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.

Protein kinase OsSAPK8 functions as an essential activator of S-type anion channel OsSLAC1, which is nitrate-selective in rice.

MAIN CONCLUSION: OsSAPK8 is an essential activator of OsSLAC1 by phosphorylation, and OsSLAC1 is a nitrate-selective anion channel. S-type anion channel AtSLAC1 and protein kinase AtOST1 have been well-characterized as two core components of ABA signaling cascade in Arabidopsis guard cells, and AtOST1 functions as a main upstream activator of AtSLAC1 for drought stress- and ABA-induced stomata closure. However, the identity of the ortholog of AtOST1 in rice, the main activator of OsSLAC1, is still unknown. Here, we report that protein kinase OsSAPK8 interacts with and activates OsSLAC1 mainly by phosphorylating serine 129 (S129) of OsSLAC1, and this phosphorylating site corresponds to the specific phosphorylating site serine 120 (S120) of AtSLAC1 for AtOST1. Additionally, we found that OsSLAC1 is a nitrate-selective anion channel without obvious permeability to chloride, malate, and sulfate, and the expression of OsSLAC1 in Arabidopsis slac1-3 (atslac1-3) mutant successfully rescued the hypersensitive phenotype of this mutant to drought stress. Together, this research suggests that OsSAPK8 is a counterpart of AtOST1 for the activation of OsSLAC1, which is a nitrate-selective anion channel.

The Os-AKT1 channel is critical for K+ uptake in rice roots and is modulated by the rice CBL1-CIPK23 complex.

Potassium (K(+)) is one of the essential nutrient elements for plant growth and development. Plants absorb K(+) ions from the environment via root cell K(+) channels and/or transporters. In this study, the Shaker K(+) channel Os-AKT1 was characterized for its function in K(+) uptake in rice (Oryza sativa) roots, and its regulation by Os-CBL1 (Calcineurin B-Like protein1) and Os-CIPK23 (CBL-Interacting Protein Kinase23) was investigated. As an inward K(+) channel, Os-AKT1 could carry out K(+) uptake and rescue the low-K(+)-sensitive phenotype of Arabidopsis thaliana akt1 mutant plants. Rice Os-akt1 mutant plants showed decreased K(+) uptake and displayed an obvious low-K(+)-sensitive phenotype. Disruption of Os-AKT1 significantly reduced the K(+) content, which resulted in inhibition of plant growth and development. Similar to the AKT1 regulation in Arabidopsis, Os-CBL1 and Os-CIPK23 were identified as the upstream regulators of Os-AKT1 in rice. The Os-CBL1-Os-CIPK23 complex could enhance Os-AKT1-mediated K(+) uptake. A phenotype test confirmed that Os-CIPK23 RNAi lines exhibited similar K(+)-deficient symptoms as the Os-akt1 mutant under low K(+) conditions. These findings demonstrate that Os-AKT1-mediated K(+) uptake in rice roots is modulated by the Os-CBL1-Os-CIPK23 complex.

Calcineurin B-like protein CBL10 directly interacts with AKT1 and modulates K+ homeostasis in Arabidopsis.

Potassium transporters and channels play crucial roles in K+ uptake and translocation in plant cells. These roles are essential for plant growth and development. AKT1 is an important K+ channel in Arabidopsis roots that is involved in K+ uptake. It is known that AKT1 is activated by a protein kinase CIPK23 interacting with two calcineurin B-like proteins CBL1/CBL9. The present study showed that another calcineurin B-like protein (CBL10) may also regulate AKT1 activity. The CBL10-over-expressing lines showed a phenotype as sensitive as that of the akt1 mutant under low-K+ conditions. In addition, the K+ content of both CBL10-over-expressing lines and akt1 mutant plants were significantly reduced compared with wild-type plants. Moreover, CBL10 directly interacted with AKT1, as verified in yeast two-hybrid, BiFC and co-immunoprecipitation experiments. The results of electrophysiological analysis in both Xenopus oocytes and Arabidopsis root cell protoplasts demonstrated that CBL10 impairs AKT1-mediated inward K+ currents. Furthermore, the results from the yeast two-hybrid competition assay indicated that CBL10 may compete with CIPK23 for binding to AKT1 and negatively modulate AKT1 activity. The present study revealed a CBL-interacting protein kinase-independent regulatory mechanism of calcineurin B-like proteins in which CBL10 directly regulates AKT1 activity and affects ion homeostasis in plant cells.