Molecular Insights into the Role of PcHSP20s in Mediating Thermotolerance in Polygonatum cyrtonema

Small heat shock proteins (HSP20s) are known to function as molecular chaperones that bind to denatured proteins under high-temperature stress and assist in their conformational recovery, thereby contributing to plant thermotolerance. In the present study, three <i>HSP20</i> genes-<i>PcHSP12.8</i>, <i>PcHSP12.9</i>, and <i>PcHSP13.4</i>-were identified in the transcriptome of <i>Polygonatum cyrtonema Hua</i>. Bioinformatics analysis indicated their phylogenetic relationships, conserved domains, and potential tertiary structures. RT-qPCR analysis revealed up-regulation of all three genes in response to heat stress. Subcellular localization studies further suggested that <i>PcHSP12.8</i>, <i>PcHSP12.9</i>, and <i>PcHSP13.4</i> are predominantly localized in the nucleus. Heterologous expression of these genes in a heat-sensitive yeast mutant appeared to improve cell survival under heat stress relative to the control strain. In <i>Arabidopsis thaliana</i> overexpressing these genes, moderate improvements in germination rate, root elongation, and stress survival were observed compared to wild-type plants under heat stress. Transgenic lines also showed a tendency toward reduced reactive oxygen species accumulation, as reflected by decreased 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, together with increased activities of catalase (CAT) and peroxidase (POD), as well as higher chlorophyll retention under thermal stress. Taken together, these findings imply that the three <i>PcHSP20</i> genes could be involved in thermotolerance in <i>P. cyrtonema</i>.