Cyclic Nucleotide-Gated Ion Channel 6 Mediates Thermotolerance in Arabidopsis Seedlings by Regulating Hydrogen Peroxide Production via Cytosolic Calcium Ions

We previously reported the involvement of cyclic nucleotide-gated ion channel 6 (CNGC6) and hydrogen peroxide (H₂O₂) in plant responses to heat shock (HS). To demonstrate their relationship with plant thermotolerance, we assessed the effect of HS on several groups of Arabidopsis (Arabidopsis thaliana) seedlings: wild-type, cngc6 mutant, and its complementation line. Under exposure to HS, the level of H₂O₂ was lower in the cngc6 mutant seedlings than in the wild-type (WT) seedlings but obviously increased in the complementation line. The treatment of Arabidopsis seeds with calcium ions (Ca²⁺) increased the H₂O₂ levels in the seedlings under HS treatment, whereas treatment with a Ca²⁺ chelator (EGTA) inhibited it, indicating that CNGC6 may stimulate the accumulation of H₂O₂ in a manner dependent on an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt). This point was verified by phenotypic observations and thermotolerance testing with transgenic plants overexpressing AtRbohB and AtRbohD (two genes involved in HS-responsive H₂O₂ production), respectively, in a cngc6 background. Real-time reverse transcription-polymerase chain reactions and Western blotting suggested that CNGC6 enhanced the gene transcription of HS factors (HSFs) and the accumulation of HS proteins (HSPs) via H₂O₂. These upon results indicate that H₂O₂ acts downstream of CNGC6 in the HS signaling pathway, increasing our understanding of the initiation of plants responses to high temperatures.