Electrophysiological Analysis Of Function And Molecular Regulatory Mechanism For Rice Potassium Channel OsAKT1

Potassium (K+) is one of the essential nutrient elements for plant growth and development. It is absorbed into the root cell through low and high affinity systems conferred by different K transporters and channels. Previous studied in our and other labs demonstrate that in model plant Arabidopsis, K+TRANSPORTER1(AKT1), belonging to Shaker K family, has both high and low affinity to K+, therefore has dominant contribution to K uptake of the root cells under both K deprivation and sufficient conditions. It is directly regulated by Arabidopsis CBL-INTERACTING PROTEIN KINASE23(CIPK23), activation of which in turn depends on interaction with Arabidopsis CALCINEURIN B-LIKE PROTEIN1(CBL1) and CBL9. This dissertation extends this key discovery found in model plant to rice, one of key staple crops in China in terms of contribution to food production.Shaker K+channels play important roles in K+uptake of plants Especially AKT1is one of the most important Shaker K+channels, and plays important role in K+uptake in the roots of Arabidopsis. Rice is one of the most important food resources. It has evolved machine to adapt low-K+environment. OsAKT1is homologous with AKT1and probobly important for K+uptake in rice root. The focus of this dissertation work is to study the function and molecular regulatory mechanism of OsAKT1in K+uptake process of rice roots by electrophysiological technology.In this dissertation work, the Shaker K+channel OsAKT1was characterized for its function in K+uptake in rice roots. Beta-glucuronidase based promoter activity analysis revealed that OsAK1primarily expresses in epidermal, cortex and xylem of the roots. The OsAKT1protein was localized in the plasma membrane. It displayed voltage-dependent inward-rectifying K-specific current while being exogenously expressed in mammalian cell HEK293but not Xenopus oocytes, as detected with patch clamp technique. The root cells of Arabidopsis AKT1knockout mutant (aktl) has no inward K current under patch clamp whole-cell configuration condition. Expressing OsAKTl in aktl rescued its inward K current. These data demonstrates that OsAKT1functions as an inward K channel like AKT1in the roots.To evaluate OsAKT1significance in K nutrient of rice, a rice mutant harboring a single T-DNA in OsAKT1was identified. The mutant osaktl had significant decreases of K uptake capacity and K-dependent growth as compared to those of wild type. Moreover, the inward K+current cross the plasma membrane of the root cell protoplasts was significant suppressed in the mutant. These data demonstrated that OsAKT1plays a crucial role in K nutrient of rice roots.In light of AKT1activity dependence on its interacting with CIPK/CBL complex, this dissertation work investigated which OsCIPK and OsCBL interact with OsAKTl with yeast two hybrid and BiFC assay. It was intriguing that OsAKTl interacts with OsCIPK3,9,19and23, all of which interact with OsCBL1. Exogenously expressing OsAKT1along with OsCIPKs and OsCBLl found that both OsCIPK23/OsCBLl and OsCIPK19/OsCBLl combination could significantly and comparably enhance OsAKT1-mediated inward K+current in HEK239Cells, which was much weaker for the OsCIPK19’s combination. These data implied that OsAKT1’s channel activity was under regulated by different OsCIPKs and OsCBL complex, which should be further investigated in vivo with genetic tools.In conclusion, this dissertation paves foundation on our understanding of OsAKT1-mediated K nutrient uptake mechanism in rice. Difference of AKT1operating machinery in Arabidopsis and rice was discussed.