Arabidopsis thaliana cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca2+/H+) antiporters that contribute to cellular Ca2+ homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remained elusive. Here, we demonstrate that expression of CAX1 and CAX3 occurs in guard cells. Additionally, CAX1 and CAX3 are co-expressed in mesophyll tissue in response to wounding or flg22 treatment, due to the induction of CAX3 expression. Having shown that the transporters can be co-expressed in the same cells, we demonstrate that CAX1 and CAX3 can form homomeric and heteromeric complexes in plants. Consistent with the formation of a functional CAX1-CAX3 complex, CAX1 and CAX3 integrated into the yeast genome suppressed a Ca2+-hypersensitive phenotype of mutants defective in vacuolar Ca2+ transport, and demonstrated enzyme kinetics different from those of either CAX protein expressed by itself. We demonstrate that the interactions between CAX proteins contribute to the functioning of stomata, because stomata were more closed in cax1-1, cax3-1, and cax1-1/cax3-1 loss-of-function mutants due to an inability to buffer Ca2+ effectively. We hypothesize that the formation of CAX1-CAX3 complexes may occur in the mesophyll to affect intracellular Ca2+ signaling during defense responses
