比野生型好一些。干旱胁迫下，叶绿体变成球形，与野生型相比，tasg1叶绿体和类囊体片层的损伤较轻。表明干旱胁迫下，突变体与野生型相比能更好的维持叶绿体以及类囊体的结构，从而可以更有助于突变体维持比较高的光合作用。我们还观察到在干旱胁迫条件下tasg1基粒融合的超微结构。2）随着干旱处理天数的增加，tasg1希尔反应活性、Ca2+-ATPase和Mg2+-ATPase的活性都比WT高，说明在干旱胁迫下滞绿突变体比野生型PSII以及类囊体膜上其它功能蛋白更稳定。3）干旱胁迫导致分子量约为25-35kDa的类囊体膜蛋白浓度增加，尤其是28kDa的多肽浓度增加明显。而且这些多肽在滞绿突变体tasg1中的表达水平始终高于WT。与小麦幼苗多肽相比，只有1个28kDa的多肽被保留在成熟的植物中。这种差异表明，一些多肽在小麦叶片发育和衰老时退化。与野生型相比，tasg1可以维持比较好的类囊体蛋白稳定性。4）干旱胁迫下，编码捕光色素复合体I（LHCI）的三个基因TaLhca1，TaLhca2和TaLhca3的转录表达在tasg1和WT中都下调，在经过48h干旱处理以后下降到最低水平。编码LHCII的TaLhcb4和TaLhcb6的基因表达在tasg1中比WT相比仍然保持比较高的水平。免疫印迹的结果进一步分析表明LHCII蛋白复合体在tasg1中比WT中更稳定。（3）大田条件下tasg1与WT开花灌浆期叶片抗氧化能力1）随着灌浆进程，突变体tasg1和野生型旗叶中氧自由基（O2ˉ）生成速率和过氧化氢（H2O2）含量、丙二醛（MDA）含量、离子渗漏、羰基蛋白含量均逐渐升高，但与野生型相比，突变体tasg1始终处于较低的水平，表明其活性氧积累较少，膜脂过氧化程度较低。2）干旱胁迫处理以后，超氧化物歧化酶（SOD），过氧化物酶（POD），过氧化氢酶（CAT）和抗坏血酸过氧化物酶（APX）的活性受到抑制，但是tasg1与野生型相比要高。与野生型相比，在抗坏血酸-谷胱甘肽循环中，tasg1的还原型抗坏血酸与氧化型抗坏血酸的比值、还原型谷胱甘肽与氧化型谷胱甘肽的比值以及该循环中的三个关键酶的活性都比野生型高。这些结果表明抗氧化能力可能在tasg1的抗旱性中起非常重要的作用。综上所述，小麦滞绿突变体tasg1在灌浆后期衰老延缓，能够保持较高的叶绿素含量和光合作用并且提高产量，属于功能型滞绿变异。与野生型相比，突变体tasg1可以维持比较好的叶绿体和类囊体结构和比较多的类囊体蛋白丰度，而且LHCII的稳定性好。突变体tasg1的抗氧化酶活性比野生型高。这些结果表明，突变体tasg1的类囊体膜蛋白稳定性的提高以及较高的氧化清除能力可能是其抗旱性高的重要原因。更多还原

【Abstract】 Drought is one of the major environment stresses during plant growth and development,and the disaster due to drought becomes more and more severe and frequent. Wheat (Triticumaesitivum L.) is one of the important food crop world widely. Water deficit is one of thecommon stresses during wheat growth, and the lost of wheat yield by water deficit is verylarge. Therefore, it is very important and significant to study the mechanism of droughttolerance in wheat and to breed the new drought tolerant wheat cultivars.Plant senescence is an important period of plant development, which is a programmedprocess that is subjected to gene regulation. The structure, function, metabolism and geneexpression of the plant cell go through a series of coordinated changes during plantsenescence, two of the distinct changes are the degradation of chlorophyll and the decline ofphotosynthesis. The grain-filling of wheat is frequently deteriorated by drought and heat stresscombination at the late stage. Senescence results in deterioration of the quality of vegetables,poor grain quality and reduced crop yield. Delay leaf senescence is a useful way to increasephotosynthetic time, which is beneficial to increase wheat yield.The wheat stay-green mutant, tasg1, was previously generated by applying the ethylmethane sulphonate (EMS) mutagen to HS2, a common wheat cultivar as the WT. In thisstudy, all the experiments were conducted in2sets of experriments: one, in the field and theother, in the laboratory. We used WT and tasg1, as materials to study the changes of plantphysiology and its stay green mechanism under water stress condition. The results willprovide a theoretical basis to wheat breeding in drought resistance and senescence delay. Themain results are as follows:(1) tasg1represent a functional stay green mutant1) Under normal field conditions, no significant difference between tasg1and WT wasobserved in plant development and phenotype before the flag leaves appeared. The stay-greenphenotype of tasg1was expressed at the beginning of anthesis and was especially apparentwith late natural senescence. Drought stress accelerated the plant senescence in both wheatvarieties, but it was delayed in tasg1compared to the WT. Compared with WT, tasg1showsan obvious stay green phenotype at later stage of nature senescence.2) No obvious difference was found in chlorophyll (Chl) a content between tasg1and WT at the initial phase of senescence. However, from the22th to the30th day after anthesis(DAA), Chl a content in tasg1was always higher than in WT, especially under drought stress(DS). Similar differences were observed in Chl b content. Net photosynthetic rate (Pn) washigher in tasg1, compared to WT, at30DAA, which was consistent with the differences inChl content. Somewhat similar differences were observed in transpiratory rate (E) andstomatal conductance (Gs), but intercellular CO2concentration (Ci) was significantly lower intasg1than WT. This may be related to the higher photosynthetic activity in tasg1.3) The yield of tasg1was9.5and7.0%higher than WT under controls (CK) and droughtstress (DS) conditions, respectively, but these differences were not significant. The greaternumber of kernels per wheat spike was the major factor contributing to the higher yield oftasg1, although the number of wheat spikes in each plot in tasg1also contributed to theincreased yield. The size and mass of each kernel was lower in tasg1than WT. From theobservations above, tasg1represents a functional stay green mutant.(2) The characteristics of the chloroplasts and thylakoid in tasg1and WT1) In normal water conditions, chloroplasts were arranged regularly along the cell wall,but their shape was slightly different in the two genotypes. Chloroplasts were approximatelyroundish in WT, but prolonged in tasg1. After drought stress, some damage to the chloroplastenvelope was found in WT, accompanied with the shift of the organelles from the cell wall tothe center of the cell. Compared to WT chloroplasts, tasg1chloroplasts showed less damageinduced by drought stress. Under normal water conditions, the thylakoid lamellae wereclosely arranged and assembled to form the grana in the WT. Lamellae were more closelyarranged in tasg1. Drought stress resulted in swollen and loosely scattered thylakoid lamellaein WT, but these changes were not obvious in tasg1. We also observed fusion of several granastacks in tasg1under drought stress.2) As the date of drought stress continued, the activity of Hill reaction, Ca2+-ATPase andMg2+-ATPase were significantly higher in tasg1than WT. The activity of thylakoid membraneproteins, including PSII and ATPase, were better maintained in tasg1than in the WT underdrought stress.3) In flag leaves under field conditions, the level of the28kDa polypeptide in tasg1washigher than that in WT under both normal and drought stress conditions on both days in thedrought of10and25d. The polypeptides were similarly detected in WT and tasg1wheatseedlings at the second-leaf stage in the laboratory, and the three observed polypeptides ofabout28,38and50kDa in tasg1were consistently higher than those in WT. This differencesuggested that some polypeptides were degraded during leaf development and senescence. Compared with WT, tasg1could maintain the thylakoid membrane protein complexes withbetter stability against damage by drought stress.4) Expression levels of genes involved in light-harvesting complex I (LHCI), namelyTaLhca1, TaLhca2and TaLhca3, were down-regulated gradually during drought stress in bothWT and tasg1. The expression levels of TaLhcb4and TaLhcb6were higher in tasg1comparedto WT, especially at the last tested time point. From these results, tasg1could increasestability of chloroplast membranes and chlorophyll-protein complexes.(3) The antioxidant activity and ascorbate-glutathione cycle in tasg1and WT duringgain-filling stage1) Superoxide radical (O2ˉ) production rate, hydrogen peroxide (H2O2) accumulation,malondialdehyde (MDA) content, relative electrical conductivity and carbonylation proteincontent in flag leaves increased significantly after anthesis in both tasg1and WT.Nevertheless, tasg1maintained lower MDA content, O2ˉproduction rate and H2O2content inflag leaves than WT. From the results above, we suggest that high antioxidative systemcompetence may be involved in the stay green characteristic of tasg1.2) The activities of several antioxidant enzymes, including superoxide dismutase (SOD),peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were mostly suppressedby drought stress, but their activities were always higher in tasg1than that in WT. Comparedwith WT, tasg1had higher reduced ascorbate/oxidized ascorbate ratio, reducedglutathione/oxidized glutathione ratio and antioxidant enzyme activities during senescenceunder both normal water and drought stress conditions. These results suggest that thecompetent antioxidative capacity may play an important role in the enhanced droughttolerance in tasg1.From the results above, we suggest that tasg1maintain higher chlorophyll content andphotosynthetic rate than WT at the gain-filling stage, indicating an obviously delaysenescence in tasg1. Compare to WT, the stay-green wheat mutant tasg1could stablymaintain chloroplast and thylakoid ultrastructure better and maintain thylakoid membranepolypeptides at high levels, while its expression of some LHCII related genes remained steadyunder drought stress. The activities of several antioxidant enzymes, including SOD, POD,CAT and APX were mostly suppressed by drought stress, but their activities were alwayshigher in tasg1than in WT. Thus, enhanced stability of thylakoid membrane proteins andantioxidant competence contribute to drought resistance in the tasg1. These data were helpfulto better understand of the stay-green mechanism and to improve the drought resistance ofwheat cultivars.更多还原