Deciphering the role of H2O2-signalling originating from different cellular compartments and cell types

MA2379/11-1项目的全基因组转录组分析表明，不同细胞区室产生的H2O2可调节特定的信号通路。首先，我们利用ros应答基因和转录因子（TF）平台，通过qRT-PCR确定了对叶绿体产生的h2o2的早期（诱导30分钟后）应答基因（Balazadeh et al., 2012）。其次，我们在诱导H2O2产生8小时后进行了广泛的转录组学分析，使我们能够鉴定出由叶绿体或过氧化物酶体产生的H2O2专门调节的诱导基因（Sewelan等，2014）。数据分析表明，叶绿体产生的H2O2具有重要的感觉和信号功能，而过氧化物酶体产生的H2O2最有可能诱导胁迫耐受反应。在接下来的一段时间里，我们将重点关注在之前的工作中确定的叶绿体h2o2信号网络的选定参与者。遗传、分子和生化方法的结合将使我们能够对最初确定的基因/遗传网络进行功能表征。一方面，我们将重点研究诱导H2O2产生后30分钟上调幅度最大的基因。该基因编码一种推测为u -box型E3泛素连接酶，其分子功能和生物学作用尚不清楚。我们将重点关注该蛋白的分子和功能特征，以揭示泛素化和细胞器h2o2信号传导之间的新联系。另一方面，我们之前的结果表明，虽然只有3个TF编码基因在过氧化物酶体中被H2O2产生特异性诱导，但在叶绿体诱导的基因中，有30个基因（14%）产生H2O2编码的TF。在GO5植物中诱导的大量tf编码基因表明，来自叶绿体的H2O2具有控制基因转录的关键特异性信号作用。由于这些TFs是GO5植物中其他表达上调基因对h2o2反应性的主要候选基因，因此我们将重点研究这些TFs在植物氧化应激反应中的作用。这个子项目的主要目的是鉴定由叶绿体产生的中等水平H2O2调控的靶基因。

The genome-wide transcriptome analysis conducted in project MA2379/11-1 indicated that H2O2 produced in different cellular compartments modulates specific signalling pathways. First, we identified early (after 30 min of induction) responding genes to chloroplastic produced-H2O2 by qRT-PCR using ROS-responsive genes and transcription factor (TF) platforms (Balazadeh et al., 2012). Second, our wide transcriptomic analysis conducted after 8 h of induction of H2O2 production, allowed us to identify induced genes exclusively modulated by chloroplastic or peroxisomal produced H2O2 (Sewelan et al., 2014). Analysis of the data suggests that chloroplastic produced H2O2 has crucial sensory and signalling functions, while peroxisomal produced H2O2 most likely induces stress tolerance responses. During the next period, we will focus on selected players of chloroplastic H2O2-signalling networks that were identified in the previous work. A combination of genetic, molecular, and biochemical approaches will enable us to functionally characterize initially identified genes/genetic networks.On the one hand, we will specifically focus on the most highly up-regulated gene at 30 min after induction of H2O2 production. This gene encodes a putative U-box-type E3 ubiquitin ligase, the molecular function and biological role of which is still unknown. We will focus on the molecular and functional characterization of this protein to shed light on a novel link between ubiquitination and organellar-H2O2 signalling.On the other hand, our previous results indicate that while only three TF encoding-genes were specifically induced by H2O2 production in peroxisomes, 30 genes (14%) of the genes induced by chloroplastic produced H2O2 encoded TFs. The relatively large number of TF-encoding genes induced in GO5 plants indicates that H2O2 originating from chloroplasts has a critical specific signalling role controlling gene transcription. As these TFs are primary candidates to govern the H2O2-responsiveness of the other genes whose expression was up-regulated in the GO5 plants, we will focus on the role of these TFs during plant oxidative stress responses. A main aim of this subproject is the identification of target genes regulated by moderate levels of H2O2 produced from chloroplasts.