图1 植物胞内蛋白亚硝基化水平的稳态平衡调控 植物体内亚硝基化水平由NO的产生及去亚硝基化的能力决定。植物体内NO可由众多途径产生, 包括NOA1途径、NR途径、质膜NR/NiNOR途径、线粒体途径、多胺途径、过氧化酶体XOR途径和质外体途径。另外, 微生物产生的NO对植物也有一定的影响(Crawford and Guo, 2005; Yu et al., 2014)。生物和非生物胁迫可诱导NO在植物体内的合成。在胁迫条件下, 细胞内NO浓度升高, NO与GSH结合形成GSNO。GSNO是亚硝基化的NO供体, 亚硝基化的实现是由GSNO而并非由NO直接介导。GSNO可将NO反式转移至靶蛋白从而改变蛋白的功能(Liu et al., 2001; Feechan et al., 2005)。GSNOR1和硫氧还蛋白(thioredoxins, TRX)是去亚硝基化的2个关键酶。GSNOR1通过还原和降低植物体内的GSNO水平间接降低蛋白亚硝基化。而TRX-h3和TRX-h5可直接催化蛋白的去亚硝基化(Tada et al., 2008)。

Figure 1 The homeostasis of cellular S-nitrosylation level in plantsThe level of S-nitrosylation in plant cells is determined by balance between the production of NO and the capacity of de-nitrosylation. There are multiple routes for NO production in plants such as NOA1-dependent pathway, NR pathway, plasma-membrane NR/NiNOR pathway, mitochondrial pathway, polyamines pathway, peroxisome (XOR) pathway and apoplast pathway. In addition, micro-organisms also contribute to the NO accumulation in plants (Crawford and Guo, 2005; Yu et al., 2014). Both biotic and abiotic stresses can induce the production of NO. Under stress conditions, NO level is increased in plant cells and as a results, the GSNO is formed in the presence of GSH; GSNO is a major donor of S-nitrosylation and S-nitrosylation is mediated by GSNO rather than by NO directly; NO moiety is transferred from GSNO to proteins in trans and thus the protein functions are altered by this modification (Liu et al., 2001; Feechan et al., 2005). GSNOR1 and thioredoxins (TRX) are two key enzymes involved in de-nitrosylation. Whereas GSNOR1 achieves the protein de-nitrosylation indirectly through reducing cellular level of GSNO, TRX (TRX-h3 and TRX-h5) catalyzes de-nitrosylation directly (Tada et al., 2008).

图2 亚硝基化调控植物细胞死亡 (A) NO通过亚硝基化抑制H2O2分解及还原相关酶造成细胞内过量积累H2O2, 从而促进细胞死亡。cAPX是亚硝基化的靶蛋白, 被亚硝基化后其活性受到抑制。另一方面亚硝基化可导致cAPX的泛素化, 加速其通过蛋白酶体的降解, 从而使细胞内H2O2含量增加, 最终导致细胞死亡(de Pinto et al., 2013)。(B) NO通过亚硝基化质膜上NADPH氧化酶复合体中AtRBOHD亚基的Cys890负反馈抑制其合成H2O2的能力, 从而降低细胞中H2O2的含量及抑制细胞过度死亡(Yun et al., 2011)。(C) NO通过亚硝基化过氧化物氧还酶PrxII E中的Cys121抑制其还原H2O2及过亚硝酸盐(ONOO-)解毒能力。过亚硝酸盐通过不可逆地硝基化(nitration)络氨酸残基而改变蛋白的功能, PrxII E具保护蛋白不被硝基化的功能(Romero-Puertas et al., 2007)。(D) GSNOR1蛋白作为正调控因子参与除草剂百草枯诱导的细胞死亡。除草剂百草枯处理可导致野生型拟南芥死亡, 但不能导致体内积累过量RNS的GSNOR1功能缺失突变体par2-1死亡。ROS具增加细胞内GSNOR1蛋白含量的效应, 而RNS则具降低GSNOR1蛋白含量的效应。GSNOR1作用于ROS的下游参与除草剂百草枯诱导的细胞死亡(Chen et al., 2009)。

Figure 2 Regulation of cell death by S-nitrosylation in plants(A) NO promotes cell death through increasing the accumulation of H2O2 excessively by inhibiting H2O2 detoxifying enzymes. cAPX is a target of S-nitrosylation and its enzyme activity is severely inhibited by S-nitrosylation. In addition, S-nitrosylation of cAPX results in its ubiquitination and degradation by proteasome and thus leads to the cell death by over-accumulation of H2O2 (de Pinto et al., 2013). (B) NO can feedback inhibit H2O2 production through S-nitrosylation of Cys890 of AtRBOHD, a subunit of NADPH oxidase on the plasmamembrane, and as a result, excessive accumulation of H2O2 is relieved and cell death is alleviated (Yun et al., 2011). (C) NO inhibits H2O2 reduction and ONOO- detoxification of PrxII E through S-nitrosylation at Cys121. ONOO- can irreversibly alter protein functions through nitration. PrxII E is an enzyme that can prevent proteins from nitration (Romero-Puertas et al., 2007). (D) GSNOR1 functions as a positive regulator that participates in the cell death induced by paraquat. Application of herbicide paraquat can kill the wild type Arabidopsis Col-0 but not par2-1, a missense mutant of GSNOR1. ROS has an effect in increasing the level of GSNOR1, whereas RSN has the opposite effect. GSNOR1 participates paraquat-induced cell death downstream of ROS (Chen et al., 2009).