Journal of Agronomy and Field Innovations

Background: Brassica napus L. is the world's second-largest oilseed crop, and rising ambient and episodic heat events increasingly compromise its flowering, silique set and seed oil yield. Plant-specific WRKY transcription factors are central hubs that couple heat perception to downstream defence gene expression, yet their role in B. napus heat tolerance remains incompletely resolved.
Objective: To identify heat-responsive BnWRKY genes at the genome-wide level, characterize their structural and evolutionary features, and functionally validate candidate genes governing thermotolerance.
Methods: Genome-wide mining of the B. napus reference genome identified WRKY loci, which were classified by conserved domain, motif and phylogenetic analysis. Heat-stressed (42°C) and control seedlings of cultivar ‘ZS11’ were profiled by qRT-PCR and RNA-seq; selected candidates were functionally validated by overexpression and CRISPR/Cas9 knockout, followed by physiological phenotyping.
Results: A total of 284 BnWRKY genes, distributed across Groups I, II and III, were identified, of which 41 were significantly heat-responsive. Five candidates, notably BnWRKY33-like and BnWRKY40-like homologs, were strongly induced within two hours of heat exposure. Overexpression lines exhibited higher chlorophyll retention, lower electrolyte leakage and malondialdehyde content, and greater survival than wild type, while knockout lines showed the reciprocal phenotype.
Conclusion: BnWRKY transcription factors, particularly heat-inducible Group II/III members, function as positive regulators of thermotolerance in B. napus through ROS-, ABA- and HSP-linked pathways, offering candidate targets for climate-resilient rapeseed breeding.