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

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.

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⁺.

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.