Integrated Analysis Of Transcriptome And Metabolome Reveals Insights For Cold Response In Rapeseed(Brassica Napus L.)

Rapeseed(Brassica napus L.)is an important oilseed crop in the world.It is the second-main edible oil source in China.Rapeseed production is of great economic importance for the sustainable edible oil supply in China.Extreme temperature variations significantly influence rapeseed growth and production.In general,cold stress thermodynamically decreases the kinetics of various physiological as well as metabolic processes occurring in plants,which accordingly seriously reduces the rate and uniformity of germination,hampers seedling vigour,and delays ontogenic plant development,resulting in serious crop yield losses.Consequently,enhancement in cold tolerance is becoming a most important area for agricultural investigations and crop improvement in China.Therefore,the current study aimed to identify the stress-responsive genes,metabolites,and metabolic pathways from the combined transcriptome and metabolome analysis to understand the cold stress responses and tolerance mechanisms at a molecular level in cold-tolerant(C18)and cold-sensitive(C6)rapeseed varieties.Three-time points,i.e.,before stress(0 d/CK),after 1 d and 7 d of stress at 4 ~oC,were set to study the effect of cold stress at transcriptome and metabolome levels.The analysis was carried out in different group comparisons in both varieties,including C18-0?vs?C18-1,C18-0?vs?C18-7,and C18-1?vs?C18-7 in C18,and C6-0?vs?C6-1,C6-0?vs?C6-7,and C6-1?vs?C6-7 in C6.Further,multi-group analysis was also carried out in both varieties at the same point in time(C6-0?vs?C18-0,C6-1?vs?C18-1,and C6-7?vs?C18-7).The metabolome data sets provided a total of 3368 differentially accumulated metabolites(DAMs),including 156 DAMs from the comparison of C6-0?vs?C6-1,357 DAMs from C6-0?vs?C6-7,358 DAMs from C6-1?vs?C6-7,respectively.For C18 cultivar,182 DAMs from C18-0?vs?C18-1,755 DAMs from C18-0?vs?C18-7,and 469 DAMs from C18-1?vs?C18-7 were detected,respectively.Furthermore,by analyzing the DAMs from the comparison between both cultivars at each time point,we identified 429 DAMs from C6-0?vs?C18-0,366 DAMs from C6-1?vs?C18-1,and 403 DAMs from C6-7?vs?C18-7.Notably,out of these,626 were known metabolites that have already been annotated.Numerous core DAMs have been recognized from the comparisons of the same time points between both varieties.Further,KEGG enrichment annotation results discovered that most DAMs were enriched in different carbohydrates and amino acid metabolisms.From the transcriptome data sets,2845 differently expressed genes(DEGs)from the comparison of C6-0?vs?C18-0,3358 DEGs from C6-1?vs?C18-1,and 2819 DEGs from C6-7?vs?C18-7 were distinguished,respectively.After that,the interaction and correlation analysis were carried out to identify the relationships between DEGs and DAMs at the same time points in both varieties.Further,by combining the transcriptome and metabolome data sets,it was identified that numerous DAMs were strongly correlated with several genes(DEGs).Functional enrichment analysis(GO and KEGG)of the metabolites-correlated DEGs specified that most DEGs were mainly enriched in diverse carbohydrates and amino acid metabolisms.Interestingly,starch and sucrose metabolism,and phenylalanine metabolism were found to be significantly enriched and playing a vital role in cold stress adaption and tolerance in rapeseed.The expression profiling of some selected DEGs from starch and sucrose metabolism and phenylalanine metabolism pathways revealed that all the genes were induced by cold stress in both varieties.The noticed expression trends were regularly constant for all the genes,indicating the reliability of the RNA-seq data.For functional validation,Arabidopsis homologous of T-DNA insertion mutants of the selected DEGs from the starch and sucrose metabolism,and phenylalanine metabolism were performed.To our best knowledge,these selected genes have not been characterized in rapeseed under cold stress.The results show that mutants of homologues of 4cl3,cel5,fruct4,ugp1,axs1,and bam2/9 were sensitive to freezing stress.The DAMs or DEGs identified here may be exploited for agronomic improvement under cold stress.The current study's outcome provided new prospects to understand the molecular basis of cold stress responses and tolerance mechanisms in rapeseed.In the near future,the candidate genes should be functionally validated under short-or/and long-term cold stress conditions in a field environment.