Identification of differentially expressed genes involved in amino acid and lipid accumulation of winter turnip rape (Brassica rapa L.) in response to cold stress.

Winter turnip rape (Brassica rapa L.) is an important overwintering oil crop that is widely planted in northwestern China. It considered to be a good genetic resource for cold-tolerant research because its roots can survive harsh winter conditions. Here, we performed comparative transcriptomics analysis of the roots of two winter turnip rape varieties, Longyou7 (L7, strong cold tolerance) and Tianyou2 (T2, low cold tolerance), under normal condition (CK) and cold stress (CT) condition. A total of 8,366 differentially expressed genes (DEGs) were detected between the two L7 root groups (L7CK_VS_L7CT), and 8,106 DEGs were detected for T2CK_VS_T2CT. Among the DEGs, two ω-3 fatty acid desaturase (FAD3), two delta-9 acyl-lipid desaturase 2 (ADS2), one diacylglycerol kinase (DGK), and one 3-ketoacyl-CoA synthase 2 (KCS2) were differentially expressed in the two varieties and identified to be related to fatty acid synthesis. Four glutamine synthetase cytosolic isozymes (GLN), serine acetyltransferase 1 (SAT1), and serine acetyltransferase 3 (SAT3) were down-regulated under cold stress, while S-adenosylmethionine decarboxylase proenzyme 1 (AMD1) had an up-regulation tendency in response to cold stress in the two samples. Moreover, the delta-1-pyrroline-5-carboxylate synthase (P5CS), δ-ornithine aminotransferase (δ-OAT), alanine-glyoxylate transaminase (AGXT), branched-chain-amino-acid transaminase (ilvE), alpha-aminoadipic semialdehyde synthase (AASS), Tyrosine aminotransferase (TAT) and arginine decarboxylase related to amino acid metabolism were identified in two cultivars variously expressed under cold stress. The above DEGs related to amino acid metabolism were suspected to the reason for amino acids content change. The RNA-seq data were validated by real-time quantitative RT-PCR of 19 randomly selected genes. The findings of our study provide the gene expression profile between two varieties of winter turnip rape, which lay the foundation for a deeper understanding of the highly complex regulatory mechanisms in plants during cold treatment.

Genome-Wide Identification and Analysis of the Ascorbate Peroxidase (APX) Gene Family of Winter Rapeseed (Brassica rapa L.) Under Abiotic Stress.

Winter Brassica rapa (B. rapa) is an important oilseed crop in northern China, but the mechanism of its cold resistance remains unclear. Ascorbate peroxidase (APX) plays important roles in the response of this plant to abiotic stress and in scavenging free radicals. In this study, the roles of APX proteins in the cold response and superoxide metabolism pathways in rapeseed species were investigated, and a comprehensive analysis of phylogeny, chromosome distribution, motif identification, sequence structure, gene duplication, and RNA-seq expression profiles in the APX gene family was conducted. Most BrAPX genes were specifically expressed under cold stress and behaved significantly differently in cold-tolerant and weakly cold-resistant varieties. Quantitative real-time-PCR (qRT-PCR) was also used to verify the differences in expression between these two varieties under cold, freezing, drought and heat stress. The expression of five BrAPX genes was significantly upregulated in growth cones at 3 h of cold stress, while their expression was significantly lower at 24 h than at 3 h. The expression of Bra015403 and Bra003918 was significantly higher in "Longyou-7" growth cones than in other treatments. Five BrAPXs (Bra035235, Bra003918, Bra033040, Bra017120, and Bra031934) were closely associated with abiotic stress responses in B. rapa. These candidate genes may play important roles in the response of B. rapa to low temperature stress and provide new information for the elucidation of the cold resistance mechanism in B. rapa.

Transcriptome Analysis Reveals Key Cold-Stress-Responsive Genes in Winter Rapeseed (Brassica rapa L.).

Low ambient air temperature limits the growth and selection of crops in cold regions, and cold tolerance is a survival strategy for overwintering plants in cold winters. Studies of differences in transcriptional levels of winter rapeseed (Brassica rapa L.) under cold stress can improve our understanding of transcript-mediated cold stress responses. In this study, two winter rapeseed varieties, Longyou-7 (cold-tolerant) and Lenox (cold-sensitive), were used to reveal morphological, physiological, and transcriptome levels after 24 h of cold stress, and 24 h at room temperature, to identify the mechanism of tolerance to cold stress. Compared to Lenox, Longyou-7 has a shorter growth period and greater belowground mass, and exhibits stronger physiological activity after cold stress. Subsequently, more complete genomic annotation was obtained by sequencing. A total of 10,251 and 10,972 differentially expressed genes (DEG) were identified in Longyou-7 and Lenox, respectively. Six terms closely related to cold stress were found by the Gene Ontology (GO) function annotation. Some of these terms had greater upregulated expression in Longyou-7, and the expression of these genes was verified by qRT-PCR. Most of these DEGs are involved in phenylpropanoid biosynthesis, plant hormone signal transduction, ribosome biogenesis, MAPK signaling pathway, basal transcription factors, and photosynthesis. Analysis of the genes involved in the peroxisome pathway revealed that Longyou-7 and Lenox may have different metabolic patterns. Some transcription factors may play an important role in winter rapeseed tolerance to cold stress, and Longyou-7 is slightly slower than Lenox. Our results provide a transcriptome database and candidate genes for further study of winter rapeseed cold stress.

iTRAQ-Based Comparative Proteomic Analysis of the Roots of TWO Winter Turnip Rapes (Brassica rapa L.) with Different Freezing-Tolerance.

The freezing tolerance of roots is crucial for winter turnip rape (Brassica rapa L.) survival in the winter in Northwest China. Cold acclimation (CA) can alleviate the root damage caused by freezing stress. To acknowledge the molecular mechanisms of freezing tolerance in winter turnip rape, two Brassica rapa genotypes, freezing stressed after the induction of cold acclimation, were used to compare the proteomic profiles of roots by isobaric tags for relative and absolute quantification (iTRAQ). Under freezing stress (-4 °C) for 8 h, 139 and 96 differentially abundant proteins (DAPs) were identified in the roots of "Longyou7" (freezing-tolerant) and "Tianyou4" (freezing-sensitive), respectively. Among these DAPs, 91 and 48 proteins were up- and down-accumulated in "Longyou7", respectively, and 46 and 50 proteins were up- and down-accumulated in "Tianyou4", respectively. Under freezing stress, 174 DAPs of two varieties were identified, including 9 proteins related to ribosome, 19 DAPs related to the biosynthesis of secondary metabolites (e.g., phenylpropanoid and the lignin pathway), and 22 down-accumulated DAPs enriched in oxidative phosphorylation, the pentose phosphate pathway, fructose and mannose metabolism, alpha-linolenic acid metabolism, carbon fixation in photosynthetic organisms, ascorbate and aldarate metabolism. The expressional pattern of the genes encoding the 15 significant DAPs were consistent with the iTRAQ data. This work indicates that protein biosynthesis, lignin synthesis, the reduction of energy consumption and a higher linolenic acid content contribute to the freezing tolerance of winter turnip rape. Functional analyses of these DAPs would be helpful in dissecting the molecular mechanisms of the stress responses in B. rapa.

iTRAQ-Based Quantitative Proteome Revealed Metabolic Changes in Winter Turnip Rape (Brassica rapa L.) under Cold Stress.

Winter turnip rape (Brassica rapa L.) is a large-scale winter-only oil crop cultivated in Northwest China. However, its cold-resistant molecular mechanism remains inadequate. Studying the cold adaptation mechanisms of winter turnip rape based on the proteomic technique of isobaric tags for relative and absolute quantification (iTRAQ) offers a solution to this problem. Under cold stress (-4 °C for eight hours), 51 and 94 differently accumulated proteins (DAPs) in Longyou 7 (cold-tolerant) and Tianyou 4 (cold-sensitive) were identified, respectively. These DAPs were classified into 38 gene ontology (GO) term categories, such as metabolic process, cellular process, catalytic activity, and binding. The 142 DAPs identified between the two cold-stressed cultivars were classified into 40 GO terms, including cellular process, metabolic process, cell, catalytic activity, and binding. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the DAPs participated in 10 pathways. The abundance of most protein functions in ribosomes, carbon metabolism, photosynthesis, and energy metabolism including the citrate cycle, pentose phosphate pathway, and glyoxylate and dicarboxylate metabolism decreased, and the proteins that participate in photosynthesis⁻antenna and isoflavonoid biosynthesis increased in cold-stressed Longyou 7 compared with those in cold-stressed Tianyou 4. The expression pattern of genes encoding the 10 significant DAPs was consistent with the iTRAQ data. This study provides new information on the proteomic differences between the leaves of Longyou 7 and Tianyou 4 plants and explains the possible molecular mechanisms of cold-stress adaptation in B. rapa.

Identification of cold stress responsive microRNAs in two winter turnip rape (Brassica rapa L.) by high throughput sequencing.

BACKGROUND: Low temperature is a major abiotic stress affecting the production of rapeseed in China by impeding plant growth and development. A comprehensive knowledge of small-RNA expression pattern in Brassica rapa under cold stress could improve our knowledge of microRNA-mediated stress responses. RESULTS: A total of 353 cold-responsive miRNAs, 84 putative novel and 269 conserved miRNAs, were identified from the leaves and roots of two winter turnip rape varieties 'Longyou 7' (cold-tolerant) and 'Tianyou 4' (cold-sensitive), which were stressed under - 4 °C for 8 h. Eight conserved (miR166h-3p-1, miR398b-3p, miR398b-3p-1, miR408d, miR156a-5p, miR396h, miR845a-1, miR166u) and two novel miRNAs (Bra-novel-miR3153-5p and Bra-novel-miR3172-5p) were differentially expressed in leaves of 'Longyou 7' under cold stress. Bra-novel-miR3936-5p was up-regulated in roots of 'Longyou 7' under cold stress. Four and five conserved miRNAs were differentially expressed in leaves and roots of 'Tianyou 4' after cold stress. Besides, we found two conserved miRNAs (miR319e and miR166m-2) were down-regulated in non-stressed roots of 'Longyou 7' compared with 'Tianyou 4'. After cold stress, we found two and eight miRNAs were differentially expressed in leaves and roots of 'Longyou 7' compared with 'Tianyou 4'. The differentially expressed miRNAs between two cultivars under cold stress include novel miRNAs and the members of the miR166 and miR319 families. A total of 211 target genes for 15 known miRNAs and two novel miRNAs were predicted by bioinformatic analysis, mainly involved in metabolic processes and stress responses. Five differentially expressed miRNAs and predicted target genes were confirmed by quantitative reverse transcription PCR, and the expressional changes of target genes were negatively correlated to differentially expressed miRNAs. Our data indicated that some candidate miRNAs (e.g., miR166e, miR319, and Bra-novel-miR3936-5p) may play important roles in plant response to cold stress. CONCLUSIONS: Our work indicates that miRNA and putative target genes mediated metabolic processes and stress responses are significant to cold tolerance in B. rapa.