Exploring The Cold Tolerance Function Of The TaMYB4-TaMDAHR Module In Winter Wheat

The winter is a long and cold in Heilongjiang Province(minimum temperature can reach-30°C),in which most economic crops cannot safely overwinter,resulting in low land utilization and economic damage.Dongnongdongmai1(Dn1)is the first winter wheat variety in Heilongjiang Province that can be safely overwintering.This variety fills the gap of winter wheat cultivation in the alpine region of China and greatly increases the land utilization.Therefore,it is particularly important to explore the Dn1 cold tolerance gene resource and understand its molecular mechanism in response to cold stress.We sampled tillering nodes,which is an important overwintering organ of winter wheat,at 5°C,0°C,-5°C,-10°C,-15°C,-20°C and-25°C for transcriptome sequencing to deeply mine the key functional genes of cold resistance.Weighted gene co-expression network(WGCNA)analysis of transcriptome data found that TaMYB4 was associated with monodehydroascorbate reductase TaMDHAR and zinc finger protein TaZFP775.Subsequently,we analyzed the expression patterns of TaMYB4,TaMDHAR and TaZFP775 under cold stress,and explored the regulatory relationship between TaMYB4 and TaMDHAR and the interaction between TaMYB4 and TaZFP775 using yeast one hybrid,yeast two hybrid,bimolecular fluorescence complementation(Bi Fc)and tobacco transient expression,respectively.OE-TaMYB4 and OETaMDHAR Arabidopsis plants were obtained by genetic transformation.The expression levels of cold tolerance related genes and physiological indexes related to cold tolerance in overexpressing plants under cold stress were measured to verify the functions of the target genes and provide a theoretical basis for crop cold tolerance breeding.The main findings are as follows:(1)Approximately 120000 genes were detected by Dn1 transcriptome sequencing under cold stress.The numbers of differentially expressed genes(DEGs)in the six comparison groups(0℃ vs.5℃,-5℃ vs.5℃,-10℃ vs.5℃,-15℃ vs.5℃,-20℃ vs.5℃ and-25℃ vs.5℃)were 11313,8313,15636,13671,14294 and 13979,respectively.Gene Ontology(GO)functional annotation suggested that the DEGs under cold stress mainly had “protein binding”,“DNA binding”,“ATP binding”,“catalytic”,“protein kinase” and other activities and were involved in “oxidation-reduction”,“protein phosphorylation”,“carbohydrate metabolic” and other processes.Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis indicated that the DEGs performed important functions in cold signal transduction and carbohydrate metabolism.In addition,important transcription factors(AP2/ERF,b ZIP,NAC,WRKY,b HLH and MYB)participating in the Dn1 cold stress response were activated by low temperature.(2)Analysis of the expression patterns of TaMYB4,TaMDAHR and TaZFP775 in the tillering nodes of Dn1 in the field showed that the expression levels of all three were significantly upregulated under freezing temperatures,and the fold change was significantly higher than that in the weakly cold resistant winter wheat cultivar Jimai 22(J22);The expression levels of TaMYB4,TaMDHAR and TaZFP775 in the tillering nodes of Dn1 in the indoor were also significantly upregulated under freezing temperatures,which were similar to the three gene expression changes of Dn1 in the field.The results of promoter activity analysis showed that the expression of TaMYB4,TaMDHAR and TaZFP775 was enhanced by cold stress.(3)Yeast one hybrid and tobacco transient expression results indicated that TaMYB4 was able to recognize the MYB binding site on the Pro MDHAR,thereby regulating the expression of TaMDHAR;Yeast two hybrid and Bi Fc results indicated that TaZFP775 and TaMYB4 interact in the nucleus.(4)The biological function of cold resistance was verified by overexpression of TaMYB4 and TaMDHAR in Arabidopsis.Measurement of physiological indexes related to cold tolerance showed that OE-TaMYB4 and OE-TaMDHAR plants had higher survival rate,proline(Pro)content,catalase(CAT)and superoxide dismutase(SOD)activities and lower electrical conductivity,malondialdehyde(MDA)content and reactive oxygen species(ROS)levels than wild type(WT)plants under cold stress.Under cold stress,the ratio of ascorbate to dehydroascorbate(As A/DHA)and the ratio of glutathione to glutathione disulfide(GSH/GSSG)in OE-TaMYB4 and OETaMDHAR plants were significantly higher than those in WT plants.And,the activities and gene expression levels of ascorbate peroxidase(APX),dehydroascorbate reductase(DHAR),monodehydroascorbate reductase(MDHAR)and glutathione reductase(GR)in OE-TaMYB4 and OE-TaMDHAR plants were also significantly higher than those in WT plants.In summary,transcriptome analysis revealed that there were a large number of cold tolerance related genes in Dn1 in response to cold stress.Among them,TaMYB4-TaMDHAR module positively regulate cold tolerance in Dn1.Reverse genetics approach proved that TaMYB4 and TaMDHAR could promote the As A-GSH cycle operation,so as to increased the ROS scavenging ability and enhanced the cold tolerance of plants.In this study,a large number of cold tolerance related genes were identified from Dn1.Exploration of cold tolerance function of TaMYB4-TaMDHAR module enriched the Dn1 cold tolerance regulatory network,which might provide a theoretical basis for cultivating new varieties of strong cold tolerance wheat.