Sivakumar Pattathil

A Comprehensive Toolkit of Plant Cell Wall Glycan-Directed Monoclonal Antibodies

A collection of 130 new plant cell wall glycan-directed monoclonal antibodies (mAbs) was generated with the aim of facilitating in-depth analysis of cell wall glycans. An enzyme-linked immunosorbent assay-based screen against a diverse panel of 54 plant polysaccharides was used to characterize the binding patterns of these new mAbs, together with 50 other previously generated mAbs, against plant cell wall glycans. Hierarchical clustering analysis was used to group these mAbs based on the polysaccharide recognition patterns observed. The mAb groupings in the resulting cladogram were further verified by immunolocalization studies in Arabidopsis (Arabidopsis thaliana) stems. The mAbs could be resolved into 19 clades of antibodies that recognize distinct epitopes present on all major classes of plant cell wall glycans, including arabinogalactans (both protein- and polysaccharide-linked), pectins (homogalacturonan, rhamnogalacturonan I), xyloglucans, xylans, mannans, and glucans. In most cases, multiple subclades of antibodies were observed to bind to each glycan class, suggesting that the mAbs in these subgroups recognize distinct epitopes present on the cell wall glycans. The epitopes recognized by many of the mAbs in the toolkit, particularly those recognizing arabinose- and/or galactose-containing structures, are present on more than one glycan class, consistent with the known structural diversity and complexity of plant cell wall glycans. Thus, these cell wall glycan-directed mAbs should be viewed and utilized as epitope-specific, rather than polymer-specific, probes. The current world-wide toolkit of approximately 180 glycan-directed antibodies from various laboratories provides a large and diverse set of probes for studies of plant cell wall structure, function, dynamics, and biosynthesis.

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

Pectin and xylan are generally considered as separate cell wall glycan networks distinct from cell wall proteins. This work describes a cell wall proteoglycan with pectin and arabinoxylan covalently attached to an arabinogalactan protein, identifying a cross-linked matrix polysaccharide wall protein architecture with implications for wall structure, function, and synthesis. Plant cell walls are comprised largely of the polysaccharides cellulose, hemicellulose, and pectin, along with ∼10% protein and up to 40% lignin. These wall polymers interact covalently and noncovalently to form the functional cell wall. Characterized cross-links in the wall include covalent linkages between wall glycoprotein extensins between rhamnogalacturonan II monomer domains and between polysaccharides and lignin phenolic residues. Here, we show that two isoforms of a purified Arabidopsis thaliana arabinogalactan protein (AGP) encoded by hydroxyproline-rich glycoprotein family protein gene At3g45230 are covalently attached to wall matrix hemicellulosic and pectic polysaccharides, with rhamnogalacturonan I (RG I)/homogalacturonan linked to the rhamnosyl residue in the arabinogalactan (AG) of the AGP and with arabinoxylan attached to either a rhamnosyl residue in the RG I domain or directly to an arabinosyl residue in the AG glycan domain. The existence of this wall structure, named ARABINOXYLAN PECTIN ARABINOGALACTAN PROTEIN1 (APAP1), is contrary to prevailing cell wall models that depict separate protein, pectin, and hemicellulose polysaccharide networks. The modified sugar composition and increased extractability of pectin and xylan immunoreactive epitopes in apap1 mutant aerial biomass support a role for the APAP1 proteoglycan in plant wall architecture and function.

Composition and structure of sugarcane cell wall polysaccharides: implications for second-generation bioethanol production.

The structure and fine structure of leaf and culm cell walls of sugarcane plants were analyzed using a combination of microscopic, chemical, biochemical, and immunological approaches. Fluorescence microscopy revealed that leaves and culm display autofluorescence and lignin distributed differently through different cell types, the former resulting from phenylpropanoids associated with vascular bundles and the latter distributed throughout all cell walls in the tissue sections. Polysaccharides in leaf and culm walls are quite similar, but differ in the proportions of xyloglucan and arabinoxylan in some fractions. In both cases, xyloglucan (XG) and arabinoxylan (AX) are closely associated with cellulose, whereas pectins, mixed-linkage-β-glucan (BG), and less branched xylans are strongly bound to cellulose. Accessibility to hydrolases of cell wall fraction increased after fractionation, suggesting that acetyl and phenolic linkages, as well as polysaccharide–polysaccharide interactions, prevented enzyme action when cell walls are assembled in its native architecture. Differently from other hemicelluloses, BG was shown to be readily accessible to lichenase when in intact walls. These results indicate that wall architecture has important implications for the development of more efficient industrial processes for second-generation bioethanol production. Considering that pretreatments such as steam explosion and alkali may lead to loss of more soluble fractions of the cell walls (BG and pectins), second-generation bioethanol, as currently proposed for sugarcane feedstock, might lead to loss of a substantial proportion of the cell wall polysaccharides, therefore decreasing the potential of sugarcane for bioethanol production in the future.

Efficient biomass pretreatment using ionic liquids derived from lignin and hemicellulose

Significance Ionic liquids (ILs) have unique properties applicable to a variety of industrial processes. Nearly universal solvating capabilities, low vapor pressures, and high thermal stabilities make these compounds ideal substitutes for a wide range of organic solvents. To date, the best performing ILs are derived from nonrenewable sources such as petroleum or natural gas. Due to their potential for large-scale deployment, ILs derived from inexpensive, renewable reagents are highly desirable. Herein, we describe a process for synthesizing ILs from materials derived from lignin and hemicellulose, major components of terrestrial plant biomass. With respect to overall sugar yield, experimental evaluation of these compounds showed that they perform comparably to traditional ILs in biomass pretreatment. Ionic liquids (ILs), solvents composed entirely of paired ions, have been used in a variety of process chemistry and renewable energy applications. Imidazolium-based ILs effectively dissolve biomass and represent a remarkable platform for biomass pretreatment. Although efficient, imidazolium cations are expensive and thus limited in their large-scale industrial deployment. To replace imidazolium-based ILs with those derived from renewable sources, we synthesized a series of tertiary amine-based ILs from aromatic aldehydes derived from lignin and hemicellulose, the major by-products of lignocellulosic biofuel production. Compositional analysis of switchgrass pretreated with ILs derived from vanillin, p-anisaldehyde, and furfural confirmed their efficacy. Enzymatic hydrolysis of pretreated switchgrass allowed for direct comparison of sugar yields and lignin removal between biomass-derived ILs and 1-ethyl-3-methylimidazolium acetate. Although the rate of cellulose hydrolysis for switchgrass pretreated with biomass-derived ILs was slightly slower than that of 1-ethyl-3-methylimidazolium acetate, 90–95% glucose and 70–75% xylose yields were obtained for these samples after 72-h incubation. Molecular modeling was used to compare IL solvent parameters with experimentally obtained compositional analysis data. Effective pretreatment of lignocellulose was further investigated by powder X-ray diffraction and glycome profiling of switchgrass cell walls. These studies showed different cellulose structural changes and differences in hemicellulose epitopes between switchgrass pretreatments with the aforementioned ILs. Our concept of deriving ILs from lignocellulosic biomass shows significant potential for the realization of a “closed-loop” process for future lignocellulosic biorefineries and has far-reaching economic impacts for other IL-based process technology currently using ILs synthesized from petroleum sources.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

The heterotrimeric G‐protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G‐protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G‐protein associates with heptahelical G‐protein‐coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G‐protein effectors and scaffold proteins, we screened a set of proteins from the G‐protein complex using two‐hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G‐protein interactome. Within this core, over half of the interactions comprising two‐thirds of the nodes were retested and validated as genuine in planta. Co‐expression analysis in combination with phenotyping of loss‐of‐function mutations in a set of core interactome genes revealed a novel role for G‐proteins in regulating cell wall modification.

Immunological Approaches to Plant Cell Wall and Biomass Characterization: Glycome Profiling

The native complexity of plant cell walls makes research on them challenging. Hence, it is advantageous to have a diversity of tools that can be used to analyze and characterize plant cell walls. In this chapter, we describe one of two immunological approaches that can be employed for screening of plant cell wall/biomass materials from diverse plants and tissues. This approach, Glycome Profiling, lends itself well to moderate to high-throughput screening of plant cell wall/biomass samples. Glycome Profiling is being further optimized to reduce the amount of sample required for the analysis, and to improve the sensitivity and throughput of the assay. We are optimistic that Glycome Profiling will prove to be a broadly applicable experimental approach that will find increasing application to a wide variety of studies on plant cell wall/biomass samples.

Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels production

To better understand how hydrothermal pretreatment reduces plant cell wall recalcitrance, we applied a high throughput approach (“glycome profiling”) using a comprehensive suite of plant glycan-directed monoclonal antibodies to monitor structural/extractability changes in Populus biomass. The results of glycome profiling studies were verified by immunolabeling using selected antibodies from the same toolkit. The array of monoclonal antibodies employed in these studies is large enough to monitor changes occurring in most plant cell wall polysaccharides. Results from these techniques demonstrate the sequence of structural changes that occur in plant cell walls during pretreatment-induced deconstruction, namely, the initial disruption of lignin-polysaccharide interactions in concert with a loss of pectins and arabinogalactans; this is followed by significant removal of xylans and xyloglucans. Additionally, this study also suggests that lignin content per se does not affect recalcitrance; instead, the integration of lignin and polysaccharides within cell walls, and their associations with one another, play a larger role.

Enhanced characteristics of genetically modified switchgrass (Panicum virgatum L.) for high biofuel production

BackgroundLignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, “absent technological breakthroughs”, it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold.ResultsWe have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance.ConclusionsOur results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Mutations in Multiple XXT Genes of Arabidopsis Reveal the Complexity of Xyloglucan Biosynthesis

Xyloglucan is an important hemicellulosic polysaccharide in dicot primary cell walls. Most of the enzymes involved in xyloglucan synthesis have been identified. However, many important details of its synthesis in vivo remain unknown. The roles of three genes encoding xylosyltransferases participating in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana) were further investigated using reverse genetic, biochemical, and immunological approaches. New double mutants (xxt1 xxt5 and xxt2 xxt5) and a triple mutant (xxt1 xxt2 xxt5) were generated, characterized, and compared with three single mutants and the xxt1 xxt2 double mutant that had been isolated previously. Antibody-based glycome profiling was applied in combination with chemical and immunohistochemical analyses for these characterizations. From the combined data, we conclude that XXT1 and XXT2 are responsible for the bulk of the xylosylation of the glucan backbone, and at least one of these proteins must be present and active for xyloglucan to be made. XXT5 plays a significant but as yet uncharacterized role in this process. The glycome profiling data demonstrate that the lack of detectable xyloglucan does not cause significant compensatory changes in other polysaccharides, although changes in nonxyloglucan polysaccharide amounts cannot be ruled out. Structural rearrangements of the polysaccharide network appear responsible for maintaining wall integrity in the absence of xyloglucan, thereby allowing nearly normal plant growth in plants lacking xyloglucan. Finally, results from immunohistochemical studies, combined with known information about expression patterns of the three genes, suggest that different combinations of xylosyltransferases contribute differently to xyloglucan biosynthesis in the various cell types found in stems, roots, and hypocotyls.