The cytoplasmic small heat shock protein, AtHsp18.5, enhances drought and Pb (lead) tolerance.

Drought and heavy metal contamination are major abiotic stresses that compromise plant growth and productivity. Small heat shock proteins (Hsp20s) act as ATP-independent molecular chaperones that are implicated in maintaining cellular proteostasis; however, their precise physiological roles and modes of action in plant stress tolerance remain poorly understood. Here, we showed that the transcript abundance of cytoplasm-localized AtHsp18.5 (AT2G19310) from Arabidopsis thaliana, one member of the CIV group, was strongly induced by both drought and lead (Pb) stress. Recombinant AtHsp18.5 exhibited chaperone activity in vitro, protecting horseradish peroxidase (POD) activity under heat stress and preserving glyceraldehyde-3-phosphate dehydrogenase (AtGAPC2, AT1G13440) function upon H2O2 exposure. AtHsp18.5 and AtHsp17.4 exhibited a synergistic protective effect on AtGAPC2. Site-directed mutagenesis identified Glu91 and the N-terminal hydrophobic patch as critical determinants for its chaperone activity. Phenotypic, physiological, and biochemical assays confirmed the positive role of AtHsp18.5 in drought and Pb tolerance, since overexpression of AtHsp18.5 (OE) enhanced tolerance, while the AtHsp18.5 mutant showed increased sensitivity. Moreover, multiple stress-responsive genes associated with reactive oxygen species (ROS) scavenging, abscisic acid (ABA) biosynthesis and signaling, and sugar transport were markedly upregulated in the OE lines and downregulated in the AtHsp18.5 mutant under stress conditions. The physical interaction of AtHsp18.5 with both AtGAPC2 and AtHsp17.4 (AT3G46230) was confirmed using yeast two-hybrid (Y2H), pull-down, bimolecular fluorescence complementation (BiFC), and isothermal titration calorimetry (ITC) assays. The positive role of AtGAPC2 in Pb tolerance was further supported by the increased sensitivity of the AtGAPC2 mutant, which had reduced transcript levels. Together with our previous findings, we proposed that AtHsp18.5 and AtHsp17.4 might assemble into a hetero-oligomeric complex that interacted with AtGAPC2 to confer drought and Pb tolerance. Collectively, our findings offer new insights into the functional diversity and regulatory network of the plant Hsp20 family and highlight potential targets for improving abiotic stress resilience.