Mechanism Of Arabidopsis SYP71 Regulating Root Development Via Regulating Apoplast PH Homeostasis

In eukaryotic cells,the material exchange between different organelles is mainly accomplished by membrane transport.Vesicle transport relies on the synergistic action of multiple regulatory factors.The main processes of Vesicle transport include budding,transport,tethering,docking and membrane fusion.The specificity of fusion of transport vesicles with target membranes depends on various regulatory factors,such as coat,tether,SNARE and Rab proteins.Among them,SNARE proteins are responsible for the docking and the membrane fusion of transport vesicles and target membranes.At SYP71 is a plant-specific Qc-SNARE localized on the endoplasmic reticulum(ER),plasma membrane(PM)and endosomes,implying its functional diversity.However,its biological functions in plant development remain unclear.In this study,we analyzed the function of At SYP71 by using approaches of genetics,cell biology,biochemistry and molecular biology,preliminarily elucidated the molecular mechanism of At SYP71 regulating apoplastic p H homeostasis and root development,which provides new ideas for understanding the adaptive mechanism of environmental stress in plants.The main results of this study are as follows:1.At SYP71 is essential for plant development: At SYP71 depletion mutant atsyp71-1 was lethal at the early seedling stage.At SYP71 down-regulated mutants,atsyp71-2 and atsyp71-3,exhibited inhibited primary root growth and retarded early development.2.At SYP71 regulates apoplastic p H homeostasis: p H 8.0 or MES treatmentment significantly alleviated the short root phenotype of atsyp71 mutants.Further analysis revealed that At SYP71 controls apoplastic p H homeostasis by regulating H+ flux in roots.3.At SYP71 controls apoplastic p H homeostasis by regulating auxin biosynthesis and polar transport: Auxin was excessively accumulated in root tips of atsyp71-2 mutant;transcriptomic,biochemical,and cell biological analyses indicated that both biosynthesis and polar transport of auxin were severely affected in atsyp71-2.The auxin inhibitor significantly alleviated the excessive rhizosphere acidification in the mutants,resulting in significant recovery of root length.4.At SYP71 affects ROS homeostasis by regulating p H homeostasis: ROS levels in atsyp71-2 roots were significantly reduced and p H 8.0 treatment significantly restored ROS levels.5.At SYP71 subcellular localization analysis: Co-localization analysis of p At SYP71::GFPAt SYP71 with organelle markers revealed that in addition to the known localization of the ER,PM,cell plate and edosomes,GFP-At SYP71 was localized at the trans-Golgi network(TGN)and prevacular compatment/multivesicular bodies(PVC/MBV),suggesting its functional diversity and complexity.GFP-At SYP71 was sensitive to BFA and wortmannin treatments,and exhibited vacuole distribution under dark conditions,suggesting that At SYP71 is involved in the endocytic cycle and vacuole degradation pathway.p H 8.0 treatment did not affect GFP-At SYP71 protein abundance nor its subcellular localization.6.At SYP71 regulates PIN2 abundance and distribution: The PIN2 vacuole/PM signal ratio was significantly lower in the atsyp71-2 mutant than that in wild type,and the negative phototropism of roots was altered.Con A treatment showed that At SYP71 may regulate PIN2 vacuole degradation by affecting vacuole p H.Taken together,At SYP71 may regulate cellular endocytosis and vacuole degradation pathways,thereby regulating auxin biosynthesis and polar transport,which controls p H homeostasis and affects ROS homeostasis.