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

## TL;DR

This study identifies and characterizes 114 heat shock protein genes in garlic, revealing their roles in helping the plant tolerate environmental stresses like heat and salinity.

## Contribution

The study provides the first comprehensive genome-wide analysis of small heat shock proteins in garlic, identifying key stress-responsive genes and their functional roles.

## Key findings

- 114 small heat shock protein genes were identified and classified into ten phylogenetic subgroups in garlic.
- Overexpression of the AsHSP20-79 gene in yeast significantly enhanced thermotolerance, highlighting its potential for improving stress resilience.
- Several HSP genes, including AsHSP20-94 and AsHSP20-79, showed strong upregulation under heat, salinity, and jasmonic acid treatments.

## Abstract

Climate change and extreme weather expose garlic plants to abiotic stresses such as heat and salinity, leading to reduced yields and compromised crop quality. To elucidate the molecular mechanisms underlying garlic’s response to environmental stress, we conducted a comprehensive genome-wide analysis to identify heat shock proteins (HSPs), a class of molecular chaperones responsible for maintaining protein homeostasis by preventing misfolding and aggregation induced by stress conditions. A total of 114 HSP genes were identified and analyzed in terms of their subcellular localization, cis-regulatory elements responsive to stress signals, and expression patterns across various tissues and stress conditions. Functional validation in Saccharomyces cerevisiae demonstrated that overexpression of one selected HSP gene significantly enhances thermotolerance. These findings provide valuable insights into the molecular basis of stress adaptation in garlic and highlight candidate genes for breeding or genetic engineering aimed at improving heat and salt tolerance. Strengthening garlic’s resilience to environmental stress will contribute to stable agricultural production and food security.

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 Allium sativum 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 (AsHSP20-80/31, and AsHSP20-81/32), 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 AsHSP20-94 and AsHSP20-79, 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 AsHSP20-79 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.