拟南芥类受体激酶FLS2调控ABA和Flg22诱导气孔关闭的机制研究

发布时间：2019-02-16 21:50

【摘要】：气孔,作为CO2同化和水分蒸腾的通道,在调控植物生长发育及应对环境胁迫的过程中发挥重要作用。拟南芥的气孔运动受到生物因素(如病原菌入侵)和非生物因素(如干旱和光照)的精细调节。例如,植物激素ABA和细菌鞭毛蛋白上的保守多肽Flg22均能有效诱导气孔关闭。迄今为止,人们在ABA及Flg22信号诱导气孔关闭机制的研究领域取得了一些进展。阴离子通道SLAC1是ABA信号转导通路下游的的关键靶标,其活性受到OST1、GHR1、CPKs等多个激酶的调控。也有文献报道Flg22信号可能通过刺激SLAC1阴离子电流进而促使气孔关闭。然而,Flg22介导SLAC1激活的具体分子机理并不清楚。同时,ABA及Flg22信号通路在保卫细胞中的互作机制仍有待研究。本研究通过筛选300个T-DNA插入的类受体激酶突变体,发现fs2突变体对干旱敏感,离体叶片失水率快于野生型,叶片表面温度较低;突变体气孔运动对ABA处理响应减弱。在转录水平上,FLS2基因表达受到ABA诱导。这些结果说明FLS2可能在ABA诱导的气孔关闭过程中发挥作用。萤光素酶互补实验、Co-IP以及Pull-down等实验分析表明,FLS2蛋白及其共受体BAK1与阴离子通道SLAC1蛋白互作。体外磷酸化实验及质谱检测证明,FLS2可磷酸化SLAC1蛋白氨基端(N-SLAC1)的Ser86、Ser113和Ser120,而BAK1可作用于Ser59、Ser86以及Thr142。这些生化结果表明SLAC1可能是FLS2和BAK1的底物蛋白。在卵母细胞体系中,Flg22诱导的FLS2-BAK1复合体能激活SLAC1的阴离子电流。位于N-SLAC1的Ser59和Ser86在FLS2-Flg22-BAK1介导的SLAC1激活过程中发挥关键作用。体外条件下,BIK1能磷酸化SLAC1的N端和C端。而电生理实验表明FLS2-Flg22-BAK1激活SLAC1的过程独立于BIK1。在fls2 bk1及fls2 slac1双突变体中,ABA及Flg22诱导的气孔关闭过程均被阻断。胶内磷酸化实验证明Flg22处理可激活OST1,这一过程部分依赖于BAK1,但不依赖FLS2。FLS2突变导致Flg22诱导的BAK1-OST1互作和OST1激活提前。在卵母细胞中,BAK1可以增强OST1激活的SLAC1的阴离子电流。ABA信号通路中的负调控因子ABI1与FLS2互作,并抑制FLS2对N-SLAC1的磷酸化作用。综上所述,本研究证明FLS2蛋白是保卫细胞中ABA信号和Flg22信号互作的节点,并初步揭示了其参与ABA及Flg22诱导气孔关闭的分子机制。在气孔免疫中,Flg22诱导的FLS2-BAK1复合物以及Flg22激活的OST1直接调控SLAC1,促进气孔关闭。在干旱胁迫下,ABA解除了 ABI1对FLS2的抑制作用,活化的FLS2可能通过作用于SLAC1调节气孔运动。这一研究有助于深刻理解生物胁迫和非生物胁迫在拟南芥保卫细胞中形成的复杂信号网络。
[Abstract]:Stomata, as a pathway of CO2 assimilation and water transpiration, play an important role in regulating plant growth and development and coping with environmental stress. The stomatal movement of Arabidopsis thaliana is regulated by biological factors (such as pathogen invasion) and abiotic factors (such as drought and light). For example, plant hormone ABA and conserved polypeptide Flg22 on bacterial flagellin can effectively induce stomatal closure. Up to now, some progress has been made in the field of stomatal closure induced by ABA and Flg22 signals. Anion channel SLAC1 is a key target downstream of ABA signal transduction pathway and its activity is regulated by several kinases such as OST1,GHR1,CPKs. It has also been reported that Flg22 signals may induce stomatal closure by stimulating SLAC1 anion currents. However, the specific molecular mechanism of SLAC1 activation mediated by Flg22 is unclear. At the same time, the interaction mechanism of ABA and Flg22 signaling pathway in guard cells remains to be studied. In this study, 300 T-DNA inserted receptor-like kinase mutants were screened. It was found that fs2 mutants were sensitive to drought, the water loss rate of leaves in vitro was faster than that of wild type, and the surface temperature of leaves was lower, and stomatal movement of fs2 mutants was less responsive to ABA treatment. At the transcriptional level, FLS2 gene expression was induced by ABA. These results suggest that FLS2 may play a role in the stomatal closure induced by ABA. Luciferase complementary assay, Co-IP and Pull-down analysis showed that FLS2 protein and its coreceptor BAK1 interacted with anion channel SLAC1 protein. In vitro phosphorylation test and mass spectrometric analysis showed that FLS2 phosphorylated SLAC1 protein amino terminal (N-SLAC1) Ser86,Ser113 and Ser120, while BAK1 could act on Ser59,Ser86 and Thr142.. These biochemical results suggest that SLAC1 may be the substrate protein of FLS2 and BAK1. In oocyte system, FLS2-BAK1 complex induced by Flg22 can activate the anion current of SLAC1. Ser59 and Ser86 located in N-SLAC1 play a key role in SLAC1 activation mediated by FLS2-Flg22-BAK1. In vitro, BIK1 can phosphorylate the N-terminal and C-terminal of SLAC1. Electrophysiological experiments show that FLS2-Flg22-BAK1 activates SLAC1 independently of BIK1.. In both fls2 bk1 and fls2 slac1 mutants, the stomatal closure induced by ABA and Flg22 was blocked. Intracellular phosphorylation experiments showed that Flg22 treatment could activate OST1, partly dependent on BAK1, but not dependent on FLS2.FLS2 mutation leading to BAK1-OST1 interaction induced by Flg22 and early activation of OST1. In oocytes, BAK1 could enhance the anion current of SLAC1 activated by OST1, the interaction of ABI1 and FLS2 in the ABA signaling pathway, and inhibit the phosphorylation of N-SLAC1 by FLS2. In conclusion, this study demonstrated that FLS2 protein is the node of interaction between ABA signal and Flg22 signal in guard cells, and preliminarily revealed the molecular mechanism of its involvement in ABA and Flg22 induced stomatal closure. In stomatal immunity, Flg22-induced FLS2-BAK1 complexes and Flg22-activated OST1 directly regulated SLAC1, to promote stomatal closure. Under drought stress, ABA removed the inhibitory effect of ABI1 on FLS2, and activated FLS2 may regulate stomatal movement by acting on SLAC1. This study contributes to a profound understanding of the complex signaling networks formed in Arabidopsis thaliana guard cells under biotic and abiotic stresses.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q945

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