OPEN STOMATA 1 phosphorylates CYCLIC NUCLEOTIDE-GATED CHANNELs to trigger Ca2+ signaling for abscisic acid-induced stomatal closure in Arabidopsis.

Multiple cyclic nucleotide-gated channels (CNGCs) are abscisic acid (ABA)-activated Ca2+ channels in Arabidopsis (Arabidopsis thaliana) guard cells. In particular, CNGC5, CNGC6, CNGC9, and CNGC12 are essential for ABA-specific cytosolic Ca2+ signaling and stomatal movements. However, the mechanisms underlying ABA-mediated regulation of CNGCs and Ca2+ signaling are still unknown. In this study, we identified the Ca2+-independent protein kinase OPEN STOMATA 1 (OST1) as a CNGC activator in Arabidopsis. OST1-targeted phosphorylation sites were identified in CNGC5, CNGC6, CNGC9, and CNGC12. These CNGCs were strongly inhibited by Ser-to-Ala mutations and fully activated by Ser-to-Asp mutations at the OST1-targeted sites. The overexpression of individual inactive CNGCs (iCNGCs) under the UBIQUITIN10 promoter in wild-type Arabidopsis conferred a strong dominant-negative-like ABA-insensitive stomatal closure phenotype. In contrast, expressing active CNGCs (aCNGCs) under their respective native promoters in the cngc5-1 cngc6-2 cngc9-1 cngc12-1 quadruple mutant fully restored ABA-activated cytosolic Ca2+ oscillations and Ca2+ currents in guard cells, and rescued the ABA-insensitive stomatal movement mutant phenotypes. Thus, we uncovered that ABA elicits cytosolic Ca2+ signaling via an OST1-CNGC module, in which OST1 functions as a convergence point of the Ca2+-dependent and -independent pathways in Arabidopsis guard cells.

Multiple cyclic nucleotide-gated channels function as ABA-activated Ca2+ channels required for ABA-induced stomatal closure in Arabidopsis.

Abscisic acid (ABA)-activated inward Ca2+-permeable channels in the plasma membrane (PM) of guard cells are required for the initiation and regulation of ABA-specific cytosolic Ca2+ signaling and stomatal closure in plants. But the identities of the PM Ca2+ channels are still unknown. We hypothesized that the ABA-activated Ca2+ channels consist of multiple CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) proteins from the CNGC family, which is known as a Ca2+-permeable channel family in Arabidopsis (Arabidopsis thaliana). In this research, we observed high expression of multiple CNGC genes in Arabidopsis guard cells, namely CNGC5, CNGC6, CNGC9, and CNGC12. The T-DNA insertional loss-of-function quadruple mutant cngc5-1 cngc6-2 cngc9-1 cngc12-1 (hereafter c5/6/9/12) showed a strong ABA-insensitive phenotype of stomatal closure. Further analysis revealed that ABA-activated Ca2+ channel currents were impaired, and ABA-specific cytosolic Ca2+ oscillation patterns were disrupted in c5/6/9/12 guard cells compared with in wild-type guard cells. All ABA-related phenotypes of the c5/6/9/12 mutant were successfully rescued by the expression of a single gene out of the four CNGCs under the respective native promoter. Thus, our findings reveal a type of ABA-activated PM Ca2+ channel comprising multiple CNGCs, which is essential for ABA-specific Ca2+ signaling of guard cells and ABA-induced stomatal closure in Arabidopsis.

Three CNGC Family Members, CNGC5, CNGC6, and CNGC9, Are Required for Constitutive Growth of Arabidopsis Root Hairs as Ca2+-Permeable Channels.

The genetic identities of Ca2+ channels in root hair (RH) tips essential for constitutive RH growth have remained elusive for decades. Here, we report the identification and characterization of three cyclic nucleotide-gated channel (CNGC) family members, CNGC5, CNGC6, and CNGC9, as Ca2+ channels essential for constitutive RH growth in Arabidopsis. We found that the cngc5-1cngc6-2cngc9-1 triple mutant (designated shrh1) showed significantly shorter and branching RH phenotypes as compared with the wild type. The defective RH growth phenotype of shrh1 could be rescued by either the expression of CNGC5, CNGC6, or CNGC9 single gene or by the supply of high external Ca2+, but could not be rescued by external K+ supply. Cytosolic Ca2+ imaging and patch-clamp data in HEK293T cells showed that these three CNGCs all function as Ca2+-permeable channels. Cytosolic Ca2+ imaging in growing RHs further showed that the Ca2+ gradients and their oscillation in RH tips were dramatically attenuated in shrh1 compared with those in the wild type. Phenotypic analysis revealed that these three CNGCs are Ca2+ channels essential for constitutive RH growth, with different roles in RHs from the conditional player CNGC14. Moreover, we found that these three CNGCs are involved in auxin signaling in RHs. Taken together, our study identified CNGC5, CNGC6, and CNGC9 as three key Ca2+ channels essential for constitutive RH growth and auxin signaling in Arabidopsis.

A cyclic nucleotide-gated channel mediates cytoplasmic calcium elevation and disease resistance in rice.

The transient elevation of cytoplasmic calcium is essential for pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). However, the calcium channels responsible for this process have remained unknown. Here, we show that rice CDS1 (CELL DEATH and SUSCEPTIBLE to BLAST 1) encoding OsCNGC9, a cyclic nucleotide-gated channel protein, positively regulates the resistance to rice blast disease. We show that OsCNGC9 mediates PAMP-induced Ca2+ influx and that this event is critical for PAMPs-triggered ROS burst and induction of PTI-related defense gene expression. We further show that a PTI-related receptor-like cytoplasmic kinase OsRLCK185 physically interacts with and phosphorylates OsCNGC9 to activate its channel activity. Our results suggest a signaling cascade linking pattern recognition to calcium channel activation, which is required for initiation of PTI and disease resistance in rice.

The S-Type Anion Channel ZmSLAC1 Plays Essential Roles in Stomatal Closure by Mediating Nitrate Efflux in Maize.

Diverse stimuli induce stomatal closure by triggering the efflux of osmotic anions, which is mainly mediated by the main anion channel SLAC1 in plants, and the anion permeability and selectivity of SLAC1 channels from several plant species have been reported to be variable. However, the genetic identity as well as the anion permeability and selectivity of the main S-type anion channel ZmSLAC1 in maize are still unknown. In this study, we identified GRMZM2G106921 as the gene encoding ZmSLAC1 in maize, and the maize mutants zmslac1-1 and zmslac1-2 harboring a mutator (Mu) transposon in ZmSLAC1 exhibited strong insensitive phenotypes of stomatal closure in response to diverse stimuli. We further found that ZmSLAC1 functions as a nitrate-selective anion channel without obvious permeability to chloride, sulfate and malate, clearly different from SLAC1 channels of Arabidopsis thaliana, Brassica rapa ssp. chinensis and Solanum lycopersicum L. Further experimental data show that the expression of ZmSLAC1 successfully rescued the stomatal movement phenotypes of the Arabidopsis double mutant atslac1-3atslah3-2 by mainly restoring nitrate-carried anion channel currents of guard cells. Together, these findings demonstrate that ZmSLAC1 is involved in stomatal closure mainly by mediating the efflux of nitrate in maize.

S-type Anion Channels SLAC1 and SLAH3 Function as Essential Negative Regulators of Inward K+ Channels and Stomatal Opening in Arabidopsis.

Drought stress induces stomatal closure and inhibits stomatal opening simultaneously. However, the underlying molecular mechanism is still largely unknown. Here we show that S-type anion channels SLAC1 and SLAH3 mainly inhibit inward K+ (K+in) channel KAT1 by protein-protein interaction, and consequently prevent stomatal opening in Arabidopsis. Voltage-clamp results demonstrated that SLAC1 inhibited KAT1 dramatically, but did not inhibit KAT2. SLAH3 inhibited KAT1 to a weaker degree relative to SLAC1. Both the N terminus and the C terminuses of SLAC1 inhibited KAT1, but the inhibition by the N terminus was stronger. The C terminus was essential for the inhibition of KAT1 by SLAC1. Furthermore, drought stress strongly up-regulated the expression of SLAC1 and SLAH3 in Arabidopsis guard cells, and the over-expression of wild type and truncated SLAC1 dramatically impaired K+in currents of guard cells and light-induced stomatal opening. Additionally, the inhibition of KAT1 by SLAC1 and KC1 only partially overlapped, suggesting that SLAC1 and KC1 inhibited K+in channels using different molecular mechanisms. Taken together, we discovered a novel regulatory mechanism for stomatal movement, in which singling pathways for stomatal closure and opening are directly coupled together by protein-protein interaction between SLAC1/SLAH3 and KAT1 in Arabidopsis.