| Rapeseed(Brassica napus L.)is an important edible oil-producing crop worldwide.However,abiotic stresses such as cold stress could adversely affect plant growth at the seedling stage and subsequently,cause poor seed yield and lower seed quality at the mature stage.To understand the molecular mechanisms of cold-stress response in rapeseed,we comprehensively analyzed the profiles of transcripts by using RNA-Sequence and proteins by iTRAQ(Isobaric Tag for Relative and Absolute Quantification)strategy,in a cold-tolerant(C18)and a cold-sensitive(Zhongshuang 6,C6)rapeseed varieties under cold stress conditions at the four-leaf stage.The main results of present study are as follows:(1)Based on the previous transcriptomics data,a total of 8485 differentially expressed genes(DEGs)were found to be differentially expressed at 4 °C for 1-d and 7-d when compared to 0-d.And 241 differentially expressed proteins(DEPs)were identified within the same comparisons.In total,10,45,and 73 DEPs were identified in C18 during early(1-d compared to 0-d),late(7-d compared to 1-d),and continuous(7-d compared to 0-d)response of cold treatment.On the contrary,23,152,and 79 DEPs were detected in C6 during the early,late,and continuous stages of response to cold treatment.Bioinformatics’ and pathway enrichment analysis showed that more stress-responsive proteins in two varieties were involved in photosynthesis and ribosome biogenesis.Photosynthesis-antenna proteins,anthocyanin biosynthesis,and thiamine metabolism were mainly enriched in C18,whereas carbon metabolism,glyoxylate,and dicarboxylate metabolism were only enriched in C6.Based on functional analysis,we concluded that the adaptive response of rapeseed to LT-stress might be related to ribosomal proteins(RPs)and heat shock proteins(HSPs),mitigation of photosynthesis,energy metabolism,and ROS scavenge.(2)Comparative dataset analysis of transcriptome and proteome revealed that 48 DEGs were correlated to 17 DEPs in C18 during cold stress treatment,whereas 82 DEGs were corresponded to 38 DEPs in C6.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of combined genes revealed that 11 key genes were involved in energy metabolism,followed by8 in photosynthesis and 3 in antioxidant enzymes.More combined DEGs were related to energy metabolism and antioxidant enzymes in C18 variety when compared to C6 variety.Furthermore,qRTPCR of nine selected combined DEGs confirmed the reliability of RNA-seq data.Based on functional annotation and enrichment analyses,it was proposed that antioxidant accumulation,photosynthesis,and energy metabolism might be the core driving forces that regulate cold response in rapeseed.(3)To verify that the candidate genes function in the process of cold response,Arabidopsis transfer DNA(T-DNA)insertion lines for homologs of the three candidates(BnNIR,BnCML,BnCAT)were obtained;the three homozygous mutant lines(nir,cml,cat)differed significantly in levels of freezing response.Freezing injury caused higher electrolyte leakage and lower chlorophyll a,b contents in three mutant lines when compared to WT,which accordingly leading to significantly lower survival rate of mutants.Besides,we found lower catalase activity and antioxidant imbalance in cat mutant,suggesting that other antioxidants participate in compensating the loss of cat function.In nir and cml mutants,we observed a strong reduction in COR genes,revealing that AtNIR or AtCML were either directly or indirectly associated with COR genes induction under stress conditions.The comparative analysis here provided insights into molecular mechanisms of the cold response in rapeseed.Moreover,nir,cml,and cat genes might play significant role in freezing stress in Arabidopsis.The results obtained in this study provided valuable information for functional characterization of coldrelated genes,furthermore,dissection the molecular mechanisms of cold response in rapeseed. |
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