CNGCs as stomatal gatekeepers during ABA signaling

Calcium (Ca2+) serves as central and ubiquitous second messenger in all eukaryotes, wherein rapid changes in free cytosolic Ca2+ levels are achieved via the coordinated action of channels, antiporters, and pumps. These changes are sensed by Ca2+ sensor proteins, which act as relays for further downstream signaling. While essentially all stimuli induce Ca2+ signals in plants, the molecular nature of relevant Ca2+-permeable channel(s) has remained stubbornly elusive in many cases. Plants lack the voltage-gated calcium channels of metazoans but do possess many nonselective, Ca2+-permeable cation channels, including those of the cyclic nucleotide-gated channel (CNGC) family. Named for their topological similarity to mammalian CNGs, CNGCs have been implicated as Ca2+ channels in diverse pathways, though extensive genetic analysis of specific CNGCs has been hindered by suspected redundancy (Jarratt-Barnham et al., 2021). In this issue of The Plant Cell, Yan-Qiu Tan and colleagues (Tan et al., 2022) have used a combination of expression profiling and dominant-negative alleles to overcome potential genetic redundancy and thereby examine the roles of several guard cell-expressed CNGCs. Perception of the phytohormone abscisic acid (ABA) initiates Ca2+ signaling within guard cells that ultimately results in anion efflux, loss of turgor, and stomatal closure. While the Ca2+-permeable channels that function in pathogen-triggered stomatal closure have been recently identified (Thor et al., 2020), those specifically involved in ABA-induced closure are poorly studied. The authors assayed the promoter activities of all 20 CNGC family members from Arabidopsis thaliana using both β-glucuronidase (GUS) reporter lines and transcriptional analyses, which revealed five members (CNGC2, 5, 6, 9, and 12) with enriched guard cell expression. In accordance with these findings, both CNGC5 and 6 had been previously implicated in guard cell signaling (Wang et al., 2013). The authors next generated higher-order knockouts of four ofthese CNGCs as well as dominant-negative CNGC6 alleles by mutating conserved residues required for channel function. The results of this combined approach indicated that these channels likely contribute to ABA-induced stomatal closure (see Figure), as both the Ca2+ signals and control of water loss compromised in the mutants. Interestingly, the Ca2+ signals induced in guard cells by reactive oxygen species (ROS) treatment were not affected in cngc5/6/9/12 mutants, indicating that these channels act downstream of ABA-induced ROS accumulation (Tan et al., 2022). CNGC5, 6, 9, and 12 function in guard cell ABA signaling. A, CNGC guard cell expression profiles as shown by promoter-GUS reporters. B, ABA-regulated water loss compromised in cngc5/6/9/12 knockout and CNGC6 dominant-negative (CNGC6-DN1) mutants. C, ABA-induced Ca2+ currents in guard cells are disrupted in knockout (cngc5/6/9/12) and dominant-negative (DEL54) mutants. Adapted from (Tan et al., 2022), Figures 5, 7, and Supplementary Figures S1 and S5. These results underscore the complexity of CNGC function, both in guard cell/ABA signaling and beyond. In addition, the identity of the CNGC family members identified in this study suggests that genetic redundancy may not be simply determined by homology: CNGC5 and 6 are close homologs; however, CNGC9 and 12 are from distinct subfamilies (Mäser et al., 2001). Given that other putatively Ca2+-permeable channels are also found in large gene families in plants, expression profiling coupled with engineered dominant-negative genetic analyses may provide a useful approach for identifying additional channel combinations that are involved in other specific tissues, cell types, and/or biological processes.

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