Abscisic acid (ABA) is an endogenous phytohormone that plays important roles in the regulation of plant growth, development, and stress responses. The pyrabactin resistance 1-like (PYR/PYL) protein is a core regulatory component of ABA signaling networks in plants. However, no details regarding this family in Brassica napus are available. Here, 46 PYLs were identified in the B. napus genome. Based on phylogenetic analysis, BnPYR1 and BnPYL1-3 belong to subfamily I, BnPYL7-10 belong to subfamily II, and BnPYL4-6 and BnPYL11-13 belong to subfamily III. Analysis of BnPYL conserved motifs showed that every subfamily contained four common motifs. By predicting cis-elements in the promoters, we found that all BnPYL members contained hormone- and stress-related elements and that expression levels of most BnPYLs were relatively higher in seeds at the germination stage than those in other organs or at other developmental stages. Gene Ontology (GO) enrichment showed that BnPYL genes mainly participate in responses to stimuli. To identify crucial PYLs mediating the response to abiotic stress in B. napus, expression changes in 14 BnPYL genes were determined by quantitative real-time RT-PCR after drought, heat, and salinity treatments, and identified BnPYR1-3, BnPYL1-2, and BnPYL7-2 in respond to abiotic stresses. The findings of this study lay a foundation for further investigations of PYL genes in B. napus.

Ferulate‐5‐hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate‐5‐hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D‐NMR profiling in KO‐7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

Drought and salinity stresses are severe and wide-ranging abiotic stresses that substantially affect crop germination, development and productivity. Seed germination is the first critical step in plant growth and development. To comprehensively investigate small-RNA targets and improve our understanding of the miRNA-mediated post-transcriptional regulation network during rapeseed seed imbibition under drought and salt stresses, we constructed three small-RNA libraries from B. napus variety ZS11 seed embryos exposed to salt (200 mM NaCl, marked as S), drought (200 g L-1 PEG-6000, marked as D), and distilled water (marked as CK) during the imbibition process and sequenced them using an Illumina Genome Analyzer. A total of 11,528,557, 12,080,081 and 12,315,608 raw reads were obtained from the CK, D and S libraries, respectively. Further analysis identified 85 known miRNAs belonging to 31 miRNA families and 882 novel miRNAs in the three libraries. Six miRNA families—miR156, miR169, miR860, miR399, miR171 and miR395—were significantly down-regulated, whereas miR172 was significantly up-regulated, in the D library compared to the CK library. Only two miRNA families—miRNA393 and miRNA399—were significantly down-regulated in the S library compared to the CK library. Furthermore, putative targets for 336, 376, and 340 novel miRNAs were successfully predicted in the CK, D, and S libraries, respectively. A total of 271 miRNA families and 20 target gene families in rapeseed (including disease resistance protein (DIRP), drought-responsive family protein (DRRP), early responsive to dehydration stress protein (ERD), stress-responsive alpha-beta barrel domain protein (SRAP), and salt tolerance homolog2 (STH2)) were confirmed to be core miRNAs and genes involved in the seed imbibition response to salt and drought stresses. The sequencing results were partially validated by quantitative RT-PCR for both conserved and novel miRNAs and the predicted target genes. Our data suggest that diverse and complex miRNAs are involved in seed imbibition, which indicates that miRNAs are involved in plant hormone regulation and may play important roles during seed germination under salt- or drought-stress conditions.

NODULE-INCEPTION-like proteins (NLPs) are conserved, plant-specific transcription factors that play crucial roles in responses to nitrogen deficiency. However, the evolutionary relationships and characteristics of NLP family genes in Brassica napus are unclear. In this study, we identified 31 NLP genes in B. napus, including 16 genes located in the A subgenome and 15 in the C subgenome. Subcellular localization predictions indicated that most BnaNLP proteins are localized to the nucleus. Phylogenetic analysis suggested that the NLP gene family could be divided into three groups and that at least three ancient copies of NLP genes existed in the ancestor of both monocots and dicots prior to their divergence. The ancestor of group III NLP genes may have experienced duplication more than once in the Brassicaceae species. Three-dimensional structural analysis suggested that 14 amino acids in BnaNLP7-1 protein are involved in DNA binding, whereas no binding sites were identified in the two RWP-RK and PB1 domains conserved in BnaNLP proteins. Expression profile analysis indicated that BnaNLP genes are expressed in most organs but tend to be highly expressed in a single organ. For example, BnaNLP6 subfamily members are primarily expressed in roots, while the four BnaNLP7 subfamily members are highly expressed in leaves. BnaNLP genes also showed different expression patterns in response to nitrogen-deficient conditions. Under nitrogen deficiency, all members of the BnaNLP1/4/5/9 subfamilies were upregulated, all BnaNLP2/6 subfamily members were downregulated, and BnaNLP7/8 subfamily members showed various expression patterns in different organs. These results provide a comprehensive evolutionary history of NLP genes in B. napus, and insight into the biological functions of BnaNLP genes in response to nitrogen deficiency.

The winter oilseed ecotype is more tolerant to low temperature than the spring ecotype. Transcriptome and metabolome analyses of leaf samples of five spring Brassica napus L. (B. napus) ecotype lines and five winter B. napus ecotype lines treated at 4 °C and 28 °C were performed. A total of 25,460 differentially expressed genes (DEGs) of the spring oilseed ecotype and 28,512 DEGs of the winter oilseed ecotype were identified after cold stress; there were 41 differentially expressed metabolites (DEMs) in the spring and 47 in the winter oilseed ecotypes. Moreover, more than 46.2% DEGs were commonly detected in both ecotypes, and the extent of the changes were much more pronounced in the winter than spring ecotype. By contrast, only six DEMs were detected in both the spring and winter oilseed ecotypes. Eighty-one DEMs mainly belonged to primary metabolites, including amino acids, organic acids and sugars. The large number of specific genes and metabolites emphasizes the complex regulatory mechanisms involved in the cold stress response in oilseed rape. Furthermore, these data suggest that lipid, ABA, secondary metabolism, signal transduction and transcription factors may play distinct roles in the spring and winter ecotypes in response to cold stress. Differences in gene expression and metabolite levels after cold stress treatment may have contributed to the cold tolerance of the different oilseed ecotypes.