Cytokinin oxidase/dehydrogenases (CKXs) play a critical role in the irreversible degradation of cytokinins, thereby regulating plant growth and development. Brassica napus is one of the most widely cultivated oilseed crops worldwide. With the completion of whole-genome sequencing of B. napus, genome-wide identification and expression analysis of the BnCKX gene family has become technically feasible. In this study, we identified 23 BnCKX genes and analyzed their phylogenetic relationships, gene structures, conserved motifs, protein subcellular localizations, and other properties. We also analyzed the expression of the 23 BnCKX genes in the B. napus cultivar Zhong Shuang 11 (‘ZS11’) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), revealing their diverse expression patterns. We selected four BnCKX genes based on the results of RNA-sequencing and qRT-PCR and compared their expression in cultivated varieties with extremely long versus short siliques. The expression levels of BnCKX5-1, 5-2, 6-1, and 7-1 significantly differed between the two lines and changed during pod development, suggesting they might play roles in determining silique length and in pod development. Finally, we investigated the effects of treatment with the synthetic cytokinin 6-benzylaminopurine (6-BA) and the auxin indole-3-acetic acid (IAA) on the expression of the four selected BnCKX genes. Our results suggest that regulating BnCKX expression is a promising way to enhance the harvest index and stress resistance in plants.

MicroRNAs (miRNAs) have important roles in regulating stress-response genes in plants. However, identification of miRNAs and the corresponding target genes that are induced in response to cadmium (Cd) stress in Brassica napus remains limited. In the current study, we sequenced three small-RNA libraries from B. napus after 0 days, 1 days, and 3 days of Cd treatment. In total, 44 known miRNAs (belonging to 27 families) and 103 novel miRNAs were identified. A comprehensive analysis of miRNA expression profiles found 39 differentially expressed miRNAs between control and Cd-treated plants; 13 differentially expressed miRNAs were confirmed by qRT-PCR. Characterization of the corresponding target genes indicated functions in processes including transcription factor regulation, biotic stress response, ion homeostasis, and secondary metabolism. Furthermore, we propose a hypothetical model of the Cd-response mechanism in B. napus. Combined with qRT-PCR confirmation, our data suggested that miRNAs were involved in the regulations of TFs, biotic stress defense, ion homeostasis and secondary metabolism synthesis to respond Cd stress in B. napus.

Trehalose-6-phosphate synthase (TPS) is an important enzyme for the synthesis of Trehalose-6-phosphate (T6P). In addition to being a signaling regulator of carbon allocation that improves crop yields, T6P also plays essential roles in desiccation tolerance. However, comprehensive studies, such as evolutionary analysis, expression analysis, and functional classification of the TPS family in rapeseed (Brassica napus L.) are lacking. Here, we identified 35 BnTPSs, 14 BoTPSs, and 17 BrTPSs in cruciferous plants, which were classified into three subfamilies. Phylogenetic and syntenic analysis of TPS genes in four cruciferous species indicated that only gene elimination occurred during their evolution. Combined phylogenetic, protein property, and expression analysis of the 35 BnTPSs suggested that changes in gene structures might have led to changes in their expression profiles and further functional differentiation during their evolution. In addition, we analyzed one set of transcriptome data from Zhongshuang11 (ZS11) and two sets of data from extreme materials associated with source-/sink-related yield traits and the drought response. The expression levels of four BnTPSs (BnTPS6, BnTPS8, BnTPS9, and BnTPS11) increased sharply after drought stress, and three differentially expressed genes (BnTPS1, BnTPS5, and BnTPS9) exhibited variable expression patterns among source and sink tissues between yield-related materials. Our findings provide a reference for fundamental studies of TPSs in rapeseed and a framework for future functional research of the roles of BnTPSs in both yield and drought resistance.

Alternative splicing (AS) is a post-transcriptional regulatory process that enhances transcriptome diversity, thereby affecting plant growth, development, and stress responses. To identify the new transcripts and changes in the isoform-level AS landscape of rapeseed (Brassica napus) infected with the fungal pathogen Leptosphaeria maculans, we compared eight RNA-seq libraries prepared from mock-inoculated and inoculated B. napus cotyledons and stems. The AS events that occurred in stems were almost the same as those in cotyledons, with intron retention representing the most common AS pattern. We identified 1892 differentially spliced genes between inoculated and uninoculated plants. We performed a weighted gene co-expression network analysis (WGCNA) to identify eight co-expression modules and their Hub genes, which are the genes most connected with other genes within each module. There are nine Hub genes, encoding nine transcription factors, which represent key regulators of each module, including members of the NAC, WRKY, TRAF, AP2/ERF-ERF, C2H2, C2C2-GATA, HMG, bHLH, and C2C2-CO-like families. Finally, 52 and 117 alternatively spliced genes in cotyledons and stems were also differentially expressed between mock-infected and infected materials, such as HMG and C2C2-Dof; which have dual regulatory mechanisms in response to L. maculans. The splicing of the candidate genes identified in this study could be exploited to improve resistance to L. maculans.

Argonautes (AGOs) interact with microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), which can posttranscriptionally regulate the expression of targeted genes. To date, however, the AGOs and their miRNA triggers remain elusive in rapeseed (Brassica napus). Here, we systematically performed a phylogenetic analysis and examined the collinear relationships of the AGOs among four Brassicaceae species. Their physicochemical properties, gene structures, and expression patterns among 81 tissues from multiple materials and developmental stages were further analyzed. Additionally, their posttranscriptional regulation was analyzed using psRNATarget prediction, miRNA-/mRNA-Seq analyses, and a qRT-PCR verification. We finally identified 10 AtAGOs, 13 BolAGOs, 11 BraAGOs, and 24 BnaAGOs. An expression analysis of the BnaAGOs in the B. napus cultivar ZS11, as well as genotypes with extreme phenotypes in various yield-related traits, revealed the conservation and diversity of these genes. Furthermore, we speculated the posttranscriptional regulation of the B. napus miR168a–AGO1s and miR403–AGO2s modules. Combining miRNA-Seq and mRNA-Seq analyses, we found that the B. napus miR168a–AGO1s module may play an essential role in negatively regulating yield traits, whereas the miR403–AGO2s module positively impacts yield. This is the first attempt to comprehensively analyze the AGOs and their miRNA triggers in B. napus and provides a theoretical basis for breeding high-yielding varieties through the manipulation of the miRNA–AGOs modules.

Sugars are important throughout a plant’s lifecycle. Monosaccharide transporters (MST) are essential sugar transporters that have been identified in many plants, but little is known about the evolution or functions of MST genes in rapeseed (Brassica napus). In this study, we identified 175 MST genes in B. napus, 87 in Brassica oleracea, and 83 in Brassica rapa. These genes were separated into the sugar transport protein (STP), polyol transporter (PLT), vacuolar glucose transporter (VGT), tonoplast monosaccharide transporter (TMT), inositol transporter (INT), plastidic glucose transporter (pGlcT), and ERD6-like subfamilies, respectively. Phylogenetic and syntenic analysis indicated that gene redundancy and gene elimination have commonly occurred in Brassica species during polyploidization. Changes in exon-intron structures during evolution likely resulted in the differences in coding regions, expression patterns, and functions seen among BnMST genes. In total, 31 differentially expressed genes (DEGs) were identified through RNA-seq among materials with high and low harvest index (HI) values, which were divided into two categories based on the qRT-PCR results, expressed more highly in source or sink organs. We finally identified four genes, including BnSTP5, BnSTP13, BnPLT5, and BnERD6-like14, which might be involved in monosaccharide uptake or unloading and further affect the HI of rapeseed. These findings provide fundamental information about MST genes in Brassica and reveal the importance of BnMST genes to high HI in B. napus.