【作者】 张书建；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2007， 博士

【摘要】 核黄素可通过生物合成、代谢与功能途径的多个环节,尤其是黄素介导的氧化还原作用,影响植物防卫和生长发育。核黄素是一种新的植物抗病激发子,外源施用核黄素能够诱导植物对多种病害的抗性,启动不同于其它已知的抗病信号通路,其机制仍不清楚。转中华鳖核黄素受体蛋白基因拟南芥体內含有高于野生型水平的核黄素,而且转基因植物也表现增强的抗病反应,但其机制尚不清楚。因此,本博士学位论文着重解析体外喷施核黄素诱导的抗病机制、转核黄素受体基因植物的抗病机制以及内源核黄素含量调控所影响的生理过程和代谢途径,为阐明核黄素参与调控植物生长和防卫反应的机制及与其他激素或非激素信号传导途径的交叉对话提供研究基础。1外源核黄素诱导的引发抗病防卫反应依赖过氧化氢和NPR1基因致坏死型病原菌接种后,植物具备了增强的防卫反应激活能力以应对生物和非生物胁迫,这一过程称为引发（priming）。引发状态（primed state）也可以被有益微生物根部定殖和许多天然或合成物质处理植物后诱导产生。近几年的研究表明,引发可能是植物诱导抗性的重要机制。核黄素处理拟南芥诱导PR-1和PR-2基因的表达,而PAL1诱导表达不显著。核黄素预先处理,再接种丁香假单胞番茄致病变种DC3000后,激发植物体內更强的细胞防卫反应并诱导防卫反应基因更强更快的表达,同时诱发过氧化氢的积累、胼胝质沉积和过敏性细胞死亡。用过氧化氢清除剂完全消弱了植物细胞和分子水平的防卫反应及其对病原菌的抗性。核黄素处理并接种病原菌诱发的细胞防卫反应在水杨酸、乙烯、茉莉酸和脱落酸信号通路的突变体上可以产生,但在npr1突变体中消失。研究结果表明,外施核黄素使植物进入引发状态,接种病原菌时,植物体內积累高浓度的过氧化氢,并激发细胞防卫反应和相关基因的快速且高强度表达以及对病原菌的抗性,这一过程依赖于过氧化氢和NPR1基因,但不依赖于水杨酸、乙烯、茉莉酸和脱落酸信号通路。2转中华鳖核黄素受体基因植物表达谱分析本实验室把中华鳖的核黄素受体基因转入拟南芥,借以调节核黄素含量及对防卫反应和有关生长发育过程的影响。转基因植物体内核黄素含量高于野生型水平,沉默核黄素受体基因后,沉默植物内的核黄素恢复到野生型水平。我们利用拟南芥基因组芯片（ATH1 Affymetrix chip）分析转基因植物表达谱的变化。预杂交实验和质量控制检验显示,表达谱芯片及样品CRNA产物质量较好,表达谱芯片实验成功,数据可靠。应用GCOS（GeneChip（?）Operating Software）软件进行数据分析获得的原始数据,再用RMA（robust multiarray analysis）进行数据预处理后,根据随机方差模型的单因素方差分析对实验组与对照进行比较,以筛选两两分组之间的差异基因。对于差异基因的筛选方法基于FDR（fdlse discovery rate）＜0.1,p＜0.05,Permutation P＜0.05.根据基因在两个分组中的几何平均数的比值倍数决定差异基因的变化方向,我们选取比值≥2的基因为显著上调基因,比值≤0.5的为显著下调基因。结果表明,共有320个基因显著上调,630个基因显著下调。我们随机选取部分基因对芯片结果进行RT-PCR和Real-time PCR验证,结果显示表达结果与芯片数据相吻合。随后,对表达差异显著的基因在拟南芥信息中心进行基因本体（Gene Ontology,GO）分析,按照GO数据库的主子集生物学过程以获取它们确切的GO分类信息。接着,我们又利用代谢途径分析程序MapMan,分析差异表达基因所属的代谢途径,以期了解转基因植物中代谢过程的变化。我们着重列举了次级代谢、物质运输、转录因子、线粒体电子传递、氧化还原调控等代谢过程的变化。同时我们选取了植物防卫反应相关基因、激素信号途径相关基因和蛋白激酶基因这三类基因及其他个别基因进行了简要的分析。结果表明,转基因植物中差异表达的基因涉及很多生理过程、代谢途径、激素及非激素信号途径。尤其令人感兴趣的是,线粒体电子传递链的部分组成基因下调表达以及氧化还原调控过程中一些清除活性氧（reacfive oxygen species,ROS）的酶基因上调表达,这暗示着转基因植株具有较高氧化态。差异表达的基因对我们进一步研究核黄素调控的代谢过程和防卫反应机制提供了线索。3转核黄素受体基因植物抗病机制解析基因表达谱的数据表明,转基因植株线粒体电子传递链的部分基因下调表达,而参与抗氧化体系中的有些基因上调表达,这暗示转基因植物可能具有较高氧化态。通过活性氧原位组织染色和叶片过氧化氢含量测定,发现转基因植株中的活性氧水平比野生型和沉默植株均高;喷施ROS供体百草枯（Paraquat）后,发现转基因植物对百草枯表现较敏感,表明转基因植物体内的氧化还原平衡被打破（具有较高的氧化态）。为了解析转基因植物抗病性与氧化还原平衡改变的关系,以及氧化平衡改变是否受体内核黄素含量升高的影响,我们通过注射过氧化氢清除剂Catalase及核黄素,并结合DC3000接种,检测不同处理中植物的氧爆发、胼胝质积累和细胞坏死这三种细胞防卫反应的产生情况,并对植物抗病性和菌落生长数量进行观测。结果表明,转基因植物体内的氧化还原改变对引发植物抗病防卫反应是必需的,而且植物体内核黄素含量的变化是引起植物体内氧化还原状态改变的原因。我们又利用本实验室产生的转基因植物与基本防卫信号通路突变体npr1-1、etr1-1、jar1-1和abi1-1杂交或转基因获得的双突变体,解析内源核黄素介导的抗病性所依赖的信号通路。结果发现,双突变体均具有较高的氧化态,但接种后,npr1-1中没有显著的活性氧积累,也没有出现胼胝质积累和细胞坏死,而其他双突变体均产生增强的细胞防卫反应,这表明內源核黄素引发抗病防卫反应需要NPR1,但不依赖于乙烯、茉莉酸和脱落酸信号通路。本章的研究结果表明,转基因植物内高水平的核黄素引起植物体内ROS积累及较高的氧化态,使得植物进入引发状态,当接种病原物时,转基因植物能够表现快速增强的细胞和分子防卫反应,从而引发对病原菌的增强抗性,而且引发防卫反应过程需要NPR1的参与。4核黄素引发抗病防卫信号传导机制研究转核黄素受体基因拟南芥RIRA11中氧化还原平衡状态的改变和ROS的积累在植物抗病性中是必需的。为了证明外施核黄素是否导致体內氧化还原态的改变并且这一改变在引发抗病防卫反应中的作用,我们检测了核黄素处理拟南芥5天内的氧化还原动态变化。结果发现,在0小时至5天內,外源核黄素处理植物激发体内的ROS积累且在2小时的达到高峰值,5天后恢复到原来水平;我们通过DAB染色、过氧化氢含量测定、百草枯（PQ）敏感性测定,发现核黄素处理拟南芥后导致氧化还原态的改变,致使体内的氧化态升高。核黄素喷施植物后再注射Catalase,体內的氧化态降低;而且5天后接种DC3000后,植物体内没有出现细胞防卫反应,其抗病性与喷施核黄素植株相比显著降低,这表明,核黄素处理拟南芥导致还原状态的改变对核黄素诱导的抗病性是必需的。根据转基因植物表达谱信息,我们选取线粒体电子传递链复合Ⅰ上的两个基因,利用Real-time PCR技术检测核黄素处理过程中的这两个基因的表达动态变化,发现两个基因的表达明显受抑制且在6小时表达量最低,这表明核黄素可能通过抑制线粒体电子传递链而导致ROS积累并打破胞内正常的氧化还原平衡,但也不能排除是线粒体行使ROS动态平衡初始变化的第一个转播站并且将信号放大的结果。核黄素处理导致体內氧化还原平衡的改变,使植物对外来刺激更加敏感并进入引发状态,当病原菌侵染时,植物能够表现快速增强的细胞防卫反应,这可能是核黄素引发防卫反应的细胞机制。5防卫反应引发激发子引发植物抗病的生产应用研究表明,水稻细菌性条斑病菌（Xanthomonas oryzae pv.oryzicola）的HpaG_xooc功能片段HpaG_10-42在引发水稻防卫反应中的活性最强。通过浓度/植物处理时期组合试验发现,在水稻苗期喷施一次和大田移栽后喷施三次浓度为6μg/ml的HpaG_10-42后,同对照处理相比,处理的籼稻和粳稻上水稻白叶枯病（X.oryzae pv.oryzae）和穗颈瘟（Magnaporthe grisea）的病情指数均显著降低;而且,该浓度的蛋白在水稻苗期,返青期、分蘖后期、始穗期单独处理一次,均可高效引发水稻在该时期的抗病性;与常规农事操作相比,水稻生长的四个时期施用6μg/ml的蛋白处理后,两种病害的病情指数显著降低,在9个测试水稻品种上的诱导抗病效果也很显著。结果表明,该激发子可以高效引发大田植物的抗病性,为其在田间作物上的大规模应用提供了基础。6总结本研究利用外源喷施核黄素及內源核黄素调控两种手段,对核黄素引发植物防卫反应的信号传导机制进行了初步解析,同时利用基因表达谱技术,分析了內源核黄素含量调控所影响的生理过程和代谢途径,还对防卫反应激发子HpaG_10-42引发田间水稻抗病性进行了研究.研究发现:第一,引发是核黄素诱导抗病防卫反应的细胞机制,引发病原菌刺激时产生的细胞和分子防卫反应依赖于NPR1,但不依赖于SA.第二,核黄素通过引起植物体內氧化还原平衡改变而使植物进入引发状态,但氧化还原状态的改变不依赖于NPR1。第三,内源核黄素影响了次级代谢、物质运输、转录调控等多种生理代谢过程以及激素、非激素信号传导信号通路,这为解析核黄素调控植物生长和防卫的机制及与激素、非激素信号通路的交叉对话提供了有益的线索。第四,核黄素可能通过作用于线粒体电子传递链而激发活性氧积累,并因此改变了植物体内的氧化还原平衡。第五,HpaG_10-42可以高效引发田间水稻的抗病性。更多还原

【Abstract】 Riboflavin （vitamin B₂） biosynthetic and functional pathways affect plant growth, development, and defensive responses by multiple mechanisms. Riboflavin is involved in anti-oxidation and peroxidation. Both processes affect the production of reactive oxygen species （ROS） in oxidative burst. Our previous studies suggest that riboflavin is a novel elicitor of systemic acquired resistance （SAR） in plants and activates a distinct defense signaling pathway from several known ones, but the underlying molecular mechanism remains unclear. Endogenous-modulated riboflavin also triggers plant disease resistance in Arabidopsis expressing the riboflavin receptor protein encoding gene of soft-shelled turtle （Trionyx sinensis japonicus）. However, the mechanism of disease resistance in transgenetic plants is still unknown. Studies in this Ph.D thesis aim at determination of signaling pathways and components in plant responses to exogenous and endogenous riboflavin, as well as the physiological processes and metabolic pathways, which are affected by endogenous riboflavin with higher levels. The results here will provide basic clues to explore the riboflavin mediated plant defense and growth pathways and crosstalk with hormonal and non-hormonal signaling pathways.1. Riboflavin-induced priming for pathogen responses in Arabidopsis requires hydrogen peroxide and NPR1Besides having a pivotal biological function as a component of coenzymes, riboflavin appears as an elicitor of systemic acquired resistance （SAR） in plants, but the underlying molecular mechanism remains unclear. SAR is associated with the ability to induce cellular defense responses more rapidly and to a greater degree than in non-induced plants, a process called "priming." Here we report that the application of riboflavin to Arabidopsis thaliana induces priming of defense responses toward infection with virulent Psedumonas syringae pv. tomato DC3000 （Pst）. Induced plant resistance to the bacterial pathogen was mechanistically connected with the expression of defense response genes and cellular defensive events, including H₂O₂ burst, HCD, and callose deposition. Riboflavin treatment and inoculation of plants with Pst were neither active but both synergized to induce priming events. The defense-priming process needed NPR1 （essential regulator of systemic acquired resistance） and maintenance of H₂O₂ burst but was independent of salicylic acid （SA）, jasmonic acid （JA）, ethylene （ET）, and abscisic acid （ABA）. Our results suggest that the role of riboflavin in priming defenses is subject to signaling process distinct from the known pathways of hormone signal transduction.2. Genome-wide transcriptional profiling and metabolism analysis of transgenetic Arabidopsis plants expressing an oviparal riboflavin receptor geneWe have cloned the riboflavin receptor protein encoding gene RIR from soft-shelled turtle and transferred it into Arabidopsis and got several homozygous lines. We also constructed RIR-silencing vector, which was able to silence RIR in RIRA11 and got SiRB11, for further study on the roles of riboflavin in plant growth and defense regulation network. We used the Arabidopsis ATH1 Affymetrix chip to analyze the global transcriptional profiling. Based on the pre-hybridization and quality control results, our microarray experiments were successful and data we got was credible. Then we used the GCOS software to analyze the original data and pre-treated with robust multiarray analysis （RMA）, and we got the differentially regulated genes between Col-0 and RIRA11 plants. Our choosing criterion was based on the FDR < 0.1, p < 0.05, Permutation P<0.05. Further data analysis was carried out by selecting for the genes exhibiting >2-fold differences. There were 320 genes found to be significantly up-regulated and 630 down-regulated. We then performed RT-PCR and Real-time PCR on a select group of genes and compared the results with those obtained from the array. Although the ratios from the PCR data are not the same as those in the chip, the correlation between the two groups of data are very good. Subsequently, Gene ontology analysis was carried out on the Arabidopsis Biological Research Center. To analyze the metabolism processes changed in RIRA11, we used the MapMan software to visualize the metabolic pathway assigned from the significantly regulated genes. The results suggested that there are some genes involved in secondary metabolism, transport, transcription and mitochondrial electron transport, redox homeostasis. Meanwhile, some genes involved in plant defense response, hormonal signaling pathway and protein kinase were analyzed in the text. To our great interesting, some genes in the mitochondrial electron transport chain were depressed but some ROS detoxification genes were up-regulated, suggesting that there is escalated oxidative state in RIRA11.These results provide us the insights into the effect of riboflavin on plants and explore a list of candidate genes that may act to regulate plant defense and development, hormonal and non-hormonal signaling pathways.3. Modulated riboflavin may activate the mitochondrial pathway of ROS signaling and primes pathogen defense response in transgenetic Arabidopsis plantsCompared with Col-0 and SiRB11 plants, RIRA11 contains higher endogenous riboflavin than that in Col-0 and SiRB11. Moreover, RIRA11 exhibits alleviative symptoms and reduced bacterial propagation after DC3000 infection, but the underlying mechanisms are unclear. Based on the microarray data, we deduced that the inhibition of the mitochondrial electron transport chain may result in the escalated oxidative state in RIRA11.Then we detected the ROS levels by DAB and DCFH-DA staining and H₂O₂ content measuring, as well as the sensitivity of the plants to the extra oxidative stresses, generated by Paraquat （PQ）, the results suggested that there was escalated oxidative state in RIRA11. Combined with catalase, riboflavin, and DC3000 inoculation, we analyzed the cellular and molecular defense responses. Thus, our results suggested that riboflavin may inactive the mitochondrial electron transport chain resulting in the escalated oxidative state and shifted the plants to the primed state, which plays a pivotal role in augmented defense response in RIRA11 when infected by DC3000. Endogenous riboflavin-induced priming is the mechanism of disease resistance in RIRA11. Then we analyzed the signaling pathways required in endogenous riboflavin-induced priming using the mutants npr1-1、etr1-1、jar1-、and abi1-1, each of which has the RIR gene by the methods of gene transferring and crossing with RIRA11. The results show that endogenous riboflavin-induced priming requires NPR1, but is independent of ET, JA and ABA signaling pathways. 4. Priming of pathogen defense signal transduction by riboflavin in ArabidopsisOur previously studies suggest that the disturbed redox homeostasis plays a pivotal role in endogenous riboflavin-mediated priming. To explore whether it also functions in the exogenous riboflavin-induced priming, we detected the ROS accumulation in plants from the beginning of riboflavin sprayed till 5 dpt. The results showed that riboflavin trigger ROS accumulation in plants during the period tested and the levels of ROS reach the highest at 2 hpt, but restore to the original level at 5dpt. Meanwhile, the riboflavin-treated plants are more sensitive to the extra oxidative stresses, generated by Paraquat, suggesting that the plants have escalated oxidative state. Combined with catalase and DC3000 inoculation, we analyzed the cellular and molecular defense responses. Catalse, which infiltrated at 30min after riboflavin treatment, nullifies the escalated oxidative state and the augmented ROS accumulation when infected by Pst. Moreover, the disease resistance is also abolished in riboflavin pretreated but catalase treated plants, which suggest that the disrupted redox homeostasis is required in riboflavin-induced priming. However, the disrupted redox homeostasis is independent of NPR1. Based on the microarray data, we then analyzed the two genes involved in mitochondrial electron transport with Real-time PCR technology. Both of the genes were depressed during the course of riboflavin treatment. Therefore, the results here suggest that disrupted redox homeostasis plays a pivotal role in riboflavin-induced priming and may connect with functional inhabitation of mitochondria.5. Practical use of defense priming by harpin in riceHpaG_10-42, which generated from truncating the Xanthomonas oryzae pv. oryzicola HpaG_Xooc protein, is active in priming defense responses in rice. When 6μg/ml HpaG_10-42 was applied once to seedling nursery and three times to transplanting fields of indica and japonica rice varieties, bacterial blight caused by X. oryzae pv. oryzae and panicle blast caused by Magnaporthe grisea were both less severe, relative to controls. Meanwhile, when HpaG_10-42 applied once at the four stages of rice growth, the effects on decreasing disease severities were also significant. HpaG_10-42 treatment was similar to local agronomic measures, including use of chemicals, in decreasing disease severities of 9 rice varieties. Hence, results here provide an example for effective use of beneficial pathogen defense priming agents to control diseases in the staple food crop. Conclusive remarksResults described above have provided us with further understanding on the mechanisms of riboflavin-mediated pathogen defense in plants. Firstly, priming is an important cellular mechanism in SAR by riboflavin and requires hydrogen peroxide and intact NPR1. Secondly, disrupted redox homeostasis induced by riboflavin, which is independent of NPR1, plays a pivotal role in shifting the plants to primed state. Thirdly, Endogenous riboflavin dose affect many physiological processes, such as secondary metabolism, transport, transcription, redox metabolism, as well as hormonal and non-hormonal signaling pathways, which provide us the cues to explore the mechanisms of riboflavin mediated plant defense and development, the crosstalk between riboflavin signaling with hormonal and non-hormonal signaling pathways. Fourthly, riboflavin may inactivate mitochondrial electron transport, resulting in ROS accumulation and disrupted redox homeostasis in plants. Fifthly, HpaG_10-42, a selected fragment of HpaG_Xooc, could prime disease resistance of rice effectively in fields.更多还原