【作者】 李兴国；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 植物学， 2008， 博士

【摘要】 细胞分裂素在植物生长发育的许多方面具有重要作用,如促进细胞分裂、延缓叶的衰老以及抑制顶端优势等。近年来,人们在模式植物拟南芥和水稻中相继克隆到细胞分裂素代谢的相关基因,初步建立了细胞分裂素信号传导途径的模型,但有关细胞分裂素调控拟南芥花序和花发育分子机理的资料仍然较少。在拟南芥中,细胞分裂素生物合成的第一步主要由7个ATP/ADP异戊烯基转移酶(AtIPT1和AtIPT3-AtIPT8)催化完成。本研究构建了花和花器官特征决定基因APETALA1(AP1)启动子驱动AtIPT4的表达载体并转化野生型拟南芥,试图通过位置特异性表达细胞分裂素合成酶基因提高植物体局部的细胞分裂素含量,探讨细胞分裂素调节拟南芥花发育的分子机理。表型分析的结果显示,AP1::IPT4转基因植株的顶端优势被强烈抑制、花序轴伸长被抑制、花和花器官数量增加、萼片上产生较多分枝的表皮毛。解剖学的证据显示,AP1::IPT4植株的花序分生组织体积明显增大,但其组织结构未受影响,细胞体积没有变化,暗示转基因表达细胞分裂素合成酶基因促进花序分生组织的细胞分裂。扫描电镜的观察结果表明,AP1::IPT4植株花原基的数量显著增加,花器官的分化延迟。GUS表达分析的结果显示,在AP1::IPT4植株的花序分生组织中,CLAVATA3(CLV3)表达的区域扩展,在花中的表达时间延长,而WUSCHEL(WUS)表达的信号减弱,表明维持干细胞活动的基因参与细胞分裂素对花序和花发育的调控。RT-PCR的结果显示,转基因植株中的AtIPT4的转录水平明显提高。为进一步阐明表型产生的生理学基础,我们利用高效液相-质谱法(LC-MS)对内源细胞分裂素的含量进行了测定。结果表明,在转基因植株花序中具生理活性的细胞分裂素异戊烯基腺嘌呤(isopentenyladenine,iP)及其直接的前体物-异戊烯基腺苷(isopentenyladenosine,iPR)的水平显著提高。上述结果提示,转基因植株的表型是由高水平细胞分裂素诱导产生的。为深入理解细胞分裂素调节花序和花发育的分子机理,我们以幼花序为材料,利用拟南芥全基因组芯片(Affymetrix Arabidopsis ATH1 GeneChips)进行了基因表达谱分析。结果显示,转基因植株中有460个基因的表达水平上升,408个基因的表达量下降。对部分基因进行的半定量RT-PCR验证结果与芯片的数据基本一致。其中,A类细胞分裂素响应调节因子ARABIDOPSIS RESPONSE REGULATOR(ARR)基因ARR5、ARR6、ARR7和ARR15、NAC基因家族成员CUP-SHAPED COTYLEDON(CUC)基因CUC2和CUC3以及LATERAL ORGAN BOUNDARIES(LOB)基因家族成员LOB domain protein(LBD)基因LBD3、LBD39和LBD41的表达水平均被显著上调。过量表达LBD3(Naito等,2007)和CUC3,导致植株矮化,这与AP1::IPT4植株的表型具有相似性;CUC3突变或过量表达反义LBD3则能够部分抑制AP1::IPT4植株表型(包括顶端优势和花节间长度等)的产生。上述结果表明LBD3和CUC3参与AP1::IPT4植株表型的形成。遗传学和RT-PCR分析的结果显示,细胞分裂素受体的突变体ahk2 ahk3几乎完全阻断AP1::IPT4植株表型,抑制细胞分裂素对CUC3和LBD3表达的上调作用。遗传学分析的结果表明ARR5突变加强AP1::IPT4植株的表型,ARR6突变能够部分抑制AP1::IPT4植株的表型,并降低LBD3的转录水平,而ARR15对AP1::IPT4植株的表型没有明显影响,提示ARR5、ARR6和ARR15在调节拟南芥花序和花发育方面具有不同的功能。本研究的结果表明细胞分裂素信号传导部分地通过介导CUC3和LBD3的表达,调控拟南芥花序和花的发育。更多还原

【Abstract】 Cytokinins are essential regulators of numerous plant growth and development processes. In Arabidopsis, genes encoding the enzymes responsible for cytokinin metabolic activity such as biosynthesis and degradation have been characterized, and the cytokinin signaling transduction was implicated in control of shoot meristem initiation, stem cell population, apical dominance and fertility. However, little are known about the mechanisms of cytokinin regulation in inflorescence and flower development.The first step of the bulk of cytokinin biosynthesis is catalyzed by seven ATP/ADP isopentenyltransferases (AtIPT1 and AtIPT3-AtIPT8) in Arabidopsis. Here, we tried to increase the cytokinin level in Arabidopsis floral tissues by transgenic expression of AtIPT4 under the control of the promoter of APETALA1 (AP1) that is known to be expressed specifically in flower primordia and perianth in Arabidopsis. Totally, 45 independent AP1::IPT4 lines obtained showed the similar phenotypical alterations with respect to the inflorescence and flower development. In AP1::IPT4 plants, the apical dominance and stem elongation of primary inflorescence were significantly inhibited, which resulted in the formation of sympodial branching architecture. Both the flower and floral organ number, however, were increased obviously in transgenic plants. In addition, many branched trichomes formed on the abaxial surface of sepals. As expected, no obvious morphological changes were observed during the vegetative development. In the longitudinal sections of primary inflorescence apex, the cell number and size of inflorescence meristem were increased in transgenic plants, whereas the meristematic cell size was not altered, indicating elevated cell division rate occurred in the transgenic inflorescence meristem. Scanning electron microscopy analyses indicated that the transgenic primary inflorescence meristem was larger in diameter than wild-type one. There were more flower primordia formed at periphery of inflorescence meristem of AP1::IPT4 plants in comparison with that of wild type plants, however, the floral organ initiation was delayed. Furthermore, GUS staining assay revealed that CLAVATA3 (CLV3) and WUSCHEL (WUS), two genes responsible for stem cell homeostasis maintenance, were implicated in cytokinin-regulated inflorescence and flower development. To uncover the physiological basis for modulating flower development in AP1::IPT4 plants, we analyzed the endogenous cytokinin levels in the young inflorescences from the homozygotic plants of two independent lines with the liquid chromatography--mass spectrometry (LC-MS) method. Compared with wild-type samples, the levels of isopentenyladenine (iP) and its precursor, isopentenyladenosine (iPR), were all elevated significantly, which is consistent with the increase of transcript level of AtIPT4 detected in transgenic lines by RT-PCR analysis. These results support our hypothesis that the phenotypical alterations of AP1::IPT4 plants should be caused by enhanced endogenous cytokinin concentration in inflorescence tissues.To explore the cytokinin-responsive genes in floral tissues, we carried out the analysis for the genome-wide expression profiling in young inflorescences using the Affymetrix Arabidopsis ATH1 GeneChips (Gene Company Limited). The results revealed that 460 gene expression levels were up-regulated and 408 genes down-regulated in floral tissues of transgenic plants. Among them, we are interested in those involved in cytokinin signaling and flower morphogenesis including the type-A ARRs (ARR5, ARR6, ARR7, and ARR15), two CUP-SHAPED COTYLEDON genes (CUC2 and CUC3), three LOB domain protein genes (LBD3, LBD39 and LBD41).Either LBD3 (Naito et al., 2007) or CUC3 overexpression led to stem elongation reduction which is similar to that of AP1::IPT4 plants. In contrast, cuc3 mutation or overexpression of anti-sense LBD3 repressed some aspects of AP1::IPT4 phenotypes including shoot apical dominance and internode length between flowers.These results revealed that both CUC3 and LBD3 contributed to the developmental changes of AP1::IPT4 plants. Genetic and molecular analyses demonstrated that combination of cytokinin receptor mutantion ahk2 and ahk3 blocked almost the phenotypes of AP1::IPT4 plants, and impaired the up-regulation of CUC3 and LBD3 in response to cytokinins. Furthermore, we examined the functions of ARR5, ARR6 and ARR15 in AP1::IPT4 inflorescences. The results suggested that mutation in ARR6 may partially inhibited the developmental alterations of AP1::IPT4 plants, and attenuated LBD3 expression level. Thus, the results indicated that proper development of inflorescence and flower might be partially controlled by cytokinin signaling-mediated CUC3 and LBD3 functions in the inflorescence and flower meristem.更多还原