【作者】 徐凡；

【导师】 王宁宁；

【作者基本信息】 南开大学 ， 植物学， 2012， 博士

【摘要】 叶片衰老是受遗传程序严格控制的、植物个体发育过程中的一个必经阶段，与其它发育过程一样，是由特殊发育信号通过一定的信号传导路径来启动和控制的。植物LRR型类受体蛋白激酶（LRR-RLKs）已被证明参与多种生命活动，并在其中发挥着重要功能。我们实验室前期研究发现，一个大豆的LRR-RLK，GmSARK，具有双底物特异性，它可以在体外自磷酸化丝/苏氨酸残基和酪氨酸残基；同时发现GmSARK参与大豆叶片衰老的调控，利用RNA干扰技术敲减GmSARK表达可以明显延缓转基因大豆衰老，而利用CaMV35S启动子驱动GmSARK组成型过表达则导致转基因大豆、番茄和拟南芥早衰致死；由于GmSARK-RNAi转基因大豆的花发育异常，具有一个细弯的柱头和不开放的花瓣而导致不育，所以不能使用上述两种转基因材料对GmSARK的功能进行具体分析。为了进一步研究GmSARK的功能及其参与衰老信号调控的分子机制，本文在以前研究的基础上，以GVG系统可诱导型启动子（诱导剂为地塞米松dexamethasone，DEX）控制GmSARK表达的转基因拟南芥为实验材料，对GmSARK所介导的叶片衰老尤其是几种不同的植物激素在其中的调控作用进行了更为深入的分析。研究发现，外源诱导GmSARK过表达可以导致转基因拟南芥幼苗产生早衰、弯曲根表型；导致成熟转基因拟南芥叶片衰老加速、产生与组成型乙烯超响应突变体类似花等表型；并导致一些衰老关键转录因子AtNAP、NAC1、NAC2和WRKY6等的表达量上升。在叶片衰老过程中，叶绿体结构与功能的破坏和叶绿素含量的下降是该过程的标志性事件；通过透射电镜观察发现，外源诱导GmSARK过表达可以导致转基因拟南芥叶绿体结构严重破坏，造成其内部简单、类囊体数目明显减少并积累大量淀粉颗粒；利用启动子-报告基因β-葡萄糖醛酸酶（β-glucuronidase，GUS）系统，对三种叶绿体功能缺陷型突变体背景下GmSARK-GUS转基因拟南芥进行分析，发现叶绿体功能缺陷又反馈促进GmSARK-GUS的活性，形成一个滚动环使叶片衰老进入不可逆转的阶段。利用基因芯片技术进行分析，发现在过表达GmSARK的转基因拟南芥中，有大量激素相关基因的表达发生变化，其中变化最明显的是生长素和乙烯。我们研究了细胞分裂素、生长素和乙烯与GmSARK之间发挥作用的关系。外源施加细胞分裂素可以有效抑制GmSARK-GUS转基因拟南芥中GmSARK启动子活性，外源施加生长素和乙烯前体ACC都可以促进GmSARK-GUS转基因拟南芥中GmSARK启动子活性；另外，外源诱导GmSARK过表达可以有效降低细胞分裂素合成和信号响应相关基因的表达，可以促进生长素和乙烯的合成和响应相关基因的表达。同时，过表达GmSARK导致转基因拟南芥对外源生长素的敏感性增强；通过检测人工合成的生长素响应元件DR5的表达情况，发现过表达GmSARK不仅改变根中生长素的浓度，还改变了其分布情况。通过外源激素处理与药物抑制剂结合实验发现，虽然外源细胞分裂素能够强烈抑制GmSARK启动子的活性，但只能部分恢复由GmSARK过表达所造成的幼苗早衰表型，而外源施加生长素作用抑制剂PCIB和乙烯合成抑制剂AVG能够有效恢复由GmSARK过表达造成的早衰表型；PCIB的衰老恢复作用可以被外源ACC所逆转，而AVG的衰老恢复作用不能被外源IAA逆转；同时，我们发现在生长素输入载体突变体aux1-7和乙烯不敏感突变体ein2-1中过表达GmSARK不仅不能引起转基因拟南芥早衰，还可以恢复由GmSARK过表达造成的花发育异常。以上结果表明，生长素和乙烯直接参与GmSARK介导的叶片衰老信号，并在其中发挥十分重要的作用，尤其是生长素促进的乙烯合成增加和信号响应增强是导致这个过程中衰老事件发生和衰老过程加速的关键环节。我们据此提出GmSARK和GmSARK类基因参与叶片衰老调控的模型，并对细胞分裂素、生长素和乙烯三种激素在GmSARK所介导的叶片衰老过程中的协同作用进行了分析和讨论。我们首次在拟南芥中分离鉴定了GmSARK的同功能基因AtSARK。实验室同期生化实验证明，AtSARK也是一个新的、定位在质膜的、具有双底物特异性的LRR-RLK。定量RT-PCR分析发现，无论是在拟南芥自然衰老还是由GmSARK过表达造成的早衰过程中，AtSARK的表达量都显著上升。通过对AtSARK T-DNA插入突变体和过表达AtSARK转基因拟南芥系统的表型分析发现，敲减AtSARK表达可以有效延缓叶片衰老，而外源诱导AtSARK过表达则可以导致转基因拟南芥早衰并产生异常的、与过表达GmSARK类似的花；同时外源抑制剂处理实验也表明生长素和乙烯直接参与由AtSARK介导的衰老信号通路。因此，我们认为由SARKs介导的衰老信号通路在高等植物中普遍存在。最后，我们又从拟南芥中分离和鉴定了一个参与叶片衰老负调控的蛋白磷酸酶编码基因，我们将其命名为SSPP。定量RT-PCR分析发现，无论是在拟南芥自然衰老还是由SARKs过表达造成的早衰过程中，SSPP的表达都显著下降。对组成型过表达SSPP的转基因拟南芥进行表型分析，发现其叶片叶绿素含量升高，具有晚花、晚衰、生活史延长等表型；令人兴奋的是，组成型过表达SSPP可以有效逆转由AtSARK过表达造成的转基因拟南芥早衰。此外，我们还发现过表达SSPP可以导致转基因拟南芥的抗干旱能力和对高温敏感性增强。综上所述，本课题研究了大豆叶片衰老相关的LRR-RLK基因GmSARK及其在拟南芥中同功能基因AtSARK参与叶片衰老调控的分子机制，结合叶片衰老负调控因子、功能未知蛋白磷酸酶编码基因SSPP的初步研究，对揭示高等植物叶片衰老的分子机制和该过程中的信号转导机制具有重要的理论意义。更多还原

【Abstract】 Leaf senescence constitutes the final stage of leaf development and is afine-tuned process regulated by interplays of multiple signaling pathways. Like otherdevelopmental processes, the onset and process of leaf senescence are influenced byspecific developmental signals.Our lab has previously shown that a soybean LRR-receptor-like protein kinase(LRR-RLK) GmSARK, positively regulate soybean leaf senescence. Biochemicalstudy revealed that GmSARK is a dual-specificity protein kinase, which couldautophosphorylate on both serine/threonine and tyrosine residues in vitro. RNAinterference (RNAi)-mediated knockdown of GmSARK expression in soybeandramatically delayed senescence, while the CaMV35S-GmSARK transgenic soybean,tomato, and Arabidopsis all exhibited a stepped-up progression of leaf senescence,leading to premature death. In the GmSARK-RNAi transgenic soybean, the flowerpetals never opened, and the stigma had a curved shape, so that the plants could notpollinate and were lethal. Since no stable transgenic soybean lines could bemaintained, I took advantage of a glucocorticoid-inducible (GVG system, the induceris dexamethasone, DEX) to ectopically express GmSARK in the model plantArabidopsis. I then analyzed GmSARK-mediated leaf senescence extensively, with afocus on how several phytohormones are involved in this process.It was found that exogenous inducible overexpression of GmSARK not only ledto growth-inhibition and precocious leaf senescence, but also produced short andcurvy roots in the GVG-GmSARK transgenic seedlings. Consistent with the soybeandata, overexpression of GmSARK in the adult transgenic Arabidopsis accelerated leafsenescence, and produced abnormal floral organs, resembling those observed inmutants with constitutive ethylene-response. Additionally, quantitative RT-PCRshowed that overexpression of GmSARK induced the expression of criticalsenescence-related transcription factors, including AtNAP, NAC1, NAC2, andWRKY6.During leaf senescence, the most prominent change in cell structure is the breakdown of the chloroplast, which has been used as a biomarker for leaf senescence.Ultrastructural morphology analyses indeed revealed a reduction in thylakoids andaccumulation of huge starch grains in GmSARK-overexpressing chloroplasts.Moreover, the GUS reporter system was used to determine the features of theGmSARK promoter in Arabidopsis. The GUS assay was taken in the GmSARK-GUStransgenic Arabidopsis in the background of three individual mutants, which havesevere defects in the structure and function of chloroplasts. The results suggested apositive feedback loop in which the expression of GmSARK was reinforced by thedisintegration of the structure and function of chloroplasts resulting from itsexpression, to further promote the senescence process during leaf senescence.Gene chip analyses showed that the expression of phytohormone-related geneschanged greatly during GmSARK-induced senescence. More than half of the changedphytohormone-related genes are auxin-and ethylene-related genes. I had focused onthe roles of and relationships among cytokinin, auxin and ethylene inGmSARK-mediated senescence in the following studies.Exogenous application of cytokinin or auxin/ethylene significantly inhibited orenhanced the activities of the GmSARK promoter in GmSARK-GUS transgenicArabidopsis seedlings, respectively. Overexpression of GmSARK also reduced orenhanced the expression level of synthesis and response genes of cytokinin or auxinand ethylene, respectively. In addition, GmSARK-overexpressing seedlings werehypersensitive to exogenous auxin. I further used a synthetic auxin-induciblepromoter, DR5, to detect auxin accumulation and distribution in the transgenicseedlings. It seemed that GmSARK overexpression affected not only in vivo levels ofauxin but also the distribution of auxin in the roots of the GVG-GmSARK transgenicArabidopsis seedlings.Although exogenous application of cytokinin could strongly reduce the activityof GmSARK promoter, it could only partially restore GmSARK-induced earlysenescence. I found that exogenous application of PCIB (p-chlorophenoxyisobutyricacid, an auxin antagonist) and AVG (aminoethoxyvinylglycine, an inhibitor ofethylene biosynthesis) could effectively inhibit GmSARK-induced precocioussenescence. Interestingly, the delay in senescence produced by PCIB was effectively inhibited by exogenous ACC, while exogenous IAA had no effect on the ability ofAVG to restore the precocious leaf senescence induced by GmSARK. Consistently,mutations of either AUX1or EIN2completely reversed the GmSARK-inducedsenescence, and their flower morphology was also indistinguishable from that of themock plants. All these results suggested that auxin and ethylene are directly involvedin the positive regulation of leaf senescence, and that the enhancement of auxin onethylene biosynthesis and response could be the critical step in GmSARK-mediatedleaf senescence. I therefore proposed a model of the leaf senescence process in whichGmSARK and GmSARK-like genes participate. It was hypothesized that the balanceamong cytokinin, auxin and ethylene plays important and synergistic roles in theinitiation and process of senescence.I have identified a functional homolog of GmSARK in Arabidopsis, and named itAtSARK. Biochemical analyses proved that AtSARK is a novel dual-specificityLRR-RLK, which localizes to the plasma membrane. Quantitative RT-PCR revealedthat the expression level of AtSARK was dramatically up-regulated in either thenatural or GmSARK-induced senescence. Leaf senescence in sark-1, a T-DNAinsertional mutant of AtSARK, was significantly delayed, while inAtSARK-overexpressing transgenic Arabidopsis plants it was strongly induced.Meanwhile, the AtSARK-overexpressing transgenic Arabidopsis plants madeabnormal floral organs, resembling those observed in mutants with constitutiveethylene-response, similar to the GmSARK-overexpressing plants. Therefore weinferred that the SARKs (e.g. GmSARK and AtSARK)-mediated pathway may be awidespread mechanism in regulating leaf senescence in higher plants.Finally, I have identified a gene encoding a novel protein phosphatase, namedSSPP (Senescence Suppressed Protein Phosphatase). Quantitative RT-PCR showedthat the expression level of SSPP was dramatically down-regulated in either thenatural or SARKs-induced senescence, suggesting that SSPP may play a negative rolein leaf senescence. Constitutive overexpression of SSPP showed asenescence-delayed phenotype, it also exhibited enhanced tolerance to drought andwas supersensitive to high temperature. It is interesting that the constitutiveoverexpression of SSPP could effectively restore AtSARK-induced senescence. In conclusion, I have investigated the molecular mechanisms of GmSARK andAtSARK involved in the regulation of leaf senescence. These results makecontributions to elucidate the molecular regulatory mechanisms of the signalingpathways involved in leaf senescence, and provide new insights into the molecularbasis of leaf development.更多还原