Salt stress, or salinity, is one of the most common environmental stresses affecting crop yield worldwide. Due to the prevalence of salinity stress, it is not surprising that plants have evolved mechanisms to tolerate osmotic and ionic stress caused by salinity. Dehydrins are intrinsically unstructured proteins that accumulate in photosynthetic organisms under dehydrating conditions, such as salinity, and are thought to confer stress tolerance through the stabilization of cellular membranes. <i>Thellungiella salsuginea</i>, a close relative of <i>Arabidopsis thaliana</i>, is a halophyte that thrives in the Canadian sub-Arctic (Yukon Territory), that is able to tolerate extreme conditions, including high salinity. TsDHN-2 is a basic dehydrin from <i>Thellungiella</i> whose transcript increases over 10-fold in response to salinity treatment. Using RNA interference (RNAi) methodology, TsDHN-2 has been silenced and these lines were used in this study to investigate the role TsDHN-2 may play in the salt tolerance of <i>Thellungiella</i>. RNAi line 7-8 presented a 41% reduced expression of TsDHN-2 in comparison to wild-type (WT). Seed of this line showed a 15% germination rate compared to 40% in WT in the presence of 100 mM NaCl. Salinity stress experiments were performed by treating the RNAi lines and WT plants with 300 mM NaCl for up to two weeks. Line 7-8 exhibited a 6.2% greater decrease in photochemical efficiency of photosystem II (PSII) as estimated by the variable to maximal fluorescence ratio (F_v/F_m) and showed 5% greater phenotypic damage than WT when estimated visually. Concentrations of the compatible osmolyte proline increased in response to salt treatment by 3.4-fold in WT and 8.1-fold in line 7-8, suggesting this compound may be a marker for salinity tolerance. Collectively, these data support the notion that TsDHN-2 plays a role in the salinity tolerance mechanisms of <i>Thellungiella</i>.

Salt stress, or salinity, is one of the most common environmental stresses affecting crop yield worldwide. Due to the prevalence of salinity stress, it is not surprising that plants have evolved mechanisms to tolerate osmotic and ionic stress caused by salinity. Dehydrins are intrinsically unstructured proteins that accumulate in photosynthetic organisms under dehydrating conditions, such as salinity, and are thought to confer stress tolerance through the stabilization of cellular membranes. <i>Thellungiella salsuginea</i>, a close relative of <i>Arabidopsis thaliana</i>, is a halophyte that thrives in the Canadian sub-Arctic (Yukon Territory), that is able to tolerate extreme conditions, including high salinity. TsDHN-2 is a basic dehydrin from <i>Thellungiella</i> whose transcript increases over 10-fold in response to salinity treatment. Using RNA interference (RNAi) methodology, TsDHN-2 has been silenced and these lines were used in this study to investigate the role TsDHN-2 may play in the salt tolerance of <i>Thellungiella</i>. RNAi line 7-8 presented a 41% reduced expression of TsDHN-2 in comparison to wild-type (WT). Seed of this line showed a 15% germination rate compared to 40% in WT in the presence of 100 mM NaCl. Salinity stress experiments were performed by treating the RNAi lines and WT plants with 300 mM NaCl for up to two weeks. Line 7-8 exhibited a 6.2% greater decrease in photochemical efficiency of photosystem II (PSII) as estimated by the variable to maximal fluorescence ratio (F_v/F_m) and showed 5% greater phenotypic damage than WT when estimated visually. Concentrations of the compatible osmolyte proline increased in response to salt treatment by 3.4-fold in WT and 8.1-fold in line 7-8, suggesting this compound may be a marker for salinity tolerance. Collectively, these data support the notion that TsDHN-2 plays a role in the salinity tolerance mechanisms of <i>Thellungiella</i>.

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Dehydrins are intrinsically disordered plant stress-proteins. Repetitively in their sequence are some highly conserved stretches of 7-17 residues, the so called K-, S-, Y- and lysine rich segments. This thesis aims to give insight into the possible role dehydrins have in the stressed plant cell with main focus on membrane interaction and protection. The work includes four recombinant dehydrins from the plant Arabidopsis thaliana: Cor47 (SK3), Lti29 (SK3), Lti30 (K6) and Rab18 (Y2SK2). Initially, we mimicked crowded cellular environment in vitro to verify that dehydrins are truly disordered proteins. Thereafter, the proposal that the compulsory K-segment determines membrane binding was tested. Experiments show that only Lti30 and Rab18 bind, whereas Cor47 and Lti29 does not. As Lti30 and Rab18 binds they assembles vesicles into clusters in vitro, a feature used to characterize the interaction. From this it was shown that membrane binding of Lti30 is electrostatic and determined by global as well as local charges. Protonation of histidine pairs flanking the K-segments works as an on/off-binding switch. By NMR studies it was shown that the K-segments form a dynamic α-helix upon binding, so called disorder-to-order behaviour. Also, dehydrins electrostatic interaction with lipids can be further tuned by posttranslational phosphorylation or coordination of calcium and zinc ions. Finally, specific binding of Rab18 to inositol lipids, mainly PI(4,5)P2, is reported. The interaction is mainly coordinated by two arginines neighboring one of the K-segments. In conclusion, the K-segments are indeed involved in the binding of dehydrins to membrane but only in combination with extensions (Lti30) or modified (Rab18). / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Strawberry (Fragaria spp.) is an agricultural crop grown often in temperate regions that has high variability in its susceptibility to freezing injury. To breed cultivars for frost and freezing tolerance, identification of molecular markers associated with low temperature tolerance is advantageous. In this work, I investigated a high-throughput method for protein assays and western blotting. Success in streamlining these processes saves an immense amount of time and allows for the processing of more samples and obtaining larger datasets. Thirty-three octoploid varieties were tested for their accumulation of five different potential biomarkers in response to cold exposure. It was found that total dehydrin content, has the strongest potential to be reliable biomarkers for breeding programs. Previous work identified seven putative dehydrins in Fragaria, where two were purified and positively identified by mass spectrometry and determined to be COR47-like (SKn) and XERO2-like (YnSKn). This work demonstrated that cold tolerance positively correlated with dehydrin protein expression levels. To understand the cold-regulated expression of dehydrins as a function of cold exposure time, the levels of transcripts and corresponding proteins were examined in strongly cold tolerant (Alta) and lesser cold tolerant (FDP817, NCGR1363) Fragaria diploid genotypes. The COR47-like (SKn) and XERO2-like (YnSKn) dehydrins both had higher transcript accumulation and protein levels in the more cold tolerant line in comparison to the two less cold tolerant lines. Lack of correlation between transcript and resulting COR47 protein level in Alta were observed at several different timepoints, where protein accumulation preceded an increase in RNA. This trend was not seen with XERO2. This initiated an investigation to discover at what level COR47 is being regulated. First, the COR47 coding region was sequenced for all the genotypes to confirm against the predicted sequence. In addition, since two isoforms of the COR47 gene exist, and could possibly explain the discrepancy in transcript counts, primers were designed for both isoforms and RT-qPCR was performed to examine the transcripts of COR47 more closely. Through examination of the non-congruence of COR47 transcripts and protein, it was found that transcriptional mechanisms of regulation are not involved, and that post transcriptional and post-RNA splicing mechanisms are likely to be responsible for the observed trend in Alta. Conclusions from this work demonstrate that dehydrin transcripts and dehydrin protein accumulations are strong potential biomarkers for identifying low temperature tolerance in diploid strawberry.

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.