Molecular Mechanisms Underlying Clathrin Regulation Of Hypocotyl Elongation And Phototropism In Arabidopsis

The hypocotyl is a very plastic organ and a excellent model system to study the molecular mechanisms of growth and environmental responses in plants.Rapid cell expansion and phototropism of the hypocotyl are important adaptive mechanisms of plants,in which cell elongation is modulated by light and plant hormones auxin,BR,and GA,whereas phototropism is predominantly regulated by blue light via BL receptor phototropins(PHOT1/2)and photo signaling and PIN3-mediated auxin transport.The previous studies have indicated that endocytosis of PHOT1 and PIN3 is dependent on clathrin-mediated endocytosis(CME).Clathrin is a highly evolutionally conserved triskelion-shaped complex consisting of three heavy chains(CHCs)and three light chains(CLCs),and CHCs form the structural backbone of the clathrin lattice,whereas CLCs have been suggested to regulate the formation and disassembly of clathrin-coated vesicles.CME is one pivotal endocytosis pathway in anminal and plant cells,it directly regulates crucial cellular processes including absorption of extracellular substance,endocytosis of plasma membrane(PM)proteins and transmission of extracellular and intracellular signals.The previous study has characterized the clc2-1clc3-1 double mutants in Arabidopsis and found that loss of CLC3 and CLC2 inhibits the endocytosis of PIN proteins and promotes hypocotyl elongation.However,how clathrin regulates cell elongation and whether it is involve in the regulation of phototropism remain to be elucidated.In this study,the biological functions of clathrin in cell elongation and phototropism were dissected using genetic,cytological and physiological strategies.The detailed results are as follows:(1)Using laser scanning confocal microscopy to analyze hypocotyl cell length and number.It was found that loss of CLC2 and CLC3 promotes hypocotyl cell elongation but not affects cell division.Using a genetic interference strategy by antisense RNA of CLC2 and CLC3,the clc2anti;clc3anti double mutants further confirmed that clathrin negatively regulates cell elongation of the hypcotyl.(2)Genetic analysis revealed that hypocotyl elongation in clc2-1clc3-1 mutants was significantly inhibited in the background of auxin nuclear signaling mutant axrl-12,BR biosynthesis mutant det2-1 and signaling mutant bril-6,GA biosynthesis mutant gal-3 and signaling mutant gai-1,in contrast,it was not affected in the cytoplasmic auxin signaling mutant abpl-5 background,indicating that clathrin regulates hypocotyl elongation downstream of ABP1 signaling and upstream of AXR-mediated signaling,BR and GA biosynthesis and signaling pathways.(3)To analyze the effect of PHOT1/2-mediated signaling on hypocotyl elongation in the clc2-1c1c3-1 mutants,the phot1-6phot2-2 double mutants were generated.Genetic analysis showed that loss of PHOT 1 and PHOT2 did not inhibit hypocotyl elongation in the clc2-1clc3-1 double mutants,indicating that clathrin regulates hypocotyl elongation downstream of PHOT-mediated photosignaling.(4)The analysis of hypocotyl phototropism showed that loss of CLC2 and CLC3 inhibited phototropic bending compared with that in WT.Genetic analysis revealed that clathrin regulates hypocotyl phototropism downstream of PHOT 1-mediated BL signaling.Cytological analysis demonstrated that loss of CLC2 and CLC3 inhibited endocytosis of PIN3 and PIN7,BL-induced PIN3/7 lateralization and lateral auxin asymmetric distribution in the hypocotyl.These results indicated that clathrin regulates BL/PHOT1-mediated lateral auxin asymmetric distribution and phototropism through mediation of endocytosis and lateral relocalization of PIN3 and PIN7.(5)BL induces CLC1-GFP relocalization and promotes PHOT1-GFP endocytosis in the hypocotyl.Genetic and cytological evidence revealed that loss of PHOT1 and PHOT2 downregulates the PM abundance of CLC1-GFP and inhibits BL-induced CLC1-GFP relocalization and endocytosis of PIN3-GFP in the hypocotyl.Similarly,loss of CLC2 and CLC3 inhibited BL-induced endocytosis and loss of PM-resident levels of PHOT1-GFP.These results together suggest that clathrin and PHOT reciprocally regulate each other in BL-induced phototropism.Based on the above data,it could be concluded that clathrin negatively regulates hypocotyl elongation downstream of light signaling and ABP1-mediated signaling and upstream of auxin nuclear signaling,BR and GA signaling.In hypocotyl phototropism,clathrin regulates blue light-triggered lateral auxin redistribution and phototropism through mediation of endocytosis of PHOT1 and PIN3/7.