Transcriptome analysis of apoplastic reactive oxygen species signaling and dissection of its regulatory pathways

As sessile organism, plants evolved a highly complicated signaling system to cope with unfavorable and fluctuating environmental conditions. Rapid and transient Reactive Oxygen Species (ROS) burst is a common response to both biotic and abiotic stresses. Plants exposed with O3 could trigger extracellular similar ROS production through cell wall peroxidases and NPADPH oxidases, resulting in changes in the gene expression and cell death. Whereas ROS induced cell death is not simply due to its toxicity, rather due to interplay with several other signaling pathways, such as salicylic acid (SA), jasmonic acid (JA) and ethylene signaling pathways. Furthermore, the three hormones have both synergistic and antagonistic interactions, where the suppression of JA signaling by SA is the mostly studied. In addition, ethylene promotes cell death while JA has a protective role upon O3 exposure. The role of SA is more complicated; depending on the genetic background it can have either cell death promoting or protecting roles. Hence, a clean system to deliver apoplastic ROS is required to study the role of ROS apart from con-current activation of other signaling pathways. Arabidopsis thaliana offer a convenient system to study apoplastic ROS signaling due to the availability of hormone signaling or biosynthesis mutants including the JA receptor mutant coi1-16 (CORONATINE INSENSITIVE1), the essential ethylene signaling mutant ein2 (ETHYLENE INSENSITIVE2), the SA biosynthesis mutant sid2 (SALICYLIC ACID INDUCTION DEFICIENT2 also known as ISOCHORISMATE SYNTHASE1), and essential regulators in SA/JA/ethylene-induced defense response triple mutant tga2 tga5 tga6 (Clade II TGA transcription factors). Here we used a combination of transcriptome analysis, cell death assays and mutant analysis to systematically quantified the contribution of hormone signaling in relation to apoplastic ROS signaling, identified transcription factors (TFs) involved in ROS regulation and dissected the components involved in defense hormones associated cell death. Overall design: Transcriptome profiling of ozone response using two arabidopsis triple mutants coi1-16 ein2 sid2 and tga2 tga5 tga6 related to Jasmonic acid, salicylic acid and ethylene signaling to identify hormone-independant apoplastic ROS signaling作为固着生物，植物演化出了极为复杂的信号系统以应对不利且波动的环境条件。快速且瞬时的活性氧（Reactive Oxygen Species, ROS）爆发是植物应对生物与非生物胁迫的常见响应。暴露于臭氧（O₃）的植物可通过细胞壁过氧化物酶和NADPH氧化酶触发胞外类似的ROS产生，进而引发基因表达改变与细胞死亡。而ROS诱导的细胞死亡并非单纯由其毒性所致，而是与水杨酸（salicylic acid, SA）、茉莉酸（jasmonic acid, JA）以及乙烯等多条信号通路相互作用的结果。此外，这三类激素之间既存在协同作用，也存在拮抗交互，其中SA对JA信号通路的抑制是研究最为广泛的机制。进一步而言，乙烯可促进细胞死亡，而JA在臭氧暴露下则发挥保护作用；SA的角色更为复杂，其可根据遗传背景的不同，分别起到促进细胞死亡或保护细胞的作用。因此，亟需一套能够精准递送质外体活性氧的实验系统，以便在不同步激活其他信号通路的前提下研究ROS的功能。拟南芥（Arabidopsis thaliana）因其可获取的激素信号或生物合成突变体，成为研究质外体ROS信号传导的理想模型系统，这些突变体包括JA受体突变体coi1-16（CORONATINE INSENSITIVE1）、核心乙烯信号通路突变体ein2（ETHYLENE INSENSITIVE2）、SA生物合成突变体sid2（SALICYLIC ACID INDUCTION DEFICIENT2，又名ISOCHORISMATE SYNTHASE1），以及参与SA/JA/乙烯诱导的防御反应的核心调控因子三重突变体tga2 tga5 tga6（Clade II TGA转录因子）。本研究结合转录组分析、细胞死亡实验与突变体分析，系统量化了激素信号通路与质外体ROS信号通路的相对贡献，鉴定了参与ROS调控的转录因子，并解析了与防御激素相关的细胞死亡所涉及的分子组分。实验设计概述：以与茉莉酸、水杨酸及乙烯信号通路相关的两株拟南芥三重突变体coi1-16 ein2 sid2与tga2 tga5 tga6为材料，开展臭氧响应的转录组分析，以鉴定不依赖于激素的质外体活性氧信号通路。