Biochemistry And Function Analysis Of Protein CAP1

Nitrogen is an essential element for plant growth.Ammonium as an important signal molecule plays a vital role in many physiological processes.Plants preferentially absorb ammonium.However,excess ammonium will lead to toxicity.It is important to reveal the mechanism of ammonium signaling,its perception and transduction.Plant receptor-like protein kinase?RLKs?belongs to protein kinases,they can feel the outside biotic and abiotic stimuli and regulate plant growth and development.RLKs percept and recognize extracellular signa molecules mainly through their extracellular receptor domain;after combining with their ligands,the intracellular kinase domain will phosphorylate/dephosphorylate a putative down-stream target,thus to activate or inhibit a certain physiological process.Our previous work identified a cytoplasmic calcium([Ca²⁺]_cyt)-associated protein kinase of Arabidopsis,At CAP1,as a member of the CrRLK1 Ls,involved in NH₄⁺-related root hair development.AtCAP1 localizes to the tonoplast,mediates NH₄⁺ homeostasis and regulates the polar growth of root hairs by maintaining tip-focused cytoplasmic Ca²⁺ gradients.Although we have demonstrated that CAP1 mediates the cytosolic ammonium homeostasis,we still don't know the mechanism of the ammonium sensing and transduction through CAP1.As a receptor-like kinases,the C-terminal of the CAP1 belongs to the kinase domain,and it is important to make sure the phosphorylate site for understanding the biochemical property.CAP1 is a Ser/Thr kinase,there are 25 Ser and Thr in its C-terminal,which consist the putative phosphorylation site.According to the published protein data of the same family and the predicted protein structure,4 amino acids of Ser and Thr have been selected as the putative phosphorylation sites for CAP1.In order to study the function of the proposed phosphorylation amino acid in the kinase activity,we constructed the point mutation and tested the kinase activity by in vitro express the related protein.We also transferred the constructs into the CAP1 deficient mutant cap1-1,and analyzed the root hair growth by the homozygous of T3 seedlings.The results of the in vitro experiments indicated that the four amino acids all contribute to the kinase activity since each protein with point mutation can show autophosphorylation;which has been testified in the transgenic homozygous,the point mutations all can rescue the root hair growth of cap1-1.