【作者】 李英丽；

【导师】 张建光；

【作者基本信息】 河北农业大学 ， 果树学， 2013， 博士

【摘要】 温度和光照是影响果实生长发育的重要环境因子。然而，由于果实发育期（甚至在贮藏期）经常遭受温度或（和）光照逆境的胁迫，致使产量和品质受到不同程度的影响。因此，研究和揭示温度与光照逆境对果实影响的内在规律和机理，对于提高果实对温度、光照逆境的抵御能力，从而保证梨果丰产、优质意义重大。本研究以鸭梨（Pyrus bretschneideri Rehd.,cv:Yali pear）为试材，采用田间和室内试验相结合的方法，探讨了不同温度和光照变化对果实活性氧含量（O-.2、H₂O₂）、抗氧化酶（SOD、POD、APX、MDHAR、GR）活性、抗坏血酸（AsA）含量以及细胞中Ca²⁺分布的影响。初步阐明了不同温度和光照条件下果实抗氧化系统以及细胞中Ca²⁺分布的变化规律。主要研究结果如下：1.高温处理显著提高了鸭梨果实中活性氧（O-.2、H₂O₂）含量，同时提高了POD和APX活性。但鸭梨果实中POD和APX对高温胁迫的响应存在时间上的差异：高温胁迫1h时显著提高了APX活性，而POD活性在处理初期无显著变化，处理后期（5h后）才显著升高。高温胁迫初期果实内H₂O₂主要由APX清除，使H₂O₂含量保持在较低水平，当胁迫变得严重时，POD开始起作用。2. AsA-GSH循环是温度逆境下清除鸭梨果实活性氧的主要途径之一。APX、GR、MDHAR作为该循环的主要抗氧化酶，对温度胁迫的响应在时间上具有先后顺序的差异：APX在胁迫1h时活性最大，而MDHAR和GR分别在处理3h和5h活性最高。3.高温处理初期显著提高了抗坏血酸合成速率，还原型抗坏血酸含量和MDHAR活性变化相一致，因此可推断：抗坏血酸主要通过AsA-GSH循环参与活性氧的清除。4.高温和强光具有胁迫增效作用，能够加重氧化胁迫的发生：高温条件下，照光处理显著提高了高温初期鸭梨果实中活性氧含量，使APX和POD活性增强，抗坏血酸合成量增加，但随着时间延长，LOX活性迅速增加，加速了果实伤害的进程。5.高温强光胁迫下，施用不同外源调节物质可以减缓胁迫对果实造成的伤害：AsA、草酸、SA、ABA处理显著提高了果实内源H₂O₂含量。当胁迫发生后，果实内大量H₂O₂积累作为信号分子诱导了AsA-GSH循环中关键酶（APX、GR）活性以及AsA含量、AsA/DHA比值有所提高，确保了AsA-GSH循环顺利运转，使LOX活性保持在较低的水平，延缓了高温强光对果实的伤害进程。6. Ca²⁺信使促进剂（CaCl₂）显著提高了胁迫初期果实内H₂O₂含量，大量的H₂O₂诱导了APX、GR活性，AsA合成量增加，使胁迫后期LOX活性保持较低水平，避免了膜磷脂过氧化，提高了果实的抗逆性。胁迫过程中Ca²⁺信使抑制剂（EGTA、LaCl3）显著抑制了APX、GR活性和AsA的合成，使AsA-GSH循环运转受到影响，LOX活性迅速提高，加剧了细胞膜磷脂的过氧化进程，加重了果实的伤害。并且从抑制剂的抑制效果来看，LaCl3较EGTA效果更为显著。7.温度能明显影响果实细胞中Ca²⁺分布。45℃处理初期，液泡中Ca²⁺开始向细胞质中移动，使细胞质中Ca²⁺浓度增加。随着处理时间的延长，液泡中Ca²⁺浓度减少，大部分Ca²⁺颗粒沉淀流向胞质中。处理7h细胞质中的Ca²⁺浓度减少，液泡中Ca²⁺颗粒增多，液泡结构完整性遭到破坏，果实细胞伤害发生。8.鸭梨果实中Ca²⁺分布受光照的影响，无光条件下果实中Ca²⁺主要分布在细胞间隙和液泡中，细胞质中几乎没有Ca²⁺分布。照光条件下，细胞间隙和液泡中的Ca²⁺向细胞质中转移，细胞质中Ca²⁺浓度急剧增加，但细胞间隙仍有少量Ca²⁺沉淀颗粒分布，而液泡中几乎没有Ca²⁺分布，由此可见：细胞质中的Ca²⁺主要来源于液泡中。9.在温度和（或）光照逆境胁迫下，果实活性氧和细胞质中Ca²⁺浓度升高，而高浓度活性氧和（或）细胞质中Ca²⁺作为信号分子，刺激或诱导细胞膜上SOD、APX、MDHAR、GR等酶活性的提高，在一定范围内消除了胁迫后期活性氧对果实细胞膜的伤害。然而，随着温度和光照胁迫加重，当果实活性氧产生速度远超过抗氧化系统清除速度时，此时液泡中的Ca²⁺全部流入细胞质中，细胞失去了自动调节能力，再不能诱导抗氧化系统做出相应的响应（或者能力达不到实际需要），从而导致果实细胞膜系统遭受伤害，直至果实出现不同程度的生理伤害。更多还原

【Abstract】 Temperature and light are very important environmental factors affecting fruit growthand development. However, fruit output and quality are influenced, more or less, due tofrequent stresses by excessive temperature and light during fruit growth （even during coldstorage）. Therefore, it is meaningful to reveal the effect of temperature and light stresses onfruits, so as to raise the ability to resist temperature and light stresses and achieve higheryield and top quality.In the present experiment, the effect of different temperature and light on fruit ROSgenerating rates, antioxidant enzymes activity, AsA contents as well as Ca²⁺distribution inpeel cells was examined under laboratory and field conditions with Yali pears. The response ofantioxidant system to different temperature and light levels and the pattern of Ca²⁺distributionin peel cells were preliminarily expounded. The main results are as follows:1. The hydrogen peroxide（H₂O₂）content, ascorbate peroxidase （APX） andperoxidase（POD） activity were improved under high temperature stress conditions.However, the susceptibility of APX and POD to high temperatures was quite different.During the incipient treatment by high temperature, APX activity was obviously increasedbut POD activity was not obviously varied. The POD activity was enhanced with time. Itwas concluded that APX was the primary H₂O₂–scavenging enzyme that could maintainH₂O₂content at a lower level. Whenever the stress became severe, POD wouldimmediately take action.2. AsA-GSH cycle was the main antioxidant pathway to scavenge ROS in fruits. APX,GR and MDHAR were the important components in the cycle and their susceptibility totemperature stress was relatively different. APX activity reached the peak at1hr, next to itwas MDHAR at3hr and the last one was GR at5hr.3. During the incipient treatment by high temperature, the contents of total ascorbicacid were significantly enhanced. The changing trend of reductive ascorbic acid （AsA） andMDHAR activity was consistent. It could be inferred that AsA scavenged ROS largelythrough the AsA-GSH cycle.4. Oxidant damage could be enhanced by double stresses from both temperature andlight. Under high temperature conditions, light could induce the ROS contents to rise, which also increased the antioxidant contents and the antioxidant enzymes activity. Butfruit injury would occur as fruit antioxidant ability declined and LOX was accelerated.5. Under temperature and light stresses，the different exogenous chemical applicationcan alleviate fruit harm. Before stresses, the different exogenous chemical application（AsA, oxalic acid, SA, ABA） significantly improved H₂O₂content. The abundant H₂O₂infruits can signale molecules to regulate the AsA-GSH cycle. Under temperature and lightstresses,the APX,GR activity are enhanced, and the reductive ascorbic acid content andthe ratio of reductive ascorbic acid of oxidative ascorbic acid （AsA/DHA） are increased,which ensure the AsA-GSH cycle to successfully run, so that the LOX activity can remainat a low level to slow down the progress of fruit injury6. Under temperature and light stresses, the effect of calcium regulators on fruitresistance was examined. At the primary stage of temperature and light stresses, theaccelerance of Ca²⁺（CaCl2） increased the H₂O₂content, which improved the activity ofAPX, GR and the AsA content. As a result, the LOX activity was inhibited, and the fruitresistibility is enhanced. The inhibitor of Ca²⁺（EGTA、LaCl3）significantly reduced theAPX and GR activity and AsA content, which affected the AsA-GSH cycle. Moreover, theactivity of LOX was rapidly improved, which aggravated the cell membrane oxidation.Compared to EGTA, LaCl3showed stronger inhibiting effect.7. Temperature could markedly affect Ca²⁺distribution within fruit peel cells. When fruitswere stressed at45℃, a great deal of Ca²⁺was transferred from vacuolar into cytosol. After fruitswere treated for7h, the cytosol was damaged and the Ca²⁺moved from cytosol into vacuolar.8. Light could also affect the Ca²⁺distribution. The Ca²⁺existed mainly in vacuolar andintercellular spaces under dark conditions. When fruits were stressed by intense light, agreat deal of Ca²⁺was transferred from vacuolar and intercellular spaces into cytosol.There was a little of Ca²⁺left in intercellular spaces and there was no Ca²⁺in vacuolar. So itwas shown that the Ca²⁺in cytosol mainly came from vacuolar.9. Under temperature and light stresses, the ROS content and Ca²⁺content in cytosolincreased. Higher concentration of ROS and Ca²⁺functioned as the signal molecules toimprove the activity of antioxidant enzymes（SOD, APX, MDHAR or GR）, protecting fruitmembranes from injury by ROS to some extent. However, when the stress aggravated,especially when the ROS generating rate exceeded the clearance rate from the antioxidantsystem, total Ca²⁺moved from vacuolar into cytosol, as a result, the cells lost theautomatically adjustable capacity, resulting in that the antioxidant system could not make acorresponding response （or the scavenging capability was insufficient to eliminate theinjury caused by free radicals）. In such a case, the membrane was destroyed, which couldcause fruit physiological injury to some extent.更多还原