S-Nitroso-Proteome in Poplar Leaves in Response to Acute Ozone Stress

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = −3.6) and caffeic acid O-methyltransferase (−3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.

蛋白质S-亚硝基化（Protein S-nitrosylation）是一氧化氮（nitric oxide，NO）与蛋白质半胱氨酸残基的共价结合反应，是动植物细胞内NO信号传导的主要机制之一。本研究通过生物素-switch实验（biotin switch assay）与无标记液相色谱-串联质谱（label-free LC-MS/MS）的联合分析，解析了木本模式植物银灰杨（Populus x canescens）的S-亚硝基化蛋白质组（S-nitroso-proteome）。在正常培养条件下，杨树叶片与愈伤组织中组成型S-亚硝基化的蛋白质涵盖了初级代谢与次级代谢的全部通路。本研究采用急性臭氧熏蒸处理，以诱导活性氧（reactive oxygen species，ROS）介导的S-亚硝基化蛋白质组变化。该处理改变了杨树叶片的总亚硝酸盐与亚硝基硫醇含量，并影响了32种S-亚硝基化蛋白质的稳态平衡。多变量数据分析结果显示，臭氧暴露对杨树叶片蛋白质的S-亚硝基化状态产生负调控作用：与对照组相比，23种蛋白质发生了去亚硝基化（de-nitrosylated）修饰，另有9种蛋白质的S-亚硝基化水平显著升高。苯丙氨酸解氨酶2（Phenylalanine ammonia-lyase 2，log₂(臭氧组/对照组) = -3.6）与咖啡酸O-甲基转移酶（caffeic acid O-methyltransferase，log₂(臭氧组/对照组) = -3.4）分别是催化苯丙烷类代谢与后续木质素生物合成通路关键步骤的核心酶，二者在臭氧胁迫下均发生了去亚硝基化修饰。通过对体内（in vivo）与体外（in vitro）苯丙氨酸解氨酶活性的检测分析发现，急性臭氧胁迫下苯丙氨酸解氨酶活性的升高部分由去亚硝基化修饰调控，这一调控机制可能在臭氧暴露后的数分钟内，促进苯丙烷类代谢通路的代谢流水平提升。