【作者】 魏晓雪；

【导师】 祖元刚；

【作者基本信息】 东北林业大学 ， 植物学， 2011， 博士

【摘要】 平流层臭氧的减少会导致到达地球表面的UV-B(280-320 nm)辐射显著增强,增强的UV-B辐射对生物体有明显的生物学效应。一氧化氮(NO)作为UV辐射的第二信使,已成为当前植物逆境生理和信号转导领域的研究热点。本文分别研究了增强UV-B辐射、施加外源NO供体及二者复合作用对红松(Pinus koraiensis)幼苗光合作用、自由基含量、抗氧化酶活性、类黄酮含量等生理生化指标的影响,以此在不同层次探讨红松幼苗对环境UV-B辐射增强、NO浓度升高的生态适应机制,探明外源NO对UV-B胁迫下红松幼苗生理特性的影响。主要结果如下：UV-B辐射增强显著提高红松针叶内羟基自由基(·OH)、过氧化氢自由基(H202)和MDA含量；UV-B辐射增强诱导过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)和超氧化物歧化酶(SOD)活性升高,显著降低过氧化物酶(POD)活性(P<0.05)；抗氧化物质中可溶性蛋白含量随UV-B胁迫程度加强而显著增加,而UV-B处理均降低了类胡萝卜素和UV-B吸收物质(总酚、黄酮、缩合单宁)的含量。经过40天UV-B辐射后红松幼苗针叶蜡质条纹状隆起断裂,破碎,条纹状隆起数量减少,蜡质颗粒增多。与对照相比,UV-B处理组红松针叶蜡质含量均降低,高剂量的UV-B辐射长期处理抑制了红松针叶蜡质的积累。红松幼苗针叶蜡质化学成分中烷类是主要成分,比重占50%以上,UV-B处理导致烷类和酚类比重随着UV-B辐射处理强度的加强而降低；酯类的比重随着UV-B辐射处理强度的加强而增加。通过喷施不同浓度外源NO对红松幼苗生理特性影响的研究中发现,SNP促进红松幼苗生长的最佳喷施浓度为0.01 mM。我们研究结果表明不同浓度外源NO对光合参数、光合色素含量、自由基含量、抗氧化酶活性影响显著不同。0.1 mM浓度SNP处理下红松光合作用的影响主要是通过气孔性限制因素引起的。光合色素含量、可溶性蛋白含量、类黄酮含量、CAT活性和POD活性的最大值都出现在0.01 mM浓度SNP处理,H202和MDA含量的最大值出现在0.1 mM浓度SNP处理,光合色素的最小值也出现在0.1 mM浓度SNP处理。本研究发现,高浓度外源NO也可能是一种胁迫因子。喷施适当浓度的外源NO能显著缓解UV-B胁迫对红松幼苗叶片的伤害,研究结果表明外源NO供体SNP通过提高4种抗氧化酶活性、降低H202和MDA含量、增加类黄酮含量等措施来抵抗UV-B胁迫引起的伤害。植物防卫反应信号分子NO广泛参与红松幼苗对UV-B胁迫的应答与防御,NO作为UV-B辐射的第二信使介导了紫外胁迫信号的转导,并诱发细胞产生抗性反应。外源NO诱导SOD、POD、CAT和APX活性增强,类黄酮含量增加,抗氧化物质和抗氧化酶类在红松幼苗体内协同作用,在胁迫条件下清除过量的活性氧,维持活性氧代谢的平衡,保护膜结构,从而使红松幼苗在一定程度上忍耐、减轻或抵御UV-B胁迫的伤害。对NO含量、NOS和NR活性测定结果表明红松幼苗体内的NO可能主要依靠NOS途径产生,而不是NR途径。综上所述,本研究证实UV-B辐射增强通过产生大量的自由基对红松幼苗的生理代谢产生影响,而红松幼苗通过提高抗氧化酶(CAT, APX, SOD)的活性和蛋白质的含量来抵抗UV-B辐射引起的损伤,但其防御机制并不能有效的缓解这个伤害。外源NO对红松幼苗生长生理的调控具有一定的剂量效应,0.01 mM SNP处理对红松幼苗生长生理的调控作用最明显。高浓度外源NO也可能是一种胁迫因子。而适当浓度的外源NO处理通过提高抗氧化酶活性、降低自由基含量、增加类黄酮含量等途径显著缓解UV-B胁迫对红松幼苗叶片的伤害。外源NO对红松幼苗在UV-B辐射胁迫下具有一定的缓解作用。红松幼苗体内的NO可能主要依靠NOS途径产生,而不是NR途径。更多还原

【Abstract】 The principal ultraviolet radiation absorbance occurs in the stratospheric ozone layer in earth’s atmosphere. The destruction of this layer, which is caused by ozone depletion, has lead to an increase in solar UV-B radiation (280-320 nm) reaching the Earth’s surface and produced potentially biological effect for plants in the earth. Nitric oxide (NO) as the second messenger of the UV radiation, has been the most activity research hotspot in plant stress physiology and signal transduction fields.In this research we studied the effects of enhanced UV-B radiation, different concentrations of exogenous NO and increased exogenous NO under enhanced UV-B radiation on Pinus koraiensis seedlings, respectively. Gas exchange parameters, the concentrations of photosynthetic pigments, contents of free radicals, activities of antioxidant enzymes, concentrations of flavonoids and other physiological indicators were determined. We revealed the adapted mechanism of Pinus koraiensis to enhanced UV-B radiation, sprayed different concentrations of exogenous NO and optimized the best UV-B radiation dose and concentration of exogenous NO to promote the growth of Pinus koraiensis seedlings. The main results are as follows:The T1, T2, T3 UV-B treatments were increased UV-B radiation by 1.40,2.81 and 4.22 kJ-m-2·d-1 compared to the control treatment (CK), respectively. Gas exchange parameters, the concentrations of photosynthetic pigments, the contents of secondary metabolites, epicuticular wax, free radical, malondialdehyde (MDA), and the activities of antioxidant enzymes were determined after 40 days exposures. Our results showed that the contents of hydrogen peroxide (H2O2) and MDA were significantly increased by enhanced UV-B radiations (P<0.05). The supplemental UV-B radiation induced the activities of catalase (CAT), ascorbate peroxide (APX), and superoxide dismutase (SOD), but significantly decreased the activity of peroxidase (POD) (P<0.05). Compared to CK, the concentration of soluble protein was increased with the exposure of UV-B radiation. The contents of flavonoids and carotenoid were significantly decreased by all levels of supplemental UV-B radiations (P<0.05). After 40 days UV-B radiation the striped uplift of wax in needle of Pinus koraiensis seedlings was broken, the number of stripe-like uplift was reduced and wax particles were increased. Compared with the CK, the wax contents were significantly decreased with the exposure UV-B radiations. The long-term treatment of high doses of UV-B radiation was inhibited the accumulation of wax in needle of Pinus koraiensis. The most prominent substance of Pinus koraiensis was alkanes. The alkanes represented more than 50% of the compounds. The percentage of alkanes and phenols in Pinus koraiensis needles were decreased by enhanced UV-B radiation. In contrast, the percentage of long chain esters was increased by enhanced UV-B radiation.The Pinus koraiensis seedlings were treated with sodium nitroprusside (SNP), a NO donor, at 5 different concentrations ranging from 0 mM to 1 mM and 0.01 mM of SNP had the most satisfactory effect. The result showed that at different concentrations exogenous NO had different effects on the photo synthetic parameters, the concentrations of photosynthetic pigments, the contents of free radical and the activities of antioxidant enzymes and photosynthetic pigments. The content of chlorophyll was maximal in the treatment with SNP at 0.01 mM. The different concentrations of SNP treatment significantly increased the content of H2O2 and MDA. Moreover, the 0.1 mM SNP treatment had the maximal concents of H2O2 and MDA. Meanwhile, the different concentrations of SNP treatment had different effects on antioxidant activity. The activities of CAT, APX, POD were maximum in the concentration of 0.1 mM SNP. The different concentrations of SNP treatments significantly decreased the activity of SOD (P＜0.05). Our results showed that high concentration of exogenous NO may be a stress factor for Pinus koraiensis seedlings.The appropriate concentration SNP treatment could significantly alleviate the damage of UV-B stress on Pinus koraiensis seedlings. The results showed that exogenous SNP, a NO donor, significantly increased activities of SOD,PQD,CAT and APX of Pinus koraiensis seedling, reduced the contents of H2O2 and MDA, and increased content of flavonoids. The results of NO content, nitric oxide synthase (NOS) and nitrate reductase (NR) activity assay showed that the NO production in vivo of Pinus koraiensis seedlings may was mainly dependent NOS pathway, but not relying on NR pathway.Our results demonstrate that Pinus koraiensis can increase of the CAT, APX and SOD activities to prevent oxidative stress by supplemental UV-B radiation, but the defense mechanism is not efficient enough to prevent the UV-B induced damage. The 0.1 mM SNP treatment significantly promoted the growth and regulated the physiology of Pinus koraiensis seedlings. The experiment proved that NO influenced the photosynthesis of Pinus koraiensis seedlings. Morevoer, the research showed that high concentrations of exogenous NO may be a stress factor for Pinus koraiensis seedlings. The appropriate concentration SNP treatment could significantly alleviate the damage of UV-B stress on Pinus koraiensis seedlings.更多还原