Multiplex Editing of BnBAK Genes Creates Compact, Semi-Dwarf Rapeseed

Plant morphology is a critical determinant of crop productivity in Brassica napus, influencing both lodging resistance and optimal planting density. Here, we investigated the genetic regulation of plant architecture by focusing on the BAK gene family. Using a multi-target sgRNA CRISPR/Cas9 strategy, we generated knockout mutants of several BAK homologs. Phenotypic characterization showed that specific higher-order mutants (Bnbak_sp and Bnbak_n) displayed significantly reduced plant height and altered silique arrangement, while maintaining normal yield-related traits. Expression and localization analyses revealed that BnBAK genes are primarily expressed in leaves and stem internodes, with proteins localizing to multiple membrane systems, including the nuclear envelope. Transcriptomic analysis of internodes indicated extensive reprogramming of photosynthesis- and defense-related pathways. Additionally, we identified TCP transcription factors as key downstream regulators, with motif enrichment confirming TCP-binding sites in promoters of differentially expressed genes. Furthermore, BnBAK proteins are required for the accumulation of BnTCP transcription factors in planta. Functional complementation in Arabidopsis further demonstrated that overexpression of TCP21 substantially rescued the dwarf phenotype of bak1-3 bkk1-1 mutants. Together, these findings elucidate the molecular mechanisms through which BnBAK genes modulate plant architecture in B. napus, providing critical insights and practical targets for breeding compact, high-yielding rapeseed varieties.