【作者】 李林；

【导师】 吴燕；

【作者基本信息】 武汉大学 ， 发育生物学， 2014， 博士

【摘要】 磷脂酶C (PLC)是介导细胞信号传导的重要媒介成员。在动物细胞中,受G蛋白偶联受体(GPCR)信号的激活,PLC能够将其底物磷脂酰肌醇(PIP2)水解为甘油二脂(DAG)和三磷酸肌醇(IP3),这些水解产物作为细胞内重要的二级信使参与了胞内各种信号传导的过程。作为PLC的水解底物,PIP2在细胞的调控过程中发挥着重要的作用,是细胞膜的重要组份之一；它不仅是细胞膜上其它磷脂的底物,同时也为诸多膜蛋白和多聚糖提供在细胞膜上的锚定位点。而作为PLC的水解产物DAG和IP3则是细胞信号传导的重要二级信使。DAG可以通过结合蛋白的C1结构域激活并调控其靶标蛋白的活性。IP3则是钙离子信号的主导者,它可以和内质网上的钙离子通道结合,调节细胞内的钙离子水平。在动物中,PLC参与了动物生长发育的方方面面,如肌肉的收缩、细胞的分裂和分化、视觉的感应、免疫反应、神经系统的发育、精卵细胞的融合和发育等等过程。相对于在动物细胞中的研究来说,人们对于PLC在植物发育中的作用了解极其有限。尽管有报道显示,植物细胞中的PLC参与了激素ABA信号的传导、气孔的运动、温度的耐受等过程；然而对于PLC在植物生长发育中的功能特点和作用机制,知之甚少；特别是PLC功能发挥与植物生殖发育的关系,迄今未见任何报道。在本论文的研究中,我们通过对拟南芥PLC2功能缺失的纯合突变体plc2/plc2的表型观察和分析,发现PLC2在拟南芥的营养生长和生殖发育的过程中发挥了极其重要的作用。如果完全失去PLC2的功能,拟南芥的雌、雄配子体和胚胎的发育均显示异常；不仅如此,拟南芥的叶片维管组织、叶片表皮细胞的形态建成、以及根与根毛的生长均表现出不同程度的异常发育。深入研究表明,在生殖发育过程中,PLC2可以通过影响YUC基因家族成员的表达水平来改变植物体内生长素的合成,从而调控雌、雄配子体的发育。在营养生长过程中,PLC2则通过影响PIN家族蛋白的定位调控生长素的运输和分布,从而调控植物组织的发育进程。本研究的主要结果总结如下：1.PLC2基因在拟南芥各个组织中均有表达。通过荧光定量PCR的检测分析,显示出9个拟南芥PLC家族成员只有PLC2的表达量在分析到的各类拟南芥组织材料中呈现出明显的优势表达。暗示了PLC2对于植物发育的重要性。2.杂合突变体plc2/PLC2受精后分离的部分后代胚胎呈现明显的异常发育。通过分析,鉴定有4.06%的纯合体后代能够从自交的杂合突变体plc2/PLC2中分离出来,而这些完全缺失PLC2基因功能的plc2/plc2胚胎的发育十分异常,最终导致部分纯合胚胎致死结局。因此,在杂合体plc2/PLC2的角果中存在少部分败育种子的现象。3.PLC2参与了花器官发育,雄配子体发育等过程,其中包括花药与花粉的发育、花药的开裂。纯合体plc2/plc2中,花冠发育异常,花丝缩短,花柱伸长；花粉粒败育。在体外花粉管萌发实验分析中,plc2/plc2的花粉粒呈现异常萌发的表型,近90%的花粉粒完全不能够萌发。通过对花粉发育过程的观察分析,发现在plc2/plc2纯合体花药中绒毡层降解不完全,花药开裂出现异常；部分花药中或产生异常发育的花粉粒,或缺乏花粉粒发生。通过对DR5:GUS的表型分析,发现在plc2/plc2纯合体背景中,花药中的DR5:GUS表达水平不仅明显高于野生型而且其持续表达的时间也明显长于野生型的,说明plc2/plc2纯合体花药中的生长素水平不仅明显高于野生型花药中的而且能够维持较长的时间。4.PLC2参与了雌配子体发育,其中包括大孢子母细胞减数分裂、大孢子有丝分裂和胚囊的形成的过程。在plc2/pc2纯合体中,胚囊的无法形成源于大孢子第一次有丝分裂的异常,大孢子在第一次有丝分裂时失去了其应有的极性；导致在plc2/plc2纯合体胚珠中无法形成“七胞八核”的胚囊结构。通过对DR5:GFP的表型分析,发现在plc2/plc2纯合体背景中,发育早期的胚珠其珠孔端的生长素浓度明显高于野生型中的,说明plc2/plc2纯合体胚珠中大孢子有丝分裂极性的丢失可能与其珠孔端集中分布的生长素含量有关。5.PLC2参与了生长素合成基因YUCs在生殖器官中的表达调控。通过荧光定量PCR检测证明在纯合体plc2/plc2花序中,生长素合成基因YUC1,YUC2,YUC4, YUC6,YUC8的表达水平明显升高；而生长素代谢基因,如GH3家族等基因的表达水平变化不明显。通过分析组织中YUCs的表达分布,进一步地验证了在plc2/plc2纯合体背景中,生殖器官中YUC2:GUS,YUC4:GUS,YUC6:GUS的表达显著上升；此外,原位杂交实验结果显示,YUC1的mRNA水平在plc2/plc2纯合体的顶端分生组织中也明显升高。这些结果揭示出,拟南芥的生殖发育与生殖器官中PLC2调节的生长素的合成水平密切相关。6.PLC2参与调控了叶片的发育过程。在plc2/plc2纯合体中,子叶和莲座叶缩小,其中的维管组织闭合不完整。通过观察叶表皮细胞的形态,发现plc2/plc2纯合体中叶表皮细胞变周长缩小,细胞上的突起变短。通过观察DR5:GFP的信号在叶片中的分布,发现在plc2/plc2纯合体中叶尖端的DR5:GFP信号消失,暗示着PLC2可能通过介导叶片组织中生长素的运输影响叶片的形态建成。7.PLC2参与调控了主根和根毛的发育过程。在plc2/plc2纯合体中,主根变短,主根的向重性减弱；随着主根发育的推进,根尖顶端分生组织结构逐渐坍塌,最后失去了正常的顶端分生组织的结构。如果完全丧失PLC2的功能,主根中生长素的浓度随着根系的发育不断降低,暗示了主根顶端分生组织结构的坍塌可能是由于生长素分布的变化引起的。在plc2/plc2纯合体中根毛的发生受到显著的抑制,外源施加NAA或NPA可以拯救plc2/plc2纯合体中根毛的发生；可是,外源施加IAA却不能够达到拯救的目的,说明plc2/plc2纯合体中根毛的发生与生长素的运输直接相关,暗示了PLC2在生长素运输中的作用。8.PLC2影响了根中PIN的极性定位。通过分析plc2/plc2纯合体背景中PIN2-GFP信号的分布,发现PIN2的极性定位在plc2/plc2纯合体中呈现异常,PIN2在根细胞侧向的非极性分布增加。9.PLC2参与了拟南芥中钙离子信号的调控。通过对根毛中的钙离子分布的分析,验证了在plc2/plc2纯合体中钙离子水平明显降低；一些与钙离子水平相关的基因的表达水平也发生了明显的变化,说明PLC2在拟南芥钙离子信号调控中的重要作用。更多还原

【Abstract】 Phospholipase C (PLC) is a phospholipid catalytic enzyme and play central role in various cellular signaling networks. In mammalian, PLC can be activated by heterotrimetic G protein coupled receptors (GPCRs), which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5-trisphosphate (IP3). Acting as the substrate of PLC, PIP2 is not only a precursor for some membrane phospholipids but also serves as a membrane anchor for many proteins. DAG can bind to the conserved C1 domains of variety enzymes and stimulate their acticities. IP3 is a major regulator for elevating the cytosolic calcium level. Through binding to the calcium channel that is localized in the endoplasmic reticulum (ER) membrane IP3 promotes releasing calcium from the ER lumen to the cytosol. In animal cells, PLCs take part in diverse cellular processes during the life cycle of animals, such as muscle contraction, cell proliferation and differentiation, visual processing, immune response, nervous system development, sperm-egg cell fusion and zygote development, etc. In plant cells, however, the involvement of a PLC in the life cycle of plants is poorly understood. Although several reports demonstrate that PLCs are crucial in the processes of abscisic acid (ABA) signaling, stomatal movement, disease resistance and thermotolerance, the mechanisms regarding to the PLCs’functions are remained largely unclear. Specifically,9 PLC family members are identified in Arabidopsis genome, but the functions of most of the PLC members such as PLC2 are undetermined. Here, we focused on analyzing the role of PLC2 in the developmental processes of plants, in particular, in the vegetative development stage and in the reproductive development stage in Arabidopsis. Through characterizing the phenotypes of loss-of-PLC2-function, the pivotal role of PLC2 in various aspects of plant development is thus determined. Results showed that PLC2 is involved in the regulations of male and female gametophytes development; moreover, it is also involved in the morphogenesis of the vine and vascular tissues of leaves and in the epidermal cells of leaves. Additionally, PLC2 plays essential role for the root including the root hair development. Further analyses identified that, in reproductive tissues, the function of PLC2 is tightly associated with the level of auxin. Through upregulating the expression levels of auxin biosynthetic YUCs genes, elevation of auxin content in the male and female gametophytes could be achieved, if the PLC2 function is completely abolished. Interestingly, in vegetative tissues, the PLC2 function is linked to the auxin transport. Via modulating the polar localizations of auxin transport PIN proteins, the development of leaves and roots could be altered. Overall, major results from this study are summarized in below paragraphs.1. Expression levels of 9 AtPLC family members were quantitatively compared in various tissues through the qRT-PCR (quantitative real time PCR). Results showed that PLC2 is predominantly expressed in all tested Arabidopsis tissues, suggesting its important roles.2. Some of progenies of T-DNA insertion heterozygous mutant plc2/PLC2 showed embryo-lethal. The resulting progenies from the self-pollinated plc2/PLC2 mutant lines were not able to complete embryogenesis, thus, they were determined as the homozygous. Only 4.06% progenies are segregated from the the self-pollinated plc2/PLC2 mutant lines. Therefore, in the siliques of plc2/PLC2 some of ovules were aborted.3. PLC2 affects the formation of floral organ and the development of male gametophytes. The petals and sepals in the flowers of plc2/plc2 homozygous plants were obviously misformed with comparing to those in the wildtype. In addition, shorten filaments and elongated styles were also observed in plc2/plc2 homozygous flowers. Defective development of anthers and pollens were also produced in plc2/plc2 plants. Incomplete degradation of tapetum and aberrant dehiscence of anthers led to aberrance in microspores and/or pollen grains maturation even pollens pollination. Although few pollen grains were produced in plc2/plc2 homozygous plants they were not able to germinate. Further analysis showed that the increased and prolonged DR5:GUS signal was detected in plc2/plc2 homozygous anther, suggesting the correlation of PLC2 function with the auxin content that might influence the male gametophyte development.4. PLC2 affects the female gametophyte development including the embryo sac development.Likewise, the female gametophyte development was totally abolished in the plc2/plc2 homozygous plants.In plc2/plc2, the first mitosis of megaspore was disoriented and lost its polarity, leading to the abolishment of embryo sac formation. Through analyzing the distribution of DR5:GFP in the ovules, it showed that intensified DR5:GFP signal was detective at the micropylar end of an ovule in which the megaspore mother cell was just developed. Thus, it is likely that the increased auxin level might be produced due to the lost of PLC2 function.5. PLC2 is involved in the regulation of expression of YUC family genes in the reproductive tissue. Auxin biosynthesis gene YUC1, YUC2, YUC4, YUC6, and YUC8 were upregulated dramatically in plc2/plc2 plants, which was further confirmed by analyzing the signal of YUC:GUS in the plc2/plc2 background and with the in situ hybridization experiment that is for characterizing the expression pattern of YUC1 in plc2/plc2 plants. Results demonstrated that the elevation of auxin content in the plc2/plc2 plants was resulted by the increased expression levels of YUCs.6. PLC2 modulates the morphogenesis of leaves.In plc2/plc2, cotyledons and rosette leaves were much smaller than those in the wildtype. The meshwork of vines and vascular tissues in leaves of plc2/plc2 plants showed defectively developed, such as that discontinuance in vascular tissue of leaves was obvious. Shorter lobes and reduced number of circularity of pavement cells were showed in the leaves of plc2/plc2. Disappearance of auxin signal in plc2/plc2 cotyledons may imply that PLC2 may be taken part in the auxin transport.7. PLC2 modulates the root growth. In plc2/plc2 shorter primary roots and decreased gravitropism were noticed. The root apical meristem was gradually collapsed in plc2/plc2 seedlings. Through analyzing the distribution of DR5:GUS it suggested that auxin accumulation in the root tip may be decreased during the growth of plc2/plc2 roots. The root hair growth was also inhibited in plc2/plc2 plants, which could be rescued by application of extracellular NAA and NPA but not by addition of IAA, thus, this result provided us another clue of that PLC2 does play pivotal role in the auxin transport.8. PLC2 plays role in PINs polar targeting to the plasma membrane. Inplc2/plc2, the basal-apical polar localization of PIN2 is affected. The increasing in the lateral membrane localization of PIN2 was detected in plc2/plc2, suggesting the role of PLC2 in the PIN protein recycling and membrane targeting.9. PLC2 modulates the calcium regulation in Arabidopsis. Through analyzing the calcium oscillation in root hairs, it showed that that the calcium level was decreased in plc2/plc2. Meanwhile, expression levels of several calcium related genes were obviously changed in plc2/plc2, suggesting that PLC2 may execute its function via downstream calcium signaling regulation.更多还原