【作者】 刘志雄；

【导师】 别之龙；

【作者基本信息】 华中农业大学 ， 设施园艺学， 2013， 博士

【摘要】 近年来,我国设施园艺发展迅速,设施内土壤次生盐渍化日益严重。土壤盐渍化问题业已成为设施蔬菜生产的主要土壤障碍因子,严重制约了设施栽培的可持续发展。黄瓜(Cucumis sativus L.)是主要的设施栽培作物之一,对盐胁迫非常敏感,但适用于黄瓜嫁接的砧木,如南瓜和葫芦等的耐盐性则相对较强。前人研究表明耐盐砧木嫁接可显著提高黄瓜耐NaCl胁迫的能力,但主要从缓解离子毒害方面揭示了其耐盐机理,而关于盐胁迫下嫁接调控植物光合作用方面的研究较少。本研究从能量耗散、光系统Ⅱ (PSⅡ)反应中心Dl蛋白合成与分解、Rubisco活性等方面出发研究嫁接黄瓜光合作用对盐胁迫的响应,探讨了耐盐砧木嫁接对盐胁迫下黄瓜光合作用的调控机制。所取得的主要结果如下：1.以盐敏感型黄瓜津春2号为接穗,耐盐性较强的南瓜(Cucurbita moschata Duch.)超级拳王为砧木,黄瓜自根和自嫁植株为对照,研究了自根、自嫁、砧木嫁接黄瓜在0和90mM NaCI下的生长情况,以及叶绿素含量、气体交换参数、叶绿素荧光的动态变化。结果表明,NaCI胁迫早期(1d,5d,10d),通过气孔限制抑制黄瓜的光合能力,而砧木嫁接在一定程度上调节了气孔的关闭而缓解NaCl胁迫的抑制作用；NaCl胁迫较长(15d)时,降解了黄瓜叶片中叶绿素,伤害了黄瓜叶片光合机构,而耐盐砧木嫁接可能通过增强黄瓜叶片中光化学猝灭和PSⅡ热量耗散过程,保护其叶片中叶绿素免遭降解,维持一定的光化学效率和CO2同化能力,从而提高其耐盐性。2.以盐敏感型黄瓜津春2号为接穗,耐盐性较强的南瓜超级拳王为砧木,黄瓜自根和自嫁植株为对照,研究了自根、自嫁、砧木嫁接黄瓜在0和90mM NaCl处理15d的气体交换参数、叶绿素荧光、叶黄素循环组分及叶绿体超微结构的变化。结果表明,利用耐盐性强的南瓜作为砧木嫁接,减轻了NaCl胁迫引起的气孔和非气孔因子对黄瓜叶片光合性能的限制作用,延缓了光抑制的发生,从而减轻了盐胁迫对黄瓜的伤害作用。自根黄瓜与自嫁黄瓜对盐胁迫的响应表现一致,这暗示了砧木嫁接黄瓜耐盐性的增强是由于砧木的作用,而非嫁接过程的影响。3.以盐敏感型黄瓜津春2号为试材,进行0、45mM、90mM NaCl及林肯霉素处理,利用Clark型液相氧电极检测黄瓜离体叶圆片的PSⅡ活性变化。结果表明,NaCl胁迫抑制了黄瓜叶圆片PSⅡ的放氧活性,而在林肯霉素的作用下,黄瓜叶圆片在1000μmolm-2s-1照射下其PSⅡ放氧活性急剧下降,而且盐胁迫对光损伤造成的PSⅡ活性的降低无影响。同时,结果发现NaCl胁迫显著减缓了黄瓜叶圆片PSⅡ放氧活性的恢复,阻碍其损伤后的修复过程。而加入林肯霉素,完全抑制PSⅡ修复过程后,不同浓度的NaCl间对其影响无显著差异。以上结果表明,NaCl胁迫对黄瓜叶片PSⅡ光损伤过程无影响,但能阻碍PSⅡ光损伤后的修复过程,从而抑制黄瓜叶片的放氧活性。4.以盐敏感型黄瓜津春2号为接穗,耐盐性较强的南瓜超级拳王为砧木,黄瓜自嫁植株为对照,研究了0和90mM NaCl对自嫁和砧木嫁接黄瓜的净光合速率、PSⅡ活性、D1蛋白含量以及根系和叶片中Na+含量影响。结果表明,利用耐盐砧木嫁接,能有效地将Nd+区域化在砧木根系中,减少黄瓜接穗叶片中Na+含量,从而减’轻Na+对PSⅡ反应中心D1蛋白修复过程的抑制作用,因此能抵御盐胁迫的负作用,较好维持叶片中PSⅡ的正常结构,从而维持叶片中PSⅡ的活性,保持黄瓜叶片光合能力。5.以盐敏感型黄瓜津春2号为接穗,以耐盐性较强的超级拳王和黑籽南瓜(Cucurbita ficifolia Bouche)为砧木,黄瓜自嫁植株为对照,研究了0和90mM NaCl对自嫁和两种砧木嫁接黄瓜的生长情况、气体交换参数、Rubisco活性及相关基因表达、氮代谢相关物质和酶活性的影响。结果表明,NaCl胁迫下利用耐盐砧木嫁接增强N代谢相关酶的活性,维持硝态氮向氨基酸转化,促进了黄瓜叶片中N代谢有效进行,从而维持Rubisco酶相关基因的表达及酶活性,这就使得黄瓜叶片光合性能得到提高。以上研究结果表明,盐胁迫通过气孔限制和非气孔限制抑制黄瓜植株的光合能力。耐盐砧木嫁接能缓解盐胁迫对黄瓜光合作用的抑制作用,其原因可能是由于：阻止短期NaCl胁迫引起的气孔限制；调控叶黄素循环组分转换耗散过剩能量,延缓光抑制的发生；阻止Na+在地上部积累,从而维持PSⅡ光损伤与修复的动态平衡；维持N代谢有效进行,促进Rubisco酶活性。更多还原

【Abstract】 In recent years, with the develop of protected horticulture, the soil secondary salinity was severe year by year. It has become the main obstacle of limiting the developing of vegetable production under protected horticulture, and seriously hampered the sustainable development of protected cultivation. Cucumber (Cucumis satuvus L.) is a salinity-sensitive plant species that is cultivated in unheated greenhouses in many areas. However, some rootstocks used for grafted-cucumber, such as pumpkin (Cucurbita moschata Duchesne ex. Poir) and bottle gourd [Lagenaria siceraria (Molina) Standl.], are more tolerant to salt stress comparatively. Recently, the use of salt-tolerant rootstock was demonstrated to be a valid strategy in increasing the salt tolerance of cucumber plants. It was suggested that the improved salt tolerance of grafted plants was related with the mitigation of the toxic effect of the ion within the plant. However, data on the rootstock-grafting regulating photosynthesis in plants exposed to salinity were limited. In present study, the mechanism of salt-tolerant rootstock-grafting on regulating photosynthesis of cucumber plants under salt stress were evaluated, by investigating the respose of photosynthesis of grafted cucumber to salt stress in energy dissipation, damage and repair of PSII, and Rubisco activity. The main results in the present study are as folloows:1. The salt-sensitive cucumber cv. Jinchun No.2was grafted onto a salt-tolerant pumpkin cv. Chaojiquanwang. The non-grafted and self-grafted cucumber plants were used as controls. The plant growth, changes in the time course of chlorophyll content, gas-exchange parameters, chlorophyll fluorescence in the leaves of non-, self-, and rootstock-grafted cucumber plants under0and90mM NaCl stress for15d were studied. The results showed that NaCl stress inhibited the photosynthetic capacity in cucumber leaves by stomatal limitation during the early phase of salinity (1d,5d,10d), whereas the use of salt-tolerant rootstocks alleviated the inhibitory effect of salt stress on photosynthesis in cucumber leaves, to some extent, by regulating stomatal activity. During the later phase of salinity (15d), NaCl stress degradated the chlorophyll, and impaired the photosynthetic apparatus in cucumber leaves, while grafting cucumber plants onto salt-tolerant rootstock protected the chlorophyll against degradation by enhancing photochemistry quenching and energy dissipation in cucumber leaves, thereby improving the photosynthetic performance of cucumber leaves.2. The salt-sensitive cucumber ’Jinchun No.2’ was used as scion, and the salt-tolerant pumpkin ’Chaojiquanwang’ was selected as rootstock. Gas exchange, photosystem II (PSII) efficiency, xanthophyll cycle, and chloroplast ultrastructure of nongrafted, self-grafted, and pumpkin-grafted cucumber plants were investigated at day15after being treated with90mM NaCl. The results showed that the use of salt-tolerant rootstock alleviates salt stress in cucumber plants by delaying photo inhibit ion, probably due to a lower incidence of both stomatal and nonstomatal factors that limit photosynthesis. Furthermore, the similar responses of the nongrafted and self-grafted cucumber plants to salt stress suggest that the improved salt tolerance of the rootstock-grafted plants was more affected by the rootstock than the grafting itself3. A salt-sensitive cucumber cultivar Jinchun No.2was used. The cucumber leaf disks were treated with0,45mM, or90mM NaCl and lincomycin, and the oxygen-evolving activity of PSII was detected by a Clark-type oxygen electrode (Chlorolab-2). The results showed that, NaCl stress inhibited the oxygen-evolving activity of PSII of the cucumber leaf disks. And lincomycin markedly accelerated the decrease of PSII activity when the cucumber leaf disks were incubated in light at1000μmol m-2s-1. The inactivation observed in the presence of lincomycin was unaffected by NaCL Meanwhile, the result showed that NaCl stress significantly inhibited the recovery of PSII activity of the cucumber leaf disks by hindering of the repair of photodamaged PSII. In the presence of lincomycin, recovery was completely blocked, no matter treated with NaCl or not. These results demonstrated that, NaCl stress inhibited the repair of the photodamaged PSII, and did not accelerate damage to PSII directly.4. The salt-sensitive cucumber’Jinchun No.2’was grafted onto the salt-tolerant pumpkin ’Chaojiquanwang’. The self-grafted cucumber plants was used as control. Net photo synthetic rate, PSⅡ activity, D1protein content in the leaves, and Na+concentration in the roots and leaves of self-grafted and pumpkin-grafted cucumber plants were investigated at day15after being treated with90mM NaCl. The results showed that, the salt-tolerant rootstock decreased the shoot Na+concentrations by retaining and accumulating Na+within the pumpkin rootstock, which alleviated the inhibitory effect of Na+on the repair of D1protein in cucumber leaves, thereby protecting the photosynthetic apparatus against NaCl stress, and enhancing the photosynthetic performance of cucumber.5. The salt-sensitive cucumber’Jinchun No.2’was grafted onto two salt-tolerant rootstocks,’Chaojiquanwang’ and figleaf gourd (Cucurbita ficifolia Bouche). Changes in the plant growth, gas-exchange parameters, Rubisco activities and its related genes expression, N metabolism-related substances and enzyme acitivies were investigated in the leaves of self-grafted and rootstock-grafted cucumber plants under0and90mM NaCl stress for15d. The results demonstrated that grafting cucumber plants onto salt-tolerant rootstocks enhances N metabolism in the leaves by increasing the N metabolism-related enzyme activities under NaCl stress, thereby maintaining Rubisco expression and activity, and improving the photosynthetic performance of cucumber leaves via higher gas exchange capacities.In conclusion, the results suggest that salt stress inhibits the photosynthetic capacity in cucumber leaves by stomatal and/or non-stomatal limitation. The use of salt-tolerant rootstocks alleviate the inhibitory effect of salt stress on photosynthesis in cucumber leaves, probably due to a lower incidence of stomatal factor that limits photosynthesis during the early phase of salinity, to the thermal dissipation of excess energy through effectively regulating of xanthophyll cyle de-epoxidation that delays photo inhibition, to restricting Na+accumulation in the shoot that maintains the dynamic balance between the photodamage of PSⅡ and the repair of damaged PSⅡ, to improving Rubisco activity by effectively maintaining N metabolism.更多还原