Action Mechnisms Of Clathrin Regulation Of Blue Light-induced Hypocotyl Phototropic Bending In Arabidopsis

Hypocotyl phototropic bending is one of the important mechanisms by which,plants adapt to the environment.Establishment of asymmetricauxin distribution is essential for blue light(BL)-induced hypocotyl phototropic bending,in which the auxin efflux transporter PIN-FORMED 3(PIN3)plays a critical role.Recent studies have showed that clathrin mediates PIN endocytosis and thereby modulates PIN polarity.Whether clathrin is involved in hypocotyl phototropic bending however is largely unknown.Our previous work has successfully isolated and genetically characterized the clc2-1clc3-1 double mutants in Arabidopsis thaliana.In this study,we used genetic,cell biological,and pharmacological strategiesto dissect the functional role of clathrin in BL-induced hypocotyl phototropic bending.The detailed results are as follows:(1)Live-cell imaging analysisshowed that BL induced the relocation of CLC1-GFP and CLC2-GFP in the hypocotyl elongation region.Genetic analysis showed that,lose of CLC2 and CLC3 functions inhibited the BL-induced hypocotyl phototropic bending,and CLC2 and CLC3 were functionally redundant during hypocotyl phototropism,suggesting the involvement of clathrin in regulation of BL-induced hypocotyl phototropic bending.(2)Analysis of auxin responsive marker DR5:GFP expression pattern revealed that loss of CLC2 and CLC3 functions inhibited BL-induced asymmetric auxin distribution between shaded and lit side,suggesting that the clathrin is involved in BL-induced asymmetric auxin distribution.(3)Live-cell imaging showed that unilateral BL triggered PIN3 lateralizationin the outer PM regions of endodermal cells on both the shaded and lit sides of the wild-type hypocotyl,while loss of CLC2 and CLC3 functions inhibited BL-induced hypocotylsPIN3 endocytosis and lateralization,indicating that clathrin regulates BL-induced PIN3 lateralization via mediation of PIN3 endocytosis.(4)Pharmacological experiments revealed that auxin efflux inhibitors NPA(1-naphthylphthalamic acid)and TIB A(2,3,5-triiodobenzoic acid)significantly inhibited unilateral BL-induced clathrin relocalization,endocytosis and relateralization of PIN3 and thereby asymmetric auxin distribution,Compared to the wild type,clc2-1clc3-1 double mutants displayed a more sensitive response to the inhibitory effects of NPA and TIBA,indicating that clathrin regulates BL-induced hypocotyl phototropic bending through modulating PIN-mediated polar auxin transport.In summary,the present study obtained the following conclusions:unilateral BL regulates clathrin relocalization and thereby PIN3 endocytosis and relateralization,finally leading to formation of asymmertric auxin distribution and phototropic bending.This study demonstrates the importance of clathrinin regulation of BL-induced hypocotyls phototropic bending,which will provides a new sight into the molecular mechanism of plant phototropic growth.