Characterization and cryoprotective activity of cold-responsive dehydrin from Citrus unshiu

Citrus unshiu dehydrin (CuCOR19) was characterized, and its cryoprotective activity and response to water stress were studied. Detached leaf of C. unshiu was treated with cold (4 degreesC), NaCl (50-200mmol/L) and abscisic acid (ABA, 0.1-10 mu mol/L). CuCOR19 mRNA or protein were detected by Northern or Western hybridization, respectively. A small amount of CuCOR19 mRNA was present in leaf when it was detached, and the mRNA level decreased during the control experiment. Cold induced the gene expression remarkably, but treatment with ABA or NaCl did so only slightly. The CuCOR19 protein accumulated only under cold stress, suggesting that cold is a crucial environmental cue for dehydrin production in the leaf of C. unshiu. In order to characterize CuCOR19 protein, we obtained the protein using the expression system of Escherichia coli and subjected it to the cryoprotection assay and circular dichroism spectrum analysis, CuCOR19 protected catalase and lactate dehydrogenase against freezing inactivation, and it was more effective than compatible solutes or BSA. The circular dichroism spectrum for CuCOR19 showed that the major secondary structure of CuCOR19 is a random coil in the solution. It is likely that the random coil structure allows CuCOR 19 to protect plant cells from cold stress, since the random coil is considered to bind water or maintain protein structure.