Search

Abstract 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 compared to endocarp tissue, which is consistent with high holocellulose relative to lignin. By contrast, greater yields of lignin-based pyrolytic products such as phenol, 2-methoxyphenol, 2-methylphenol, 2-methoxy-4-methylphenol and 4-ethyl-2-methoxyphenol arising from drupe endocarp tissue are documented. Conclusions Differences in product yield, thermal decomposition rates and molecular species distribution among the feedstocks illustrate the potential of high-lignin endocarp feedstocks to generate valuable chemicals by thermochemical deconstruction.

White oak (Quercus alba) is an abundant forest tree species across eastern North America that is ecologically, culturally, and economically important. We report the first haplotype-resolved chromosome-scale genome assembly of Q. alba and conduct comparative analyses of genome structure and gene content against other published Fagaceae genomes. We investigate the genetic diversity of this widespread species and the phylogenetic relationships among oaks using whole genome data. Despite strongly conserved chromosome synteny and genome size across Quercus, certain gene families have undergone rapid changes in size, including defense genes. Unbiased annotation of resistance (R) genes across oaks revealed that the overall number of R genes is similar across species - as are the chromosomal locations of R gene clusters - but, gene number within clusters is more labile. We found that Q. alba has high genetic diversity, much of which predates its divergence from other oaks and likely impacts divergence time estimations. Our phylogenetic results highlight widespread phylogenetic discordance across the genus. The white oak genome represents a major new resource for studying genome diversity and evolution in Quercus. Additionally, we show that unbiased gene annotation is key to accurately assessing R gene evolution in Quercus., (© 2025 The Author(s). New Phytologist © 2025 New Phytologist Foundation.)

American white (Quercus alba L.) oak casks have been used for liquid storage for centuries. Their use in aged spirits is critical to imparting flavor and mouthfeel to the final product. The reason that barrels retain liquid has been hypothesized to be the result of abundant physiological structures called tyloses in parenchyma tissues and medullary rays in white oak. Using non-destructive X-ray computed tomography (XRCT) imaging, we reveal an unprecedented view of tylose structure and quantify the pore-filling capacity of tyloses in white oak that underscores the liquid retention we observe in casks. We show that pores of white oaks are filled with sevenfold higher tylose volume compared to northern red oak (Q. rubra), consistent with prior literature that casks made from white oak retain liquid while red oak fails to do so. We propose that XRCT represents a methodological standard for observing these complex structures and should be employed to understand the many questions related to liquid losses from casks, cultural treatment of casks, and the influence of climate change on oak tyloses in the future., (© 2024. The Author(s).)

In this study, we investigated the interplay between the spermosphere inoculum, host plant physiology, and endophytic compartment (EC) microbial community. Using 16S ribosomal RNA gene sequencing of root, stem, and leaf endophytic compartment communities, we established a baseline microbiome for Nicotiana sp. Phenotypic differences were observed due to the addition of some bacterial inoculants, correlated with endogenous auxin loads using transgenic plants expressing the auxin reporter pB-GFP::P87. When applied as spermosphere inoculants, select bacteria were found to create reproducible variation within the root EC microbiome and, more systematically, the host plant physiology. Our findings support the assertion that the spermosphere of plants is a zone that can influence the EC microbiome when applied in a greenhouse setting.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}- N -(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis ( Arabidopsis thaliana ). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

Stalk lodging (structural failure crops prior to harvest) significantly reduces annual yields of vital grain crops. The lack of standardized, high throughput phenotyping methods capable of quantifying biomechanical plant traits prevents comprehensive understanding of the genetic architecture of stalk lodging resistance. A phenotyping pipeline developed to enable higher throughput biomechanical measurements of plant traits related to stalk lodging is presented. The methods were developed using principles from the fields of engineering mechanics and metrology and they enable retention of plant-specific data instead of averaging data across plots as is typical in most phenotyping studies. This pipeline was specifically designed to be implemented in large experimental studies and has been used to phenotype over 40,000 maize stalks. The pipeline includes both lab- and field-based phenotyping methodologies and enables the collection of metadata. Best practices learned by implementing this pipeline over the past three years are presented. The specific instruments (including model numbers and manufacturers) that work well for these methods are presented, however comparable instruments may be used in conjunction with these methods as seen fit.•Efficient methods to measure biomechanical traits and record metadata related to stalk lodging.•Can be used in studies with large sample sizes (i.e., > 1,000)., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Whiskey production originated in Scotland in the 15th century and was based on malted barley. As Scotch-Irish settlers came into the Ohio river valley, they began fermenting and distilling the primary grain of North America, maize. These earlier settlers started a heritage; they created American Whiskey. The bourbon industry in Kentucky had tremendous growth in the last 20 years, and currently, distilleries have a broad increase in product innovation, new raw materials, improved sustainability, efficient processes, and product diversification. Our study presents a new lab-scale method for new-make bourbon whiskey production. It was developed to mimic distilleries' processes; therefore, results can be extrapolated and adopted by commercial distilleries. The method focused on reproducibility with consistency from batch to batch when handled by an operator or small crew in a university lab. The method consisted of a first cooking step to make a "mash", a fermentation phase of 96 h, a first distillation accomplished with a copper pot still to obtain the "low wines" and a second distillation carried out with an air still to collect the "hearts". The method produced a final distillate of 500-700 mL for further sensory analysis and tasting. This lab-scale method showed consistency between samples in the different parameters quantified and will be also used to train students in fermentation and distillation studies.

This study presents a methodology for a high-throughput digitization and quantification process of plant cell walls characterization, including the automated development of two-dimensional finite element models. Custom algorithms based on machine learning can also analyze the cellular microstructure for phenotypes such as cell size, cell wall curvature, and cell wall orientation. To demonstrate the utility of these models, a series of compound microscope images of both herbaceous and woody representatives were observed and processed. In addition, parametric analyses were performed on the resulting finite element models. Sensitivity analyses of the structural stiffness of the resulting tissue based on the cell wall elastic modulus and the cell wall thickness; demonstrated that the cell wall thickness has a three-fold larger impact of tissue stiffness than cell wall elastic modulus., (© 2023. The Author(s).)

The cell wall compositional (lignin and polysaccharides) variation of two sweet sorghum varieties, Della (D) and its variant REDforGREEN (RG), was evaluated at internodes (IN) and nodes (N) using high-performance liquid chromatography (HPLC), pyrolysis-gas chromatography-mass spectrometry (Py-GCMS), X-ray diffraction (XRD), and two-dimensional (2D) 1 H- 13 C nuclear magnetic resonance (NMR). The stalks were grown in 2018 (D1 and RG1) and 2019 (D2 and RG2) seasons. In RG1, Klason lignin reductions by 16-44 and 2-26% were detected in IN and N, respectively. The analyses also revealed that lignin from the sorghum stalks was enriched in guaiacyl units and the syringyl/guaiacyl ratio was increased in RG1 and RG2, respectively, by 96% and more than 2-fold at IN and 61 and 23% at N. The glucan content was reduced by 23-27% for RG1 and by 17-22% for RG2 at internodes. Structural variations due to changes in both cellulose- and hemicellulose-based sugars were detected. The nonacylated and γ-acylated β-O-4 linkages were the main interunit linkages detected in lignin. These results indicate compositional variation of stalks due to the RG variation, and the growing season could influence their mechanical and lodging behavior.

We report here the genome sequence of Bacillus sp. RRD69, a plant-growth-promoting bacterial endophyte isolated from switchgrass plants grown on a reclaimed coal-mining site in Kentucky. RRD69 is predicted to contain 3,758 protein-coding genes, with a genome size of 3.715 Mbp and a 41.41% GC content.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Although microbes influence plant growth, little is known about the impact of microbial diversity on plant fitness trade-offs, intraspecific-interactions, and soil nutrient dynamics in the context of biodiversity-ecosystem functioning (BEF) research. The BEF theory states that higher species richness can enhance ecosystem functioning. Thus, we hypothesize that rhizobacterial species richness will alter sorghum (Sorghum bicolor L.) growth, soil nutrient dynamics and interactions (antagonism or synergism) in a nutrient-poor greenhouse soil. Using six rhizobacterial species in a BEF experiment, we tested the impact of a species richness gradient (0, 1, 3, 5 or 6 species per community) on plant growth, nutrient assimilation, and soil nutrient dynamics via seed-inoculation. Our experiment included, one un-inoculated control, six rhizobacterial monoculture (Pseudomonas poae, Pseudomonas sp., Bacillus pumilus., Pantoea agglomerance., Microbacterium sp., and Serratia marcescens), and their nine mixture treatments in triplicate (48). Rhizobacterial species richness enhanced per pot above- or below-ground dry mass. However, the per plant growth and plant nutrient assimilation declined, most likely, due to microbial-driven competitive interactions among sorghum plants. But nevertheless, some rhizobacterial monoculture and mixture treatments improved per plant (shoot and root) growth and nutrient assimilation as well. Soil nutrient contents were mostly lower at higher plant-associated rhizobacterial diversity; among these, the soil Zn contents decreased significantly across the rhizobacterial diversity gradient. Rhizobacterial diversity promoted synergistic interactions among soil nutrients and improved root-soil interactions. Overall, our results suggest that a higher rhizobacterial diversity may enhance soil-plant interactions and total productivity under resource limited conditions.

We report here the genome assembly and analysis of Microbacterium strain sp. LKL04, a Gram-positive bacterial endophyte isolated from switchgrass plants ( Panicum virgatum ) grown on a reclaimed coal-mining site. The 2.9-Mbp genome of this bacterium was assembled into a single contig encoding 2,806 protein coding genes., (Copyright © 2019 Sahib et al.)

Here, we present a study into the mechanisms of primary cell wall cellulose formation in grasses, using the model cereal grass Brachypodium distachyon . The exon found adjacent to the Bd CESA1 glycosyltransferase QXXRW motif was targeted using Targeting Induced Local Lesions in Genomes (TILLING) and sequencing candidate amplicons in multiple parallel reactions (SCAMPRing) leading to the identification of the Bd cesa1 S830N allele. Plants carrying this missense mutation exhibited a significant reduction in crystalline cellulose content in tissues that rely on the primary cell wall for biomechanical support. However, Bdcesa1 S830N plants failed to exhibit the predicted reduction in plant height. In a mechanism unavailable to eudicotyledons, B. distachyon plants homozygous for the Bdcesa1 S830N allele appear to overcome the loss of internode expansion anatomically by increasing the number of nodes along the stem. Stem biomechanics were resultantly compromised in Bd cesa1 S830N . The Bdcesa1 S830N missense mutation did not interfere with BdCESA1 gene expression. However, molecular dynamic simulations of the CELLULOSE SYNTHASE A (CESA) structure with modelled membrane interactions illustrated that Bd cesa1 S830N exhibited structural changes in the translated gene product responsible for reduced cellulose biosynthesis. Molecular dynamic simulations showed that substituting S830N resulted in a stabilizing shift in the flexibility of the class specific region arm of the core catalytic domain of CESA, revealing the importance of this motion to protein function., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company.)

We report here the improved draft genome sequence of Bacillus sp. strain YF23, a bacterium originally isolated from switchgrass ( Panicum virgatum ) plants and shown to exhibit plant growth-promoting activity. The genome comprised 5.82 Mbp, containing 5,933 genes, with 193 as RNA genes, and a GC content of 35.10%., (Copyright © 2019 Xia et al.)

We report here the improved draft genome sequence of Pseudomonas poae strain A2-S9, a bacterium that was originally isolated from switchgrass plants and exhibited the capacity for plant growth promotion. Its genome has a size of 6.68 Mbp and a GC content of 61.3%. The genome encodes 6,022 predicted protein-coding genes., (Copyright © 2019 Xia et al.)

Background: Lignin is a promising source of building blocks for upgrading to valuable aromatic chemicals and materials. Endocarp biomass represents a non-edible crop residue in an existing agricultural setting which cannot be used as animal feed nor soil amendment. With significantly higher lignin content and bulk energy density, endocarps have significant advantages to be converted into both biofuel and bioproducts as compared to other biomass resources. Deep eutectic solvent (DES) is highly effective in fractionating lignin from a variety of biomass feedstocks with high yield and purity while at lower cost comparing to certain ionic liquids., Results: In the present study, the structural and compositional features of peach and walnut endocarp cells were characterized. Compared to typical woody and herbaceous biomass, endocarp biomass exhibits significantly higher bulk density and hardness due to its high cellular density. The sugar yields of DES (1:2 choline chloride: lactic acid) pretreated peach pit ( Prunus persica ) and walnut shell ( Juglans nigra ) were determined and the impacts of DES pretreatment on the physical and chemical properties of extracted lignin were characterized. Enzymatic saccharification of DES pretreated walnut and peach endocarps gave high glucose yields (over 90%); meanwhile, compared with dilute acid and alkaline pretreatment, DES pretreatment led to significantly higher lignin removal (64.3% and 70.2% for walnut and peach endocarps, respectively). The molecular weights of the extracted lignin from DES pretreated endocarp biomass were significantly reduced. 1 H- 13 C HSQC NMR results demonstrate that the native endocarp lignins were SGH type lignins with dominant G-unit (86.7% and 80.5% for walnut and peach endocarps lignins, respectively). DES pretreatment decreased the S and H-unit while led to an increase in condensed G-units, which may contribute to a higher thermal stability of the isolated lignin. Nearly all β- O -4' and a large portion of β-5' linkages were removed during DES pretreatment., Conclusions: The high lignin content endocarps have unique cell wall characteristics when compared to the other lignocellulosic biomass feedstocks. DES pretreatment was highly effective in fractionating high lignin content endocarps to produce both sugar and lignin streams while the DES extracted lignins underwent significant changes in SGH ratio, interunit linkages, and molecular sizes.

Oak barrels have been used by humans for thousands of years to store and transport valuable materials. Early settlers of the United States in Kentucky began charring the interior of new white oak barrels prior to aging distillate to create the distinctively flavored spirit we know as bourbon whiskey. Despite the unique flavor and cultural significance of "America's Spirit", little is known about the wood-distillate interaction that shapes bourbon whiskey. Here, we employed an inverse method to measure the loss of specific wood polysaccharides in the oak cask during aging for up to ten years. We found that the structural cell wall wood biopolymer, cellulose, was partially decrystallized by the charring process. This pyrolytic fracturing and subsequent exposure to the distillate was accompanied by a steady loss of sugars from the cellulose and hemicellulose fractions of the oak cask. Distinct layers of structural degradation and product release from within the barrel stave are formed over time as the distillate expands into and contracts from the barrel staves. This complex, wood-sugar release process is likely associated with the time-dependent generation of the unique palate of bourbon whiskey.

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Background: Cellulose biosynthesis inhibitors (CBIs) are pre-emergence herbicides that inhibit anisotropic cell expansion resulting in a severely swollen and stunted growth phenotype. Resistance to group 21 CBIs, such as isoxaben, is conferred by missense mutations in CELLOSE SYNTHASE A (CesA) genes required for primary cell wall synthesis, concluding that this is their in vivo target., Results: Herein, we show that grasses exhibit tolerance to group 21 CBIs and explore the mechanism of tolerance to isoxaben in the grass Brachypodium distachyon (L.). Comparative genomics failed to identify synonymous point mutations that have been found to confer isoxaben resistance in the dicot Arabidopsis thaliana (L.). Brachypodium did not metabolize 14 C-isoxaben. We next explored the role of grass-specific non-cellulosic cell wall components, specifically the hemicellulose polysaccharide mix linkage glucans (MLG), as a potential tolerance mechanism by compensating for the loss of cellulose during cell elongation. A partial-transcriptional knockdown T-DNA insertion was found in a key MLG synthesis gene, Cellulose synthase-like F6 (CslF6) and this mutant was found to be 2.1 times more sensitive to isoxaben than wild-type plants., Conclusion: These data suggest that the composition and compensatory response of grass cell walls may be a factor in conferring tolerance to group 21 CBIs. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The biological function of sterol glucosides (SGs), the most abundant sterol derivatives in higher plants, remains uncertain. In an effort to improve our understanding of these membrane lipids we examined phenotypes exhibited by the roots of Arabidopsis (Arabidopsis thaliana) lines carrying insertions in the UDP-Glc:sterol glucosyltransferase genes, UGT80A2 and UGT80B1. We show that although ugt80A2 mutants exhibit significantly lower levels of total SGs they are morphologically indistinguishable from wild-type plants. In contrast, the roots of ugt80B1 mutants are only deficient in stigmasteryl glucosides but exhibit a significant reduction in root hairs. Sub-cellular investigations reveal that the plasma membrane cell fate regulator, SCRAMBLED (SCM), is mislocalized in ugt80B1 mutants, underscoring the aberrant root epidermal cell patterning. Live imaging of roots indicates that SCM:GFP is localized to the cytoplasm in a non cell type dependent manner instead of the hair (H) cell plasma membrane in these mutants. In addition, we provide evidence for the localization of the UGT80B1 enzyme in the plasma membrane. These data lend further support to the notion that deficiencies in specific SGs are sufficient to disrupt normal cell function and point to a possible role for SGs in cargo transport and/or protein targeting to the plasma membrane.

In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Global grape production could generate up to 13 Mt/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 400 L/t. However, bioethanol from only soluble carbohydrates could yield 270 L/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.)

Here, we show a mechanism for expansion regulation through mutations in the green revolution gene gibberellin20 (GA20)-oxidase and show that GAs control biosynthesis of the plants main structural polymer cellulose. Within a 12,000 mutagenized Sorghum bicolor plant population, we identified a single cellulose-deficient and male gametophyte-dysfunctional mutant named dwarf1-1 (dwf1-1). Through the Sorghum propinquum male/dwf1-1 female F2 population, we mapped dwf1-1 to a frameshift in GA20-oxidase. Assessment of GAs in dwf1-1 revealed ablation of GA. GA ablation was antagonistic to the expression of three specific cellulose synthase genes resulting in cellulose deficiency and growth dwarfism, which were complemented by exogenous bioactive gibberellic acid application. Using quantitative polymerase chain reaction, we found that GA was positively regulating the expression of a subset of specific cellulose synthase genes. To cross reference data from our mapped Sorghum sp. allele with another monocotyledonous plant, a series of rice (Oryza sativa) mutants involved in GA biosynthesis and signaling were isolated, and these too displayed cellulose deficit. Taken together, data support a model whereby suppressed expansion in green revolution GA genes involves regulation of cellulose biosynthesis., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

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, soluble sugars and pectin. The dry leaf fiber was composed of crystalline cellulose (47-50% w/w) and non-cellulosic polysaccharides (16-22% w/w), and whole leaves were low in lignin (9-13% w/w). Of the dry mass of whole Agave leaves, 85-95% consisted of soluble sugars, cellulose, non-cellulosic polysaccharides, lignin, acetate, protein and minerals. Juice pressed from the Agave leaves accounted for 69% of the fresh weight and was rich in glucose and fructose. Hydrolysis of the fructan oligosaccharides doubled the amount of fermentable fructose in A. tequilana leaf juice samples and the concentration of fermentable hexose sugars was 41-48 g/L. In agricultural production systems such as the tequila making, Agave leaves are discarded as waste. Theoretically, up to 4000 L/ha/yr of bioethanol could be produced from juice extracted from waste Agave leaves. Using standard Saccharomyces cerevisiae strains to ferment Agave juice, we observed ethanol yields that were 66% of the theoretical yields. These data indicate that Agave could rival currently used bioethanol feedstocks, particularly if the fermentation organisms and conditions were adapted to suit Agave leaf composition.

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 or conventional practices. Endophytic bacteria were isolated from surface-sterilized shoot, root, and seed tissues and sequence identified. A total of 336 bacterial isolates were identified, and grouped into 32 species and five phyla. Among these, 239 isolates were from organically grown plants and 97 from those grown conventionally. Although a diverse range of bacteria were documented, 186 were from the Phylum Firmicutes, representing 55% of all isolates. Using the Shannon diversity index, we observed a gradation of diversity in tissues, with shoots and roots having a similar value, and seeds having the least diversity. Importantly, endophytic microbial species abundance and diversity was significantly higher in the organically grown plants compared to those grown using conventional practices, potentially indicating that organic management practices may increase endophyte presence and diversity. The impact that these endophytes could have on plant growth and yield was evaluated by reintroducing them into tomato plants in a greenhouse environment. Of the bacterial isolates tested, 61% were found to promote tomato plant growth and 50-64% were shown to enhance biomass accumulation, illustrating their potential agroecosystem application.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are 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 resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move 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 and 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., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

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 h of treatment and in a dose-dependent manner. Unlike the CBI isoxaben, indaziflam had strong CBI activity in both a monocotylonous plant (Poa annua) and a dicotyledonous plant (Arabidopsis [Arabidopsis thaliana]). 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 YELLOW FLUORESCENT PROTEIN:CESA6 particles at the plasma membrane focal plane compared with controls. Microtubule morphology and motility were not altered after indaziflam treatment. In the hypocotyl expansion zone, indaziflam caused an atypical increase in the density of plasma membrane-localized CESA particles. Interestingly, this was accompanied by a cellulose synthase interacting1-independent reduction in the normal coincidence rate between microtubules and CESA particles. 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., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

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.

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.

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.

Natural food colorants with functional properties are of increasing interest. Prior papers indicate the chemical suitability of sorghum leaf 3-deoxyanthocyanidins as natural food colorants. Via mutagenesis-assisted breeding, a sorghum variety that greatly overaccumulates 3-deoxyanthocyanidins of leaf tissue, named REDforGREEN (RG), has been isolated and characterized. 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 levels in the mutant leaves. Thus, RG represents a useful sorghum mutagenesis variant to develop as a functionalized food colorant.

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 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., Conclusion: 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.

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.

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.

Manipulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create modified cellulose for anthropogenic use. Evidence exists that cellulose microfibril structure and its recalcitrance to enzymatic digestion can ameliorated via mis-sense mutation in the primary cell wall-specific gene AtCELLULOSE SYNTHASE (CESA)3. This mis-sense mutation has been identified based on conferring drug resistance to the cellulose inhibitory herbicide isoxaben. To examine whether it would be possible to introduce mutant CESA alleles via a transgenic approach, we overexpressed a modified version of CESA3, AtCESA3(ixr1-2) derived from Arabidopsis thaliana L. Heynh into a different plant family, the Solanceae dicotyledon tobacco (Nicotiana tabacum L. variety Samsun NN). Specifically, a chimeric gene construct of CESA3(ixr1-2) , codon optimized for tobacco, was placed between the heterologous M24 promoter and the rbcSE9 gene terminator. The results demonstrated that the tobacco plants expressing M24-CESA3(ixr1-2) displayed isoxaben resistance, consistent with functionality of the mutated AtCESA3(ixr1-2) in tobacco. Secondly, during enzymatic saccharification, transgenic leaf- and stem-derived cellulose is 54%-66% and 40%-51% more efficient, respectively, compared to the wild type, illustrating translational potential of modified CESA loci. Moreover, the introduction of M24-AtCESA3(ixr1-2) caused aberrant spatial distribution of lignified secondary cell wall tissue and a reduction in the zone occupied by parenchyma cells., (© 2012 The Authors Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.)

Long-term efforts to decode plant cellulose biosynthesis via molecular genetics and biochemical strategies are being enhanced by the ever-expanding scale of omics technologies. An alternative approach to consider are the prospects for inducing change in plant metabolism using exogenously supplied chemical ligands. Cellulose biosynthesis inhibitors (CBIs) have been identified among known herbicides, during diverse combinatorial chemical libraries screens, and natural chemical screens from microbial agents. In this review, we summarize the current knowledge of the inhibitory effects of CBIs and further group them by how they influence fluorescently tagged cellulose synthase A proteins. Additional attention is paid to the continuing development of the CBI toolbox to explore the cell biology and genetic mechanisms underpinning effector molecule activity.

Sterols play vital roles in plant growth and development, as components of membranes and as precursors to steroid hormones. Analysis of Arabidopsis mutants indicates that sterol composition is crucial for cellulose biosynthesis. Sterols are widespread in the plasma membrane (PM), suggesting a possible link between sterols and the multimeric cellulose synthase complex. In one possible scenario, molecular interactions in sterol-rich PM microdomains or another form of sterol-dependent membrane scaffolding may be critical for maintaining the correct subcellular localization, structural integrity and/or activity of the cellulose synthase machinery. Another possible link may be through steryl glucosides, which could act as primers for the attachment of glucose monomers during the synthesis of β-(1 → 4) glucan chains that form the cellulose microfibrils. This mini-review examines genetic and biochemical data supporting the link between sterols and cellulose biosynthesis in cell wall formation and explores potential approaches to elucidate the mechanism of this association.

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Almost one-quarter of the world's population has basic energy needs that are not being met. Efforts to increase renewable energy resources in developing countries where per capita energy availability is low are needed. Herein, we examine integrated dual use farming for sustained food security and agro-bioenergy development. Many nonedible crop residues are used for animal feed or reincorporated into the soil to maintain fertility. By contrast, drupe endocarp biomass represents a high-lignin feedstock that is a waste stream from food crops, such as coconut (Cocos nucifera) shell, which is nonedible, not of use for livestock feed, and not reintegrated into soil in an agricultural setting. Because of high-lignin content, endocarp biomass has optimal energy-to-weight returns, applicable to small-scale gasification for bioelectricity. Using spatial datasets for 12 principal drupe commodity groups that have notable endocarp byproduct, we examine both their potential energy contribution by decentralized gasification and relationship to regions of energy poverty. Globally, between 24 million and 31 million tons of drupe endocarp biomass is available per year, primarily driven by coconut production. Endocarp biomass used in small-scale decentralized gasification systems (15-40% efficiency) could contribute to the total energy requirement of several countries, the highest being Sri Lanka (8-30%) followed by Philippines (7-25%), Indonesia (4-13%), and India (1-3%). While representing a modest gain in global energy resources, mitigating energy poverty via decentralized renewable energy sources is proposed for rural communities in developing countries, where the greatest disparity between societal allowances exist.