Functional divergence of BnaWRKY7 homologs drives phytosterol variations in polyploid Brassica napus

Gene duplication and subsequent functional divergence drive species evolution, adaptability, and biodiversity, particularly in polyploids. The polyploid Brassica napus has abundant phytosterols, which are crucial for plant growth and development, and human health. However, the regulatory mechanism of phytosterol biosynthesis remains poorly understood in polyploid systems.

This study aims to provide a novel method for analyzing the functions of multi-copy homologous genes and to dissect the genetic and molecular basis of phytosterol biosynthesis in B. napus.

Genome-wide association studies (GWAS), temporal transcriptome and phytosterol profiles were employed to identify phytosterol-regulatory genes. Transgenic validation (B. napus) combined with yeast one-hybrid assay, dual-luciferase reporter assay and electrophoretic mobility shift assay was used to reveal phytosterol biosynthesis mechanisms.

Through dynamic gene expression analysis and phytosterol profiles, 103 transcription factors (TFs) were preliminarily screened out by coordinating an intricate transcriptional program. Then, GWAS revealed two highly significant loci on chromosomes A03 and C07, located in syntenic regions between the A_n- and C_n-subgenomes. By integrating a synteny-based candidate gene identification approach and comparative expression analysis, we pinpointed two WRKY7 homologs as key TF regulators of phytosterol biosynthesis. The sequences, structures, and expression patterns of these two WRKY7 homologs were highly similar but significantly different from other homologs, indicating that the functional divergence of WRKY7 homologs drives natural variation in phytosterols. Transgenic validation, combined with phytosterol rate calculations and molecular interaction assays, mechanistically confirmed their regulatory roles in shaping phytosterol variations.

In this study, we established a multi-copy gene co-screening strategy and elucidated the molecular mechanism. BnaWRKY7 regulates phytosterol variations by interacting with four key phytosterol biosynthesis genes. The findings enhance our understanding of the temporal regulation of phytosterols in polyploids, establish a foundation for future research on duplicated genes and genome evolution, and propose targets for high-phytosterol crops.