Botrytis cinerea infection accelerates ripening and cell wall disassembly to promote disease in tomato fruit.

Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry.

Novel Immunological and Mass Spectrometry Methods for Comprehensive Analysis of Recalcitrant Oligosaccharides in AFEX Pretreated Corn Stover. Lignocellulosic biomass is a sustainable alternative to fossil fuel and is extensively used for developing bio-based technologies to produce products such as food, feed, fuel, and chemicals. The key to these technologies is to develop cost competitive processes to convert complex carbohydrates present in plant cell wall to simple sugars such as glucose, xylose, and arabinose. Since lignocellulosic biomass is highly recalcitrant, it must undergo a combination of thermochemical treatment such as Ammonia Fiber Expansion (AFEX), dilute acid (DA), Ionic Liquid (IL) and biological treatment such as enzyme hydrolysis and microbial fermentation to produce desired products. However, when using commercial fungal enzymes during hydrolysis, only 75-85% of the soluble sugars generated are monomeric sugars, while the remaining 15-25% are soluble recalcitrant oligosaccharides that cannot be easily utilized by microorganisms. Previously, we successfully separated and purified the soluble recalcitrant oligosaccharides using a combination of charcoal and celite-based separation followed by size exclusion chromatography and studies their inhibitory properties on enzymes. We discovered that the oligosaccharides with higher degree of polymerization (DP) containing methylated uronic acid substitutions were more recalcitrant towards commercial enzyme mixtures than lower DP and neutral oligosaccharides. Here, we report the use of several complementary techniques that include glycome profiling using plant biomass glycan specific monoclonal antibodies (mAbs) to characterize sugar linkages in plant cell walls and enzymatic hydrolysate, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using structurally-informative diagnostic peaks offered by negative ion post-secondary decay spectra, gas chromatography followed by mass spectrometry (GC-MS) to characterize oligosaccharide sugar linkages with and without derivatization. Since oligosaccharides (DP 4-20) are small, it is challenging to mobilize these molecules for mAbs binding and characterization. To overcome this problem, we have applied a new biotin-coupling based oligosaccharide immobilization method that successfully tagged most of the low DP soluble oligosaccharides on to a micro-plate surface followed by specific linkage analysis using mAbs in a high-throughput system. This new approach will help develop more advanced versions of future high throughput glycome profiling methods that can be used to separate and characterize oligosaccharides present in biomarkers for diagnostic applications.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1) releases latent defense signals in stems with reduced lignin content.

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

Identification and analysis of cell wall glycan epitopes and polyphenol oxidase in pawpaw (Asimina triloba [L.] Dunal) fruit pulp as affected by high pressure processing and refrigerated storage.

This research explores the cell wall composition and polyphenol oxidase activity of two pawpaw (Asimina triloba) fruit varieties, Susquehanna and Green River Belle, that were subjected to high pressure processing and 45 days of refrigerated storage. We hypothesize that high pressure processing may inhibit enzymatic action responsible for pawpaw's deleterious postharvest tissue softening and browning. Glycome profiling uses mAb groupings that recognize 19 groups of glycan epitopes present in most major classes of cell wall glycans and was used to determine cell wall composition. Results show that both varieties have typical type I primary cell walls of flowering dicots. However, differences in the fine cell wall structure between the varieties can be inferred and the varieties behaved differently during refrigerated storage, likely indicating of a difference in cell wall-modifying enzymes present in the primary cell walls. High pressure processing treatment does not seem to be effective at eliminating polyphenol oxidase activity.

A two-phase model for the non-processive biosynthesis of homogalacturonan polysaccharides by the GAUT1:GAUT7 complex.

Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Insights into cell wall structure of Sida hermaphrodita and its influence on recalcitrance.

The perennial plant Sida hermaphrodita (Sida) is attracting attention as potential energy crop. Here, the first detailed view on non-cellulosic Sida cell wall polysaccharide composition, structure and architecture is given. Cell walls were prepared from Sida stems and sequentially extracted with aqueous buffers and alkali. The structures of the quantitatively predominant polysaccharides present in each fraction were determined by biochemical characterization, glycome profiling and mass spectrometry. The amounts of glucose released by Accellerase-1500® treatment of the cell wall and the cell wall residue remaining after each extraction were used to assess the roles of pectin and hemicellulose in the recalcitrance of Sida biomass. 4-O-Methyl glucuronoxylan with a low proportion of side substitutions was identified as the major non-cellulosic glycan component of Sida stem cell walls. Pectic polysaccharides and xylans were found to be associated with lignin, suggesting that these polysaccharides have roles in Sida cell wall recalcitrance to enzymatic hydrolysis.

Cell wall composition and penetration resistance against the fungal pathogen Colletotrichum higginsianum are affected by impaired starch turnover in Arabidopsis mutants.

Penetration resistance represents the first level of plant defense against phytopathogenic fungi. Here, we report that the starch-deficient Arabidopsis thaliana phosphoglucomutase (pgm) mutant has impaired penetration resistance against the hemibiotrophic fungus Colletotrichum higginsianum. We could not determine any changes in leaf cutin and epicuticular wax composition or indolic glucosinolate levels, but detected complex alterations in the cell wall monosaccharide composition of pgm. Notably, other mutants deficient in starch biosynthesis (adg1) or mobilization (sex1) had similarly affected cell wall composition and penetration resistance. Glycome profiling analysis showed that both overall cell wall polysaccharide extractability and relative extractability of specific pectin and xylan epitopes were affected in pgm, suggesting extensive structural changes in pgm cell walls. Screening of mutants with alterations in content or modification of specific cell wall monosaccharides indicated an important function of pectic polymers for penetration resistance and hyphal growth of C. higginsianum during the biotrophic interaction phase. While mutants with affected pectic rhamnogalacturonan-I (mur8) were hypersusceptible, penetration frequency and morphology of fungal hyphae were impaired on pmr5 pmr6 mutants with increased pectin levels. Our results reveal a strong impact of starch metabolism on cell wall composition and suggest a link between carbohydrate availability, cell wall pectin and penetration resistance.

S-nitrosoglutathione promotes cell wall remodelling, alters the transcriptional profile and induces root hair formation in the hairless root hair defective 6 (rhd6) mutant of Arabidopsis thaliana.

Nitric oxide (NO) exerts pleiotropic effects on plant development; however, its involvement in cell wall modification during root hair formation (RHF) has not yet been addressed. Here, mutants of Arabidopsis thaliana with altered root hair phenotypes were used to assess the involvement of S-nitrosoglutathione (GSNO), the primary NO source, in cell wall dynamics and gene expression in roots induced to form hairs. GSNO and auxin restored the root hair phenotype of the hairless root hair defective 6 (rhd6) mutant. A positive correlation was observed between increased NO production and RHF induced by auxin in rhd6 and transparent testa glabra (ttg) mutants. Deposition of an epitope within rhamnogalacturonan-I recognized by the CCRC-M2 antibody was delayed in root hair cells (trichoblasts) compared with nonhair cells (atrichoblasts). GSNO, but not auxin, restored the wild-type root glycome and transcriptome profiles in rhd6, modulating the expression of a large number of genes related to cell wall composition and metabolism, as well as those encoding ribosomal proteins, DNA and histone-modifying enzymes and proteins involved in post-translational modification. Our results demonstrate that NO plays a key role in cell wall remodelling in trichoblasts and suggest that it also participates in chromatin modification in root cells of A. thaliana.

A DUF-246 family glycosyltransferase-like gene affects male fertility and the biosynthesis of pectic arabinogalactans.

BACKGROUND: Pectins are a group of structurally complex plant cell wall polysaccharides whose biosynthesis and function remain poorly understood. The pectic polysaccharide rhamnogalacturonan-I (RG-I) has two types of arabinogalactan side chains, type-I and type-II arabinogalactans. To date few enzymes involved in the biosynthesis of pectin have been described. Here we report the identification of a highly conserved putative glycosyltransferase encoding gene, Pectic ArabinoGalactan synthesis-Related (PAGR), affecting the biosynthesis of RG-I arabinogalactans and critical for pollen tube growth. RESULTS: T-DNA insertions in PAGR were identified in Arabidopsis thaliana and were found to segregate at a 1:1 ratio of heterozygotes to wild type. We were unable to isolate homozygous pagr mutants as pagr mutant alleles were not transmitted via pollen. In vitro pollen germination assays revealed reduced rates of pollen tube formation in pollen from pagr heterozygotes. To characterize a loss-of-function phenotype for PAGR, the Nicotiana benthamiana orthologs, NbPAGR-A and B, were transiently silenced using Virus Induced Gene Silencing. NbPAGR-silenced plants exhibited reduced internode and petiole expansion. Cell wall materials from NbPAGR-silenced plants had reduced galactose content compared to the control. Immunological and linkage analyses support that RG-I has reduced type-I arabinogalactan content and reduced branching of the RG-I backbone in NbPAGR-silenced plants. Arabidopsis lines overexpressing PAGR exhibit pleiotropic developmental phenotypes and the loss of apical dominance as well as an increase in RG-I type-II arabinogalactan content. CONCLUSIONS: Together, results support a function for PAGR in the biosynthesis of RG-I arabinogalactans and illustrate the essential roles of these polysaccharides in vegetative and reproductive plant growth.

Xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I do not have identical structures in soybean root and root hair cell walls.

MAIN CONCLUSION: Chemical analyses and glycome profiling demonstrate differences in the structures of the xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I isolated from soybean ( Glycine max ) roots and root hair cell walls. The root hair is a plant cell that extends only at its tip. All other root cells have the ability to grow in different directions (diffuse growth). Although both growth modes require controlled expansion of the cell wall, the types and structures of polysaccharides in the walls of diffuse and tip-growing cells from the same plant have not been determined. Soybean (Glycine max) is one of the few plants whose root hairs can be isolated in amounts sufficient for cell wall chemical characterization. Here, we describe the structural features of rhamnogalacturonan I, rhamnogalacturonan II, xyloglucan, glucomannan, and 4-O-methyl glucuronoxylan present in the cell walls of soybean root hairs and roots stripped of root hairs. Irrespective of cell type, rhamnogalacturonan II exists as a dimer that is cross-linked by a borate ester. Root hair rhamnogalacturonan I contains more neutral oligosaccharide side chains than its root counterpart. At least 90% of the glucuronic acid is 4-O-methylated in root glucuronoxylan. Only 50% of this glycose is 4-O-methylated in the root hair counterpart. Mono O-acetylated fucose-containing subunits account for at least 60% of the neutral xyloglucan from root and root hair walls. By contrast, a galacturonic acid-containing xyloglucan was detected only in root hair cell walls. Soybean homologs of the Arabidopsis xyloglucan-specific galacturonosyltransferase are highly expressed only in root hairs. A mannose-rich polysaccharide was also detected only in root hair cell walls. Our data demonstrate that the walls of tip-growing root hairs cells have structural features that distinguish them from the walls of other roots cells.

Galactose-depleted xyloglucan is dysfunctional and leads to dwarfism in Arabidopsis.

Xyloglucan is a polysaccharide that has important roles in the formation and function of the walls that surround growing land plant cells. Many of these plants synthesize xyloglucan that contains galactose in two different side chains (L and F), which exist in distinct molecular environments. However, little is known about the contribution of these side chains to xyloglucan function. Here, we show that Arabidopsis (Arabidopsis thaliana) mutants devoid of the F side chain galactosyltransferase MURUS3 (MUR3) form xyloglucan that lacks F side chains and contains much less galactosylated xylose than its wild-type counterpart. The galactose-depleted xyloglucan is dysfunctional, as it leads to mutants that are dwarfed with curled rosette leaves, short petioles, and short inflorescence stems. Moreover, cell wall matrix polysaccharides, including xyloglucan and pectin, are not properly secreted and instead accumulate within intracellular aggregates. Near-normal growth is restored by generating mur3 mutants that produce no detectable amounts of xyloglucan. Thus, cellular processes are affected more by the presence of the dysfunctional xyloglucan than by eliminating xyloglucan altogether. To identify structural features responsible for xyloglucan dysfunction, xyloglucan structure was modified in situ by generating mur3 mutants that lack specific xyloglucan xylosyltransferases (XXTs) or that overexpress the XYLOGLUCAN L-SIDE CHAIN GALACTOSYLTRANSFERASE2 (XLT2) gene. Normal growth was restored in the mur3-3 mutant overexpressing XLT2 and in mur3-3 xxt double mutants when the dysfunctional xyloglucan was modified by doubling the amounts of galactosylated side chains. Our study assigns a role for galactosylation in normal xyloglucan function and demonstrates that altering xyloglucan side chain structure disturbs diverse cellular and physiological processes.

An Arabidopsis cell wall proteoglycan consists of pectin and arabinoxylan covalently linked to an arabinogalactan protein.

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.

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.

Arabidopsis thaliana T-DNA mutants implicate GAUT genes in the biosynthesis of pectin and xylan in cell walls and seed testa.

Galacturonosyltransferase 1 (GAUT1) is an alpha1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan (Sterling et al., 2006). The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like (GATL) proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades: A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades: clade A-1 (GAUTs 1 to 3); A-2 (GAUT4); A-3 (GAUTs 5 and 6); A-4 (GAUT7); B-1 (GAUTs 8 and 9); B-2 (GAUTs 10 and 11); and clade C (GAUTs 12 to 15). The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with either Arabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUT genes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUT genes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.