Exploration Of Mechanism Underlying The Freezing Tolerance In Actinidia Arguta And Functional Identification Of Two Cold Responsive Genes

Kiwifruit belongs to the genus Actinidia which consists of 75 taxa(54 species and 21 varieties).Currently,commercial kiwifruit species include Actinidia chinensis(Actinidia chinensis Planchon and Actinidia chinensis var.deliciosa),Actinidia eriantha and Actinidia arguta.Among them,both Actinidia chinensis and Actinidia eriantha are the most important in Chinese kiwifruit industry.They are suitable for growth in the south of the Yellow-River,where the temperature in winter is normally above-10 °C.Recently,the kiwifruit planting area is increasing and towards to the northern China.In addition,the frequent occurrence of extremely low temperatures makes kiwifruit vulnerable in recent years,which can lead to a decrease in the yield or the death of the whole plant.A.arguta is a special species with widely geographical distribution among the genus Actinidia,it has stronger freezing tolerance compared with A.chinensis.The ultimate method to solve the low-temperature stress problems of kiwifruit is to explore the molecular mechanism of freezing tolerance and screen out the main genes related to freezing tolerance.In this study,we combined the NGS transcriptomics,SMRT transcriptomics and metabolomics to identify the key pathways and genes involved in freezing tolerance,and the function of the two candidate genes was verified.The main results were as follows:1.To obtain further knowledge of the mechanism of freezing tolerance,we carried out full-length transcriptome analysis of two A.arguta genotypes,KL with stronger tolerance and RB with weak tolerance,respectively.Both genotypes were subjected to-25 °C for 0 h,1 h,and 4 h.The SMRT(single-molecule real-time)RNA-Seq data were assembled using the de novo method,and produced 24,306 Unigenes with an N50 value of 1,834 bp.The KEGG enrichment analysis of DEGs indicated that ‘starch and sucrose metabolism’,‘MAPK signaling pathway’,‘phosphatidylinositol signaling system’,‘inositol phosphate metabolism’,and ‘plant hormone signal transduction’ were enriched.Especially,for ‘starch and sucrose metabolism’,we identified 3 key genes involved in cellulose degradation,trehalose synthesis,and starch degradationprocesses.Moreover,beta-GC(beta-glucosidase),TPS(trehalose-6-phosphate synthase),and BAM(beta-amylase)activities,encoded by the above-mentioned3 key genes,were enhanced by cold stress.Three transcription factors(TFs)belong to the AP2/ERF,b HLH(basic helix-leucine-helix),and MYB families were involved in the low temperature response.Furthermore,weighted gene co-expression network analysis(WGCNA)results indicated that beta-GC,TPS,and BAM genes were the key genes involved in starch-sucrose pathway,and showed a high co-expression model with CBF,MYC2,and MYB44 genes.Hence,the ‘starch and sucrose metabolism’ pathway plays an important role in responding to low temperature in A.arguta.2.We combined metabolome and transcriptome to identify the main pathways and important metabolites related to the freezing tolerance.A total of 565 metabolites were detected by a wide-targeting metabolomics method.Under(-25 °C)cold stress,KEGG pathway annotations showed that the flavonoid metabolic pathways were specifically upregulated in KL.Nucleotides metabolism and phenolic acids metabolism pathways were specifically upregulated in RB,which indicated that RB had a higher energy metabolism and weaker dormancy ability.Since the LPCs(Lyso PC),LPEs(Lyso PE)and free fatty acids were accumulated simultaneously in both genotypes,these could serve as biomarkers of cold-induced frost damage.These key metabolism components participated in the regulation of freezing tolerance of both kiwifruit genotypes.In conclusion,the results of this study demonstrated the kiwifruit can reduce the energy metabolism by reducing phenolic metabolites and enhance the antioxidant capacity by accumulating flavonoid metabolites to improve freezing tolerance during the dormant period.3.Beta-amylase(BAM)is an important enzyme to enhance freezing tolerance.Sixteen BAMs were identified from the kiwifruit ‘Red5’ genome.BAMs were distributed in 11 chromosomes.The 5 genes were found to be replicative accounting for 31.25% of the total genes,which indicated that gene replication events were important to the expansion of the family.furthermore,thephysicochemical properties of their proteins are conserved and their amino acid sequences are relatively similar.A total of 16 Aa BAM genes were obtained by BLAST in the full-length transcriptome database of Actinidia arguta.Combining with the NGS-transcriptome data,the expressional data of the 16 Aa BAM genes under low temperature stress were obtained,in which Aa BAM3.1 strongly responded to low temperature stress.Aa BAM3.1-overexpressing kiwifruit lines showed increased freezing tolerance,and the heterologous overexpression of Aa BAM3.1 in Arabidopsis thaliana resulted in a similar phenotype.The REL and content of MDA,H2O2,and O2? in over-expressed lines were significantly lower than that of the wild types.However,the soluble sugar and BAM enzyme activity in the over-expressed lines were higher than those in the WT plants.Above-mentioned results indicated that Aa BAM3.1 could decrease osmotic stress and oxidative stress to increase the freezing tolerance.The results of promoter GUS activity and cis-element analyses predicted Aa CBF4 to be an upstream transcription factor that could regulate Aa BAM3.1.The further investigation of protein-DNA interactions by using yeast one-hybrid,GUS coexpression,and dual luciferase reporter assays confirmed that Aa CBF4 directly regulated Aa BAM3.1.In addition,kiwifruit Aa BAM3.1 and Aa CBF4 both showed a positive response to cold stress.Hence,we conclude that the Aa CBF-Aa BAM module is involved in the positive regulation of freezing tolerance in kiwifruit.4.Leucoanthocyanidin reductase(LAR)plays a key role in plant tolerance to cold stress.Under low temperature treatment,the proanthocyanidin contents of the kiwifruit shoots were significantly increased.The q RT-PCR was used to measure the expressional level of the structural genes related to proanthocyanidin synthesis,and results showed that the expression level of Aa LAR was also significantly up-regulated under cold treatment.Moreover,Aa LAR of KL had a higher expression level than it of RB.By comparing the sequence differences of Aa LAR between tolerant and sensitive genotypes,the results showed that there was a 60 bp insertion or deletion in the Aa LAR promoter sequence,and this important sequence mutation region contained an MYC-core(CANNTG)motif,which may be the binding site of MYC transcription factor.Yeast and dual luciferase reporter gene experiments showed that Aa MYC could bind to the MYC-core element of the Aa LAR promoter with the assistance of Aa MYB,which promoted the accumulation of proanthocyanidins in the shoots of kiwifruit,and the accumulated proanthocyanidins ultimately enhanced freezing tolerance of kiwifruit by improving ROS scavenging ability.Therefore,we conclude that the Aa MYB-Aa MYC-Aa LAR module is involved in the freezing tolerance enhancement in kiwifruit.This study used multi-omics analysis to explore the freezing tolerance mechanism of A.arguta.We demonstrated that cold-responsive gene modules including Aa CBF-Aa BAM and Aa MYB-Aa MYC-Aa LAR were involved in freezing tolerance in kiwifruit.These results deepen our understanding of the complicated mechanisms involved in freezing tolerance in kiwifruit under cold stress and identify a series of candidate genes for breeding new cultivars with strong freezing tolerance.