The Molecular Mechanism Of Clatnrin Regulating Hypocotyl Elongation In Arabidopsis Thaliana

The rapid elongation of hypocotyl ensured that plant will receive sunlight for photosynthesis as soon as they break the soil and thus plays critical roles in survival and adaptation of plant.It was known that the elongation of hypocotyl was a comprehensive result of environmental factors and phytohormones by interaction with related transcription factors.Recent research had indicated that clathrin light chain was involved in the regulation of hypocotyl elongation.However,the underlying regulatory mechanism remained unclear.The preliminary work of the laboratory successfully isolated and identified clathrin light chain clc2-1 clc3-1 double mutants in Arabidopsis,showing that loss of CLC2 and CLC3 promoted elongation of hypocotyl.Here,we demonstrated that the molecular mechanism of clathrin light chain in hypocotyl elongation using genetics,cell biology and pharmacology combined with live-cell imaging.The main research results were as follows:(1)The hypocotyl length of clc2-1 clc3-1 double mutants was much longer than that of the wild type when grown in normal light conditions(16 h light/8 h dark);in addition,SEM results indicated that hypocotyl elongation of clc2-1 clc3-1 double mutants was mainly due to the cell elongation,not the increase in the number of cell,compared to the wild type.Meanwhile,complementation experiments demenstrated that the hypocotyl elongation of clc2-1 clc3-1 double mutants were indeed caused by knocking out CLC2 and CLC3.(2)Live-cell imaging showed that the transverse arrangement of cortical microtubules increased significantly,and the longitudinal arrangement decreased in the hypocotyl cells of clc2-1 clc3-1 double mutants;Moreover,the actin filaments were bundled to a larger extent in hypocotyl epidermal cells of clc2-1 clc3-1 double mutants compared to the wild-type.The transverse arrangement of the microtubules and the bundling of microfilaments are considered to be beneficial for cell elongation,suggesting that the loss of CLC2 and CLC3 promoted hypocotyl elongation by regulating cytoskeletal changes.(3)Subcellular localization analysis,showing that the loss of CLC2 and CLC3 function affected relocation of PIN3 in hypocotyl stele area and the plasma membrane abundance of PIN3 in epidermal cells;Furthermore,important signal was detected in stele and epidermal cells of clc2-1clc3-1/DR5-GFP hypocotyl compared to the wild type.These results suggested that clathrin mediated the elongation of hypocotyl through modulating the auxin redistribution by regulating the polar localization of PIN3.(4)Pharmacological analysis showed that the auxin transport inhibitors TIBA,NPA and 1-NOA inhibited the hypocotyl elongation,meantime,the hypocotyl length of clc2-1 clc3-1 double mutants was suppressed more seriously than wild-type.Additionally,the hypocotyl length of clc2-1 clc3-1 double mutants with loss of auxin synthesis,signal transduction and other functions was inhibited in Arabidopsis.These results suggested that clathrin may regulate hypocotyl elongation by affecting auxin polar transport and related signaling pathways.(5)Confocal microscopy analysis showed that the transverse arrangement of cortical microtubules in the hypocotyl epidermal cells of clc2-1 clc3-1 double mutants were significantly smaller than wild type under the MAP4-GFP background after treatment with the auxin transport inhibitor TIBA.This indicated that clathrin was involved in the process of auxin regulating microtubule arrangement.(6)q RT-PCR analysis of PIFs gene expression levels in wild type and clc2-1clc3-1 double mutants grown 5-d-old seedlings,it was found that PIF3 expression was upregulated for loss of the CLC2 and CLC3.And using genetics to knock out PIF3 in the context of the clc2-1 clc3-1 double mutants,the long hypocotyl phenotype of the clc2-1 clc3-1 double mutants can be inhibited accordingly.This result indicated that clathrin regulated PIF3 expression mediating hypocotyl elongation.In summary,these findings proved that clathrin regulated auxin redistribution by regulating the polar localization of PIN3,regulation of reorganizing microtubules,changing bundling of the microfilaments and influencing PIF3 expression level,revealing clathrin regulation hypocotyl elongation in Arabidopsis.It provided new insights for further analysis of the regulation mechanism of hypocotyl elongation.