Cyclic Nucleotide-Gated Ion Channel 6 Mediates Thermotolerance in Arabidopsis Seedlings by Regulating Hydrogen Peroxide Production via Cytosolic Calcium Ions
Wen-Xu Wang, Jiaojiao Zhang, Lijuan Ai, Dan Wu, Bing Li, Lingang Zhang, Liqun Zhao
Abstract
We previously reported the involvement of cyclic nucleotide-gated ion channel 6 (CNGC6) and hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 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 (Ca 2+ ) increased the H 2 O 2 levels in the seedlings under HS treatment, whereas treatment with a Ca 2+ chelator (EGTA) inhibited it, indicating that CNGC6 may stimulate the accumulation of H 2 O 2 in a manner dependent on an increase in cytosolic Ca 2+ ([Ca 2+ ] cyt ). This point was verified by phenotypic observations and thermotolerance testing with transgenic plants overexpressing AtRbohB and AtRbohD (two genes involved in HS-responsive H 2 O 2 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 H 2 O 2 . These upon results indicate that H 2 O 2 acts downstream of CNGC6 in the HS signaling pathway, increasing our understanding of the initiation of plants responses to high temperatures.