Integrated In Silico Characterization of Quinoa Hsp20 Genes Reveals Preferential Responsiveness to Drought and Salinity over Heat Stress

Abstract

The Hsp20 protein family, essential in heat stress responses across all organisms, is part of the heat shock protein (Hsp) superfamily, recognized for its conserved alpha-crystallin domain (ACD). Hsp20s are the smallest proteins in the superfamily and primarily assist in protein refolding during stress and developmental processes. We present an in silico characterization of the Hsp20 gene family in Chenopodium quinoa (2n = 4x = 36) using an integrative approach. Quinoa is well known for its global contributions to food production and tolerance to various abiotic stresses. We identified 69 CqHsp20 genes that exhibit a well-conserved evolutionary pattern, characterized by a balanced copy number distributed symmetrically across 19 homeologous pairs in both subgenomes (A and B), with localized expansions driven by tandem duplications on eight chromosomes. High sequence identity in contiguous gene pairs and Ka/Ks ratios consistently below 1 (0.14–0.84) mathematically demonstrate that strict purifying selection has maintained the structural and sequence integrity of these genes since the ancestral polyploidization event. The phylogenetic analysis grouped CqHsp20 into two main clusters, splitted into four sub-clusters based on peptides’ cellular localization, consistent with a characteristic gene structure and conserved motif analysis, which may reflect the evolutionary trajectory and functional specialization of the Hsp20 family in plants. The integration of transcriptomic data from published experiments enabled us to detect a cluster of putatively ubiquitously expressed CqHsp20, as well as other groups that showed differential responses across abiotic stress conditions. The pattern shows that more genes exhibit higher transcription abundance under drought and salinity than under heat, key adaptive traits underlying quinoa’s known ecological versatility. Some of these genes, which are undetectable or have low abundance under heat stress, encode organelle-targeting peptides, a phenomenon not reported in other model plant studies. Differential expression analysis revealed a highly transcribed sub-cluster where six out of seven of nuclear CqHsp20 genes were active in aerial tissue during initial heat stress, with a specific cohort of four genes (CQ025082, CQ031384, CQ041158, and CQ055373) maintaining significant upregulation (|log2⁡Fold⁢Change|≥1.0, padj<0.05) under prolonged and simultaneous shoot/root exposure. Varying expression within CqHsp20 homologous and paralogs supports the idea that gene duplication creates genomic diversity, facilitating adaptation to variable extreme environments. However, while theoretical and in silico analysis provide valuable insight into quinoa Hsp20 response, empirical data are essential to unequivocally understand how these gene expression variations affect quinoa response to abiotic stressors.