Functional Study Of AtHSPR Gene In Stress Tolerance And Development In Arabidopsis

Salt stress is part of the important adversity factors limiting plant growth and development.As an abiotic stress,excessive salts are harmful to plants due to toxicity of ions,osmotic and secondary oxidative stresses that damage many physiological processes.To endure a saline environment,plants have evolved various morphological,physiological,biochemical and molecular regulatory mechanisms.Plants alleviate the adverse effects of salinity stress through removal of excess reactive oxygen species(ROS)and enhancement of antioxidant enzyme activities,accumulation of both low-molecular-weight organic compounds(sugars,glycine betaine and proline)and molecular chaperones(heat shock proteins,HSPs),alteration of ion transport,vacuolar compartmentalization and induction of the phytohormones abscisic acid and ethylene.At present,high salinity in soils dramatically affects the yield and quality of agricultural crops throughout the world,and it is a significant and important project to explore novel genes and to dissect their molecular mechanism resistant to high salinity.A T-DNA insertion mutant in C24 ecotype was screened by our lab,named athspr(Arabidopsis thaliana heat shock protein-related),and showed several phenotypes,including reduced organ size and enhanced sensitivity to environmental cues.In our study,wild type(C24),athspr mutant,complementation(COM)and overexpression(OE)transgenic lines were used as materials,and we focused on the function of AtHSPR gene by morphological comparison,physiological and biochemical techniques,genetic engineering,RNA-seq and bioinformatics,q RT-PCR,Western-blot,yeast two hybrid system,GST pull-down and Co-IP,which could systematically elucidate the pivotal role of AtHSPR in salt and drought stress tolerance in Arabidopsis.Moreover,we explored the function of AtHSPR gene in regulating plant growth and development,which will provide important and valuable information for the plant how to resist against stress and regulate development.The main results of this work are as follows:1.AtHSPR was specifically expressed in the vascular system of Arabidopsis,including in cotyledons,leaves,roots,shoot apical meristem and cambium and phloem of inflorescence stem.2.AtHSPR encoded a nuclear-localized protein with ATPase activity,and its expression was enhanced by high salinity and abscisic acid(ABA).3.Disruption of AtHSPR results in reduced salt and drought tolerance in athspr mutant.Over-accumulated ROS is responsible for the salt-induced cell death in athspr.4.Overexpression(OE)of AtHSPR significantly enhanced tolerance to salt stress by increasing the activities of the antioxidant system and maintaining K~+/Na~+homeostasis.And OE of AtHSPR increased the expression of ABA/stress-responsive,salt-overly sensitive(SOS)-related and antioxidant-related genes.5.ABA content was reduced in athspr plants with or without salt stress,and exogenous ABA restored WT-like salt tolerance to athspr plants.athspr exhibited increased leaf stomatal density and stomatal index,slower ABA-induced stomatal closure relative to WT.The results indicated that AtHSPR links ABA-dependent salt tolerance.AtHSPR-OE enhanced drought tolerance by reducing leaf water loss and stomatal aperture.6.Transcript profiling in athspr showed a differential salt stress response for genes involved in ROS accumulation,ABA signaling,cell death,stress response and photosynthesis.AtHSPR may be functionally involved in response to stress by regulating these signaling pathways.7.Mutation of AtHSPR altered plant cell size and morphology,and delayed the development of shoot apical meristem(SAM)in Arabidopsis.AtHSPR-overexpressing transgenic Arabidopsis increased biomass.AtHSPR gene may be involved in regulating the expansion of plant cells.8.By yeast two-hybrid screening,11 candidate proteins that interact with AtHSPR were obtained.Further more,we confirmed that AtHSPR directly interacts with IAA27 protein by GST pull-down and Co-IP analysis,which is a development-related auxin response factor.9.athspr mutant and AtHSPR-overexpressing transgenic seedlings showed different sensitivity to auxin and auxin transport inhibitors,and changed the expression of auxin homeostasis related(such as GH3 family genes)and auxin transport related genes(such as PIN family genes).AtHSPR gene may involve in regulating the auxin homeostasis by regulating the GH3 family genes expression that involved in IAA conjugation,and in regulating the auxin polar transport by regulating PIN family genes expression.Taken together,our results suggested that AtHSPR is involved in salt tolerance in Arabidopsis through modulation of ROS levels,ABA-dependent stomatal closure,photosynthesis,and K~+/Na~+homeostasis.In addition,AtHSPR may interact with IAA27 to control plant development and cell expansion by regulating auxin signal,auxin homeostasis and auxin polar transport.This study not only has important academic value,but also the potential application in improving the salt and drought tolerance of crops.