Functional Analysis Of Clathrin Light Chains In Arabidopsis

Endocytosis is a key way for a cell to take up extracellular materials such as nutrients, transduct extra-or intracellular signals, and regulate the abundance of plasma membrane proteins and thus plays critical roles in survival and adaptation of unicellular and multicellular organisms. Clathrin-mediated endocytosis (CME) is a major endocytic pathway in animals and plants.CME has been proposed to participate in many plant developmental processes and responses to various environmental stimuli such as gravity and light. Bioinformatic analysis showed that two clathrin heavy chains(CHC1and CHC2), three clathrin light chains(CLC1, CLC2and CLC3) in Arabidopsis genome. It has been shown that15%-20%of chc2seedlings mutated by T-DNA insertion displays abnormal cotyledons such as stick-shaped, collar-shaped, and trumpet-shaped but other seedlings showed WT phenotypes. chc1-1chc2-1double mutant was not recovered due to embryonic lethality, which hinders from genetically dissecting their functions in planta. However, the roles of CLCs are not still identified in plants.In this study, two T-DNA insertion mutants for clc1-1and clc2-1were successfully isolated and genetically characterized and showed similar phenotyopes with short roots and long hypocotyls. clc3-1single homozygous mutant was not recovered due to embryonic lethality. To further dissect CLC function in plants, clc1-1clc2-1double mutant was generated and displays shorter roots and longer hypocotyls. Complementary test shows that double mutant phenotypes were fully rescued by CLC1and CLC2.clc1-1clc2-1double mutant was used to study biological functions of CLCs in details using cell biological, physiological, and biochemical strategies. Confocal data show:(1) Endocytic rates of FM4-64and PIN2-GFP were dramatically reduced in the double mutant compared with the WT, leading to significantly increased their accumulation at the plasma membrane (PM), however, PM-associated CHC was significantly descreased;(2) auxin and A23effectively blocked endocytosis of PM-localized PINs in the WT but not in the double mutant;(4) and meanwile increased auxin transport activity, changed auxin distribution pattern and gravity response in the roots.Above data demonstrate that CLCs play a critical role in auxin regutation of PIN endocytosis and thus participate in the regulation of primary root development, lateral root and root hair iniatiation, and root gravitropism.