Identification And Functional Characterization Of Potassium And Anion Channels In The Plasma Membrane Of Maize Guard Cells

Leaf stomatal pores are the main gateways for plant water and gas (C02, O2) exchange with the atmosphere. Stomatal aperture is finely controlled through osmotic regulation of guard cells to prevent excessive water loss for better adaptation to drought stress, and regulation of ion channels, such as potassium channels and anion channels in the plasma membrane (PM) of guard cells, plays important roles in regulation of stomatal movement.Vicia faba and Arabidopsis thaliana are two plant species that were used in most previous investigations on stomatal regulation, resulting in a number of important progresses in last two decades. However, regulatory mechanism of stomatal movement of all important crop plants, such as corm, wheat, rice, and soybean, remains unclear. In the present work, function and regulation of potassium and anion channels in maize stomatal movement are analyzed.In maize (Zea mays), a pair of dumbbell-shaped guard cells together with the flanking subsidiary cells form stomatal complex. Whole-cell patch clamping results showed that inward K+ (Kin) channels in the PM of stomatal guard cells were sensitive to apoplastic K+ concentration, apoplastic/cytolplastic pH, and were also regulated by ABA and Ca2+; Outward K+ channels were sensitive to apoplastic K+concentration and cytolplastic pH.KZM2 and KZM3 in maize plants are two ortholog Kin channels of Arabidopsis guard cell KAT1.They were mainly expressed in maize leaves, leaf epidermis, and guard cells. Both KZM2 and KZM3 were Kin channels and localized in the PM of stomatal guard cells. When expressed in Xenopus oocytes,functional silent KZM2 could form heterormeric channel with KZM3, negatively shift half activation potential (V1/2) of KZM3, and change its sensitivity to apoplastic channel blockers and pH. In KZM2 RNAi-1?3 lines, light induced stomatal opening was accelerated. The Kin channel activity was significantly increased in guard cells of KZM2 RNAi-1-3 plants, suggesting that at least one fast-activated Kin channel component in maize guard cell is negatively regulated by KZM2. Further analysis indicates that KZM3 is the fast-activated component in maize plants.ZmSLAC1 has high sequence similarity with Arabidopsis anion channel AtSLAC1, and was expressed in maize guard cells and localized at the PM. Expression of ZmSLAC1 in the atslacl mutant recovered its stomatal closure. Similar to the atslacl mutant, the zmslacl mutant was deficient in stomatal closure and drought sensitive. Arabidopsis AtCPK8 is the ortholog of ZmCPK35 or ZmCPK37.The atcpk8 mutant showed drought sensitive phenotype, and expression of either ZmCPK35 or ZmCPK37 in the atcpk8 mutant complemented its phenotype. ZmCPK35 or ZmCPK37 were localized in the PM and activated ZmSLAC 1. In addition, overexpression of ZmCPK35 or ZmCPK37 gave rise to ABA and Ca2+ hypersensitivity of stomata, reduced leaf water-loss, and increased maize drought tolerance.The experimental results presented in this dissertation demonstrate that Kin channels in the PM of maize guard cells are finely regulated, and both KZM2 and KZM3 are involved in regulation of stomatal opening through heterotetramerization. ZmCPK35 or ZmCPK37 in the PM of maize guard cells functions in activation of anion channel ZmSLAC1, involving in regulation of stomatal closure.Further investigation in regulatory mechanisms of maize stomatal movement may provide valuable information for genetic improvement of maize drought stress tolerance.