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.

The β-glucosidases (BGLUs) of Glycoside Hydrolase Family 1 (GH1) exhibit essential functions in plant secondary metabolism and stress responses, mediated by their dual catalytic capabilities in hydrolysis and transglycosylation. This study identified 149 BGLU family members within B. napus (Brassica napus L.), which were systematically categorized into 10 distinct subgroups. Subsequent characterization encompassed detailed examination of their motif composition, chromosomal distribution, gene collinearity, selection pressure, and expression profiling. Transient overexpression of BnBGLU77 in N. benthamiana (Nicotiana benthamiana), combined with untargeted metabolomics analysis, revealed pronounced modulatory effects on the degradation and accumulation of β-glucosidic compounds, suggesting potential roles of the protein encoded by BnBGLU77 in metabolic homeostasis and stress response mechanisms. These experimental results first validated the bidirectional catalytic activity of a BGLU enzyme in B. napus, while simultaneously advancing fundamental understanding of BnBGLU gene functions and providing new insights for developing stress-resistant rapeseed cultivars through targeted genetic improvement.

The BAHD acyltransferase family plays a critical role in plant secondary metabolism by catalyzing acyl transfer reactions that are essential for synthesizing metabolites involved in environmental adaptation. However, systematic investigation of this superfamily in Brassica napus has not been reported. In this study, 158 BnaBAHD genes were identified by comprehensive analyses of evolutionary relationships, motif structures, chromosomal distribution, gene collinearity, and selection pressures, and these genes were phylogenetically classified into five clades harboring conserved catalytic domains (HXXXD and DFGWG). Transient overexpression combined with metabolomic profiling demonstrated that two homologous seed-specific Clade V members, BnaBAHD040 and BnaBAHD120, which exhibited elevated expression during late seed development, significantly enhanced the accumulation of acylated metabolites contributing to biotic/abiotic stress resistance. This study provides the first experimental validation of the catalytic functions of BAHD enzymes in B. napus, establishing a theoretical foundation for leveraging this gene family in genetic improvement to develop novel rapeseed cultivars with enhanced stress tolerance and yield.

Sclerotinia sclerotiorum is a highly destructive phytopathogenic fungus that poses a significant threat to a wide array of crops. The current constraints in genetic manipulation techniques impede a thorough comprehension of its pathogenic mechanisms and the development of effective control strategies. Herein, we present a highly efficient genetic transformation system for S. sclerotiorum, leveraging the use of fusiform nanoparticles, which are synthesized with FeCl₃ and 2,6-diaminopyrimidine (DAP). These nanoparticles, with an average longitude length of 59.00 nm and a positively charged surface, facilitate the direct delivery of exogenous DNA into the mycelial cells of S. sclerotiorum, as well as successful integration with stable expression. Notably, this system circumvents fungal protoplast preparation and tedious recovery processes, streamlining the transformation process considerably. Furthermore, we successfully employed this system to generate S. sclerotiorum strains with silenced oxaloacetate acetylhydrolase-encoding gene Ss-oah1. Our findings demonstrate the feasibility of using nanoparticle-mediated delivery as a rapid and reliable tool for genetic modification in S. sclerotiorum. Given its simplicity and high efficiency, it has the potential to significantly propel genetic research in filamentous fungi, offering new avenues for elucidating the intricacies of pathogenicity and developing innovative disease management strategies.

Calmodulin-binding transcription activators (CAMTAs) are pivotal regulators decoding calcium signals, with crucial roles in plant development, hormone responses, and adaptation to abiotic stresses. Although extensive research has been conducted on CAMTAs in model plants such as Arabidopsis thaliana, a comprehensive genome-wide analysis of the CAMTA gene family across the economically important Brassica U-triangle species has not been performed. In this study, we systematically identified and characterized 64 CAMTA genes from the genomes of Brassica U-triangle species. Phylogenetic analysis classified these genes into four conserved groups, a finding corroborated by analyses of gene structure and conserved motifs. These analyses revealed strong evolutionary preservation of functional domains, especially the calmodulin-binding domain (CaMBD). Chromosomal distribution and collinearity assessment highlighted the significant impact of polyploidization on the expansion of the CAMTA family, with most orthologous pairs being under purifying selection. Cis-element analysis in promoters uncovered an abundance of stress- and hormone-related elements, suggesting diverse regulatory roles for these genes. Furthermore, RNA-Seq and RT-qPCR expression profiling demonstrated that BnaCAMTA genes exhibit tissue-specific expression and are dynamically responsive to various phytohormones (ABA, JA, and GA) and abiotic stresses (salt and drought), particularly in the root. Notably, BnaCAMTA5.2, which was prioritized among several validated candidates, mediates the antagonistic regulation of hypocotyl and root growth under GA and salt stress, indicating its key role in balancing growth promotion and stress adaptation. Additionally, we identified a set of stress-related miRNAs that potentially target BnaCAMTAs, suggesting a potential layer of post-transcriptional regulation. Our results provide valuable insights into the evolutionary and functional diversity of CAMTA genes in Brassica U-triangle species and lay a foundation for further research into their roles in enhancing stress resistance in B. napus.