GSNOR参与生长素信号调控番茄侧枝发生的生理与分子机制

The control of plants’ branching by auxin-mediated cytokinin and strigolactone has been extensively studied. However, little has been known about the cross-talk between auxin and other signal molecules in regulating axillary bud outgrowth. Previously, we found that S-nitrosoglutathione reductase (GSNOR)-mediated nitric oxide (NO) can be induced in the process of axillary bud outgrowth involving auxin signaling in tomato plants. And over-expression (or suppressed-expression) of GSNOR can inhibit (or promote) the axillary bud outgrowth of tomato plants. Additionally, we also identified 17 NO-mediated proteins involving auxin signaling by nitrosoproteomic analysis. Based on these results, the expression of GSNOR promoter, as well as the signaling accumulation of NO, the content of auxin, the expression of related genes, the expression and subcellular localization of polar auxin transport proteins, as well as pharmacological and genetic experiments will be performed to explain the cross-talk between GSNOR and auxin signaling in the process of axillary bud outgrowth. Also, we will reveal the molecular mechanism of interaction between GSNOR and auxin signaling in the process of axillary bud outgrowth by analyzing nitrosoproteomic, verification of site-directed mutagenesis, and interaction of protein-protein or protein-DNA. Taken together, we want to explain the physiological and molecular mechanism of GSNOR-mediated auxin signaling in controlling tomato axillary bud outgrowth, which can provide target genes and theoretical basis for the breeding of less lateral branch tomato plants.

生长素调控细胞分裂素和独脚金内酯影响植物分枝的研究已取得长足进展，但生长素与其它信号分子互作，调控侧枝发生的研究相对匮乏。先前，我们发现硫代亚硝基谷胱甘肽还原酶（GSNOR）调控一氧化氮（NO）介导生长素信号影响番茄侧枝发生，且过量（或抑制）表达GSNOR能抑制（或促进）番茄侧枝生长，并通过蛋白质亚硝基化分析发现17个可被NO靶向修饰的生长素信号相关蛋白。在此基础上，本研究拟针对番茄侧枝发生过程，分析GSNOR启动子原位表达和NO信号、生长素代谢和相关基因表达、极性运输蛋白表达及亚细胞极性定位、辅以药理学和遗传学验证，阐明GSNOR和生长素间的信号交流机理；通过蛋白质亚硝基化组学分析及定点突变验证、蛋白-蛋白及蛋白-核酸互作分析，揭示GSNOR与生长素信号相关基因的分子互作机制。旨在阐明GSNOR参与生长素信号调控番茄侧枝发生的生理与分子机制，为少侧枝番茄的选育提供目标基因和理论基础。

探明番茄侧枝发生及调控机制，创制少侧枝番茄种质，具有重要的理论与实际意义。本项目研究发现，硫代亚硝基谷胱甘肽还原酶（GSNOR）调控一氧化氮（NO）介导生长素信号影响番茄侧枝发生，且过量（或抑制）表达GSNOR能抑制（或促进）番茄侧枝生长。与WT相比，GSNOR-RNAi表现为矮化、多侧枝、促进种子发芽、抑制根和下胚轴生长、降低光合作用、抑制植株生长和花粉发育、造成产量降低等农艺性状。通过对GSNOR介导的蛋白质亚硝基化组学分析，鉴定到395个亚硝基化修饰蛋白，这些被修饰的蛋白富集在能量代谢、刺激应答和激素代谢等通路。在此基础上，申请人采用WT、GSNOR-OE和GSNOR-RNAi株系，结合外源药物学手段，研究发现GSNOR通过负调节NO稳态，建立番茄顶端优势。抑制表达GSNOR显著降低了IAA在顶芽和腋芽中的合成。与WT相比，GSNOR-RNAi株系顶芽中生IAA转运能力下降，而腋芽中IAA的转运能力提高，导致其顶芽和腋芽中均显示IAA积累减少的特征。通过异位嫁接试验，申请人发现GSNOR介导的NO和IAA变化影响CKs的生物合成、转运和信号传导，这些结果综合导致GSNOR-RNAi株系腋芽中的IAA:CKs的比例降低，促进腋芽休眠解除。针对番茄腋芽本底NO来源的研究，发现最初的NO信号是由硝酸还原酶（NR）催化硝酸盐过程产生的。NR介导的低浓度NO通过对GSNOR的Cys10进行蛋白质亚硝基化修饰，抑制GSNOR酶活性，进一步诱导NO的爆发，形成NO级联激活信号，并通过IAA-CK激素调控途径促进侧枝生长。综上所述，本项目构建了番茄的蛋白质亚硝基化修饰网络，并揭示了番茄侧枝生长过程中NO的来源和信号放大机理，及其通过改变腋芽IAA和CKs的信号稳态控制侧枝生长方面的生理分子机制，为少侧枝番茄的选育提供目标基因和理论基础。