Search

Pectin is a major component of primary cell walls and performs a plethora of functions crucial for plant growth, development and plant-defense responses. Despite the importance of pectic polysaccharides their biosynthesis is poorly understood. Several genes have been implicated in pectin biosynthesis by mutant analysis, but biochemical activity has been shown for very few. We used reverse genetics and biochemical analysis to study members of Glycosyltransferase Family 92 (GT92) in Arabidopsis thaliana. Biochemical analysis gave detailed insight into the properties of GALS1 (Galactan synthase 1) and showed galactan synthase activity of GALS2 and GALS3. All proteins are responsible for adding galactose onto existing galactose residues attached to the rhamnogalacturonan-I (RG-I) backbone. Significant GALS activity was observed with galactopentaose as acceptor but longer acceptors are favored. Overexpression of the GALS proteins in Arabidopsis resulted in accumulation of unbranched β-1, 4-galactan. Plants in which all three genes were inactivated had no detectable β-1, 4-galactan, and surprisingly these plants exhibited no obvious developmental phenotypes under standard growth conditions. RG-I in the triple mutants retained branching indicating that the initial Gal substitutions on the RG-I backbone are added by enzymes different from GALS.

Glycosylation requires activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational protein modifications and glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi, requiring cytosolic-derived nucleotide sugars, which need to be actively transferred into the Golgi lumen by nucleotide sugar transporters. We identified a Golgi-localized nucleotide sugar transporter from Arabidopsis thaliana with affinity for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and assigned it UDP-GlcNAc transporter 1 (UGNT1). Profiles of N-glycopeptides revealed that plants carrying the ugnt1 loss-of-function allele are virtually devoid of complex and hybrid N-glycans. Instead, the N-glycopeptide population from these alleles exhibited high-mannose structures, representing structures prior to the addition of the first GlcNAc in the Golgi. Concomitantly, sphingolipid profiling revealed that the biosynthesis of GlcNAc-containing glycosyl inositol phosphorylceramides (GIPCs) is also reliant on this transporter. By contrast, plants carrying the loss-of-function alleles affecting ROCK1, which has been reported to transport UDP-GlcNAc and UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC profiles. Our findings reveal that plants contain a single UDP-GlcNAc transporter that delivers an essential substrate for the maturation of N-glycans and the GIPC class of sphingolipids.

Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combined with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).

In plants, L-arabinose (Ara) is a key component of cell wall polymers, glycoproteins, as well as flavonoids, and signaling peptides. Whereas the majority of Ara found in plant glycans occurs as a furanose ring (Araf), the activated precursor has a pyranose ring configuration (UDP-Arap). The biosynthesis of UDP-Arap mainly occurs via the epimerization of UDP-xylose (UDP-Xyl) in the Golgi lumen. Given that the predominant Ara form found in plants is Araf, UDP-Arap must exit the Golgi to be interconverted into UDP-Araf by UDP-Ara mutases that are located outside on the cytosolic surface of the Golgi. Subsequently, UDP-Araf must be transported back into the lumen. This step is vital because glycosyltransferases, the enzymes mediating the glycosylation reactions, are located within the Golgi lumen, and UDP-Arap, synthesized within the Golgi, is not their preferred substrate. Thus, the transport of UDP-Araf into the Golgi is a prerequisite. Although this step is critical for cell wall biosynthesis and the glycosylation of proteins and signaling peptides, the identification of these transporters has remained elusive. In this study, we present data demonstrating the identification and characterization of a family of Golgi-localized UDP-Araf transporters in Arabidopsis The application of a proteoliposome-based transport assay revealed that four members of the nucleotide sugar transporter (NST) family can efficiently transport UDP-Araf in vitro. Subsequent analysis of mutant lines affected in the function of these NSTs confirmed their role as UDP-Araf transporters in vivo.

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.

Aliphatic and aromatic lipids are both essential structural components of the plant cuticle, an important interface between the plant and environment. Although cross links between aromatic and aliphatic or other moieties are known to be associated with the formation of leaf cutin and root and seed suberin, the contribution of aromatic lipids to the biosynthesis of anther cuticles and pollen walls remains elusive. In this study, we characterized the rice (Oryza sativa) male sterile mutant, defective pollen wall 2 (dpw2), which showed an abnormal anther cuticle, a defective pollen wall, and complete male sterility. Compared with the wild type, dpw2 anthers have increased amounts of cutin and waxes and decreased levels of lipidic and phenolic compounds. DPW2 encodes a cytoplasmically localized BAHD acyltransferase. In vitro assays demonstrated that recombinant DPW2 specifically transfers hydroxycinnamic acid moieties, using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs as acyl donors. Thus, The cytoplasmic hydroxycinnamoyl-CoA:ω-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components of the anther cuticle and pollen wall.

BackgroundPectins 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.ResultsT-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.ConclusionsTogether, 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.

BackgroundBAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals.ResultsFor the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate.ConclusionWe demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates.

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

Reduced cell wall recalcitrance and increased C6 monosaccharide content are desirable traits for future biofuel crops, as long as these biomass modifications do not significantly alter normal growth and development. Mixed-linkage glucan (MLG), a cell wall polysaccharide only present in grasses and related species among flowering plants, is comprised of glucose monomers linked by both β-1,3 and β-1,4 bonds. Previous data have shown that constitutive production of MLG in barley (Hordeum vulgare) severely compromises growth and development. Here, we used spatio-temporal strategies to engineer Arabidopsis thaliana plants to accumulate significant amounts of MLG in the cell wall by expressing the rice CslF6 MLG synthase using secondary cell wall and senescence-associated promoters. Results using secondary wall promoters were suboptimal. When the rice MLG synthase was expressed under the control of a senescence-associated promoter, we obtained up to four times more glucose in the matrix cell wall fraction and up to a 42% increase in saccharification compared to control lines. Importantly, these plants grew and developed normally. The induction of MLG deposition at senescence correlated with an increase of gluconic acid in cell wall extracts of transgenic plants in contrast to the other approaches presented in this study. MLG produced in Arabidopsis has an altered structure compared to the grass glucan, which likely affects its solubility, while its molecular size is unaffected. The induction of cell wall polysaccharide biosynthesis in senescing tissues offers a novel engineering alternative to enhance cell wall properties of lignocellulosic biofuel crops.

Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21-amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals.

The cost-effective production of biofuels from lignocellulosic material will likely require manipulation of plant biomass, specifically cell walls. The North American native prairie grass Panicum virgatum (switchgrass) is seen as a potential biofuel crop with an array of genetic resources currently being developed. We have characterized the endomembrane proteome of switchgrass coleoptiles to provide additional information to the switchgrass community. In total, we identified 1750 unique proteins from two biological replicates. These data have been deposited in the ProteomeXchange with the identifier PXD001351 (http://proteomecentral.proteomexchange.org/dataset/PXD001351).

Most glycosylation reactions require activated glycosyl donors in the form of nucleotide sugars to drive processes such as posttranslational modifications and polysaccharide biosynthesis. Most plant cell wall polysaccharides are biosynthesized in the Golgi apparatus from cytosolic-derived nucleotide sugars, which are actively transferred into the Golgi lumen by nucleotide sugar transporters (NSTs). An exception is UDP-xylose, which is biosynthesized in both the cytosol and the Golgi lumen by a family of UDP-xylose synthases. The NST-based transport of UDP-xylose into the Golgi lumen would appear to be redundant. However, employing a recently developed approach, we identified three UDP-xylose transporters in the Arabidopsis thaliana NST family and designated them UDP-XYLOSE TRANSPORTER1 (UXT1) to UXT3. All three transporters localize to the Golgi apparatus, and UXT1 also localizes to the endoplasmic reticulum. Mutants in UXT1 exhibit ∼30% reduction in xylose in stem cell walls. These findings support the importance of the cytosolic UDP-xylose pool and UDP-xylose transporters in cell wall biosynthesis.

[This corrects the article DOI: 10.1371/journal.ppat.1004809.].

Plant plasma membrane localized pattern recognition receptors (PRRs) detect extracellular pathogen-associated molecules. PRRs such as Arabidopsis EFR and rice XA21 are taxonomically restricted and are absent from most plant genomes. Here we show that rice plants expressing EFR or the chimeric receptor EFR::XA21, containing the EFR ectodomain and the XA21 intracellular domain, sense both Escherichia coli- and Xanthomonas oryzae pv. oryzae (Xoo)-derived elf18 peptides at sub-nanomolar concentrations. Treatment of EFR and EFR::XA21 rice leaf tissue with elf18 leads to MAP kinase activation, reactive oxygen production and defense gene expression. Although expression of EFR does not lead to robust enhanced resistance to fully virulent Xoo isolates, it does lead to quantitatively enhanced resistance to weakly virulent Xoo isolates. EFR interacts with OsSERK2 and the XA21 binding protein 24 (XB24), two key components of the rice XA21-mediated immune response. Rice-EFR plants silenced for OsSERK2, or overexpressing rice XB24 are compromised in elf18-induced reactive oxygen production and defense gene expression indicating that these proteins are also important for EFR-mediated signaling in transgenic rice. Taken together, our results demonstrate the potential feasibility of enhancing disease resistance in rice and possibly other monocotyledonous crop species by expression of dicotyledonous PRRs. Our results also suggest that Arabidopsis EFR utilizes at least a subset of the known endogenous rice XA21 signaling components.

The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

The rice XA21 immune receptor kinase and the structurally related XA3 receptor confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast two-hybrid system in a kinase activity-dependent manner. OsSERK2 undergoes bidirectional transphosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3, and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR.

Eucalypt species are a group of flowering trees widely used in pulp production for paper manufacture. For several decades, the wood pulp industry has focused research and development efforts on improving yields, growth rates and pulp quality through breeding and the genetic improvement of key tree species. Recently, this focus has shifted from the production of high quality pulps to the investigation of the use of eucalypts as feedstocks for biofuel production. Here the structure and chemical composition of the heartwood and sapwood of Eucalyptus dunnii, E. globulus, E. pillularis, E. urophylla, an E. urophylla-E. grandis cross, Corymbia citriodora ssp. variegata, and Acacia mangium were compared using nuclear magnetic resonance spectroscopy (NMR), X-ray diffraction (XRD) and biochemical composition analysis. Some trends relating to these compositions were also identified by Fourier transform near infrared (FT-NIR) spectroscopy. These results will serve as a foundation for a more comprehensive database of wood properties that will help develop criteria for the selection of tree species for use as biorefinery feedstocks.

We outline a high throughput procedure that improves outlier detection in cell wall screens using FT-NIR spectroscopy of plant leaves. The improvement relies on generating a calibration set from a subset of a mutant population by taking advantage of the Mahalanobis distance outlier scheme to construct a monosaccharide range predictive model using PLS regression. This model was then used to identify specific monosaccharide outliers from the mutant population.

The process of plant cell wall biosynthesis involves a complex series of biochemical processes involving many hundreds of proteins. Determining the function of a specific gene through functional genomics techniques have proved problematic due to genetic redundancies and undetectable changes. Common techniques have used mutant collections in forward genetic screens or reverse genetics to directly target and disrupt genes of interest. Since such techniques are heavily reliant on some phenotypic discrimination to asses gene function, the absence of a measureable difference often results in little useful information. Furthermore the complete absence of many genes produced by such techniques results in a lethal phenotype as the gene is absolutely required for normal function of the plant. A more subtle approach utilizes genetic differences in naturally occurring variants to provide important information about gene function and genetic diversity. In collaboration with the Joint Genome Institute we have sequenced two Arabidopsis accessions (Bay-0 and Sha-0) that have previously been shown to have measureable differences in Ara-Rha ratios in cell wall extracts. This genetic information and the utilization of recombinant inbred lines (RIL?s) will be used to map QTL?s identified in these and other Arabidopsis accessions to identify loci that contribute to functional differences in plant cell walls.

Genetic variation in different accessions of Arabidopsis thaliana has occurred through thousands of years of geographic spread and adaptation. As a result of this diversity, disparate accessions have phenotypic differences that can be used to identify genes that contribute to the biosynthesis and composition of cell walls. Forward and reverse genetic screens to identify single mutants often prove difficult for detection of genetic differences that may lead to more subtle phenotypes. Exploiting the inherent genetic variation in Arabidopsis accessions through quantitative trait analysis will allow for the detection of variation in cell wall biosynthesis and composition. We selected two methods for screening parental accessions: measuring monosaccharide composition by HPAEC and structural changes by Near Infrared (NIR) spectroscopy. From these analyses, we selected Ri-0 as the most different accession from the reference accession Col-0. Recombinant inbred lines (RILs) derived from Col-0 and Ri-0 parents were used to determine quantitative trait loci (QTL) that contribute to the differences observed in monosaccharide and pectin content and NIR spectroscopy. Two putative candidate genes encoding enzymes involved in nucleotide sugar conversion have been identified. We are currently determining differences between the parental versions of the genes and how any differences contribute to the observed phenotypes. Additionally, we have re-sequenced two Arabidopsis accessions, Bay-0 and Shahdara, in collaboration with the Joint Genome Institute. This effort has resulted in a collection of SNPs between these accessions and the reference Arabidopsis accession, Col-0. Re-seqeuencing efforts are being expanded to other accessions, beginning with Ri-0. Information obtained from resequencing will aid in QTL analysis and be of service to the general Arabidopsis community.