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

Ca 2+ 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 Ca 2+ /H + transporters CATION EXCHANGER1 (CAX1) and CAX3 sequester Ca 2+ from the cytosol into the vacuole, but it is not known what transporter mediates the initial Ca 2+ influx from the apoplast to the cytosol. Here, we report that Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL2 ( CNGC2 ) encodes a protein with Ca 2+ influx channel activity and is expressed in the leaf areas surrounding the free endings of minor veins, which is the primary site for Ca 2+ unloading from the vasculature and influx into leaf cells. Under hydroponic growth conditions, with 0.1 mm Ca 2+ , both Arabidopsis cngc2 and cax1cax3 loss-of-function mutants grew normally. Increasing the Ca 2+ concentration to 10 mm induced H 2 O 2 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 Ca 2+ overaccumulation was found in the leaves of cngc2 and cax1cax 3 but not the wild type under the 10 mm Ca 2+ condition, as monitored by Oregon Green BAPTA 488 5N, a low-affinity and membrane-impermeable Ca 2+ probe. Our results indicate that CNGC2 likely has no direct roles in leaf development or the hypersensitive response but, instead, that CNGC2 could mediate Ca 2+ influx into leaf cells. Finally, the in vivo extracellular Ca 2+ imaging method developed in this study provides a new tool for investigating Ca 2+ dynamics in plant cells.