【作者】 李萍；

【导师】 梁永超；

【作者基本信息】 中国农业科学院 ， 植物营养， 2012， 博士

【摘要】 本研究旨在揭示硅调控水稻锰营养的生理效应及分子机制，为减轻水稻的锰毒害、提高水稻产量并促进水稻生产的持续发展提供理论依据。通过水培试验，采用两个对Mn耐性不同的水稻品种，XXY640（锰敏感型）和ZLY99（耐锰型）作为试验材料，研究了硅（1.5mmol·L-1）对高锰（2mmol·L-1）胁迫下水稻的生长、元素吸收、运输及分布、抗氧化体统和光合作用的影响；利用敏感型品种为材料，采用Solexa高通量测序技术获得了高锰胁迫下加硅和不加硅处理的水稻基因表达图谱；根据测序结果，通过Gene Ontology和Pathway显著性富集分析，得到有关光合作用过程中差异表达显著基因；并利用实时荧光定量PCR技术研究这些关键基因相对表达量的影响。主要结果如下：（1）两个水稻品种对高锰胁迫反应存在巨大差异，高锰胁迫严重抑制了敏感品种的干物质重，使其叶片毒害症状明显。加硅处理增强了两个品种对锰毒的抗性，硅抑制了锰从敏感品种地下部到地上部的转移，而对耐性品种来说，硅则抑制了锰的吸收，从而降低了锰的毒害。（2）高锰胁迫下，耐性品种叶片和根系的Mn含量都显著高于敏感品种。高锰胁迫抑制了敏感品种K元素从根部向叶片的转移，而降低了耐性品种的根部吸收K元素的能力。高锰胁迫下，施硅处理，增加了敏感品种叶片中K、Fe和Zn的相对含量；显著增加耐性品种叶片中K和Zn的相对含量，显著降低其Ca和Fe的相对含量。高锰胁迫下施硅可以促进敏感品种K元素的转运。Si对耐性品种根系中各元素含量保持相对平衡具有重要作用。（3）在Mn胁迫下，敏感品种根系的超氧化物歧化酶（SOD）活性、抗坏血酸（AsA）和非蛋白巯基（NPT）含量显著增加，但过氧化物酶（CAT）和抗坏血酸过氧化物酶（APX）酶活性显著降低，谷胱甘肽（GSH）含量也显著降低，最终导致膜脂过氧化加剧，丙二醛（MDA）含量升高，质膜完整性遭到破坏，根系受到伤害。Mn胁迫下，耐性品种根系的GSH和AsA含量增加，但CAT酶活性和NPT含量降低，导致MDA含量升高，根系受到伤害。加Si后增强了水稻根系抗氧化系统物质活性，降低MDA和过氧化氢（H2O2）含量，缓解水稻根系受到的伤害。且这种缓解效应在敏感品种根系的更为显著。高锰胁迫显著增加了敏感品叶片的SOD、CAT和APX活性，但是显著降低了NPT和GSH含量，因此导致了H2O2和MDA大量累积，植株受到伤害。加硅后显著降低了其MDA和H2O2含量，促进了植物生长。对于耐性品种来说，高锰胁迫下，叶片中SOD活性和和GSH含量显著增加，增强了其清除自由基的能力，导致低的氧化胁迫。高锰胁迫下，加硅处理显著影响了敏感品种非酶抗氧化物质的活性。（4）锰胁迫下，敏感品种的叶绿素a、叶绿素b、类胡萝卜素和叶绿素总量显著降低，而耐性品种仅类胡萝卜素含量显著下降。两个品种的光合速率在锰胁迫下都显著降低。扫描电镜和透射电镜结果表明，锰胁迫使敏感品种叶片气孔关闭，基粒片层紊乱，而耐性品种气孔开度略有减小，叶绿体片层结构基本不受影响。加硅之后，锰胁迫下敏感品种的叶绿素总量和类胡萝卜素含量显著增加，耐性品种的类胡萝卜素含量也增加，敏感品种的光合速率也显著增加，气孔开度增大。（5）高锰胁迫下有效差异表达(fdr≤0.001和|log2Ratio|≥1)的基因为2831个，其中上调1336个，下调1495个。高锰胁迫下施硅后差异表达基因上调表达647个，下调表达892个。正常锰浓度下，施硅后基因表达16525个，其中上调表达1558个，下调2028个。同时进行硅处理和锰处理时与单硅处理相比，表达基因16273个，上调表达320个，下调表达172个。锰胁迫下施硅后差异表达基因功能分析表明：差异表达基因涉及到转录因子，转运子，转移酶蛋白等基因，涉及到植物体内的初生代谢和次生代谢等过程。研究结果表明水稻应答锰胁迫的机理非常复杂，涉及到代谢调节、离子转运、信号传导、转录调节和逆境应答等大量基因协调表达结果。（6）根据高通量测序结果，通过实时荧光定量PCR分析了敏感品种中光合作用显著差异表达基因表达量变化情况。结果表明，高锰胁迫下，叶绿素合成受阻，天线色素捕光过程受影响，PSI结构受损，卡尔文循环过程CO2固定的受体大大减少，这些因素综合导致植物体的光合作用下降。锰胁迫下加硅后，可以增加叶绿素含量、光能利用率和ATP数量，稳定PSI结构，促进CO2的同化，因此减轻了作物锰毒害的程度。更多还原

【Abstract】 This study aimed to explore the physiological and molecular mechanisms of Si-enhanced toleranceto Mn toxicity, and improve rice growth and yield to sustain rice production. The roles of Si inenhancing tolerance to manganese (Mn) toxicity were studied in two rice (Oryza sativa L.) cultivars: i.e.cv. Xinxiangyou640(XXY), a Mn-sensitive cultivar and cv. Zhuliangyou99(ZLY), a Mn-tolerantcultivar. Plants were cultured in nutrient solution containing normal Mn (6.7μM) or high Mn (2.0mM),both with or without Si supply at1.5mM Si. We investigated the effects of Si on plant growth, elementsuptake, transport, distribution, antioxidative defense capacity and photosynthetic parameters of bothcultivars under high Mn stress; Using high-throughput sequencing, we performed a comprehensiveanalysis of the influence of Mn on gene expression of the sensitive rice with or without Si. Using thesignificant enrichment analysis of Gene Ontology and Pathway combined with the analysis ofphysiological indicators, we obtained the key genes related to photosynthesis; Using real-timequantitative PCR technique, we further studied the relative expression levels of the key genes ofinterest.The main results are presented as follows:1. Plant growth was severely inhibited by high Mn stress in cv. XXY, but was enhanced by Sisupply. Mn toxicity symptoms observed in leaves were more severe in the Mn-sensitive cultivar (XXY)than in the Mn-resistant cultivar (ZLY) under high Mn stress. In the high Mn treatment, greater Mnconcentrations in shoots and roots respectively were found in the Mn-tolerant cultivar than in theMn-sensitive cultivar. In cv. XXY, Si-enhanced tolerance resulted from a restriction of Mn transport,whereas Mn uptake was depressed in cv. ZLY.2. Mn concentration in foliage and roots of Mn-tolerant cultivar was much greater than that ofMn-sensitive cultivar at the high Mn level. In cv. XXY, the suppression of Mn resulted from arestriction of K transport, whereas K uptake was depressed in cv. ZLY. Supply with Si significantlyincreased the relative contents of K, Fe and Zn in foliage at the high Mn level compared with theSi-untreated plants in the Mn-sensitive cultivar. Supply with Si significantly increased the relativecontents of K and Zn in foliage at the high Mn level, whereas considerably decreased the relativecontents of Ca and Fe compared with the Si-untreated plants in the Mn-sensitive cultivar. Supply withSi considerably decreased the relative content of K in roots of Mn-sensitive cultivar, but significantlyincreased it in foliage at the high Mn level. This suggests that Si can promote K transport from roots toshoots under high Mn stress. This indicates that Si plays an important role in keeping the relativebalance between elements in roots of Mn-tolerant cultivar.3. For the Mn–sensitive cultivar, high Mn significantly increased the activities of superoxidedismutase (SOD), the concentrations of ascorbate (AsA) and non-protein thiols (NPT), while itsignificantly decreased the activities of catalase (CAT) and ascorbate peroxidase (APX) and theconcentrations of glutathione (GSH), thus leading to accumulation of high levels of malondialdehyde(MDA) in root tissues and destruction of plasma membrane integrity. However, high Mn significantlyincreased the concentrations of GSH and AsA, but significantly decreased the activities of CAT and the concentrations of NPT, thus leading to accumulation of high level of MDA in the Mn-tolerant cultivar(ZLY). The addition of Si significantly decreased the concentrations of MDA and H2O2in Mn-stressedroot tissues, thereby mitigating the damage. Under high Mn stress, application of silicon enhances theantioxidant activities of rice roots, and reduces lipid peroxidation and damage to the integrity of theapical membrane, thereby enhancing rice resistance to manganese toxicity. This alleviative effect ismore pronounced in Mn-sensitive rice (XXY) than in Mn-tolerant rice (ZLY). In cv. XXY, high Mnsignificantly increased SOD, CATand APX activities but decreased NPT and GSH concentrations,leading to accumulation of H2O2and MDA in leaves of rice. The addition of Si significantlycounteracted high Mn-elevated MDA and H2O2concentrations and enhanced plant growth. In cv. ZLY,high Mn considerably raised SOD activities and GSH concentrations in leaves of rice, thus leading torelatively low oxidative damage. Silicon mainly influenced non-enzymatic antioxidants in Mn-sensitiverice cultivars under high Mn stress.4. High Mn stress considerably decreased the content of chlorophyll‘a’, chlorophyll‘b’,carotenoids and Chlorophyll‘a+b’ in cv. XXY, while only carotenoids content was decreased in cv. ZLY.Net photosynthetic rates (Pn) of the two rice cultivars tested were all decreased under high Mn stress.By scanning electron microscopy and transmission electron microscopy, high Mn stress made leafstomata closed and grana lamellae disorder in cv. XXY, while stomatal aperture was slightly decreasedand the chloroplast lamellar structure was not affected in cv. ZLY. By the addition of silicon thecontent of carotenoids and Chlorophyll‘a+b’ were significantly increased in cv. XXY, also thecarotenoid content was significantly increased in cv. ZLY. The addition of Si improved thephotosynthetic efficiency and alleviated the chloroplast ultrastructure under high Mn level in cv. XXY.5. There were about lots of differently expressed genes (The false discovery rate (FDR)≤0.001and|log2ratio(Mn/CK)|≥1) in which1336appeared to be up-regulated and1495appeared to bedown-regulated in rice treated with high level of Mn compared with the normal level of Mn. Under highMn stress, Si addtion induced647up-regulated genes，892down-regulated genes compared with theMn-treated plants. Under the normal Mn level, Si addtion induced1558up-regulated genes among16525genes, and2028down-regulated genes. In the high Mn-treated plants amended with Si,16273genes were expressed, with320genes up-regulated and172genes down-regulated compared with theSi-treated plants. The differentially expressed genes were relating to various transcription factors (TFs),large number of transporters, numerous transferase proteins, etc, involving in the major primary andsecondary metabolisms. Functional analysis showed that these differentially expressed genes wereinvolved in metabolism, ion transport, signal transduction, transcription regulation, and stress responsegenes etc. Manganese resistance mechanism in rice is very complex and is a consequence ofcoordinated expression of a large number of genes.6. Mn-induced inhibition of photosynthesis can be attributed to the suppressed chlorophyllbiosynthesis, light-harvesting process and ATP synthesis, the impaired stability of PSI structure and theimpaired regeneration of the acceptor molecule for CO2fixation of the Calvin cycle. Si apparentlyallows plants to respond to Mn toxicity more efficiently by increasing chlorophyll content, light-use-efficiency and ATP quantity, stabilizing the structure of PSI, and promoting CO2assimilation.Our findings suggest active involvement of Si in Mn detoxification ranging from physiologicalresponses to gene expression.更多还原