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. uns hiu was treated with cold (4 degreesC), NaCl (50-200mmol/L) and abscisic ac id (ABA, 0.1-10 mu mol/L). CuCOR19 mRNA or protein were detected by Norther n or Western hybridization, respectively. A small amount of CuCOR19 mRNA wa s present in leaf when it was detached, and the mRNA level decreased during the control experiment. Cold induced the gene expression remarkably, but t reatment with ABA or NaCl did so only slightly. The CuCOR19 protein accumul ated only under cold stress, suggesting that cold is a crucial environmenta l cue for dehydrin production in the leaf of C. unshiu. In order to charact erize CuCOR19 protein, we obtained the protein using the expression system of Escherichia coli and subjected it to the cryoprotection assay and circul ar dichroism spectrum analysis, CuCOR19 protected catalase and lactate dehy drogenase against freezing inactivation, and it was more effective than com patible solutes or BSA. The circular dichroism spectrum for CuCOR19 showed that the major secondary structure of CuCOR19 is a random coil in the solut ion. 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.