Functions And Mechanism Of Protein Kinase SnRKl In Arabidopsis Thaliana To Adapt To Low-potassium And-energy Environments

As its content is the the highest among the metal mineral elements,potassium(K)accounts for 10%of the dry weight of most plants.K is also an essential nutrient element for plant growth and development,and determines the yield and quality of the crop.Uptake and translocation of K+from soils by plants are mainly mediated by transporters or ion channels.Plants uses mainly K+channel AKT1,a Shaker-type channel with six transmembrane domains to adapt to the low-K environments.Our group previously discovered that a calcium signaling pathway,CBL-CIPK23 contributes to AKT1 activation for low-K response in Arabidopsis.Protein kinase CIPK is named as Calcineurin B-like Interracting Protein Kinase,and belongs to 3-subfamily of SnRK(sucrose non-fermenting-1-related protein kinase)family.SnRKs are highly homologous to yeast SNF1(non-fermenting-1 sucrose,SNF1)and mammalian AMPK.SnRK family contains 38 members,and is divided into three sub groups:SnRK1,SnRK2 and SnRK3 in Arabidopsis.SnRK3 subfamily has 3 members,SnRK1.1,SnRK1.2 and SnRK1.3.SnRK1 functions similarly to yeast SNF1 with a key regulator of carbon metabolism.In particular,SnRKl.1 is an important regulator of sugar,metabolism,stress and development.Once exposed to low-energy,SnRK1.1 will trigger various reactions,including the increase in gene transcription and anaerobic metabolism,and the decrease in synthetic metabolism,thereby to enhance starvation endurance and prolong the life cycle of the plant.Here,we found that aktl mutants of Arabidopsis thaliana were sensitive to light cycle,while cipk23 mutants did not displayed this phenotype,suggesting that AKT1 activity might be regulated by the energy-related mechanisms except for CBL-CIPK23.Considering the important roles of SnRK1 plays in the process of energy metabolism in plant,we applied plant molecular genetics,protein immunofluorescence and yeast-two hybrid assay to study the potential functions of SnRK1 for Arabidopsis thaliana to adapt to low-potassium and-energy environments.The main results are as follows:1.Assay the subcellular localization of SnRK1.1.We expressed the fusion of GFP and SnRK1.1 in Arabidopsis PSBD suspension cells,and found the SnRK1.1 was localized in the cytoplasm using laser scanning miscrocopy.2.The physical interactions between SnRK1.1 and AKT1 in yeast two-hybrid assay.After comfirming a physical interaction with SnRK1.1 and C-terminal non transmembrane fragment of AKT1,we prepared the various fragments containing the KHA,Ankyin,and cNMP binding domain,and found that the Ankyin domain is the main target for SnRK1.1 and SnRKl.2 to anchor AKT1 protein.3.preparation the trangenic SnRK1.1 over-expressing Arabidopsis thaliana(SnRK1.1-OE).SnRK1.1-OE is more telearant to low-light stress comared with the wild-type,while akt1 mutants is more sensitive to low-light stress comared "with the wild-type.4.The trangenic SnRK1.1 over-expressing Arabidopsis thaliana(SnRKl.1-OE)displayed a phenotype resistant to low-K.SnRK1.1-OE showed the significant phenotype resistant to low-potassium under the K+concentration equal to or less than 0.05mM in 1/2 MS containing 1%sucrose compared with the wild-type.5.The tolearance of the trangenic SnRK1.1 over-expressing Arabidopsis thaliana(SnRK1.1-OE)of low-potassium relates to the cellular energy status.Compared with the wild-type,SnRK1.1-OE displayed a tolerance to low potassium with the concentrations less than or equal to 0.1 mM phenotype grown in the medium containing no sugar,indicating that SnRK1 promotes the plants to adapt to a more extensive low potassium environments under the low-energy status.In summary,our results show that exposed to the low-energy status,Arabidopsis plants use SnRK1.1 response to K+ channel AKT1 activation,in addition to the CBL-CIPK23 pathway,to adapt to the low-energy environments.Therefore,the findings reveals that the molecular mechanism that plants adapt to the environments whose K-deficient soil and insufficient light,and can provide theoretical guidance and experimental basis for the molecular breeding of crops with high resistance to these environments.