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

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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.