A cold-inducible MYB like transcription factor, CsHHO2, positively regulates chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression

Plant HRS1 (hypersensitive to low phosphate (Pi)-elicited primary root shortening 1) and its homologs have been primarily characterized in Pi and nitrogen responses. However, their regulatory roles in cold responses remain largely unexplored. Here, we characterized the functional roles of HRS1 homolog 2 (CsHHO2) in regulating cold resistance in harvested cucumber fruit and examined the dependency of the role on glycine-rich RNA-binding protein 3 (CsGR-RBP3) gene expression. Cold treatments strongly induced CsHHO2 expression in cucumber fruit. GUS staining and expression analysis revealed that cold treatment stimulated CsHHO2 expression, at least partially, by enhancing the promoter activity. Subcellular localization results indicated that the green fluorescent protein (GFP)-tagged CsHHO2 proteins were localized to both the nucleus and the cytoplasm (potentially the plasma membrane), with confirmed nuclear localization when co-overexpressed with mCherry. Reducing CsHHO2 expression in cucumber fruit through VIGS significantly compromised the chilling tolerance, while its overexpression in cucumber fruit and Arabidopsis plants enhanced cold tolerance. The results of electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H) assay and dual-luciferase reporter (DLR) assay indicated that CsHHO2 specifically bound to the promoter of CsGR-RBP3, and enhanced its transcription. Transcriptomic analysis indicated that most differentially expressed genes (DEGs) modulated by CsHHO2 and CsGR-RBP3 proteins were identical, and the common DEGs were mainly enriched in several stress defense pathways, such as plant-pathogen interaction, phenylpropanoid biosynthesis, and plant hormone signal transduction. Functional complementation analysis of CsHHO2 and CsGR-RBP3 in cucumber fruit revealed that CsGR-RBP3 expression were indispensable for CsHHO2-modulated cold resistance. These results collectively suggest that CsHHO2, activated by cold signals, positively regulates the chilling tolerance of cucumber fruit by enhancing CsGR-RBP3 expression. Overall, this work suggests CsHHO2 as a key regulator of cold tolerance in cucumber fruit, provides key genetic resources for breeding cold-resistant cucumbers, and sheds novel insight into the roles of HRS1 family members in regulating plant development under environmental stresses.