Cyclic nucleotide-gated ion channel 6 mediates thermotolerance in Arabidopsis seedlings by regulating nitric oxide production via cytosolic calcium ions

Abstract

Background We previously reported the involvement of nitric oxide (NO) and cyclic nucleotide-gated ion channel 6 (CNGC6) in the responses of plants to heat shock (HS) exposure. To elucidate their relationship with heat tolerance in Arabidopsis thaliana , we examined the effects of HS on several groups of seedlings: wild type, cngc6 , and cngc6 complementation and overexpression lines. Results After HS exposure, the level of NO was lower in cngc6 seedlings than in wild-type seedlings but significantly elevated in the transgenic lines depending on CNGC6 expression level. The treatment of seeds with calcium ions (Ca 2+ ) enhanced the NO level in Arabidopsis seedlings under HS conditions, whereas treatment with EGTA (a Ca 2+ chelator) reduced it, implicating that CNGC6 stimulates the accumulation of NO depending on an increase in cytosolic Ca 2+ ([Ca 2+ ] cyt ). This idea was proved by phenotypic observations and thermotolerance testing of transgenic plants overexpressing NIA2 and NOA1, respectively, in a cngc6 background. Western blotting indicated that CNGC6 stimulated the accumulation of HS proteins via NO. Conclusion These data indicate that CNGC6 acts upstream of NO in the HS pathway, which improves our insufficient knowledge of the initiation of plant responses to high temerature. Electronic supplementary material The online version of this article (10.1186/s12870-019-1974-9) contains supplementary material, which is available to authorized users.

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

“…Under HS at 37°C, the inward Ca 2+ current was swiftly elevated to −375 pA in the wild-type within 1 min. However, only a slight increase (to −171 pA) was observed in cngc6 (Figures 6A,B), in accordance with our previous reports (Gao et al, 2012;Peng et al, 2019;Niu et al, 2020). In the rbohB/D double mutant with low internal H 2 O 2 levels, the Ca 2+ currents exhibited similar changing trends to those in the wild-type under both of normal and HS conditions (Figure 6C).…”

“…For Western blotting, 10-day-old seedlings were kept at 37°C for 2 h and collected for the analyses of HSP accumulation. All the experiments were repeated at least three times, and there were three independent biological replicates in each repeat (Peng et al, 2019).…”

“…Protoplasts were isolated as described previously (Demidchik and Tester, 2002) from 1 cm long of root tips of Arabidopsis seedlings cultivated vertically at 22°C for 8 days. Whole-cell voltage patchclamping was carried out as described previously (Gao et al, 2012;Peng et al, 2019;Niu et al, 2020) with minor modification. Patch-clamp pipettes were pulled on a vertical electrode puller.…”

“…To test this hypothesis, the H 2 O 2 levels were examined in the wild-type, cngc6, and COM12 seedlings pre-treated with 5 mM CaCl 2 or 2 mM EGTA (a Ca 2+ chelator) before germination as described previously (Liu et al, 2005;Peng et al, 2019). Fluorescence analysis showed that under normal growth conditions, the H 2 O 2 levels in wild-type, cngc6, and COM12 seedlings were rather stable.…”

“…To confirm whether H 2 O 2 influences the action of heatresponsive Ca 2+ -permeable channels, we determined the effects of internal H 2 O 2 on the function of CNCG6 in the PM of root protoplasts of Arabidopsis with the whole-cell patchclamp technique (Gao et al, 2012;Peng et al, 2019). Under normal conditions at 22°C, the Ca 2+ current in cngc6 (−136 pA) was lower than in the wild-type (−178 pA) at −200 mV.…”