Seth Debolt

University of Kentucky, Lexington, Kentucky, United States

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Publications (54)253.3 Total impact

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    ABSTRACT: Global grape production could generate up to 13Mt/yr of wasted biomass. The compositions of Cabernet Sauvignon (red marc) and Sauvignon Blanc (white marc) were analyzed with a view to using marc as raw material for biofuel production. On a dry weight basis, 31-54% w/w of the grape marc consisted of carbohydrate, of which 47-80% was soluble in aqueous media. Ethanol insoluble residues consisted mainly of polyphenols, pectic polysaccharides, heteroxylans and cellulose. Acid and thermal pre-treatments were investigated for their effects on subsequent cellulose saccharification. A 0.5M sulfuric acid pre-treatment yielded a 10% increase in the amount of liberated glucose after enzymatic saccharification. The theoretical amount of bioethanol that could be produced by fermentation of grape marc was up to 400L/t. However, bioethanol from only soluble carbohydrates could yield 270L/t, leaving a polyphenol enriched fraction that may be used in animal feed or as fertilizer. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.
    Bioresource Technology 06/2015; 193:76-83. DOI:10.1016/j.biortech.2015.06.030 · 5.04 Impact Factor
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    ABSTRACT: CESA5 synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis thaliana. The involvement of additional CESA proteins in this process and details concerning the manner in which the cellulose is deposited in the mucilage pocket are unknown. Here we show that both CESA3 and CESA10 are also highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant 1 (ixr1-1 and ixr1-2) mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and GFP-tagged CESA5, CESA3, and CESA10 proteins moved in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion as well as mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence. Copyright © 2015, Plant Physiology.
    Plant physiology 04/2015; 168(2). DOI:10.1104/pp.15.00478 · 7.39 Impact Factor
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    Conference Paper: Methiozolin & TAT enzymes
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    ABSTRACT: Methiozolin is a recently introduced herbicide for selective control of Poa annua in golf greens. The objective of this study is to characterize the herbicide symptomologies exhibited by Arabidopsis thaliana treated with a range of methiozolin rates (5nM to 50uM) and to provide further insight into its potential mode of action. Treated seedlings exhibited a range of unique visual characteristics. Root growth was inhibited by methiozolin at 5 nM but this rate had little effect on chlorophyll content at 7 or 14 days after treatment (DAT). Chlorophyll content was significantly different between 7 and 14 DAT and was significantly reduced at rates greater than 5 nM at 14 DAT. Analysis of cell expansion in methiozolin treated Arabidopsis seedlings were inconsistent with the radial cell swelling symptomology indicative of cellulose biosynthesis inhibitors. Methiozolin has also been proposed to be a pigment inhibitor by inhibiting the conversion of tyrosine to 4-hydroxyphenylpyruvate by the enzyme tyrosine aminotransferase (TAT). Arabidopsis has six predicted TATs enzyme and we test five out of the six gene knockouts to screen for their increased susceptibility to methiozolin.
    Weed Scince Society of America, Lexington Kentucky; 02/2015
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    ABSTRACT: In a screen for root hair morphogenesis mutants in Arabidopsis thaliana L. we identified a T-DNA insertion within a type III J-protein AtDjC17 caused altered root hair development and reduced hair length. Root hairs were observed to develop from trichoblast and atrichoblast cell files in both Atdjc17 and 35S::AtDJC17. Localization of gene expression in the root using transgenic plants expressing proAtDjC17::GUS revealed constitutive expression in stele cells. No AtDJC17 expression was observed in epidermal, endodermal, or cortical layers. To explore the contrast between gene expression in the stele and epidermal phenotype, hand cut transverse sections of Atdjc17 roots were examined showing that the endodermal and cortical cell layers displayed increased anticlinal cell divisions. Aberrant cortical cell division in Atdjc17 is proposed as causal in ectopic root hair formation via the positional cue requirement that exists between cortical and epidermal cell in hair cell fate determination. Results indicate a requirement for AtDJC17 in position-dependent cell fate determination and illustrate an intriguing requirement for molecular co-chaperone activity during root development.
    Frontiers in Plant Science 10/2014; 5:532. DOI:10.3389/fpls.2014.00532 · 3.64 Impact Factor
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    Ye Xia, Williams MA Petti C, Seth DeBolt
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    ABSTRACT: Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques
    Frontiers in Plant Science 09/2014; 5. DOI:10.3389/fpls.2014.00540 · 3.64 Impact Factor
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    ABSTRACT: Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) have a potentially broad acting herbicidal mode of action and are also useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam as a CBI and provide insight into its inhibitory mechanism. Indaziflam treated seedlings exhibited the CBI-like symptomologies of radial swelling and ectopic lignification. Furthermore, indaziflam inhibited the production of cellulose within < 1 hour of treatment and in a dose dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous (Poa annua L.) and a dicotyledonous plant (Arabidopsis thaliana L.). Arabidopsis mutants resistant to known CBIs, isoxaben or quinoxyphen, were not cross-resistant to indaziflam suggesting a different molecular target for indaziflam. To explore this further, we monitored the distribution and mobility of fluorescently labeled CELLULOSE SYNTHASE A (CESA) proteins in living cells of Arabidopsis during indaziflam exposure. Indaziflam caused a reduction in the velocity of YFP:CESA6 particles at the plasma membrane (PM) focal plane when compared to controls. Microtubule (MT) morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of PM localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting 1 (CSI1) independent reduction in the normal coincidence rate between MT and CESA. As a CBI, for which there is little evidence of evolved weed resistance, indaziflam represents an important addition to the action mechanisms available for weed management.
    Plant physiology 07/2014; DOI:10.1104/pp.114.241950 · 7.39 Impact Factor
  • PLoS ONE 04/2014; 9(4). · 3.53 Impact Factor
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    ABSTRACT: In plants, cellulose biosynthesis is an essential process for anisotropic growth and therefore is an ideal target for inhibition. Based on the documented utility of small-molecule inhibitors to dissect complex cellular processes we identified a cellulose biosynthesis inhibitor (CBI), named acetobixan, by bio-prospecting among compounds secreted by endophytic microorganisms. Acetobixan was identified using a drug-gene interaction screen to sift through hundreds of endophytic microbial secretions for one that caused synergistic reduction in root expansion of the leaky AtcesA6prc1-1 mutant. We then mined this microbial secretion for compounds that were differentially abundant compared with Bacilli that failed to mimic CBI action to isolate a lead pharmacophore. Analogs of this lead compound were screened for CBI activity, and the most potent analog was named acetobixan. In living Arabidopsis cells visualized by confocal microscopy, acetobixan treatment caused CESA particles localized at the plasma membrane (PM) to rapidly re-localize to cytoplasmic vesicles. Acetobixan inhibited 14C-Glc uptake into crystalline cellulose. Moreover, cortical microtubule dynamics were not disrupted by acetobixan, suggesting specific activity towards cellulose synthesis. Previous CBI resistant mutants such as ixr1-2, ixr2-1 or aegeus were not cross resistant to acetobixan indicating that acetobixan targets a different aspect of cellulose biosynthesis.
    PLoS ONE 04/2014; 9(4):e95245. DOI:10.1371/journal.pone.0095245 · 3.53 Impact Factor
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    ABSTRACT: Morlin (7-ethoxy-4-methyl chromen-2-one) is coumarin and acts as a cellulose biosynthesis inhibitor (CBI). Plants treated with morlin display radially swollen organs and at a cellular level morlin slows down the velocity of the CESA complex and changes the rate at which microtubules grow and shrink. Despite its potential as a herbicide, the mode of action of this chemical remains poorly understood. To attempt to elucidate further details on how morlin impacts cell biology, a whole genome microarray was performed on wild type Arabidopsis treated with morlin. Transcript abundance data was screened for genes that could be influenced by the presence of the compound. Genes that were differentially expressed after morlin treatment were selected as candidate interactors. To study these further, homozygous T-DNA knockout alleles were isolated and each allele was examined for resistance or sensitivity to morlin. Employing a series of dual drug experiments combining morlin with several other CBIs was conducted to establish similarity of mechanism. These data confirm the uniqueness of morlin action mechanism.
    National Conference on Undergraduate Research; 04/2014
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    ABSTRACT: Pyrolysis-GC/mass spectrometry (Py-GC/MS) and thermogravimetric analysis (TGA) was used to analyze the thermal decomposition of several endocarp sources, namely, coconut shells, walnut shells, peach pits, and olive pits, as well as their respective lignin fractions. To determine whether extraction procedures influenced pyrolysate composition and thermal decomposition processes, lignin was extracted from these feedstocks using two different procedures based on the use of formic acid and sulfuric acid (National Renewable Energy Laboratory (NREL) laboratory analytical procedure), after which the lignin-derived pyrolysates and TGA profiles were compared. Qualitative analysis of the distribution of pyrolysates provided predictive information about the structure and composition of the lignin in each sample. Results suggest that the lignin extract pyrolysates contained a different distribution of linkages and monomers in comparison to the non-extracted biomass, suggesting that lignin processing can influence bio-oil composition. Moreover, we identify the types of products obtainable by pyrolysis of these feedstocks and their lignin extracts. Heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC NMR) and Fourier transform infrared spectroscopy (FTIR) were also used to elucidate the structures of the extracted lignin samples.
    BioEnergy Research 03/2014; 8(1):1-19. DOI:10.1007/s12155-014-9526-5 · 3.40 Impact Factor
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    ABSTRACT: Natural food colorants with functional properties are of increasing interest. Prior reports indicate the chemical suitability of sorghum leaf 3-deoxyanthocyanidins as natural food colorants. Via mutagenesis assisted breeding, we isolated and characterized a sorghum variety that greatly over-accumulates 3-deoxyanthocyanidins of leaf tissue, named REDforGREEN (RG). Interestingly, RG not only caused increased 3-deoxyanthocyanidins, but also caused increased tannins, chlorogenic acid and total phenolics in the leaf tissue. Chemical composition of pigments was established through high performance liquid chromatography (HPLC) that identified luteolinidin (LUT) and apigeninidin (APG) as the main 3-deoxyanthocianidin species. Specifically, 3-deoxyanthocianidin levels were 1768 μg.g-1 LUT and 421 μg.g-1 APG in RG leaves compared with trace amounts in wild type representing 1000-fold greater in the mutant leaves. Thus RG represents a useful sorghum mutagenesis variant to develop as a functionalized food colorant.
    Journal of Agricultural and Food Chemistry 01/2014; 62(6). DOI:10.1021/jf405324j · 3.11 Impact Factor
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    ABSTRACT: Herein we report the fast pyrolysis of dried, ground Scenedesmus sp. at two different reactor scales. Pyrolysis was performed at 480 degrees C and 1 bar in both an isothermal spouted bed reactor and a dynamic pyrolysis-GC/MS unit, each with 2 s vapor residence times. Bio-oil products were characterized on the basis of GC-MS, simulated distillation GC, elemental analysis, calorific content and total acid number. The ratio of crude oil: char obtained from the spouted bed reactor was 3.76 by weight, the average calorific content of the oil being 18.4 MJ/kg. The average total acid number (68 mg KOH/g) was lower than typical bio-oil produced via wood pyrolysis. Simulated distillation results indicated that a significant proportion of the oil corresponded to the boiling range typical for heavy gas oil (343 degrees C-524 degrees C). Elemental analysis showed the oil contained an average of 27.6 wt.% oxygen and 8.6 wt.% nitrogen, the relatively high nitrogen content being a consequence of the high protein content of the algae. According to GC-MS data, the oil consisted of various hydrocarbons as well as oxygenated and nitrogenous species, including indoles, fatty acids and amides. Pyrolysis-GC-MS was also performed on Scenedesmus sp. in order to provide insights into the nature of the primary pyrolysis products.
    Renewable Energy 12/2013; 60:625-632. DOI:10.1016/j.renene.2013.06.016 · 3.36 Impact Factor
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    ABSTRACT: Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.
    The Plant Cell 11/2013; 25(11). DOI:10.1105/tpc.113.119115 · 9.58 Impact Factor
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    ABSTRACT: ORCID ID: 0000-0002-5234-8346 (R.L.H.). Calmodulin N-methyltransferase (CaM KMT) is an evolutionarily conserved enzyme in eukaryotes that transfers three methyl groups to a highly conserved lysyl residue at position 115 in calmodulin (CaM). We sought to elucidate whether the methylation status of CaM plays a role in CaM-mediated signaling pathways by gene expression analyses of CaM KMT and phenotypic characterization of Arabidopsis thaliana lines wherein CaM KMT was overexpressed (OX), partially silenced, or knocked out. CaM KMT was expressed in discreet spatial and tissue-specific patterns, most notably in root tips, floral buds, stamens, apical meristems, and germinating seeds. Analysis of transgenic plants with genetic dysfunction in CaM KMT revealed a link between the methylation status of CaM and root length. Plants with suppressed CaM methylation had longer roots and CaM KMT OX lines had shorter roots than wild type (Columbia-0). CaM KMT was also found to influence the root radial developmental program. Protein microarray analyses revealed a number of proteins with specificity for methylated forms of CaM, providing candidate functional intermediates between the observed phenotypes and the target pathways. This work demonstrates that the functionality of the large CaM family in plants is fine-tuned by an overarching methylation mechanism.
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    ABSTRACT: Improving saccharification efficiency in bioenergy crop species remains an important challenge. Here, we report the characterization of a Sorghum (Sorghum bicolor L.) mutant, named REDforGREEN (RG), as a bioenergy feedstock. It was found that RG displayed increased accumulation of lignin in leaves and depletion in the stems, antithetic to the trend observed in wild type. Consistent with these measurements, the RG leaf tissue displayed reduced saccharification efficiency whereas the stem saccharification efficiency increased relative to wild type. Reduced lignin was linked to improved saccharification in RG stems, but a chemical shift to greater S:G ratios in RG stem lignin was also observed. Similarities in cellulose content and structure by XRD-analysis support the correlation between increased saccharification properties and reduced lignin instead of changes in the cellulose composition and/or structure. Antithetic lignin accumulation was observed in the RG mutant leaf-and stem-tissue, which resulted in greater saccharification efficiency in the RG stem and differential thermochemical product yield in high lignin leaves. Thus, the red leaf coloration of the RG mutant represents a potential marker for improved conversion of stem cellulose to fermentable sugars in the C4 grass Sorghum.
    Biotechnology for Biofuels 10/2013; 6(1):146. DOI:10.1186/1754-6834-6-146 · 6.22 Impact Factor
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    ABSTRACT: Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus×giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.
    Frontiers in Plant Science 06/2013; 4:181. DOI:10.3389/fpls.2013.00181 · 3.64 Impact Factor
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    ABSTRACT: Second generation feedstocks for bioethanol will likely include a sizable proportion of perennial C4 grasses, principally in the Panicoideae clade. The Panicoideae contain agronomically important annual grasses including Zea mays L. (maize), Sorghum bicolor (L.) Moench (sorghum), and Saccharum officinarum L. (sugar cane) as well as promising second generation perennial feedstocks including Miscanthus × giganteus and Panicum virgatum L. (switchgrass). The underlying complexity of these polyploid grass genomes is a major limitation for their direct manipulation and thus driving a need for rapidly cycling comparative model. Setaria viridis (green millet) is a rapid cycling C4 panicoid grass with a relatively small and sequenced diploid genome and abundant seed production. Stable, transient, and protoplast transformation technologies have also been developed for Setaria viridis making it a potentially excellent model for other C4 bioenergy grasses. Here, the lignocellulosic feedstock composition, cellulose biosynthesis inhibitor response and saccharification dynamics of Setaria viridis are compared with the annual sorghum and maize and the perennial switchgrass bioenergy crops as a baseline study into the applicability for translational research. A genome-wide systematic investigation of the cellulose synthase-A genes was performed identifying eight candidate sequences. Two developmental stages; (a) metabolically active young tissue and (b) metabolically plateaued (mature) material are examined to compare biomass performance metrics.
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    2013 IN VITRO BIOLOGY MEETING, 2013 Meeting of the Society for In Vitro Biology; 06/2013
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    ABSTRACT: A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose-synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs.
    Proceedings of the National Academy of Sciences 04/2013; DOI:10.1073/pnas.1301027110 · 9.81 Impact Factor

Publication Stats

911 Citations
253.30 Total Impact Points

Institutions

  • 2008–2014
    • University of Kentucky
      • Department of Horticulture
      Lexington, Kentucky, United States
  • 2006–2008
    • University of Adelaide
      • School of Agriculture, Food and Wine
      Adelaide, South Australia, Australia
  • 2007
    • Carnegie Institution for Science
      • Department of Plant Biology
      Washington, West Virginia, United States