Study Of Abscisic Acid’s Role In Ameliorative Effects Of 24-epibrassinolide On Grape Seedlings Under Cold Stress

Grapes in northwestern China is often threatened by the extreme low temperature in winter and spring frost due to the influence of continental climate.Cold stress could causethe damage to the cell membrane system and disturbances in the metabolismsin plant cells.This not only affects the yield and quality of the grapes,but also poses a threat to the survival and the growth of the vines.Abscisic acid（ABA）is an important hormone in plants which can enhance the resistance of plants to a variety of abiotic and biotic stresses.ABA is also an important stress signal molecule in plants,participating in the transmission of cold stress signals.Brassinolide（BRs）is a new type of plant hormone.Previous studies in the laboratory found that exogenous BRs treatment can improve the resistance of grape seedlings to cold stress by alleviating oxidative stress.However,whether ABA is involved in the resistance of grape seedlings induced by exogenous BRs to low temperature stress needs further study.In the experiment,we applied ABA,EBR and EBR+NDGA（ABA synthesis inhibitor）to grape seedlings under cold stress,and observed the chlorophyll fluorescence parameters,reactive oxygen species,antioxidant system and the endogenous ABA and BRs levels from12 to 96 hours.In addition,we also analyzed the relative expression levels of CBF family genes from 1 to 96 hours.It was found that the cold-stress resistance induced by ABA and EBR treatment of grape seedlings had little relationship with CBF family genes.Therefore,the seedlings treated with ABA and EBR were separately subjected to RNA sequencing to investigate the effects of ABA and BR on the signal transduction under cold stress in grape seedlings.The main conclusions are as follows:（1）Compared with the control,ABA and EBR treatment obviously alleviated the cold injury of grape leavesand seedling’s tip under cold stress,while NDGA+EBR treatment only alleviated the cold damageof that to some extent;（2）Compared with the control,ABA,EBR and NDGA+EBR treatment significantly increased the endogenous ABA,BL（brassinolide）,CS（castasterone）and 6-deoxo CS（6-deoxocastasterone）contents in grape leaves under cold stress.Endogenous ABA and BRs contents in NDGA+EBR treated-leaves were significantly lower than that in EBR-treatedleaves during the experimental stage,which indicated that NDGA could inhibit the accumulation of endogenous ABA content to a degree and the inhibition of ABA de novo synthesiscould inhibit the content of endogenous BRs;（3）Compared with the control,ABA and EBR treatment increased the chlorophyll fluorescence parameters NPQ andφPSII as well as reducing the content of H₂O₂（hydrogen peroxide）and O₂^·-（superoxide anion）under cold stress.At the most sampling time points,NPQ,φPSII,H₂O₂and O₂^·-in EBR-treated leaves showed no significant differences than that in NDGA+EBR treated leaves,indicating that under low temperature,EBR increased the two chlorophyll fluorescence parameters and reduced the over-accumulation of ROS（reactive oxygen species）may not depend on ABA;（4）Compared with the control,ABA and EBR treatment increased the activity of antioxidant enzymes（CAT,SOD,APX,GR,DHAR and MDHAR）and the contents of antioxidant substances（As A and GSH）.Comparing with the EBR treatment,EBR+NDGA showed different significance in different antioxidase and antioxidants at different time points,which indicated that ABA may participate in EBR-regulated antioxidant systems at different stages during cold stress;（5）The relative expression levels of Vvi CBF family genes in grapes seedlings from 1to 96 hours under cold stress were analyzed.It was found that exogenous hormone treatments did not significantly increase the transcription level of Vvi CBF family genes,indicating that there exsits a cold stress signal transduction pathway independent of CBF pathway in grape seedlings.（6）Differential expression genes（DEGs）responded to cold stress were analyzed and found that the most DEGs were enriched in plant hormone signal transduction,cell metabolism and secondary metabolite synthesis pathways,indicating that these processes are closely related to cold tolerance in grape seedlings;（7）Up-regulated DEGs both responded to ABA and EBR treatment under cold stress were analyzed and found 30 transcription factors（TFs）;Up-regulated DEGs only responded to ABA treatment under cold stress were analyzed and found 5 mitogen-activated protein kinase（MAPK）cascade-related candidate genes and 11 TFs;Up-regulated DEGs only responded to EBR treatment were analyzed and found that 14 candidate genes related to calcium signaling pathway,5 candidate genes encodingstilbene synthase and 79 TFs.All transcription factor families were analyzed and found ABA and BR can not only jointly regulate transcription factor families such as AP2/ERF-ERF and b HLH,but also has their own unique way in regulting transcription factors under cold stress.In summary,in terms of long-run cold stress,ABA,EBR and NDGA+EBR treatment can balance the endogenous phytohormones,increase the chlorophyll fluorescence parameters,reduce the over-accumulation of ROS,enhance the antioxidant enzyme activity and increase antioxidant contents to alleviate the cold damage to grape seedlings.ABA is involved in BR-induced cold tolerance in grape seedlings.In terms of short-term cold stress,in grape seedlings,ABA has some common in transcription regulation with BR under cold stress while the two hormones also possess their own unique patterns in response to low temperature.