Figure 2: RHC1 physically interacts with CAs and HT1 and functions upstream of HT1.
From: A molecular pathway for CO2 response in Arabidopsis guard cells
(a) RHC1 interaction with CA4 and HT1 as assayed in BiFC using Arabidopsis mesophyll protoplasts. Protoplasts were transfected with different combinations of expression vectors encoding RHC1-nVenus, CA4-cCFP, HT1-cCFP, OST1-cCFP, nVenus or cCFP. Scale bars, 5 μm. (b) GST pull-down assay. Immobilized GST, or RHC1 N- or C-terminal region fused to GST (RHC1(NT)–GST or RHC1(CT)–GST, respectively) was incubated with his-tagged CA4 (CA4-His). After washing, CA4 bound to fusion proteins was eluted and resolved by SDS–PAGE and visualized by western blot analysis with anti-His antibody. (c) Interaction of RHC1 and HT1 in a yeast-based split-ubiquitin system. pTSU2-APP/pNubG-Fe65 and pBT3-RHC1/pPR3 were used as positive and negative controls, respectively. (d) Selection of rhc1 ht1-2 double-mutant. Gel image shows genotyping by PCR of WT, rhc1, ht1-2 and rhc1 ht1-2 plants. (e) Time-resolved stomatal conductance analyses in WT, rhc1, ht1-2 and rhc1 ht1-2 double-mutant leaves in response to changes of CO2 concentrations (n=3). The data represent means±s.e.m.
