Genome Sequence Resource of Bacillus sp. RRD69, a Beneficial Bacterial Endophyte Isolated from Switchgrass Plants.

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.

Mapping of a Cellulose-Deficient Mutant Named dwarf1-1 in Sorghum bicolor to the Green Revolution Gene gibberellin20-oxidase Reveals a Positive Regulatory Association between Gibberellin and Cellulose Biosynthesis.

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.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

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.

Cellulose biosynthesis inhibitors - a multifunctional toolbox.

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.

Calmodulin-mediated signal transduction pathways in Arabidopsis are fine-tuned by methylation.

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.

Tertiary model of a plant cellulose synthase.

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 ß-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.

Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor.

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.

Global bioenergy potential from high-lignin agricultural residue.

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.

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase.

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.

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis.

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.

Bacterial Spermosphere Inoculants Alter N. benthamiana-Plant Physiology and Host Bacterial Microbiome.

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.

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

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.

Biomechanical phenotyping pipeline for stalk lodging resistance in maize.

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).

Manipulating cellulose biosynthesis by expression of mutant Arabidopsis proM24::CESA3(ixr1-2) gene in transgenic tobacco.

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.

Lab-Scale Methodology for New-Make Bourbon Whiskey Production.

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.

The semi-automated development of plant cell wall finite element models.

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.

Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment.

Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes. Here, we investigated the role of cellulose synthase (CESA) subunits CESA2, CESA5, and CESA9 in the seed coat epidermis. We characterized the roles of these CESA proteins in the seed coat by analyzing cell wall composition and morphology in cesa mutant lines. Mutations in any one of these three genes resulted in lower cellulose content, a loss of cell shape uniformity, and reduced radial wall integrity. In addition, we found that attachment of the mucilage halo to the parent seed following extrusion is maintained by cellulose-based connections requiring CESA5. Hence, we show that cellulose fulfills an adhesion role between the extracellular mucilage matrix and the parent cell in seed coat epidermal cells. We propose that mucilage remains attached to the seed coat through interactions between components in the seed mucilage and cellulose. Our data suggest that CESA2 and CESA9 serve in radial wall reinforcement, as does CESA5, but CESA5 also functions in mucilage biosynthesis. These data suggest unique roles for different CESA subunits in one cell type and illustrate a complex role for cellulose biosynthesis in plant developmental biology.

Evaluation of Cell Wall Chemistry of Della and Its Mutant Sweet Sorghum Stalks.

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) 1H-13C 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 ß-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.

CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell wall synthesis in Arabidopsis epidermal testa cells.

Herein, we sought to explore the contribution of cellulose biosynthesis to the shape and morphogenesis of hexagonal seed coat cells in Arabidopsis (Arabidopsis thaliana). Consistent with seed preferential expression of CELLULOSE SYNTHASE9 (CESA9), null mutations in CESA9 caused no change in cellulose content in leaves or stems, but caused a 25% reduction in seeds. Compositional studies of cesa9 seeds uncovered substantial proportional increases in cell wall neutral sugars and in several monomers of cell wall-associated polyesters. Despite these metabolic compensations, cesa9 seeds were permeable to tetrazolium salt, implying that cellulose biosynthesis, via CESA9, is required for correct barrier function of the seed coat. A syndrome of depleted radial wall, altered seed coat cell size, shape, and internal angle uniformity was quantified using scanning electron micrographs in cesa9 epidermal cells. By contrast, morphological defects were absent in cesa9 embryos, visually inspected from torpedo to bent cotyledon, consistent with no reduction in postgermination radical or hypocotyl elongation. These data implied that CESA9 was seed coat specific or functionally redundant in other tissues. Assessment of sections from glutaraldehyde fixed wild-type and cesa9 mature seeds supported results of scanning electron micrographs and quantitatively showed depletion of secondary cell wall synthesis in the radial cell wall. Herein, we show a nonredundant role for CESA9 in secondary cell wall biosynthesis in radial cell walls of epidermal seed coats and document its importance for cell morphogenesis and barrier function of the seed coat.

Prefoldin 6 is required for normal microtubule dynamics and organization in Arabidopsis.

Newly translated tubulin molecules undergo a series of complex interactions with nascent chain-binding chaperones, including prefoldin (PFD) and chaperonin-containing TCP-1 (CCT). By screening for oryzalin hypersensitivity, we identified several mutants of Arabidopsis that have lesions in PFD subunits. The pfd6-1 mutant exhibits a range of microtubule defects, including hypersensitivity to oryzalin, defects in cell division, cortical array organization, and microtubule dynamicity. Consistent with phenotypic analysis, proteomic analysis indicates several isoforms of tubulins were reduced in pfd6-1. These results support the concept that the function of microtubules is critically dependent on the absolute amount of tubulins.