Heat stress disrupts protein homeostasis, yet the thermoregulatory roles of HSP10/CPN10 co-chaperonins in polyploid crops remain unexplored. This study conducted the first genome-wide characterization of the HSP10 gene family in allotetraploid Brassica juncea to identify heat tolerance candidates. Genome-wide identification of BjuHSP10 genes was performed across A and B subgenomes, followed by phylogenetic, gene structure, duplication, synteny, promoter, and structural analyses. Transcriptional responses were evaluated under moderate (28/24 °C) and severe (36/32 °C) heat stress conditions. Ten BjuHSP10 genes were identified, encoding six mitochondrial, three chloroplast-targeted, and one cytoplasmic protein. Mitochondrial members were small (~10.7 kDa) with three-exon structures, whereas chloroplast members were larger (15.5–27.1 kDa) with complex exon–intron organization. Segmental duplication drove family expansion (19 duplicated pairs), with differential retention of Arabidopsis homologs. Promoter regions contained 391 putative cis-regulatory elements associated with stress, hormonal, and developmental regulation. Under heat stress, BjuHSP10-4 and BjuHSP10-5 were induced under both treatments; BjuHSP10-1/3/6/9 responded only to severe stress, while four genes were repressed. Structural modeling confirmed conserved β-rich GroES/CPN10-like folds. These findings identify candidate BjuHSP10 genes, particularly BjuHSP10-4 and BjuHSP10-5, as promising targets for heat-tolerance improvement in mustard, providing a foundation for future breeding and functional studies in polyploid crops.

Background/Objectives: Dehydrins (DHNs) are late embryogenesis abundant proteins that play protective roles under water-deficit conditions; however, their organization and function remain unexplored in sesame (Sesamum indicum), an important oilseed crop frequently cultivated in arid and semi-arid regions. This study aimed to identify and characterize the DHN gene family in sesame and evaluate the expression of its members under drought stress. Methods: Genome-wide identification was performed using the Dehydrin domain HMM profile, followed by phylogenetic analysis, conserved motif and gene structure characterization, synteny analysis, promoter cis-element profiling, and secondary/tertiary structure prediction. Transcriptional responses were profiled by qPCR in two sesame cultivars (drought-sensitive and drought-tolerant) under PEG-induced osmotic stress at germination and seedling stages. Results: Four DHN genes were identified, spanning three phylogenetic subfamilies (I–III) and three DHN subclasses: SK_n (SiDHN1/2), Y_nK_n (SiDHN3), and Y_nSK_n (SiDHN4). SiDHN1 and SiDHN2 likely arose from a segmental duplication, and a single conserved syntenic pair was found between SiDHN4 and olive (Olea europaea). Secondary structure predictions uncovered contrasting structural propensities: SiDHN1/2 are predicted to be α-helix-rich, partially ordered proteins (41–44% predicted α-helix), whereas SiDHN3/4 are predicted to be predominantly intrinsically disordered (~75–80% random coil). SiDHN3 was consistently upregulated across all conditions (1.84–9.12-fold), while SiDHN4 exhibited strong genotype-specific induction of 9.39-fold exclusively in the drought-tolerant cultivar during germination. Conclusions: The sesame DHN family achieves functional breadth through structural diversification—an ordered–disordered continuum mirrored by divergent expression programming—rather than through numerical expansion. SiDHN3 and SiDHN4 are identified as primary candidates for drought tolerance improvement in sesame.