PDK1亚硝基化与一氧化氮诱导的植物细胞死亡间关系的研究

It has been known for over a decade that nitric oxide (NO) can trigger cell death in plants. However, the molecular mechanism of the NO-triggered cell death remains elusive. Our preliminary data showed that silencing a gene encoding S-nitrosoglutathione reductase1 (GSNOR1), which maintains the equilibrium of cellular level of protein S-nitrosylation, induced spontaneous cell death on the leaves of tobacco, implying that the cell death triggered by exogenous NO application and the cell death induced by silencing GSNOR1 share the same molecular mechanism. We therefore hypothesize that the cell death triggered by NO application or by silencing GSNOR1 is a consequence resulted from functional inhibition of a key negative regulator of cell death by S-nitrosylation. Based on the candidate predictions and preliminary results, we showed that tomato 3-phosphoinositide-dependent protein kinase-1 (SlPDK1), a negative regulator of cell death， whose function is conserved among yeast, mammals, and plants, is a target of S-nitrosylation and its kinase activity was inhibited by GSNO in a concentration-dependent manner. Based on these preliminary results, we come up with a hypothesis: the cell death triggered by NO is achieved through inhibiting kinase activity of PDK1 by S-nitrosylation. In this proposal, we firstly will confirm that the inhibiting effect of S-nitrosylation on kinase activity of SlPDK1 in vivo or in planta; Through in vitro site-directed mutagenesis combining with biotin switch assay and nanoLC-MS/MS, we will identify the target Cys residue(s) being S-nitrosylated and examine the effect of S-nitrosylation on kinase activity of mutant version of SlPDK1, whose target Cys is mutated to Ala. We expect to create a mutant SlPDK1, whose kinase activity will not be inhibited by S-nitrosylation. Through transforming this mutant version of SlPDK1 into tomato and tobacco, we expect to generate transgenic tomato and tobacco plants, on which the cell death can no longer be triggered by NO. Together, our proposed experiments will help to gain insight into the molecular mechanism of NO triggered cell death.

一氧化氮（NO）诱导的植物细胞死亡是目前植物抗病信号传导领域中的研究热点之一。但其分子机理仍未被揭示。本申请在发现沉默烟草GSNOR1后诱导细胞死亡及番茄中负调控细胞死亡的PDK1是亚硝基化的靶蛋白且GSNO以浓度依赖方式抑制PDK1激酶活性的基础上，拟证明NO诱导细胞死亡是通过亚硝基化PDK1从而抑制其激酶活性而实现的。主要研究内容包括：1.确定沉默GSNOR1和沉默PDK1诱导的细胞死亡类型及与抗病性的关系;2.利用生物素置换法结合离体定点突变技术及nanoLC/MS/MS技术，鉴定出PDK1的亚硝基化位点；3.分析PDK1亚硝基化位点突变对其激酶活性的影响；4.期望通过定点突变技术创制出消除亚硝基化修饰但激酶活性不受亚硝基化抑制的PDK1突变体; 5. 将此PDK1突变体转化至番茄和烟草中以期创制不能被NO诱导死亡的转基因株系。通过本项目的实施期望揭示NO诱导植物细胞死亡的

一氧化氮(NO)可诱导植物细胞死亡，但其诱导细胞死亡的分子机理仍不清楚。我们前期的研究表明，在烟草与番茄中沉默亚硝基化谷胱甘肽还原酶(Nitrosoglutathione reductase, GSNOR1)可导致沉默植株叶片上出现自发性的细胞死亡, 暗示细胞中积累过多的GSNO是导致细胞死亡的可能原因。过量的GSNO可将其NO基团转至某些蛋白的半胱氨酸残基上导致蛋白的亚硝基化。蛋白的亚硝基化可增强或减弱蛋白的功能，因而在众多生物学过程中起着重要的调控作用。我们推测沉默GSNOR1导致细胞死亡可能是亚硝基化某个细胞死亡的负调控因子所致。PDK1-PKB1激酶途径在酵母及动物中高度保守，是细胞凋亡的负调控因子。有研究表明在番茄中沉默PDK1或PKB1/Adi3均可导致细胞死亡。此外，小鼠中PKB1是亚硝基化的靶蛋白，亚硝基化可抑制其激酶活性。我们在检测番茄PKB1/Adi3是否同样可被亚硝基化时意外发现PKB1/Adi3不能被亚硝基化，但作用于其上游的PDK1却可被亚硝基化。GSNO以浓度依赖方式抑制PDK1激酶活性，这种抑制可被还原剂DTT恢复，但却被H2O2增强，说明PDK1的亚硝基化是可逆的，NO与H2O2可协同诱导细胞死亡。PDK1中有4个半胱氨酸残基。通过Lc-MS/MS分析，我们鉴定出PDK1中128位及466位的半胱氨酸残基是亚硝基化的靶位点。通过定点突变技术我们创制出每个半胱氨酸残基突变为丝氨酸的PDK1突变体。激酶分析结果表明， 214及466位的突变对PDK1的激酶活性并无影响，244位的突变导致PDK1的激酶活性显著降低(>90%), 而128位的突变则导致PDK1的激酶活性彻底丧失。 以上结果表明在番茄中沉默GSNOR1导致的自发性细胞死亡可能是通过亚硝基化PDK1中128位半胱氨酸残基从而抑制其激酶活性所致 (Liu 等，2017， JBC)。..在项目执行过程中我们偶然发现拟南芥gsnor1-3突变体表现出生长素突变体的表型，继而我们发现该突变体对生长素敏感性及极性运输显著降低; 生长素极性运输的降低与一系列PIN蛋白的积累水平的降低及内吞的下降所致 (Shi 等, 2015, Molecular Plant；Ni 等，2017， Frontiers in Plant Science)。这些研究揭示了NO可通过与生长素互作调控植物生长发育。