Abstract
Brassica juncea (L.) Coss. var. foliosa Bailey contains high glucosinolate (GSL) levels that define its flavor and defense properties. However, the regulatory mechanisms controlling GSL biosynthesis in Brassica crops remain unclear. Here, four MYB29 homologs were identified in allotetraploid Brassica juncea. These BjuMYB29 proteins localize to the nucleus and possess transcriptional activation activity. Evolutionary analysis suggests polyploidization-driven expansion of MYB genes contributed to GLS diversification in Brassica species. Expression profiling showed distinct spatiotemporal and herbivory-responsive patterns among BjuMYB29 homologs. Heterologous expression of BjuA03.MYB29 and BjuA10.MYB29 in Arabidopsis enhanced insect resistance via GSL accumulation. Although both homologs upregulate aliphatic GSL biosynthetic genes, they differentially regulate indolic GSLs, with BjuA03.MYB29 suppressing and BjuA10.MYB29 enhancing their accumulation, potentially through differential control of CYP79B2. These results reveal subfunctionalization among MYB29 homologs in GSL regulation. This functional diversification of MYB29 homologs offers novel targets for precision breeding of Brassica crops with customized GSL profiles to optimize pest resistance and nutritional quality.