| Plant hypocotyls are important channels for plants to transport organic nutrients,water,inorganic salts and plant hormones.Meanwhile,it responds quickly to changes in gravity to ensure the correct direction for growth.The elongation of plant hypocotyl is regulated by factors such as light,hormones,and vacuole inflation.However,most hypocotyl cells in Arabidopsis are divided and formed during the embryonic period,and only a few cells are formed by cell division after seed germination.Hence cell elongation is the main cause of hypocotyl elongation in Arabidopsis.Therefore,exocytosis of intracellular signaling molecules that affects cell activity is also likely to affect elongation of hypocotyl cells.In our research,we found that the hypocotyls of exo70b1-3 and exo70b1-3exo70b2-1 mutants of Arabidopsis exocytosis complex subunits were defective in their response to auxin inhibitors.exo70b1-3 is a published T-DNA insertion mutant.exo70b2-1 is a crisper mutant with the background of DR5::GFP.The exocytosis complex is composed of highly conserved octamers,and tethers Golgi-derived vesicles to target membrane to complete exocytosis in eukaryotic cells.In Arabidopsis the exocytosis complex consists of eight subunits,including EXO70 s.Our research results are as follows:The hypocotyls of the exo70b1-3 and exo70b1-3exo70b2-1(DR5::GFP)mutants grown in dark conditions were significantly longer compared to the wild type.The epidermal cells of their hypocotyls have DR5::GFP signal distribution.In NPA treatment experiments,the hypocotyl elongation inhibition rate of the mutants of exo70b1-3 and exo70b1-3exo70b2-1was significantly lower than that of the control group.In particular,light can induce EXO70B1 expression in hypocotyls.This indicates that the function of EXO7OB1 is to negatively regulate hypocotyl elongation.The hypocotyls of exo70b1-3 and exo70b1-3exo70b2-1 mutants under the stimulation of gravity reset have a faster response to gravity than the wild type.Through analyzing the DR5::GFP fluorescence intensity ratio of the upper and lower sides of the hypocotyl curve in gravity treated exo70b1-3 and exo70b1-3exo70b2-1,we found that rapid redistribution of auxin in hypocotyl curve is the reason for a faster response to gravity in the mutants than the wild type.This indicates that the function of EXO70B1 is necessary for maintaining the normal distribution and polar transport of auxin in hypocotyls.PIN3 protein mediates the lateral transport of auxin in hypocotyl.The distribution of PIN3-GFP protein in the cell membrane of cortex,endodermis and stele of hypocotyl in the mutants was significantly more than the wild type.In particular,after the 2-hour gravity stimulation,the cell membrane distribution of PIN3 on the upper side of the hypocotyl curve was significantly less than that of the lower side in the mutants,but not in the wild type.In BFA and NAA treatment experiments,NAA could not inhibit the compartment formation of PIN mediated by BFA in exo70b1-3,indicating EXO70B1 positively regulates auxin promoted exocytosis of PIN protein.We further verified the interaction between EXO70B1 and IAA5,IAA17,IAA18 of the Aux/IAA protein family in vitro through Bi FC experiments.We obtained gain-of-function mutants of IAA18 by mutating IAA18 domain II.The hypocotyls of iaa18 grown under dark conditions were significantly longer than the wild type.NPA cannot inhibit the elongation of hypocotyl of iaa18.These results suggest that AUX/IAAs proteins may inhibit the polar transport of auxin by interacting with exocytosis complex proteins. |
