CNGC2 Is a Ca²⁺ Influx Channel That Prevents Accumulation of Apoplastic Ca²⁺ in the Leaf

Ca²⁺ is absorbed by roots and transported upward through the xylem to the apoplastic space of the leaf, after which it is deposited into the leaf cell. In Arabidopsis (Arabidopsis thaliana), the tonoplast-localized Ca²⁺/H⁺ transporters CATION EXCHANGER1 (CAX1) and CAX3 sequester Ca²⁺ from the cytosol into the vacuole, but it is not known what transporter mediates the initial Ca²⁺ influx from the apoplast to the cytosol. Here, we report that Arabidopsis CYCLIC NUCLEOTIDEGATED CHANNEL2 (CNGC2) encodes a protein with Ca²⁺ influx channel activity and is expressed in the leaf areas surrounding the free endings of minor veins, which is the primary site for Ca²⁺ unloading from the vasculature and influx into leaf cells. Under hydroponic growth conditions, with 0.1 mM Ca²⁺, both Arabidopsis cngc2 and cax1cax3 loss-of-function mutants grew normally. Increasing the Ca²⁺ concentration to 10 mM induced H₂O₂ accumulation, cell death, and leaf senescence and partially suppressed the hypersensitive response to avirulent pathogens in the mutants but not in the wild type. In vivo apoplastic Ca²⁺ overaccumulation was found in the leaves of cngc2 and cax1cax3 but not the wild type under the 10 mM Ca²⁺ condition, as monitored by Oregon Green BAPTA 488 5N, a low-affinity and membrane-impermeable Ca²⁺ probe. Our results indicate that CNGC2 likely has no direct roles in leaf development or the hypersensitive response but, instead, that CNGC2 could mediate Ca²⁺ influx into leaf cells. Finally, the in vivo extracellular Ca²⁺ imaging method developed in this study provides a new tool for investigating Ca²⁺ dynamics in plant cells.