Browsing by Subject "Cold stress"

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    Functional dissection of ERD14 phosphorylation-dependent calcium binding activity
    (2014-12-11) Chacha, Allen R.; Randall, Stephen Karl, 1953-; Watson, John; Kusmierczyk, Andrew
    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.
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    Molecular and Physiological Responses of Soybean (Glycine max) to Cold and the Stress Hormone Ethylene
    (2019-05) Robison, Jennifer Dawn; Randall, Stephen K.; Balakrishnan, Lata; Watson, John C.; Blacklock, Brenda J.
    Abiotic stresses, such as cold, are serious agricultural problems resulting in substantial crop and revenue losses. Soybean (Glycine max) is an important worldwide crop for food, feed, fuel, and other products. Soybean has long been considered to be cold-intolerant and incapable of cold acclimation. In contrast to these reports, this study demonstrates that cold acclimation improved freezing tolerance in the domestic soybean cultivar ‘Williams 82’ with 50% enhancement of freezing tolerance after 5.2 +\- 0.6 days of cold exposure. Decreases in light dependent photosynthetic function and efficiency accompanied cold treatment. These decreases were due to an increase in photon dissipation likely driven by a decrease in plastoquinone (PQ) pool size limiting electron flow from photosystem II (PSII) to photosystem I (PSI). Cold-induced damage to operational photosynthesis began at 25 minutes of cold exposure and maximal photosynthesis was disrupted after 6 to 7 hours of cold exposure. Cold exposure caused severe photodamage leading to the loss of PSII reaction centers and photosynthetic efficiency. Comparisons of eight cultivars of G. max demonstrated a weak correlation between cold acclimation and northern cultivars versus southern cultivars. In the non-domesticated soybean species Glycine soja, the germination rate after cold imbibition was positively correlated with seedling cold acclimation potential. However, the overall cold acclimation potential in G. soja was equal to that of domestic soybean G. max reducing the enthusiasm for the “wild” soybean as an additional source of genetic diversity for cold tolerance. Despite being relatively cold intolerant, the soybean genome possesses homologs of the major cold responsive CBF/DREB1 transcription factors. These genes are cold-induced in soybean in a similar pattern to that of the cold tolerant model plant species Arabidopsis thaliana. In Arabidopsis, EIN3, a major component of the ethylene signaling pathway, is a negative transcriptional regulator of CBF/DREB1. In contrast to AtEIN3 transcript levels which do not change during cold treatment in Arabidopsis, we observed a cold-dependent 3.6 fold increase in GmEIN3 transcript levels in soybean. We hypothesized that this increase could prevent effective CBF/DREB1 cold regulation in soybean. Analysis of our newly developed cold responsive reporter (AtRD29Aprom::GFP/GUS) soybean transgenic lines demonstrated that inhibition of the ethylene pathway via foliar sprays (AVG, 1-MCP, and silver nitrate) resulted in significant cold-induced GUS activity. Transcripts of GmEIN3A;1 increased in response to ethylene pathway stimulation (ACC and ethephon) and decreased in response to ethylene pathway inhibition in the cold. Additionally, in the cold, inhibition of the ethylene pathway resulted in a significant increase in transcripts of GmDREB1A;1 and GmDREB1A;2 and stimulation of the ethylene pathway led to a decrease in GmDREB1A;1 and GmDREB1B;1 transcripts. To assess the physiological effects of these transcriptional changes; electrolyte leakage, lipid oxidation, free proline content, and photosynthesis were examined. Improvement in electrolyte leakage, a measure of freezing tolerance, was seen only under silver nitrate treatment. Only 1-MCP treatment resulted in significantly decreased lipid oxidation. Transcripts for CBF/DREB1 downstream targets (containing the consensus CRT/DRE motifs) significantly decreased in plants treated with ethylene pathway stimulators in the cold; however, ethylene pathway inhibition generally produced no increase over basal cold levels. To identify if GmEIN3A;1 was capable of binding to GmDREB1 promoters, the negative regulator GmEIN3A;1 and the positive regulator GmICE1A were cloned and expressed in Escherichia coli (E. coli). Preliminary binding results indicated that GmEIN3A;1 can bind to a double stranded section of the GmDREB1A;1 promoter containing putative EIN3 and ICE1 binding sites. GmICE1A is capable of binding to the same section of the GmDREB1A;1 promoter, though only when single stranded. Additional experiments will be required to demonstrate that GmEIN3A;1 and GmICE1A are capable of binding to the GmDREB1A;1 promoter and this work provides the tools to answer these questions. Overall, this work provides evidence that the ethylene pathway transcriptionally inhibits the CBF/DREB1 pathway in soybean through the action of GmEIN3A;1. Yet when GmCBF/DREB1 transcripts are upregulated by ethylene pathway inhibition, no consistent change in downstream targets was observed. These data indicate that the limitation in cold tolerance in soybean is due to a yet unidentified target downstream of CBF/DREB1 transcription.