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Item Characterization of a cold-responsive dehydrin promoter(2014-08-27) Osadczuk, Elizabeth A.; Randall, Stephen Karl, 1953-; Watson, John C., 1953-; Picard, Christine; Atkinson, SimonDehydrins 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.Item Cold response biomarker identification in strawberry(2018-07-17) Deitch, Zachary M.; Randall, StephenStrawberry (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.Item Daily Situational Brief, January 15, 2015(MESH Coalition, 1/15/2015) MESH CoalitionItem The Ethylene Signaling Pathway Negatively Impacts CBF/DREB-Regulated Cold Response in Soybean (Glycine max)(Frontiers, 2019) Robison, Jennifer D.; Yamasaki, Yuji; Randall, Stephen K.; Biology, School of ScienceDuring cold stress, soybean CBF/DREB1 transcript levels increase rapidly; however, expected downstream targets appear unresponsive. Here we asked whether the ethylene signaling pathway, which is enhanced in the cold can negatively regulate the soybean CBF/DREB1 cold responsive pathway; thus contributing to the relatively poor cold tolerance of soybean. Inhibition of the ethylene signaling pathway resulted in a significant increase in GmDREB1A;1 and GmDREB1A;2 transcripts, while stimulation led to decreased GmDREB1A;1 and GmDREB1B;1 transcripts. A cold responsive reporter construct (AtRD29Aprom::GFP/GUS), as well as predicted downstream targets of soybean CBF/DREB1 [ Glyma.12g015100 (ADH), Glyma.14g212200 (ubiquitin ligase), Glyma.05g186700 (AP2), and Glyma.19g014600 (CYP)] were impacted by the modulation of the ethylene signaling pathway. Photosynthetic parameters were affected by ethylene pathway stimulation, but only at control temperatures. Freezing tolerance (as measured by electrolyte leakage), free proline, and MDA; in both acclimated and non-acclimated plants were increased by silver nitrate but not by other ethylene pathway inhibitors. This work provides evidence that the ethylene signaling pathway, possibly through the action of EIN3, transcriptionally inhibits the CBF/DREB1 pathway in soybean.Item Integrative "omic" analysis reveals distinctive cold responses in leaves and roots of strawberry, Fragaria × ananassa 'Korona'(Frontiers Media SA, 2015) Koehler, Gage; Rohloff, Jens; Wilson, Robert C.; Kopka, Joachim; Erban, Alexander; Winge, Per; Bones, Atle M.; Davik, Jahn; Alsheikh, Muath K.; Randall, Stephen K.; Department of Biology, School of ScienceTo assess underlying metabolic processes and regulatory mechanisms during cold exposure of strawberry, integrative "omic" approaches were applied to Fragaria × ananassa Duch. 'Korona.' Both root and leaf tissues were examined for responses to the cold acclimation processes. Levels of metabolites, proteins, and transcripts in tissues from plants grown at 18°C were compared to those following 1-10 days of cold (2°C) exposure. When leaves and roots were subjected to GC/TOF-MS-based metabolite profiling, about 160 compounds comprising mostly structurally annotated primary and secondary metabolites, were found. Overall, 'Korona' showed a modest increase of protective metabolites such as amino acids (aspartic acid, leucine, isoleucine, and valine), pentoses, phosphorylated and non-phosphorylated hexoses, and distinct compounds of the raffinose pathway (galactinol and raffinose). Distinctive responses were observed in roots and leaves. By 2DE proteomics a total of 845 spots were observed in leaves; 4.6% changed significantly in response to cold. Twenty-one proteins were identified, many of which were associated with general metabolism or photosynthesis. Transcript levels in leaves were determined by microarray, where dozens of cold associated transcripts were quantitatively characterized, and levels of several potential key contributors (e.g., the dehydrin COR47 and GADb) to cold tolerance were confirmed by qRT-PCR. Cold responses are placed within the existing knowledge base of low temperature-induced changes in plants, allowing an evaluation of the uniqueness or generality of Fragaria responses in photosynthetic tissues. Overall, the cold response characteristics of 'Korona' are consistent with a moderately cold tolerant plant.Item Is it a cold or the flu?(2005-09) National Institute of Allergy and Infectious Diseases (U.S.)Information sheet comparing the common cold with the flu.