Multi-genome comparison reveals a conserved 14–3-3 core and type-specific cold responses in Brassica juncea

Mustard ( Brassica juncea (L.) Czern. & Coss.) is cultivated as an industrial oilseed, while its lignocellulosic biomass and glucosinolate-rich seed meal provide additional feedstocks for energy production and biofumigation. Its allotetraploid genome retains extensive variation generated by polyploidization and domestication. To determine how this variation has shaped a conserved regulatory family, we curated 14–3–3 genes in six B. juncea genomes and integrated phylogenetic, orthogroup, duplication, sequence, transposable-element, promoter, candidate-interactor, and cold-response analyses. The 262 genes (38–48 per accession) formed 40 orthogroups (OGs). Thirty OGs were shared by all six accessions, including 25 that remained single copy, whereas the 15 copy-variable OGs were concentrated in seven GF14-like types. Whole-genome or segmental duplication accounted for 240 genes, and all evaluable syntenic pairs were under purifying selection. Most sequence divergence occurred near the C terminus, while local regulatory environments differed through promoter and downstream transposable elements and variable cold-responsive cis-elements. Projection from experimentally supported Arabidopsis interactions yielded 2326 candidate interactor groups whose functional composition differed among GF14-like types. During a 4 °C leaf time course in T84–66, GF14ψ-like was the only type containing strongly induced genes. Its more highly expressed member, BjuVB06G51590, showed co-localization with an endoplasmic reticulum marker in tobacco leaves, with additional non-overlapping fluorescence. Under prolonged cold, Arabidopsis expressing BjuVB06G51590 accumulated more hydrogen peroxide and malondialdehyde but showed lower antioxidant enzyme activities and reduced proline and soluble-sugar accumulation than wild-type plants. These results resolve the conserved and variable components of the B. juncea 14–3–3 family and associate a GF14ψ-like member with greater physiological injury during prolonged cold.