Arabidopsis cyclic nucleotide-gated channel 6 is negatively modulated by multiple calmodulin isoforms during heat shock

Abstract

Arabidopsis calmodulin isoforms CaM2/3/5 and CaM7 negatively regulate Ca2+ conductivity during heat shock by binding to an atypical IQ motif in cyclic nucleotide-gated ion channel 6.

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).…”

“…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.…”

Arabidopsis mtHSC70-1 physically interacts with the Cox2 subunit of cytochrome c oxidase

“…The glutathione S-transferase (GST)-mtHSC70-1 fusion proteins and maltose-binding protein (MBP)-Cox2 fusion proteins were expressed in Escherichia coli BL21, and purified using glutathione agarose (Sigma-Aldrich, St Louis, MO, USA) or amylose resin (NEB, Ipswich, MA, England). Pull-down experiment was performed according to the protocol of Niu, et al 15 The pull-down product was analyzed by immunoblotting using a mouse monoclonal anti-GST antibody (Sigma-Aldrich) or a mouse monoclonal anti-MBP antibody (Sigma-Aldrich). The result showed that GST-mtHSC70-1 were pulled-down by amylose resin with MBP-Cox2 but were not pulled-down by amylose resin with MBP alone (Figure 2(a)), indicating that mtHSC70-1 is able to directly bind to Cox2.…”

“…Meanwhile, the interaction between mtHSC70-2 and Cox2 (Figure 2(b)) and the interaction between mtHSC70-1 and Cox1 (Figure 2(c)) were also tested; however, no positive reactivity was found. In addition, the binding of mtHSC70-1 or mtHSC70-2 to Cox1 or Cox2 was also examined through firefly luciferase complementation imaging (LCI) assay in tobacco leaves according to the protocol of Niu, et al 15 The mtHSC70-1/2 and Cox1/2 coding sequence were cloned into the pCAMBIA-NLuc and pCAMBIA-CLuc vectors. The resulting constructs were separately introduced into the Agrobacterium tumefaciens strain GV3101 and then infiltrated into tobacco (N. benthamiana) leaves.…”

Plant Cyclic Nucleotide-Gated Channels: New Insights on Their Functions and Regulation

“…1). Structural modeling using solved animal CNGC/HCN structures has improved our understanding (Hua et al, 2003b;Baxter et al, 2008;Niu et al, 2020) but cannot accurately predict the structure of the important CT CaM binding domains. Therefore, resolving high-resolution structures of plant CNGCs will help our understanding of their gating and regulation mechanisms.…”