Action Mechanisms Of Blue Light-mediated Phototropism And Gravitropism In Arabidopsis Hypocotyls

Photoropism and gravitropism are one of the important response mechanisms,which playing critical roles in helping the plants to adapt to the external environment.Phototropin?PHOT?is essential for blue light?BL?-triggered hypocotyl phototropism in plants.Recent studies have shown that clathrin regulates BL-triggered hypocotyl phototropism in Arabidopsis.However,whether clathrin is involved in PHOT-mediated hypocotyl phototropism and the regulatory relationship between clathrin and BL/PHOT signaling are poorly understood.It has also been reported that the phot1 mutants are defective in hypocotyl negative gravitropism under weak BL conditions,but little is known about detailed mechanisms.Our previous work has successfully generated the double mutants clc2 clc3,phot1-6 phot2-2 and the quadruple mutants phot1 phot2 clc2 clc3 in Arabidopsis.In this study,T-DNA insertional,mutants for phot1-7 and phot2-3 were isolated and the double mutants phot1-7 phot2-3 was generated.This study used genetics,cell biology and pharmacology to examine the roles of clathrin and PHOT in hypocotyl phototropism and/or in hypocotyl negative gravitropism.The detailed results are as follows:?1?Under high unilateral blue light(10?mol m^-2 s^-1)conditions,phot1 clc2 clc3and phot2 clc2 clc3 triple mutants displayed significant reduction in phototropic bending relative to phot1 and phot2 single mutants,respectively.However,there was no significant difference between phot1 phot2 and phot1 phot2 clc2 clc3,indicating the involvement of clathrin in regulating hypocotyl phototropism downstream of PHOT-mediated BL signaling pathway.?2?Analysis of auxin responsive marker DR5:GFP expression pattern revealed that loss of CLC and PHOT function inhibited BL-induced asymmetric auxin distribution between shaded and lit side,which in turn affected hypocotyl phototropic bending,suggesting that the clathrin is involved in BL/PHOT mediated asymmetric auxin distribution.?3?Live-cell imaging analysis showed that loss of CLC and PHOT function inhibited PIN3 relocalization and endocytosis in the outer PM regions of the endodermal cells,suggesting that clathrin and PHOT are likely to regulate the hypocotyl phototropism by mediating the lateralization of PIN3.?4?FM4-64 uptake assay showed that the uptake of FM4-64 was significantly reduced in phot1-6 phot2-2 and phot1-7 phot2-3 double mutants compared to wild type,indicating that loss of PHOT1 and PHOT2 function inhibits PM endocytosis.Confocal microscopy analysis showed that loss of PHOT1 and PHOT2 function inhibited PM-associated levels of CLC-GFP,AP2?2-YFP,TPLATE-GFP and TML-YFP.These results suggesting that PHOT mediates membrane proteins endocytosis by regulating the recruitment of clathrin and its adaptor proteins.?5?Analysis and identification the phenotype showed that under blue light conditions,phot1-6,phot1-7,phot1-6 phot2-2 and phot1-7 phot2-3 mutants were defective in hypocotyl negative gravitropism,however,phot2-2 and phot2-3 mutants did not.Further analysis found that phot1-6 phot2-2 and phot1-7 phot2-3 mutants hypocotyl endodermal starch granules significantly reduced compared to wild type.These results indicating that loss of PHOT1 function promotes converting starch-filled gravity-sensing endodermal amyloplasts,resulting in altering hypocotyl gravisensing,which leading to a loss of hypocotyl negative gravitropism;demonstrating that PHOT1 is necessary to maintain hypocotyl negative gravitropism,on the contrary,PHOT2 did not participate in this process.?6?Pharmacological experiments revealed that under blue light,auxin efflux transport inhibitor NPA could restore phot1-6 phot2-2 and phot1-7 phot2-3 mutants hypocotyl negative gravitropism,while auxin influx transport inhibitor could not,suggesting that PHOT1-mediated auxin efflux during hypocotyl negative gravitropism.In summary,the study preliminarily obtained the following conclusions:?1?clathrin is involved in regulating BL/PHOT-mediated hypocotyl phototropism.PHOT may regulate the relocalization of PIN3 by regulating the recruitment of clathrin and its adaptor proteins,then leading to formation of asymmetric auxin distribution and phototropic bending.?2?PHOT1 mediates BL-triggered hypocotyl negative gravitropism by regulating the converting starch-filled gravity-sensing endodermal amyloplasts and auxin efflux transport.