ALA6,a P₄-type ATPase,is Involved In Heat Stress Responses In Arabidopsis Thaliana

Heat stress,as an important factor of adversity,is affecting the normal growth and development of plants.High temperature can directly cause the plant protein denaturation and aggregation,increase the liquidity of cell membrane,and affect the stability of cell membrane,finally seriously damaging plant normal growth and development,and even leading to the death of plants.However the plant itself has a variety of mechanisms that can resist moderately heat stress,such as maintenance of membrane stability,elimination of reactive oxygen species(ROS)and accumulation of antioxidant compounds,as well as induction of MAPK(Mitogen Activated Protein Kinase)and CDPK(Calcium-Dependent Protein Kinase)signal and cellular responses,in this case,activation of transcription factors and induced molecular chaperone signal response is the key to heat stress response.At present researchs are mostly focused on heat shock proteins(HSPs)and heat shock transcription factors(HSFs).However,the studies on functional proteins in plant heat stress response are relatively few reported.According to the report,Phospholipid molecules carrying amino acid transported by P4 type ATPase family are the main component of membrane lipid bilayer.Inference that P4 type ATPases are responsible for flipping and stabilizing asymmetric phospholipids in membrane systems probably related to the plant's response to stress.Aminophospholipid ATPase 6(ALA6)is one of12 members of P4 type ATPase family in Arabidopsis thaliana,and it's function have not been fully understood yet.In this research,Araidopsis wild type(WT),P4-ATPases' s ALA6 gene T-DNA insertion mutant(ala6-1),complement line(COM),overexpression line(OE)and the phosphorylated function site mutation(D426A)of ALA6 gene were used as materials,and the molecular mechanism of ALA6 participating plant heat stress response was studied by means of bioinformatics,molecular biology and physiology methods.The main results of this study are as follows.1.Compared with wild-type plants,ALA6 T-DNA insertion mutant(ala6-1)exhibits obvioussensitivity to heat stress.ala6-1 mutant displays a higher conductivity(electrolyte leakage)during heat stress compared with WT plants,suggesting the involvement of ALA6 in the regulation of cell membrane stability.Chlorophyll contents of both WT and ala6-1 mutant decreased during heat stress treatments,indicating a possible heat-induced damage to the photosynthetic activity of chloroplasts.In addition,the chl b/a ratio also increased dramatically in ala6-1 mutants after heat treatment.These results indicate that the missing of ALA6 may increase the damage of membranous organelles caused by heat stress.2.Both the overexpression line and complement line of ALA6 were obtained.The overexpression of ALA6 gene can obviously improve the resistance of seedling to heat stress increase.After the conserved functional sitse of ALA6 were mutated through point mutation approach and integrated into ala6-1 mutant,the ALA6 complement plants were still susceptible to heat stress like ala6-1 mutant.This result implies that the activity of phospholipid translocation enzyme is involved in the heat stress response.3.GUS histochemical staining found that ALA6 showed an expression in hypocotyl,cotyledon,root,petals,pollen and silique especially in the hypocotyl and root transition region of2-6 day old seedling.Its expression is controlled by plant development and can be induced by high temperature.4.Transcriptome analysis revealed that there was certain differences in gene expression between ala6-1 mutant and WT plants with or without heat stress.The differentially expressed genes were involved primarily in physiological processes related to stress response,including cellular compartment maintenance,macromolecule stability and energy production.ALA6 gene is involved in heat stress response of plant by regulating these signaling pathways.In conclusion,the ALA6 is critical for the stability of membrane when plants suffer from high temperature stress.This study not only has important academic value for understanding the mechanism of ALA6 in regulating plant response to high temperature stress,but also has important advising interests for breeding new crop varieties with high resistence to to environmental stress using ALA6.