Genome-Wide Analysis and Functional Characterization of Small Heat Shock Proteins in Allium sativum L. Under Multiple Abiotic Stresses

Small heat shock proteins play a pivotal role in maintaining protein homeostasis under abiotic stress conditions and are indispensable for plant viability. In the present study, a comprehensive characterization of this gene family in <i>Allium sativum</i> was conducted through genome-wide sequence identification, phylogenetic reconstruction, conserved motif analysis, promoter cis-element profiling, transcriptomic investigation, quantitative real-time PCR, subcellular localization, and yeast-based functional assays. A total of 114 small heat shock protein genes were identified across eight chromosomes and subsequently classified into ten phylogenetic subgroups. All encoded proteins conserved the α-crystallin domain, whereas their exon-intron architectures and promoter elements responsive to environmental stress or phytohormones exhibited considerable diversity. The predicted proteins range from 130 to 364 amino acids, with isoelectric points (pI) spanning 3.97 to 9.95 and GRAVY values from -1.131 to -0.014, indicating predominantly hydrophilic characteristics. Subcellular localization analysis revealed a broad distribution across the cytoplasm, chloroplasts, mitochondria, and other compartments, with the majority (74 proteins) localized in the cytoplasm. Synteny analysis uncovered two segmentally duplicated gene pairs (<i>AsHSP20-80/31,</i> and <i>AsHSP20-81/32</i>), both showing strong purifying selection (Ka/Ks = 0.0459 and 0.2545, respectively), suggesting functional conservation. Expression profiling demonstrated predominant transcript accumulation in bulbs and floral organs, with significant induction under heat, salinity, and jasmonic acid treatments. qRT-PCR validation further confirmed that several candidate genes, notably <i>AsHSP20-94</i> and <i>AsHSP20-79</i>, were strongly and consistently upregulated across multiple stress conditions, underscoring their roles as core stress-responsive regulators. Subcellular localization experiments demonstrated that representative proteins are targeted to the cytoplasm, nucleus and chloroplasts. Furthermore, heterologous expression of <i>AsHSP20-79</i> in yeast conferred marked thermotolerance. Collectively, these findings reveal extensive expansion and functional divergence of the small heat shock protein gene family in garlic and provide valuable candidate genes for improving stress resilience in this important crop species.