Cinnamyl alcohol dehydrogenase (CAD) is essential for lignin precursor synthesis and responses to various abiotic stresses in plants. However, the functions of CAD in Brassica species, especially in Brassica napus, remain poorly characterized. In the present study, we identified a total of 90 CAD genes across the Brassica U-triangle species, including B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. Comprehensive analyses of phylogenetic relationships, sequence identity, conserved motifs, gene structure, chromosomal distribution, collinearity, and cis-acting elements were performed. Based on phylogenetic analysis, these genes were categorized into four groups, designated as groups I to IV. Most of the CAD genes were implicated in mediating responses to abiotic stresses and phytohormones. Notably, members in group III, containing the bona fide CAD genes, were directly involved in lignin synthesis. Furthermore, the expression profiles of BnaCAD genes exhibited differential responses to drought, osmotic, and ABA treatments. The expression levels of the BnaCAD4a, BnaCAD4b, BnaCAD5b, and BnaCAD5d genes were detected and found to be significantly lower in yellow-seeded B. napus compared to the black-seeded ones. This study provides a comprehensive characterization of CAD genes in Brassica U-triangle species and partially validates their functions in B. napus, thereby contributing to a better understanding of their roles. The insights gained are expected to facilitate the breeding of yellow-seeded B. napus cultivars with enhanced stress tolerance and desirable agronomic traits.

4-Coumarate: CoA ligase (4CL) is a key branch point enzyme at the end of the phenylpropanoid metabolic pathway. It regulates the synthesis of various metabolites and participates in plant growth and development by catalyzing the formation of CoA ester compounds. However, 4CL family members have not been identified and analyzed among U’s triangle species in Brassica. In this study, 53 4CL genes were identified in Brassica U’s triangle species and divided into 4 groups (group I, II, III and IV) according to phylogenetic relationship. Based on phylogenetics, gene structure, conserved motifs, chromosome localization and collinearity analysis, 4CLs were relatively conserved in the evolution of Brassica U’s triangle species. The promoter region contains a large number of cis-acting elements, implying the functional diversity of 4CLs. Further combining transcriptome data and reverse transcription quantitative PCR (RT-qPCR), we found that Bna4CLs have tissue specificity and can not only respond to exogenous phytohormone changes but also regulate the flavonoid biosynthetic pathway in the yellow- and black-seeded B. napus. Our results complement the lack of research on the 4CL gene family in Brassica, clarify the sequence characteristics and functional diversity of these genes and lay a foundation for further exploration of 4CL genes in response to abiotic stress and regulation of seed coat flavonoid accumulation.

Members of the high-affinity potassium transporter (HKT) protein family regulate the uptake and homeostasis of sodium and potassium ions, but little research describes their roles in response to abiotic stresses in rapeseed (Brassica napus L.). In this study, we identified and characterized a total of 36 HKT genes from the species comprising the triangle of U model (U-triangle species): B. rapa, B. nigra, B. oleracea, B. juncea, B. napus, and B. carinata. We analyzed the phylogenetic relationships, gene structures, motif compositions, and chromosomal distributions of the HKT family members of rapeseed. Based on their phylogenetic relationships and assemblage of functional domains, we classified the HKT members into four subgroups, HKT1;1 to HKT1;4. Analysis of the nonsynonymous substitutions (Ka), synonymous substitutions (Ks), and the Ka/Ks ratios of HKT gene pairs suggested that these genes have experienced strong purifying selective pressure after duplication, with their evolutionary relationships supporting the U-triangle theory. Furthermore, the expression profiles of BnaHKT genes varies among potassium, phytohormone and heavy-metal treatment. Their repression provides resistance to heavy-metal stress, possibly by limiting uptake. Our results systematically reveal the characteristics of HKT family proteins and their encoding genes in six Brassica species and lay a foundation for further exploration of the role of HKT family genes in heavy-metal tolerance.