【作者】 蔡悦；

【导师】 邬飞波；

【作者基本信息】 浙江大学 ， 作物学， 2010， 博士

【摘要】 农田遭受镉(cadmium, Cd)污染,不仅对作物生长发育造成伤害,导致产量下降,更有可能造成作物产品中镉积累过多,导致粮食卫生品质的下降。本研究利用水培试验,系统地研究了镉抑制水稻生长及Cd耐性和吸收积累的基因型差异的生理机理,并探索了以谷胱甘肽(GSH)为主的外源物对水稻镉毒害的缓解效果及作用机理。主要研究结果如下：1不同浓度外源谷胱甘肽和植物激素对水稻镉毒害的缓解效应以水稻品种秀水63为材料,水培试验,比较研究了外源GSH、吲哚-3-乙酸(IAA)、6-苄氨基嘌呤(6-BA)和赤霉素(GA3)对镉胁迫下对水稻生长的影响。结果表明,5μM Cd显著降低株高和地上部干重和鲜重。与5μM Cd对照相比,不同浓度GSH显著提高水稻幼苗的株高和地上部千重,其中尤以50GSH处理对地上部鲜重的恢复作用最明显。低浓度IAA显著提高了地下部鲜重。而各个浓度的6-BA和GA3均没有显著的缓解镉对水稻幼苗生长的抑制。5μM Cd胁迫添加不同浓度GSH和IAA处理试验结果表明,与对照5μM Cd相比,不同浓度的GSH(除50 gM外)和0.1μM与10μM IAA可以显著提高叶绿素含量(SPAD值)。各浓度GSH和10 IAA均可显著降低地上部与地下部镉浓度。综合比较分析结果表明,对镉毒害缓解效果以50μM和200μM GSH最好,其次是10μM IAA。2镉对水稻超微结构与抗氧化系统影响的基因型差异及外源GSH和IAA的缓解效应水培试验,研究了外源GSH和IAA对镉胁迫下2个耐镉性不同水稻基因型(秀水63,镉敏感基因型；丙97252,耐镉基因型)生长、超微结构、抗氧化系统活性的影响。结果表明：5μM Cd胁迫显著影响叶肉与根尖细胞结构,使细胞基质变稀，叶绿体片层结构松散,液泡变多变大,根系电子富集颗粒(EDG)增加,其中敏感基因型受到更严重的抑制。镉胁迫提高地上部超氧化物歧化酶(SOD)(主要是Mn-SOD)和谷胱甘肽S转移酶(GST)活性、GSH含量、和镉处理10d以前过氧化物酶(POD)活性；但降低抗坏血酸过氧化物酶(APX)活性、地下部POD、过氧化氢酶(CAT)活性,地上部抗坏血酸(AsA)含量和AsA/DHA比值。但是镉处理后丙97252的地上部CAT活性上升,而秀水63则下降；而膜脂过氧化产物丙二醛(MDA)的积累则是秀水63增加的多。说明了丙97252有较强耐性的可能机制是将镉储存在液泡以及更高的CAT活性。镉胁迫条件下添加GSH,镉中毒现象得到明显缓解：生长和细胞结构恢复、减少MDA积累,并且丙97252恢复相对更好。GSH显著提高了耐镉基因型根系POD、CAT、APX活性、地上部AsA含量及AsA/DHA比值,显著降低两基因型根系GST活性、地上部GSH含量以及15 d时Cu/Zn-SOD活性。镉胁迫条件下添加IAA,镉中毒现象也得到一定程度缓解：显著提高根系干重、细胞结构恢复、减少地上部MDA积累、减少植株中镉积累。IAA提高镉胁迫后根系AsA含量,降低GSH含量、GST活性和地上部POD活性。比较GSH和IAA的缓解效果,GSH要更显著。镉胁迫条件下同时添加GSH与IAA,也可以明显缓解镉中毒症状,对生长的影响与单一用GSH缓解相近。其对SOD活性的影响也更接近于单一用GSH来缓解。不同的是,在SOD同工酶中,Cu/Zn-SOD活性更低。并且复合处理后的GSH含量、GST活性、地上部AsA和AsA/DHA值,均低于单一用GSH或者IAA缓解。这些说明外源GSH和IAA可能都有参与螯合作用,GSH还与抗氧化系统活性的恢复有关系,而IAA对抗氧化系统可能不起作用。3外源GSH影响镉胁迫水稻苗生长及元素吸收积累的基因型差异外源GSH对不同浓度镉(5和50μM)胁迫下水稻矿质元素浓度和积累量的基因型差异。结果表明,50μM Cd处理后水稻生长相比5μM Cd受到更严重抑制,对秀水63的影响尤为严重。镉胁迫显著减少根系与地上部的Mn和地上部的Zn的浓度和积累量,降低根系与地上部的Cu浓度。地上部Zn浓度与地上部／根系镉浓度之间,以及根系中Cd和Mn浓度之间均存在显著的负相关。外源GSH显著降低根系和地上部中镉浓度。同时,在5μM Cd处理中,GSH提高了丙97252中Ca和Mn的浓度以及秀水63根系中Ca和Zn的浓度；而在50μM Cd处理中,GSH增加了Zn的转运。启示外源GSH的效果具有基因型和镉处理浓度的差异。4 GSH对镉胁迫水稻光合作用和植物螯合肽分泌的影响基因型差异外源GSH对镉胁迫下光合特性和植物螯合肽(PCs)的影响及基因型差异。结果表明,镉(5和50μM)胁迫显著降低叶绿素含量、净光合速率和生物学产量,其中秀水63受到抑制尤为严重。镉降低PSⅡ最大光化学效率(Fν/Fm)和有效PSⅡ量子产量(Y(Ⅱ)),增加能量规则损耗量子产量(Y(NPQ)),镉敏感基因型受影响也更明显。镉诱导根系产生PCs,增加GSH和半胱氨酸(Cys)的含量。其中,丙97252在5d时诱导的PCs和GSH含量均高于秀水63。GSH显著增加叶绿素含量,Pn, Fν/Fm和Y(Ⅱ),降低Y(NPQ)和非光化学猝灭系数(qN)。除50μM Cd处理5d外,添加GSH显著提高两基因型根系中GSH水平；GSH还上调5μM Cd处理下丙97252的PCs水平和秀水63在5d时的水平。因此,GSH对镉毒害的缓解效应与叶绿素含量的提高、光合系统的恢复和根系GSH水平有关。5水稻蛋白质组对低浓度镉毒害响应的基因型差异及外源谷胱甘肽的影响研究了50μM GSH对镉胁迫下水稻蛋白质谱表达的影响及基因型差异。结果表明,秀水63的生长受到更明显的抑制。鉴定到基因型差异表达的27个蛋白点,质谱分析鉴定出21个差异蛋白点。在根系中,参与抗氧化系统、碳水化合物循环、膜保护、蛋白质合成、DNA表达和防御的蛋白；和在叶中参与光合系统、ROS信号、能量和蛋白降解的蛋白应该与镉耐性有关。而在添加GSH后,在根系中参与抗氧化系统、碳水化合物循环、膜保护、蛋白质合成、DNA表达和防御的蛋白；和叶片中参与卡尔文循环、蛋白和核酸代谢、热激反应的蛋白,这些蛋白的表达均得到显著提升。结果暗示了GSH在这些方面的作用起到了提高水稻镉耐性。更多还原

【Abstract】 Farmland suffers from cadmium (Cd) pollution, causing crop growth inhibition, yield reduction, and more likely to result in excessive accumulation of Cd in crop products, affecting food quality. This investigation was carried out to study physiological mechanism of genotypic differences in Cd-toxicity, uptake and accumulation, and explored with the mitigation effect and mechanism of exogenous GSH-based materials on Cd toxicity of rice.1 Mitigation effect and optimal concentration of exogenous GSH and plant hormones on Cd toxicity in riceHydroponic experiment was performed to study the exogenous reduced glutathione (GSH), indole-3-acetic acid (IAA),6-benzylaminopurine (6-BA) and gibberellin (GA3) on the alleviation of Cd toxicity and optimize the concentration of alleviation. The results showed that 5μM Cd significantly reduced plant height and shoot dry weight and fresh weight. Different concentrations of GSH significantly elevated plant height and shoot dry weight of rice seedlings. FiftyμM GSH mostly resumed the shoot fresh weight. Low concentrations of IAA significantly increased the root fresh weight. The various concentrations of 6-BA and GA3 did not significantly alleviate the Cd induced growth inhibition in rice seedling. Further research showed that all the concentrations of GSH (except for 50μM), as well as 0.1 and 10μM IAA, significantly increased the chlorophyll content (SPAD value). The variety of concentrations of GSH and IAA could reduce the shoot/root Cd concentration. Comprehensive comparative analysis of results showed that concentrations of 50 and 200μM GSH and 10μM IAA concentrations could serve as an effective mitigation factor in Cd toxicity.2 Genotypic differences of Cd stress on the ultrastructure and antioxidative system of rice seedlings and mitigation effects of exogenous GSH and IAAHydroponic experiment was performed to study the effects of exogenous GSH and IAA on Cd stress on growth, ultrastructure. and antioxidant system activity in two different Cd-resistance rice genotypes (Xiushui63, cadmium-sensitive genotype; Bing97252, cadmium-resistant genotype). The results showed that 5μM Cd stress significantly inhibited the growth of rice seedlings, destroyed the structure of mesophyll and root tip cells, with cell matrix thinning, chloroplast lamellae loose, vacuole larger and increased, root EDG increased, and more severely inhibition was observed in sensitive genotype. Cd also increased shoot SOD (mainly Mn-SOD), and GST activity, GSH content, and enhanced the POD activity during 10 d treatment, while reduced the APX activity, root POD, CAT activity, shoot AsA content and AsA/DHA ratio. The shoot CAT was elvated in Bing97252, but decreased in Xiushui63 after Cd exposure. Moreover, the increased of accumulation of membrane lipid peroxidation product MDA was more in Xiushui63. Thus, the possible mechanism of Bing97252 showing stronger potential tolerance to Cd might be that it has the ability to transfer and held Cd in the vacuole and has an effective antioxidant system. Addition of GSH significantly relieved of Cd toxicity in rice with plant growth and cell structure restored, MDA accumulation reduced, and the recovery was better in Bing97252. GSH significantly increased root POD, CAT, APX activity, shoot AsA content and AsA/DHA ratios in the cadmium-resistant genotype, significantly reduced GST activity in roots, GSH content in shoots and Cu/Zn-SOD activity at thel5th d in the both genotypes. Addition of IAA also alleviated the Cd toxicity to some extent with significantly increase of root dry weight, cell structure recovery, reducing shoot MDA accumulation and Cd accumulation in plants. IAA increased root AsA content, decreased the GSH content, GST activity and shoot POD activity. Combined GSH and IAA treatment can also significantly alleviate the symptoms of cadmium toxicity on the growth of rice; the effect was similar to single addition of GSH. The differences are that Cu/Zn-SOD activity, GSH content, GST activity, shoot AsA content and AsA/DHA values were lower with Cd+GSH+IAA treatment. These results showed that exogenous GSH, IAA may both take part in chelation, the effect of GSH should be related to restoration of antioxidant system, but IAA appeared to have no effect on antioxidant system.3 Genotypic dependent effect of exogenous glutathione on Cd-induced changes in cadmium and mineral uptake and accumulation in rice seedlingsWe evaluated how the different rice genotypes respond to Cd toxicity in the presence of GSH. We studied the effects on growth, Cd and mineral uptake and accumulation. The results showed that Cd stress (5,50μM Cd) decreased plant fresh weight, reduced content of chlorophyll a, b and total carotenoids, with Cd-sensitive genotype being much sever than tolerant one. Cd significantly decreased concentration and accumulation of Mn in root/shoot, and Zn in shoot, but increased Cu concentration in root/shoot. Significantly negative correlation was discovered between shoot Zn concentration and shoot/root Cd concentration, and between root Cd and Mn concentration. Exogenous GSH significantly alleviated Cd-induced growth inhibition in both genotypes, and significantly reduced Cd concentration in roots and shoots. Meanwhile, in 5μM Cd, the concentrations of Ca, Mn in Bing97252, and Ca, Zn in Xiushui63 in roots were elevated by addition of GSH. In 50dM Cd, external GSH enhanced the translocation of Zn. The effects of GSH were different between genotypes and dose of Cd treatment.4 Difference in response to Cd toxicity in photosynthesis and phytochelatins between Cd tolerant and sensitive rice genotypes and as affected by exogenous glutathioneWe evaluated how the different genotypes responded in photosynthesis and phytochelatins to Cd toxicity in the presence of GSH. Plant height, chlorophyll content, net photosynthetic rate (Pn) were reduced in the treatments of 5 and 50μM Cd and Cd-sensitive genotype showed much severer in the reduction than tolerant one. Cd stress caused decrease in maximal photochemical efficiency of PSⅡ(Fν/Fm) and effective PSⅡquantum yield (Y(Ⅱ)) and increase in quantum yield of regulated energy dissipation (Y(NPQ)), with Cd-sensitive genotype being more evident. Cd-induced phytochelatins (PCs), GSH and Cysteine (Cys) accumulation was observed in roots of both genotypes, with a markedly higher level in PCs and GSH on day 5 in Bing97252 compared with that in Xiushui63. Exogenous GSH significantly alleviated growth inhibition in Xiushui63 under 5μM Cd, and both genotypes in 50μM Cd. External GSH significant enhanced chlorophyll content, Pn, Fv/Fm and Y(II) of the plants exposed to Cd, but decreased Y(NPQ) and the coefficient of nonphotochemical quenching (qN). GSH addition significantly increased root GSH levels of both genotypes during 15 d Cd exposure, except day 5 of 50μM Cd; induced up-regulation in PCs of 5μM Cd treated Bing97252 thoughtout 15 d and Xiushui63 of 5 d exposure. The results suggest that alleviation of Cd toxicity by GSH is related to a significant improvement in chlorophyll content, photosynthetic performance and root GSH levels.5 Proteomic analysis to unravel mild cadmium stress response in rice seedlings and as affected by exogenous glutathioneWe applied 5μM Cd to investigate the effect of environmentally realistic Cd concentration on proteomics in rice. A comparative analysis between two genotypes with proteomic approach revealed that proteins involved in anti-oxidative system, carbohydrate metabolism, membrane protection, protein synthesis, DNA expression and defense in roots; photosystem, ROS signaling, energy, and protein degradations in leaves are important in Cd tolerance. We also applied GSH to exam the alleviating effects on Cd toxicity. GSH enhanced the expressions of proteins involved in anti-oxidative system, carbohydrate metabolism, membrane protection, protein synthesis, DNA expression and defense in roots; Calvin cycle, protein and amino acid metabolism, heat shock responses in leaves. The results suggested GSH play an important role in elevating Cd tolerance of rice in these processes.更多还原