The bermudagrass CdbZIPs-CdsHSP16.970 regulatory module enhances osmotic stress tolerance in Arabidopsis

Small heat shock proteins (sHSPs) act as molecular chaperones that protect other proteins from damage caused by stress-induced denaturation. Bermudagrass (<i>Cynodon dactylon</i> L.) is a broadly adopted forage and turfgrass known for its capability to withstand various abiotic stresses. However, the biological pathways by which sHSPs promote drought tolerance in bermudagrass remain unclear. In this study, 99 sHSPs were characterized in the bermudagrass genome. Drought stress led to the induction of the majority of these genes with <i>CdsHSP16.970</i> showing the most significant induction. Overexpression (OE) of <i>CdsHSP16.970</i> promoted root elongation and improved seedling growth performance in transgenic <i>Arabidopsis</i> lines under osmotic stress, with reduced electrolyte leakage (EL) and lower malondialdehyde (MDA) deposition compared with the control. Meanwhile, several stress-related genes were significantly induced in <i>CdsHSP16.970</i>-OE plants when subjected to osmotic stress compared to the control group. Two basic leucine zipper transcription factors, CdbZIP04 and CdbZIP65, were also induced by drought stress in bermudagrass. Further investigation using electrophoretic mobility shift assay, yeast one-hybrid and dual-LUC assays revealed that they directly and specifically bind to the upstream regulatory region of <i>CdsHSP16.970</i>, consequently promoting its expression. In summary, our results suggest that the CdbZIPs–<i>CdsHSP16.970</i> cascade positively regulates the osmotic stress signaling pathway in bermudagrass.