Horticulture Commons^™

[14C] Glucose Cell Wall Incorporation Assay For The Estimation Of Cellulose Biosynthesis, Chad Brabham, Jozsef Stork, Seth Debolt

Horticulture Faculty Publications

Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining …

Prospecting For Energy-Rich Renewable Raw Materials: Agave Leaf Case Study, Kendall R. Corbin, Caitlin S. Byrt, Stefan Bauer, Seth Debolt, Don Chambers, Joseph A. M. Holtum, Ghazwan Karem, Marilyn Henderson, Jelle Lahnstein, Cherie T. Beahan, Antony Bacic, Geoffrey B. Fincher, Natalie S. Betts, Rachel A. Burton

Horticulture Faculty Publications

Plant biomass from different species is heterogeneous, and this diversity in composition can be mined to identify materials of value to fuel and chemical industries. Agave produces high yields of energy-rich biomass, and the sugar-rich stem tissue has traditionally been used to make alcoholic beverages. Here, the compositions of Agave americana and Agave tequilana leaves are determined, particularly in the context of bioethanol production. Agave leaf cell wall polysaccharide content was characterized by linkage analysis, non-cellulosic polysaccharides such as pectins were observed by immuno-microscopy, and leaf juice composition was determined by liquid chromatography. Agave leaves are fruit-like--rich in moisture, …

Calmodulin Methyltransferase Is Required For Growth, Muscle Strength, Somatosensory Development And Brain Function, Sitvanit Haziza, Roberta Magnani, Dima Lan, Omer Keinan, Ann Saada, Eli Hershkovitz, Nurit Yanay, Yoram Cohen, Yoram Nevo, Robert L. Houtz, Val C. Sheffield, Hava Golan, Ruti Parvari

Horticulture Faculty Publications

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute …

Engineering Plant Biomass Lignin Content And Composition For Biofuels And Bioproducts, Cassie Marie Welker, Vimal Kumar Balasubramanian, Carloalberto Petti, Krishan Mohan Rai, Seth Debolt, Venugopal Mendu

Horticulture Faculty Publications

Lignin is an aromatic biopolymer involved in providing structural support to plant cell walls. Compared to the other cell wall polymers, i.e., cellulose and hemicelluloses, lignin has been considered a hindrance in cellulosic bioethanol production due to the complexity involved in its separation from other polymers of various biomass feedstocks. Nevertheless, lignin is a potential source of valuable aromatic chemical compounds and upgradable building blocks. Though the biosynthetic pathway of lignin has been elucidated in great detail, the random nature of the polymerization (free radical coupling) process poses challenges for its depolymerization into valuable bioproducts. The absence of specific …

Characterization Of Culturable Bacterial Endophytes And Their Capacity To Promote Plant Growth From Plants Grown Using Organic Or Conventional Practices, Ye Xia, Seth Debolt, Jamin Dreyer, Delia Scott, Mark A. Williams

Horticulture Faculty Publications

Plants have a diverse internal microbial biota that has been shown to have an important influence on a range of plant health attributes. Although these endophytes have been found to be widely occurring, few studies have correlated agricultural production practices with endophyte community structure and function. One agricultural system that focuses on preserving and enhancing soil microbial abundance and biodiversity is organic farming, and numerous studies have shown that organically managed system have increased microbial community characteristics. Herein, the diversity and specificity of culturable bacterial endophytes were evaluated in four vegetable crops: corn, tomato, melon, and pepper grown under organic …

Horticulturae — An International, Multidisciplinary, Open Access Journal, Douglas D. Archbold

Horticulture Faculty Publications

No abstract provided.

Identification Of Micrornas In Two Species Of Tomato, Solanum Lycopersicum And Solanum Habrochaites, By Deep Sequencing, Shan-Shan Fan, Qian-Nan Li, Guang-Jun Guo, Jian-Chang Gao, Xiao-Xuan Wang, Yan-Mei Guo, John C. Snyder, Yong-Chen Du

Horticulture Faculty Publications

MicroRNAs (miRNAs) are ~21 nucleotide (nt), endogenous RNAs that regulate gene expression in plants. Increasing evidence suggests that miRNAs play an important role in species-specific development in plants. However, the detailed miRNA profile divergence has not been performed among tomato species. In this study, the small RNA (sRNA) profiles of Solanum lycopersicum cultivar 9706 and Solanum habrochaites species PI 134417 were obtained by deep sequencing. Sixty-three known miRNA families were identified from these two species, of which 39 were common. Further miRNA profile comparison showed that 24 known non-conserved miRNA families were species-specific between these two tomato species. In addition, …

Experimental Approaches To Study Plant Cell Walls During Plant-Microbe Interactions, Ye Xia, Carloalberto Petti, Mark A. Williams, Seth Debolt

Horticulture Faculty Publications

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.

The Involvement Of J-Protein Atdjc17 In Root Development In Arabidopsis, Carloalberto Petti, Meera Nair, Seth Debolt

Horticulture Faculty Publications

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 …

Haploid Induction Via In Vitro Gynogenesis In Tomato (Solanum Lycopersicum L.), He Zhao, Xiao-Xuan Wang, Yong-Cheng Du, De-Wei Zhu, Yan-Mei Guo, Jian-Chang Gao, Fei Li, John C. Snyder

Horticulture Faculty Publications

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely …

Acetobixan, An Inhibitor Of Cellulose Synthesis Identified By Microbial Bioprospecting, Ye Xia, Lei Lei, Chad Brabham, Jozsef Stork, James R. Strickland, Adam Ladak, Ying Gu, Ian Wallace, Seth Debolt

Horticulture Faculty Publications

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 …

Sorghum Mutant Rg Displays Antithetic Leaf Shoot Lignin Accumulation Resulting In Improved Stem Saccharification Properties, Carloalberto Petti, Anne E. Harman-Ware, Mizuki Tateno, Rekha Kushwaha, Andrew Shearer, A. Bruce Downie, Mark Crocker, Seth Debolt

Horticulture Faculty Publications

BACKGROUND: 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.

RESULTS: 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 …

Comparative Feedstock Analysis In Setaria Viridis L. As A Model For C4 Bioenergy Grasses And Panicoid Crop Species, Carloalberto Petti, Andrew Shearer, Mizuki Tateno, Mathew J. Ruwaya, Sue E. Nokes, Tom Brutnell, Seth Debolt

Horticulture Faculty Publications

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 …

Uses Of Phage Display In Agriculture: Sequence Analysis And Comparative Modeling Of Late Embryogenesis Abundant Client Proteins Suggest Protein-Nucleic Acid Binding Functionality, Rekha Kushwaha, A. Bruce Downie, Christina M. Payne

Horticulture Faculty Publications

A group of intrinsically disordered, hydrophilic proteins-Late Embryogenesis Abundant (LEA) proteins-has been linked to survival in plants and animals in periods of stress, putatively through safeguarding enzymatic function and prevention of aggregation in times of dehydration/heat. Yet despite decades of effort, the molecular-level mechanisms defining this protective function remain unknown. A recent effort to understand LEA functionality began with the unique application of phage display, wherein phage display and biopanning over recombinant Seed Maturation Protein homologs from Arabidopsis thaliana and Glycine max were used to retrieve client proteins at two different temperatures, with one intended to represent heat stress. From …

Uses Of Phage Display In Agriculture: A Review Of Food-Related Protein-Protein Interactions Discovered By Biopanning Over Diverse Baits, Rekha Kushwaha, Christina M. Payne, A. Bruce Downie

Horticulture Faculty Publications

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction …

Human Calmodulin Methyltransferase: Expression, Activity On Calmodulin, And Hsp90 Dependence, Sophia Magen, Roberta Magnani, Sitvanit Haziza, Eli Hershkovitz, Robert Houtz, Franca Cambi, Ruti Parvari

Horticulture Faculty Publications

Deletion of the first exon of calmodulin-lysine N-methyltransferase (CaM KMT, previously C2orf34) has been reported in two multigene deletion syndromes, but additional studies on the gene have not been reported. Here we show that in the cells from 2p21 deletion patients the loss of CaM KMT expression results in accumulation of hypomethylated calmodulin compared to normal controls, suggesting that CaM KMT is essential for calmodulin methylation and there are no compensatory mechanisms for CaM methylation in humans. We have further studied the expression of this gene at the transcript and protein levels. We have identified 2 additional …

Identification Of Late Embryogenesis Abundant (Lea) Protein Putative Interactors Using Phage Display, Rekha Kushwaha, Taylor D. Lloyd, Kim R. Schäfermeyer, Santosh Kumar, A. Bruce Downie

Horticulture Faculty Publications

Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 °C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom …

Sustainable Agriculture Undergraduate Degree Programs: A Land-Grant University Mission, Krista L. Jacobsen, Kim L. Niewolny, Michelle S. Schroeder-Moreno, Mark Van Horn, Alison H. Harmon, Yolanda H. Chen Fanslow, Mark A. Williams, Damian Parr

Horticulture Faculty Publications

There has been considerable growth in the number undergraduate degree programs in sustainable agriculture (SA) in universities and colleges across the country in the past 25 years. As a subset of this national trend, land-grant universities (LGUs) are emerging as catalysts in innovative SA program development, in part due to the LGU tripartite mission of education, extension, and research. This mission compels LGUs to develop undergraduate degree offerings to engage student, faculty, and community stakeholders who are increasingly interested in SA. In this article, which is an outcome of a gathering of faculty, staff and students from SA programs at …

Identification And Thermochemical Analysis Of High-Lignin Feedstocks For Biofuel And Biochemical Production, Venugopal Mendu, Anne E. Harman-Ware, Mark Crocker, Jungho Jae, Jozsef Stork, Samuel Morton, Andrew Placido, George Huber, Seth Debolt

Horticulture Faculty Publications

BACKGROUND: Lignin is a highly abundant biopolymer synthesized by plants as a complex component of plant secondary cell walls. Efforts to utilize lignin-based bioproducts are needed.

RESULTS: Herein we identify and characterize the composition and pyrolytic deconstruction characteristics of high-lignin feedstocks. Feedstocks displaying the highest levels of lignin were identified as drupe endocarp biomass arising as agricultural waste from horticultural crops. By performing pyrolysis coupled to gas chromatography-mass spectrometry, we characterized lignin-derived deconstruction products from endocarp biomass and compared these with switchgrass. By comparing individual pyrolytic products, we document higher amounts of acetic acid, 1-hydroxy-2-propanone, acetone and furfural in switchgrass …

Substrates Of The Arabidopsis Thaliana Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display And Biopanning, Tingsu Chen, Nihar Nayak, Susmita Maitra Majee, Jonathan Lowenson, Kim R. Schäfermeyer, Alyssa C. Eliopoulos, Taylor D. Lloyd, Randy Dinkins, Sharyn E. Perry, Nancy R. Forsthoefel, Steven G. Clarke, Daniel M. Vernon, Zhaohui Sunny Zhou, Tomas Rejtar, A. Bruce Downie

Horticulture Faculty Publications

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when …

Copy Number Variation Shapes Genome Diversity In Arabidopsis Over Immediate Family Generational Scales, Seth Debolt

Horticulture Faculty Publications

Arabidopsis thaliana is the model plant and is grown worldwide by plant biologists seeking to dissect the molecular underpinning of plant growth and development. Gene copy number variation (CNV) is a common form of genome natural diversity that is currently poorly studied in plants and may have broad implications for model organism research, evolutionary biology, and crop science. Herein, comparative genomic hybridization (CGH) was used to identify and interrogate regions of gene CNV across the A. thaliana genome. A common temperature condition used for growth of A. thaliana in our laboratory and many around the globe is 22 degrees C. …

Relative Crystallinity Of Plant Biomass: Studies On Assembly, Adaptation And Acclimation, Darby Harris, Seth Debolt

Horticulture Faculty Publications

Plant biomechanical design is central to cell shape, morphogenesis, reproductive performance and protection against environmental and mechanical stress. The cell wall forms the central load bearing support structure for plant design, yet a mechanistic understanding of its synthesis is incomplete. A key tool for studying the structure of cellulose polymorphs has been x-ray diffraction and fourier transform infrared spectroscopy (FTIR). Relative crystallinity index (RCI) is based on the x-ray diffraction characteristics of two signature peaks and we used this technique to probe plant assembly, adaptation and acclimation. Confocal microscopy was used to visualize the dynamics of cellulose synthase in transgenic …