【作者】 孙枫；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2009， 博士

【摘要】 核黄素可通过生物合成、代谢等多个环节,尤其是黄素介导的氧化还原过程,影响植物防卫和生长发育。异源表达中华鳖核黄素受体蛋白转基因拟南芥体内内源核黄素含量高于野生型,同时转基因植物也表现增强的抗病反应,但其机制不清楚。因此,本博士论文着重解析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提供研究基础。生物胁迫与非生物胁迫影响植物的正常生长与发育,植物对生物胁迫和非生物胁迫的响应称为植物防卫反应。本文的研究目标是鉴定参与调控植物防卫反应中新的因子。1.核黄素受体蛋白调控拟南芥过氧化氢(H2O2)信号及对病原细菌的防卫反应核黄素(维生素B2)在生物体内以黄素单核苷酸(flavin mononucleotide, FMN)和黄素腺嘌呤二核苷酸(flavin adenine dinucleotide, FAD)的形式作为辅基,与黄素蛋白(flavoprotein)或金属黄素蛋白(metalloflavoprotein)组成复合酶,参与生物体内以氧化-还原反应为特征的五十多个生理生化过程。我们的研究表明,核黄素在拟南芥体内引发H2O2信号,调控对病原细菌的抗性。为了调控植物体内核黄素的含量,我们通过转基因手段,在拟南芥过量表达中华鳖(Trionyx sinensis japonicus)的核黄素受体(riboflavin receptor)基因,得到转基因拟南芥品系RIRA11 (riboflavin receptor expression Arabidopsis)。拟南芥转基因植株(RIRA11)与野生型相比,在营养生长期间,体内核黄素含量增加、黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)含量降低,增强对病原细菌的抗性。转录组学分析表明,转基因拟南芥(RIRA11)中涉及多个细胞进程的950个基因的表达发生了改变。其中线粒体电子传递有关的13个基因表达显著下降,导致植株体内H2O2含量上升；RT-PCR和real-time PCR分析表明,此13个基因表达水平与芯片数据一致。转基因植株中核黄素受体基因沉默后,其黄素含量和抗病表型回复到野生型的表型。我们的研究表明,通过调控增加植物体内黄素含量,抑制线粒体电子传递链基因表达,促进H2O2的产生,这些细胞生理代谢的变化,最后导致植物体内H2O2含量增加,增强植物抗病性。2.外源核黄素受体蛋白调控拟南芥开花转型核黄素可通过生物合成、代谢等多个环节,尤其是黄素介导的氧化还原过程,影响植物防卫和生长发育。我们把中华鳖的核黄素受体基因转入拟南芥中,借以调控植物体内的黄素含量,研究黄素对于植物防卫与生长发育的调控作用。异源表达核黄素受体蛋白拟南芥品系RIRA11 (riboflavin receptor expression Arabidopsis)体内总的黄素含量增加,线粒体电子传递链主要基因转录水平下降,细胞氧化还原水平上升,开花提前。用RNAi方法沉默转基因植株中的外源核黄素受体基因后,沉默植物恢复到野生型的表型。与野生型或基因沉默植株相比,过量表达核黄素受体拟南芥(RIRA11)开花转型相关基因表达增强,顶端分生组织中开花标志基因AP1表达增强,开花提前。外源施用H202能诱导植物开花,但依赖于植株体内H2O2含量。我们的研究表明,过量表达核黄素受体拟南芥开花提前,H2O2在植物开花转型过程中起重要作用。3.昼夜节律调控拟南芥核黄素信号传导反应植物昼夜节律钟在调控植物生理进程、对环境的响应和发育中起及其重要的作用。与此相似,核黄素信号途径广泛参与调控植物生长与发育。核黄素是黄素单核苷酸(FMN)和黄素腺嘌呤二核苷酸(FAD)的前体。FMN, FAD是生物体黄素酶辅基,参与植物体内线粒体电子传递、光合作用、脂肪酸氧化、维生素B6、维生素B12、叶酸代谢等多个生化过程。本研究中,我们的研究结果表明,植物昼夜节律调控核黄素信号传导途径。我们通过异源表达中华鳖核黄素受体蛋白的拟南芥,来调控植物内源核黄素含量。异源表达核黄素受体拟南芥和野生型相似,内源核黄素含量在24 h周期内呈节律波动,但异源表达核黄素受体拟南芥植株内源核黄素含量显著高于野生型植株。RT-PCR分析结果表明,植物内源核黄素含量升高影响了昼夜节律钟输入途径和中心震荡器有关基因的表达模式。另外,外源核黄素诱导或抑制基因的表达模式也受昼夜节律调控。外源核黄素施用植物能诱导植物产生防卫反应,该过程也受昼夜节律调控。因此,我们的研究表明,植物昼夜节律钟调控核黄素信号传导反应。4.拟南芥RIR类似蛋白(AtRIR)在非生物胁迫中的功能分析渗透压、氧和盐等非生物胁迫影响植物的正常生长和发育。在非生物胁迫环境下植物体内产生、积累的活性氧对植物造成了氧胁迫。AtRIR (At5G27830)是一个功能未知蛋白,序列推测可能是核黄素受体(RIR)类似蛋白。RT-PCR和AtRIR启动子控制的GUS活性检测表明,AtRIR在植物所有主要器官包括根、茎、叶、花中表达。通过根长测定表明,与野生型拟南芥相比,AtRIR T-DNA插入突变体对渗透压、氧和盐胁迫更敏感；过量表达AtRIR转基因拟南芥(AtRIR-OE)增强对渗透压、氧胁迫的抗性。转基因植株在盐、氧、渗透压胁迫和脱落酸处理下,与野生型相比,体内活性氧的含量降低,氧胁迫反应基因(APX1和FSD1)表达减弱；但非生物胁迫反应基因(COR47, RD29B, RD29A)表达增强。这些研究表明,AtRIR通过调控植物体内活性氧的含量,介导植物对非生物胁迫的抗性。5.拟南芥RAP2.6L转录因子突变体增强对Pseudomonas syringae的抗性植物受病原菌侵染或用病原物激发子处理植物时,激活的植物防卫反应中涉及大量基因的转录调控。目前许多研究表明,ERF类转录因子在植物受病原物侵染后,通过调控寄主植物基因的表达参与植物防卫反应。然而,特异的ERF转录因子在植物防卫中的作用还知之甚少。我们分析了RAP2.6L转录因子在拟南芥对Pst DC3000基本防卫中的作用。RAP2.6L偶联GFP的融合蛋白在洋葱表皮细胞中定位于细胞核。RAP2.6L受环境胁迫(水杨酸、茉莉酸、黄酮)的诱导表达。通过分析RAP2.6L在基本防卫信号通路突变abi1-1, jar1-1, ein2-1和npr1-1上的受病原细菌诱导表达模式,表明水杨酸和茉莉酸信号通路正调控RAP2.6L表达,而乙烯信号通路抑制其表达。RAP2.6L T-DNA插入突变体rap2.6L接种Pst DC3000后,与野生型相比,3d后叶片细菌数量减少约10倍,病害症状显著减轻,水杨酸调控的PR1, PR2基因表达增强。基于rap2.6L突变体的分析表明,RAP2.6L负调控植物对Pst DC3000的基本防卫反应。RAP2.6L具有转录激活功能,在植物防卫反应中调控了许多下游因子,参与植物对病原细菌的抗性。总结本研究利用核黄素受体蛋白转基因拟南芥为主要研究材料,对内源核黄素变化对于植物防卫反应信号传导机制及其开花转型进行了解析。本文还对鉴定了调控拟南芥防卫反应的两个因子。研究发现：第一,核黄素受体转基因拟南芥体内核黄素含量升高,转录组学分析影响了次级代谢、物质运输、转录调控等多种生理代谢过程。核黄素通过抑制线粒体电子传递链基因表达,致使植株内源过氧化氢水平升高。第二,过氧化氢是拟南芥防卫的重要信号分子,抑制植物过氧化氢含量同时也削弱了植物的防卫能力。过氧化氢介导核黄素受体转基因拟南芥对病原细菌的抗性。第三,核黄素受体蛋白转基因拟南芥开花提前,过氧化氢在此过程中起重要作用,体外施用过氧化氢能诱导拟南芥提前开花。第四,野生型拟南芥叶片中核黄素含量受光周期调控,昼夜节律调控核黄素信号传导。第五,鉴定了AtRIR在拟南芥抗非生物胁迫中的作用。第六,拟南芥RAP2.6L T-DNA突变体增强对病原细菌的抗性。更多还原

【Abstract】 Riboflavin (vitamin B2) biosynthetic and functional pathways affect plant growth, development, and defensive responses by multiple mechanisms. Endogenous-modulated riboflavin also triggers plant disease resistance in Arabidopsis expressing the riboflavin receptor protein encoding gene from soft-shelled turtle(Trionyx sinensis japonicus). However, the mechanism of disease resistance in transgenic plants is still unknown. Studies in this Ph.D thesis aim at determination of signaling pathways and components in plant responses to endogenous riboflavin, as well as the physiological processes and metabolic pathways, which are affected by endogenous riboflavin with higher levels. Biotic stress and abiotic stresses such as osmotic, oxidative, and salinity affect normal growth and development in plants. Our objective is identifying new genes involved in biotic stress and abiotic stress.1. Riboflavin modulation of hydrogen peroxide signaling that regulates Arabidopsis resistance to a bacterial pathogenRiboflavin (RIB) mediates multiple bioprocesses through flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Here we show that RIB plays an initial role in hydrogen peroxide (H2O2) signaling that regulates pathogen defense in Arabidopsis {Arabidopsis thaliana). We transformed Arabidopsis with TsRIR, a RIB receptor gene from soft-shelled turtle (Trionyx sinensis japonicus), and characterized the RIR-expressing Arabidopsis (RIRA) line RIRA11. Resistance to a bacterial pathogen was promoted coincidently with increased contents of RIB, decreased contents of FMN and FAD in RIRA11 compared to wild-type (Col-0) plant during vegetative growth. Transcriptomics analysis revealed that 950 genes assorted in various cellular processes were altered in RIRA11. Significantly down-regulated profiles included 13 genes critically functional in the mitochondrial electron transport chain (METC), indicating a facilitated production of H2O2 via METC. The transcriptomics information was confirmed by RT-PCR and Real-Time PCR. Silencing the riboflavin receptor protein (RIR) in transgenic plant (RIRA) restored WT characters. Our results uncover a novel signaling contrail:the modulation of RIB reduces FMN and FAD contents and thus depresses METC flux but promotes peroxidation; these changes in METC result in H2O2 burst, which in turn stimulates defense response.2. A riboflavin receptor protein modulates the floral transition in ArabidopsisPlants flower in response to specific signals. Hydrogen peroxide from flavin-dependent redox mediates many bioprocesses but is unclear for effects on the floral transition. In early-flowering Arabidopsis plants expressing turtle riboflavin receptor protein, the protein localizes to chloroplasts, alters flavin form conversion, and represses the mitochondrial electron transport chain. Cellular hydrogen peroxide is thus elevated to promote the function of the FT protein in activating API, an integrator of the floral transition pathways. Comparing wild-type or silencing Arabidopsis, the transcription levels of genes involved in flowering were increased in over-expression riboflavin receptor protein Arabidopsis (RIRA). RIRA plants show early flowering phenotype. Our results demonstrate a molecular basis by which H2O2 promotes flower development.3. The circadian clock regulates riboflavin responses in ArabidopsisThe circadian clock plays a pervasive role in the temporal regulation of plant physiology, environmental responsiveness, and development. In contrast, the riboflavin play a similarly far-reaching role in the spatial regulation of plant growth and development. Here we present work that the circadian clock regulates riboflavin signal transduction. Using transgenic Arabidopsis plants (RIRA) which ectopic express riboflavin receptor protein from turtle (TsRIR), we found that endogenous riboflavin levels showed circadian rhythms in both wild-type and RIRA. RIRA plants with high concentration of endogenous riboflavin affected the circadian input and central genes expression pattern. By RT-PCR assay, we verified that riboflavin-mediate transcriptional response are circadian regulated. Exogenous riboflavin can induce priming of defense toward infection by Pseudomonas syringae pv. tomato DC3000 (Pst), but the clock controls plant sensitivity to applied riboflavin. Thus the circadian clock regulates some riboflavin response. 4. Functional characterization of Arabidopsis RIR-like protein (AtRIR) in abiotic stressesAbiotic stresses such as osmotic, oxidative, and salinity affect normal growth and development in plants. The production and accumulation of reactive oxygen species (ROS) cause oxidative stress under these abiotic conditions. AtRIR (At5G27830) was identified as a unknown function protein that contain no previously defined domains or motifs, a putative riboflavin receptor protein (RIR) like protein. AtRIR transcripts are found in all major organs including root, leaf and flower by RT-PCR and AtRIR promoter controlled GUS activity. The AtRIR T-DNA insertion mutant (Atrir) showed a higher sensitivity to osmotic, oxidative and salinity stress than the wild-type (Col-0), as revealed by root growth length. Over expression of AtRIR protein in Arabidopsis (AtRIR-OE) was found to enhance of plants to osmotic, oxidative stress. AtRIR-OE reduces the content of ROS and transcripts of oxidative responsive genes (APX1, FSD1) under salt, oxidant, osmotic and abscisic acid treatment. The induced expression of stress-responsive genes (COR47, RD29B, RD29A) was more sensitive to abiotic stress and exogenous ABA in AtRIR-OE than in the wild-type. This indicates that AtRIR modulates the abiotic stress response via the regulation of oxidative stress response.5. Mutant in Arabidopsis RAP2.6L transcription factor enhancing plant defense against Pseudomonas syringaeA common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant ERF transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific ERF DNA-binding transcription factors in plant defense. We analyzed the role of the RAP2.6L transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. RAP2.6L translational fusion with green fluorescent protein is localized to the nucleus. RAP2.6L expression is responsive to general environmental stress. Analysis of Pst DC3000-induced RAP2.6L in the defense signaling mutants abi1-1,jar1-1, ein2-1 and npr1-1 further indicated that this gene is positively regulated by the salicylic acid (SA) and jasmonic acid (JA) signaling pathway and negatively regulated by ethylene signaling pathway. T-DNA insertion mutants for RAP2.6L reduced growth of Pst DC3000 and displayed reduced disease symptom severity as compared to wild-type plants. The rap2.6L mutant plants also displayed increased expression of the SA-regulated PR1 and PR2 genes after the pathogen infection. Based on analysis of T-DNA insertion mutants, stress-induced RAP2.6L functions as a negative regulator of SA-mediated defense responses to P. syringae. RAP2.6L was a transcriptional activator and was able to activate the expression of genes involved in plant defense.Conclusive remarksData obtained from studies in RIR (riboflavin receptor protein) transgenic Arabidopsis have provided us the further understanding on the mechanisms of riboflavin mediated pathogen defense and floral transition in Arabidopsis. Firstly, RIR transgenic Arabidopsis increased riboflavin content in leaves affecting many physiological processes, which provide us the cues to explore the mechanisms of riboflavin mediated plant defense and development. Secondly, the modulation of RIB reduces FMN and FAD contents and thus depresses METC flux, these changes in METC result in H2O2 burst, which in turn stimulates defense response. Thirdly, cellular hydrogen peroxide is elevated to promote flowering in RIR transgenic Arabidopsis and the circadian clock regulates some riboflavin response in wild type Arabidopsis. Fourthly, we have identified Arabidopsis AtRIR function in abiotic stresses. Fifthly, we have identified a new transcription factor which regulates plane defense in Arabidopsis.更多还原