Browsing by Subject "Photosynthesis"

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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.
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    Molecular cloning of the soybean phototropins
    (2014) Roy, Pallabi; Watson, John C., 1953-; Randall, Stephen Karl, 1953-; Anderson, Gregory G.; Atkinson, Simon
    The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.