【作者】 焦彦生；

【导师】 郭世荣；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2007， 博士

【摘要】 黄瓜（Cucumis sativus L．）是我国设施栽培的重要蔬菜作物之一。但在生产实践中，水培营养液供氧不足或基质培根垫的形成均会产生低氧逆境，这已成为黄瓜生产的重要限制性因子之一，严重影响了黄瓜植株的生长发育，探索简易、经济、实用的减缓低氧胁迫危害的方法对黄瓜无土栽培技术的提高具有重要的意义。Ca²⁺作为植物生长所必需的大量元素和偶联胞外信号与胞内生理反应的第二信使，在逆境胁迫下细胞内的含量会显著提高，与胞内CaM形成Ca²⁺·CaM复合物，能维持细胞壁、细胞膜及膜蛋白的稳定性；Ca²⁺还可调节气孔运动和放氧活性，可活化ATP酶，促进光合产物的运转，从而缓解多种逆境胁迫对植物的伤害。低氧逆境胁迫下植物会形成适应低氧逆境的生理代谢反应，活性氧和保护酶，以及多胺的含量处于动态变化之中，共同参与植物对逆境胁迫的适应性调节，但外源钙和钙调素拮抗剂处理对低氧胁迫下黄瓜活性氧及多胺代谢的影响还未有系统性研究报道。本文以耐低氧性具有显著差异的两个黄瓜品种（耐低氧性较强的‘绿霸春四号’和耐低氧性较弱的‘中农八号’）为试材，采用营养液水培法，运用溶氧浓度调节仪精确控制营养液溶氧浓度，研究了喷施和营养液添加钙的适宜浓度，外源钙对低氧胁迫下活性氧代谢、多胺代谢的影响和钙调素拮抗剂W7对活性氧、多胺、光合代谢的影响，探讨了Ca²⁺和CaM在黄瓜适应低氧胁迫过程中的生理调节功能。主要研究结果如下：1．根际低氧胁迫下叶面喷施10mmol·L^-1 CaCl₂或营养液Ca²⁺浓度提高到8mmol·L^-1后，黄瓜植株的干鲜重均高于单纯低氧胁迫处理，增强了地上部和根系的生长，外源钙减缓了低氧胁迫对黄瓜植株生长的抑制作用，可促进光合色素的增加，Pn、Gs、Ci和Tr均得到提高。而叶面喷施CaCl₂20 mmol·L（-1）以上高浓度容易导致叶面损伤和生长受阻，根际营养液Ca²⁺浓度超过16mmol·L^-1的以上的处理也容易导致黄瓜生长变弱。表明适当浓度的钙处理可以缓解低氧胁迫对黄瓜植株的伤害。2．低氧胁迫下，黄瓜幼苗根系和叶片中游离态、结合态、束缚态多胺含量均有不同程度的增加。低氧胁迫营养液Ca²⁺浓度提高到8mmol·L^-1后黄瓜幼苗根系和叶片的游离态、结合态、束缚态的Put、Spm、Spd含量会进一步提高；而PAO活性比低氧常钙(2mmol·L^-1)处理低，多胺合成酶ADC、ODC、SAMDC活性明显有所提高，表明低氧胁迫下营养液加钙通过调控多胺的合成和降解，促使植株体内多胺含量增加，以提高黄瓜幼苗对低氧的适应性，表明钙参与了黄瓜幼苗体内多胺的代谢过程。3．低氧胁迫下，黄瓜幼苗根系和叶片中抗氧化酶活性显著增强，活性氧和MDA的含量明显增加，耐低氧品种比不耐低氧品种抗氧化酶活性增强幅度大，而活性氧和MDA含量增加幅度较小；低氧胁迫营养液8mmol·L^-1Ca²⁺处理显著降低了根际低氧胁迫下黄瓜幼苗体内MDA、H₂O₂含量和O_2·^-产生速率，提高了SOD、POD、CAT、APX、GR活性，降低了膜脂过氧化水平，从而减缓了低氧胁迫的伤害，增强了幼苗对低氧胁迫的适应性。4．用50mmol·L^-1钙调素拮抗剂W7预处理黄瓜幼苗24h后进行低氧胁迫处理，与单纯低氧胁迫处理相比，黄瓜植株的生长明显受到抑制，表现为地上部生长变缓，根系生长较弱；黄瓜根系和叶片中的SOD、POD、CAT、APX、GR、DHAR、MDAR的活性降低，AsA、DAsA、GSH、GSSG的含量减少，而O_2·^-产生速率、H₂O₂和MDA的含量均高于单纯低氧胁迫处理；低氧加W7预处理后黄瓜体内的游离态、结合态、束缚态的Put、Spm、Spd含量和Pn、Gs、Ci和Tr等光合作用指标均低于单纯低氧胁迫处理，表明钙通过Ca²⁺·CaM复合体的形成，通过Ca₂₊·CaM信使系统影响黄瓜幼苗体内的多胺代谢、活性氧代谢和光合作用来调节黄瓜对低氧胁迫的适应。5．低氧胁迫下，‘中农八号’和‘绿霸春四号’两品种由于基因型的差异，处理后生理指标存在显著差异，耐低氧性强的‘绿霸春四号’在生长、保护酶、多胺、光合等方面比‘中农八号’表现出更强的低氧适应性。同时根系和叶片适应低氧的生理代谢途径不同。综上所述，叶面喷施或营养液中添加钙可以适当缓解低氧胁迫对植株的伤害，通过外源钙处理和钙调素拮抗剂W7对低氧胁迫下黄瓜幼苗的试验，从正反两个方面表明钙通过调节黄瓜幼苗保护酶系统和多胺代谢等来缓解低氧胁迫对黄瓜幼苗的伤害。更多还原

【Abstract】 Cucumber （Cucumis sativus L. ） is an important vegetable for greenhouseproduction in China. Oxygen deficiency in the root-zone and root-cushion formationhave become a limiting factor in soilless culture, which influences the growth anddevelopment of cucumber, therefore it is essential to develop some simple,economical and practical methods to alleviate injurous effects of hypoxia stress,which would be helpful in improving soilless culture of cucumber.Calcium is an essential element in plant growth and a second messenger ofcoupling extracellular signal with intracellular physiological reaction. It has beenknown that [Ca2+]cyt increases rapidly under environmental stress, and formsCa2+.CaM compound, which plays an important role in stabilizing cell wall, cellmembrane and membrane protein; Ca2+ activates ATPase and facilitates transport ofassimilates, regulates stoma movement and oxygen-evolving activity, therefore, Ca2+can alleviate damage caused by various environmental stresses. Plants can producesome physiological metabolism changes in response to the hypoxia stress, the level ofreactive oxygen species, antioxidative enzyme activities, the content of polyaminesare under dynamic change. All these are involved in the regulation of plant adaption toenvironmental stress. However, there has been no systematic work on the effect ofexogenous Ca2+ on reactive oxygen species and polyamines metabolism in cucumberseedlings under hypoxia stress.Using two cucumber cultivars with different resistance to hypoxia stress,’Zhongnong No. 8’ （a hypoxia-sensitive cultivar） and ’Lvbachun No.4’ （ahypoxia-resistant） as materials and precise control of dissolved oxygen in nutrientsolution through dissolved oxygen controller, we selected suitable Ca2+ concentrationsfor foliar spray or by adding to the nutrient solution, we investigated the effects ofexogenous calcium on reactive oxygen species and polyamines metabolism, effects of calmodulin antagonist W7 on reactive oxygen species, polyamines metabolism andphotosynthesis, and explored physiological function of calcium in cucumber seedlingsunder hypoxia stress. Main research findings were as follow:1. Foliar spraying 10 mmol·L-1 CaCI2 or maintaining 8mmol·L-1 Ca2+concentration in the nutrient solution increased the fresh and dry weight of cucumberseedlings, improved the growth of shoot and root, alleviated the growth inhibition ofcucumber seedlings under hypoxia stress. Exogenous Ca2+ increased Pn, Gs, Ci, Tr ofleaves. But foliar spray of higher concentration CaCI2 (more than 20 mmol·L-1)aggravated leaf damage and growth inhibition. Higher concentrations Ca2+（more than16 mmol·Ll） in the nutritient solution also resulted in growth inhibition. These resultsindicated that appropriate concentration of Ca2+ could alleviate the damagements ofhypoxia stress to cucumber.2. Under hypoxia stress, the contents of free, conjugated, bound polyamine inleaves and roots of the two cucumber cultivars increased to different degrees. Afterexogenous Ca2+ treatment, the contents of Put, Spm, Spd were higher than thosewithout Ca2+ treatment, PAO activity was lower than those without Ca2+ treatment,but the activities of polyamine synthetases such as ADC, ODC, SAMDC were higherthan those of treatments without Ca2+. These observation indicated that exogenousCa2+ adjusted polyamines synthesis and degradation, promoted the increase ofpolyamines content, calcium is involved in polyamines metabolism in cucumberseedlings under hypoxia stress.3. Under hypoxia stress, the activities of antioxidative enzymes in roots andleaves of cucumber seedlings increased significantly, the contents of ROS and MDAalso obviously increased, the increased range of antioxidative enzymes activities werehigher in ’Zhongnong No. 8’ than those of ’Lvbachun No.4’, but the increased rangeof the contents of ROS and MDA range were much lower in ’Zhongnong No. 8’ thanthose of ’Lvbachun No.4’. After the exogenous Ca2+ treatment, the MDA, H3O2contents and O2.- production rate decreased in cucumber seedlings, the activities ofSOD, POD, CAT, APX, and GR increased, the level of membrane lipid peroxidationdecreased, indicating that exogenous Ca2+ treatment alleviated injury due to hypoxiastress, and increased the adaptability of cucumber seedlings to hypoxia stress.4. Compared with the hypoxia treatment, the growth of cucumber leaves androots of pretreated cucumber seedlings with calmodulin antagonist W7 weresignificantly inhibited. The activities of SOD, POD, CAT, APX, GR, DHAR, and MDAR decreased and the contents of AsA, DAsA, GSH, GSSG decreased, but O2.-production rate, the contents of H2O2 and MDA in leaves and roots of cucumberseedling were higher than those without W7 treatment. Hypoxia stress andpretreatment with W7 decreased the contents of free, conjugated, bound polyaminesand Pn, Gs, Ci, and Tr, leading to the decrease of the adaptability of seedlings tohypoxia stress. These results indicated that Ca2+ modified the adaptability of seedlingsto hypoxia stress by the formation of Ca2+. CaM, and therefore regulating polyaminesmetabolism, reactive oxygen species metabolism and photosynthesis of cucumberseedlings.5. Under hypoxia stress, there were significant difference in all observationdetermined between ’Zhongnong No.8’ and ’Lvbachun No.4’ in all treatmentsbecause of the their different genetic backgrounds of the two cucumber cultivars.Hypoxia-resistant cultivar ’Lvbachun No.4’ exhibited better adaptability thanhypoxia-sensitive cultivar ’Zhongnong No.8’. in growth, antioxidative enzymes,polyamines metabolism and photosynthesis. Physiological metabolism paths in rootsand leaves of the same cultivar in adaptation to hypoxia stress were different.In addition, Foliar spraying of CaCl2 or addition of CaCl2 to nutrient solutioncould alleviate the harm of hypoxia stress to cucumber. Two experiments usingexogenous Ca2+ （positive aspect） and calmodulin antagonist W7 （negative aspect） incucumber seedlings under hypoxia stress were carried out. Results indicated that Ca2+alleviated the injury due to hypoxia stress to the cucumber seedlings. by adjusting theantioxidant systems and polyamines metabolism.更多还原