Clathrin Regulates Hypocotyl Hook Development And Light-Stimulated Hook Opening In Arabidopsis

In plants, dark/soil-induced apical hook at the top of the hypocotyl is formed to protect the two cotyledons and shoot meristem from damage by soil particles. Recent research has indicated that the establishment of auxin maxima by PIN-FORMED 3 (PIN3)-and AUXIN RESISTANT 1/LIKE AUX1 (LAX) 3 (AUX1/LAX3)-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, however, the underlying regulatory mechanism remains poorly understood.The preliminary work of this study successfully isolated and genetically characterized the clc2-1 clc3-1 double mutants in Arabidopsis thaliana, suggesting that loss of CLC2 and CLC3 impairs PIN proteins endocytosis. In this study, we characterized the functional roles of clathrin in hook formation and blue light-stimulated hook opening using genetical, cell biological, pharmacological and other strategies. The main results in this study were as follows:(1) In darkness, loss of CLC2 and CLC3 function enhanced auxin maxima formation at the concave side of the hook, thereby, hook formation, however, inhibited the decreased auxin asymmetric distribution and hook opening, suggesting clathrin negatively regulates hypocotyl hook formation, whereas positively regulates hook opening.(2) Auxin overproduction disrupts the establishment of auxin maxima in the hook region. Loss of CLC2 and CLC3 function partially restored the inhibitory effects of auxin overproduction on asymmetric auxin distribution and hook formation. Taken together, these results further confirm that loss of clathrin funtion enhances the asymmetric auxin distribution and hook formation. Moreover, the establishment of auxin maxima is essential for hook formation.(3) Pharmacological data, showing that clc2 clc3 double mutants were more sensitive to NPA and TIBA but not to 1-NOA than WT, support that clathrin regulates auxin efflux but does not directly affect auxin influx during hook formation.(4) Blue light can effectively stimulate the decreased auxin asymmetric distribution and hook opening. Loss of CLC2 and CLC3 function inhibited BL-induced hook opening and diminishment of auxin maxima, demonstrating the importance of clathrin in BL-triggered hook opening, suggesting that clathrin is functionally required for BL-triggered hook opening.(5) Live-cell imaging analysis, showing that loss of CLC2 and CLC3 function alleviated the endocytosis and basal localization of PIN3 in the cortical cells but did’t affect AUX1 localization in the hook region, suggest loss of CLC2 and CLC3 function impairs PIN3 endocytosis and subsequently its lateralization. Taken together, these results further confirm that clathrin regulates hook formation and BL-triggered hook opening through modulating PIN3 trafficking.In summary, the study uncovered the following conclusions:clathrin regulates auxin maxima, hook formation and BL-triggered hook opening through modulating PIN3 localization and auxin efflux, revealing an important role for clathrin regulation during plant skotomophogenesis and photomorphogenesis. The study provides a new sight into the molecular mechanism of auxin regulation in hook development, allowing a better understanding of molecular mechanisms of plant skotomorphogenesis and photomorphogenesis.