Indiana University-Purdue University Indianapolis (IUPUI) / Drought and cold conditions are among the major factors affecting plant growth and crop production globally. Dehydrins are group II late embryogenesis abundant (LEA) proteins characterized by a conserved K-region (EKKGIMDKIKEKLPG consensus sequence) that accumulate in many plants during drought, low temperature, and high salinity to confer stress tolerance. While it has been demonstrated that overexpression of dehydrins improves cold tolerance in various crop plants, the mechanism leading to cold tolerance is still unclear. Previous studies reported phosphorylation of AtERD14 dehydrin by casein kinase II (CKII) led to an increase in calcium binding activity. Mass spectroscopy analysis determined that the phosphorylation was localized to a poly-serine (S) region. To further characterize the S-region, GST fused ERD14 mutants were created via site-directed mutagenesis and deletion of either the amino or carboxyl ends of ERD14 via the QuickChange® Multi Site-Directed Mutagenesis Kit. Phosphorylation of purified mutant proteins by CKII was analyzed via gel shift and direct phosphorylation assays. The effect of phosphorylation on calcium binding activity was also analyzed. Results showed the serine (S) residue at position 83 was crucial to phosphorylation-dependent molecular mass shift and Ca2+-binding activities followed by the serine residue at position 85 in importance. Mutation of serines at positions 83, 84, and 85 completely eliminated the phosphorylation-dependent gel shift and calcium binding. Examination of truncation mutants determined the N-terminal was an important region for protein structure modification and phosphorylation ability leading to Ca2+ activation. Calcium binding activity of the truncated mutants indicated the calcium binding site was localized in the region between the S-region and the K-region near the C-terminal end. To characterize the acidic dehydrins contribution to cold tolerance in vivo, three single (erd10, erd14, cor47) knockouts (KOs) were characterized. Single KOs produced no cold sensitive phenotype indicating the need for multiple dehydrin KOs in Arabidopsis in order to potentially produce a cold sensitive phenotype.

Indiana University-Purdue University Indianapolis (IUPUI) / Dehydrins are type II LEA proteins induced in many plants during drought, low temperature, and high salinity to confer stress tolerance. AtERD14 is an Arabidopsis thaliana dehydrin that functions in part of the cold stress pathway. AtERD14 has chaperone-like capabilities that allow it to bind and protect various proteins from dehydration stresses. In order to determine the necessary components for cold induction of AtERD14, AtERD14prom::GFP/GUS and AtERD14prom::AtERD14 in AtERD14 KO constructs were created and stably transformed into A. thaliana. Analysis of the constructs showed the AtERD14 promoter alone was insufficient to respond to cold, and it was necessary to attach the AtERD14 coding region to the promoter to induce a cold response in ERD14. On the other hand, the RD29aprom::GFP/GUS promoter did respond to cold stress, indicating that RD29a does not require its coding region to support an increased amount of reporter activity after cold stress. The protoplast transformation system, while capable of transient expression of introduced constructs in protoplasts, was difficult for use for cold-inducible expression.