Ca2+-dependent H2O2 response in roots and leaves of barley - a transcriptomic investigation

Background Ca2+ and H2O2 are second messengers that regulate a wide range of cellular events in response to different environmental and developmental cues. In plants, stress-induced H2O2 has been shown to initiate characteristic Ca2+ signatures; however, a clear picture of the molecular connection between H2O2-induced Ca2+ signals and H2O2-induced cellular responses is missing, particularly in cereal crops such as barley. Here, we employed RNA-seq analyses to identify transcriptome changes in roots and leaves of barley after H2O2 treatment under conditions that inhibited the formation of cytosolic Ca2+ transients. To that end, plasma membrane Ca2+ channels were blocked by LaCl3 application prior to stimulation of barley tissues with H2O2. Results We examined the expression patterns of 4246 genes that had previously been shown to be differentially expressed upon H2O2 application. Here, we further compared their expression between H2O2 and LaCl3 + H2O2 treatment. Genes showing expression patterns different to the previous study were considered to be Ca2+-dependent H2O2-responsive genes. These genes, numbering 331 in leaves and 1320 in roots, could be classified in five and four clusters, respectively. Expression patterns of several genes from each cluster were confirmed by RT-qPCR. We furthermore performed a network analysis to identify potential regulatory paths from known Ca2+-related genes to the newly identified Ca2+-dependent H2O2 responsive genes, using the recently described Stress Knowledge Map. This analysis indicated several transcription factors as key points of the responses mediated by the cross-talk between H2O2 and Ca2+. Conclusion Our study indicates that about 70% of the H2O2-responsive genes in barley roots require a transient increase in cytosolic Ca2+ concentrations for alteration in their transcript abundance, whereas in leaves, the Ca2+ dependency was much lower at about 33%. Targeted gene analysis and pathway modeling identified not only known components of the Ca2+ signaling cascade in plants but also genes that are not yet connected to stimuli-associated signaling. Potential key transcription factors identified in this study can be further analyzed in barley and other crops to ultimately disentangle the underlying mechanisms of H2O2-associated signal transduction mechanisms. This could aid breeding for improved stress resistance to optimize performance and productivity under increasing climate challenges.