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A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP-glucose 4-epimerases (UGEs) are NSEs that function in the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant (bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins. Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map-based cloning demonstrated that OsBP1 encodes a UDP-glucose 4-epimerase (OsUGE2), a cytosolic protein. We also show that BP1 can form homo- and hetero-protein complexes with other UGE family members and with UDP-galactose transporters 2 (OsUGT2) and 3 (OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.

The evolution of membrane-bound organelles among eukaryotes led to a highly compartmentalized metabolism. As a compartment of the central carbon metabolism, mitochondria must be connected to the cytosol by molecular gates that facilitate a myriad of cellular processes. Members of the mitochondrial carrier family function to mediate the transport of metabolites across the impermeable inner mitochondrial membrane and, thus, are potentially crucial for metabolic control and regulation. Here, we focus on members of this family that might impact intracellular central plant carbon metabolism. We summarize and review what is currently known about these transporters from in vitro transport assays and in planta physiological functions, whenever available. From the biochemical and molecular data, we hypothesize how these relevant transporters might play a role in the shuttling of organic acids in the various flux modes of the TCA cycle. Furthermore, we also review relevant mitochondrial carriers that may be vital in mitochondrial oxidative phosphorylation. Lastly, we survey novel experimental approaches that could possibly extend and/or complement the widely accepted proteoliposome reconstitution approach.

Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.

We report the case of a consanguineous couple who lost four pregnancies associated with skeletal dysplasia. Radiological examination of one fetus was inconclusive. Parental exome sequencing showed that both parents were heterozygous for a novel missense variant, p.(Pro133Leu), in the SLC35D1 gene encoding a nucleotide sugar transporter. The affected fetus was homozygous for the variant. The radiological features were reviewed, and being similar, but atypical, the phenotype was classified as a 'Schneckenbecken-like dysplasia.' The effect of the missense change was assessed using protein modelling techniques and indicated alterations in the mouth of the solute channel. A detailed biochemical investigation of SLC35D1 transport function and that of the missense variant p.(Pro133Leu) revealed that SLC35D1 acts as a general UDP-sugar transporter and that the p.(Pro133Leu) mutation resulted in a significant decrease in transport activity. The reduced transport activity observed for p.(Pro133Leu) was contrasted with in vitro activity for SLC35D1 p.(Thr65Pro), the loss-of-function mutation was associated with Schneckenbecken dysplasia. The functional classification of SLC35D1 as a general nucleotide sugar transporter of the endoplasmic reticulum suggests an expanded role for this transporter beyond chondroitin sulfate biosynthesis to a variety of important glycosylation reactions occurring in the endoplasmic reticulum.

The plant endoplasmic reticulum-Golgi apparatus is the site of synthesis, assembly, and trafficking of all noncellulosic polysaccharides, proteoglycans, and proteins destined for the cell wall. As grass species make cell walls distinct from those of dicots and noncommelinid monocots, it has been assumed that the differences in cell-wall composition stem from differences in biosynthetic capacities of their respective Golgi. However, immunosorbence-based screens and carbohydrate linkage analysis of polysaccharides in Golgi membranes, enriched by flotation centrifugation from etiolated coleoptiles of maize (Zea mays) and leaves of Arabidopsis (Arabidopsis thaliana), showed that arabinogalactan-proteins and arabinans represent substantial portions of the Golgi-resident polysaccharides not typically found in high abundance in cell walls of either species. Further, hemicelluloses accumulated in Golgi at levels that contrasted with those found in their respective cell walls, with xyloglucans enriched in maize Golgi, and xylans enriched in Arabidopsis. Consistent with this finding, maize Golgi membranes isolated by flotation centrifugation and enriched further by free-flow electrophoresis, yielded >200 proteins known to function in the biosynthesis and metabolism of cell-wall polysaccharides common to all angiosperms, and not just those specific to cell-wall type. We propose that the distinctive compositions of grass primary cell walls compared with other angiosperms result from differential gating or metabolism of secreted polysaccharides post-Golgi by an as-yet unknown mechanism, and not necessarily by differential expression of genes encoding specific synthase complexes.

Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions. By NMR and live cell analyses we reveal that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction. The microtubule-binding mechanism of CC1 is reminiscent to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.

The cell wall is an intricate mesh largely composed of polysaccharides that vary in structure and abundance. Apart from cellulose biosynthesis, the assembly of matrix polysaccharides such as pectin and hemicellulose occur in the Golgi apparatus before being transported via vesicles to the cell wall. Matrix polysaccharides are biosynthesized from activated precursors or nucleotide sugars. The composition and assembly of the cell wall is an important aspect in plant development and plant biomass utilization. The application of anion-exchange chromatography to determine the monosaccharide composition of the insoluble matrix polysaccharides enables a complete profile of all major sugars in the cell wall from a single run. While porous carbon graphite chromatography and tandem mass spectrometry delivers a sensitive and robust nucleotide sugar profile from plant extracts. Here we describe detailed methodology to quantify nucleotide sugars within the cell and profile the non-cellulosic monosaccharide composition of the cell wall.

Glycans play diverse biological roles, ranging from structural and regulatory functions to mediating cellular interactions. For pathogens, they are also often required for virulence and survival in the host. In Cryptococcus neoformans, an opportunistic pathogen of humans, the acidic monosaccharide glucuronic acid (GlcA) is a critical component of multiple essential glycoconjugates. One of these glycoconjugates is the polysaccharide capsule, a major virulence factor that enables this yeast to modulate the host immune response and resist antimicrobial defenses. This allows cryptococci to colonize the lung and brain, leading to hundreds of thousands of deaths each year worldwide. Synthesis of most glycans, including capsule polysaccharides, occurs in the secretory pathway. However, the activated precursors for this process, nucleotide sugars, are made primarily in the cytosol. This topological problem is resolved by the action of nucleotide sugar transporters (NSTs). We discovered that Uut1 is the sole UDP-GlcA transporter in C. neoformans and is unique among NSTs for its narrow substrate range and high affinity for UDP-GlcA. Mutant cells with UUT1 deleted lack capsule polysaccharides and are highly sensitive to environmental stress. As a result, the deletion mutant is internalized and cleared by phagocytes more readily than wild-type cells are and is completely avirulent in mice. These findings expand our understanding of the requirements for capsule synthesis and cryptococcal virulence and elucidate a critical protein family.IMPORTANCECryptococcus neoformans causes lethal meningitis in almost two hundred thousand immunocompromised patients each year. Much of this fungal pathogen's ability to resist host defenses and cause disease is mediated by carbohydrate structures, including a complex polysaccharide capsule around the cell. Like most eukaryotic glycoconjugates, capsule polysaccharides are made within the secretory pathway, although their precursors are generated

The purification of a functional soluble protein from biological or in vitro expression systems can be problematic and the enrichment of a functional membrane protein for biochemical analyses can be a serious technical challenge. Recently we have been characterizing plant endomembrane nucleotide sugar transporters using a yeast expression system. However, rather than enriching these in vitro expressed proteins to homogeneity, we have been conducting biochemical characterization of these transport proteins in yeast microsomal fractions. While this approach has enabled us to estimate a variety of kinetic parameters, the accurate determination of the turnover number of an enzyme-substrate complex (k cat) requires that the catalytic site concentration (amount of protein) in the total reaction volume is known. As a result, we have been employing targeted proteomics (multiple reaction monitoring) with peptide standards and a triple quadrupole mass spectrometer to estimate the absolute amount of protein in a mixed protein microsomal fraction. The following method details the steps required to define the absolute quantitation of an in vitro expressed membrane protein to define complete kinetic parameters.

Cryptococcus neoformans, an AIDS-defining opportunistic pathogen, is the leading cause of fungal meningitis worldwide and is responsible for hundreds of thousands of deaths annually. Cryptococcal glycans are required for fungal survival in the host and for pathogenesis. Most glycans are made in the secretory pathway, although the activated precursors for their synthesis, nucleotide sugars, are made primarily in the cytosol. Nucleotide sugar transporters are membrane proteins that solve this topological problem, by exchanging nucleotide sugars for the corresponding nucleoside phosphates. The major virulence factor of C. neoformans is an anti-phagocytic polysaccharide capsule that is displayed on the cell surface; capsule polysaccharides are also shed from the cell and impede the host immune response. Xylose, a neutral monosaccharide that is absent from model yeast, is a significant capsule component. Here we show that Uxt1 and Uxt2 are both transporters specific for the xylose donor, UDP-xylose, although they exhibit distinct subcellular localization, expression patterns, and kinetic parameters. Both proteins also transport the galactofuranose donor, UDP-galactofuranose. We further show that Uxt1 and Uxt2 are required for xylose incorporation into capsule and protein; they are also necessary for C. neoformans to cause disease in mice, although surprisingly not for fungal viability in the context of infection. These findings provide a starting point for deciphering the substrate specificity of an important class of transporters, elucidate a synthetic pathway that may be productively targeted for therapy, and contribute to our understanding of fundamental glycobiology.

UDP-xylose (UDP-Xyl) is synthesized by UDP-glucuronic acid decarboxylases, also termed UDP-Xyl synthases (UXSs). The Arabidopsis genome encodes six UXSs, which fall into two groups based upon their subcellular location: the Golgi lumen and the cytosol. The latter group appears to play an important role in xylan biosynthesis. Cytosolic UDP-Xyl is transported into the Golgi lumen by three UDP-Xyl transporters (UXT1, 2, and 3). However, while single mutants affected in the UDP-Xyl transporter 1 (UXT1) showed a substantial reduction in cell wall xylose content, a double mutant affected in UXT2 and UXT3 had no obvious effect on cell wall xylose deposition. This prompted us to further investigate redundancy among the members of the UXT family. Multiple uxt mutants were generated, including a triple mutant, which exhibited collapsed vessels and reduced cell wall thickness in interfascicular fiber cells. Monosaccharide composition, molecular weight, nuclear magnetic resonance, and immunolabeling studies demonstrated that both xylan biosynthesis (content) and fine structure were significantly affected in the uxt triple mutant, leading to phenotypes resembling those of the irx mutants. Pollination was also impaired in the uxt triple mutant, likely due to reduced filament growth and anther dehiscence caused by alterations in the composition of the cell walls. Moreover, analysis of the nucleotide sugar composition of the uxt mutants indicated that nucleotide sugar interconversion is influenced by the cytosolic UDP-Xyl pool within the cell. Taken together, our results underpin the physiological roles of the UXT family in xylan biosynthesis and provide novel insights into the nucleotide sugar metabolism and trafficking in plants.

The cytosol is at the core of cellular metabolism and contains many important metabolic pathways, including glycolysis, gluconeogenesis, and the pentose phosphate pathway. Despite the importance of this matrix, few attempts have sought to specifically enrich this compartment from plants. Although a variety of biochemical pathways and signaling cascades pass through the cytosol, much of the focus has usually been targeted at the reactions that occur within membrane-bound organelles of the plant cell. In this chapter, we outline a method for the enrichment of the cytosol from rice suspension cell cultures which includes sample preparation and enrichment as well as validation using immunoblotting and fluorescence-tagged proteins.

Mapping of the human proteome has advanced significantly in recent years and will provide a knowledge base to accelerate our understanding of how proteins and protein networks can affect human health and disease. However, providing solutions to human health challenges will likely fail if insights are exclusively based on studies of human samples and human proteomes. In recent years, it has become evident that human health depends on an integrated understanding of the many species that make human life possible. These include the commensal microorganisms that are essential to human life, pathogens, and food species as well as the classic model organisms that enable studies of biological mechanisms. The Human Proteome Organization (HUPO) initiative on multiorganism proteomes (iMOP) works to support proteome research undertaken on nonhuman species that remain widely under-studied compared with the progress in human proteome research. This perspective argues the need for further research on multiple species that impact human life. We also present an update on recent progress in model organisms, microbiota, and food species, address the emerging problem of antibiotics resistance, and outline how iMOP activities could lead to a more inclusive approach for the human proteome project (HPP) to better support proteome research aimed at improving human health and furthering knowledge on human biology.

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).

The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.

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.

The plant plasma membrane is the interface between the cell and its environment undertaking a range of important functions related to transport, signaling, cell wall biosynthesis, and secretion. Multiple proteomic studies have attempted to capture the diversity of proteins in the plasma membrane using biochemical fractionation techniques. In this study, two-phase partitioning was combined with free-flow electrophoresis to produce a population of highly purified plasma membrane vesicles that were subsequently characterized by tandem mass spectroscopy. This combined high-quality plasma membrane isolation technique produced a reproducible proteomic library of over 1000 proteins with an extended dynamic range including plasma membrane-associated proteins. The approach enabled the detection of a number of putative plasma membrane proteins not previously identified by other studies, including peripheral membrane proteins. Utilizing multiple data sources, we developed a PM-confidence score to provide a value indicating association to the plasma membrane. This study highlights over 700 proteins that, while seemingly abundant at the plasma membrane, are mostly unstudied. To validate this data set, we selected 14 candidates and transiently localized 13 to the plasma membrane using a fluorescent tag. Given the importance of the plasma membrane, this data set provides a valuable tool to further investigate important proteins. The mass spectrometry data are available via ProteomeXchange, identifier PXD001795.

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.

The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes from an Arabidopsis cell suspension culture that can be used to investigate the proteome of this organelle. We also provide a useful workflow for the examination of proteomic data as the result of multiple analyses. Finally, we highlight a simple technique to validate the subcellular localization of proteins by fluorescent tags after their identification by tandem mass spectrometry.

BACKGROUND: BAHD 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. RESULTS: For 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

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.

Over the past 10 years, the bioenergy field has realized significant achievements that have encouraged many follow on efforts centered on biosynthetic production of fuel-like compounds. Key to the success of these efforts has been transformational developments in feedstock characterization and metabolic engineering of biofuel-producing microbes. Lagging far behind these advancements are analytical methods to characterize and quantify systems of interest to the bioenergy field. In particular, the utilization of proteomics, while valuable for identifying novel enzymes and diagnosing problems associated with biofuel-producing microbes, is limited by a lack of robustness and limited throughput. Nano-flow liquid chromatography coupled to high-mass accuracy, high-resolution mass spectrometers has become the dominant approach for the analysis of complex proteomic samples, yet such assays still require dedicated experts for data acquisition, analysis, and instrument upkeep. The recent adoption of standard flow chromatography (ca. 0.5 mL/min) for targeted proteomics has highlighted the robust nature and increased throughput of this approach for sample analysis. Consequently, we assessed the applicability of standard flow liquid chromatography for shotgun proteomics using samples from Escherichia coli and Arabidopsis thaliana, organisms commonly used as model systems for lignocellulosic biofuels research. Employing 120 min gradients with standard flow chromatography, we were able to routinely identify nearly 800 proteins from E. coli samples; while for samples from Arabidopsis, over 1,000 proteins could be reliably identified. An examination of identified peptides indicated that the method was suitable for reproducible applications in shotgun proteomics. Standard flow liquid chromatography for shotgun proteomics provides a robust approach for the analysis of complex samples. To the best of our knowledge, this study represents the first attempt to validate the standard flow ap

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 analysis of post-translational modifications (PTMs) by proteomics is regarded as a technically challenging undertaking. While in recent years approaches to examine and quantify protein phosphorylation have greatly improved, the analysis of many protein modifications, such as glycosylation, are still regarded as problematic. Limitations in the standard proteomics workflow, such as use of suboptimal peptide fragmentation methods, can significantly prevent the identification of glycopeptides. The current generation of tandem mass spectrometers has made available a variety of fragmentation options, many of which are becoming standard features on these instruments. We have used three common fragmentation techniques, namely CID, HCD, and ETD, to analyze a glycopeptide and highlight how an integrated fragmentation approach can be used to identify the modified residue and characterize the N-glycan on a peptide.

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

The application of westerns or immunoblotting techniques for assessing the composition, dynamics, and purity of protein extracts from plant material has become common practice. While the approach is reproducible, can be readily applied and is generally considered robust, the field of plant science suffers from a lack of antibody variety against plant proteins. The development of approaches that employ mass spectrometry to enable both relative and absolute quantification of many hundreds of proteins in a single sample from a single analysis provides a mechanism to overcome the expensive impediment in having to develop antibodies in plant science. We consider it an opportune moment to consider and better develop the adoption of multiple reaction monitoring (MRM)-based analyses in plant biochemistry.

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 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).

The plant cytosol is the major intracellular fluid that acts as the medium for inter-organellar crosstalk and where a plethora of important biological reactions take place. These include its involvement in protein synthesis and degradation, stress response signaling, carbon metabolism, biosynthesis of secondary metabolites, and accumulation of enzymes for defense and detoxification. This central role is highlighted by estimates indicating that the majority of eukaryotic proteins are cytosolic. Arabidopsis thaliana has been the subject of numerous proteomic studies on its different subcellular compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted proteomic characterizations of complexes is included. Despite this, few groups are currently applying advanced proteomic approaches to this important metabolic space. This review will highlight the current state of the Arabidopsis cytosolic proteome since its initial characterization a few years ago.

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.

Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin) indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.

A key challenge in the area of bioinformatics in the coming decades is the ability to manage the wealth of information that is being generated from the variety of high throughput methodologies currently being undertaken in laboratories across the world. While these approaches have made available large volumes of data to the research community, less attention has been given to the problem of how to intuitively present the data to enable greater biological insights. Recently, an attempt was made to tackle this problem in the area of Arabidopsis proteomics. The model plant has been the target of countless proteomics surveys producing an exhaustive array of data and online repositories. The MASCP Gator is an aggregation portal for proteomic data currently being produced by the community and unites a large collection of specialized resources to a single portal (http://gator.masc-proteomics.org/). Here we describe the latest additions, upgrades and features to this resource further expanding its role into protein modifications and genome sequence variations.

The plant Golgi apparatus and trans-Golgi network are major endomembrane trafficking hubs within the plant cell and are involved in a diverse and vital series of functions to maintain plant growth and development. Recently, a series of disparate technical approaches have been used to isolate and characterize components of these complex organelles by mass spectrometry in the model plant Arabidopsis thaliana. Collectively, these studies have increased the number of Golgi and vesicular localized proteins identified by mass spectrometry to nearly 500 proteins. We have sought to provide a brief overview of these technical approaches and bring the datasets together to examine how they can reveal insights into the secretory pathway.

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 PhosPhAt database of Arabidopsis phosphorylation sites was initially launched in August 2007. Since then, along with 10-fold increase in database entries, functionality of PhosPhAt (phosphat.mpimp-golm.mpg.de) has been considerably upgraded and re-designed. PhosPhAt is now more of a web application with the inclusion of advanced search functions allowing combinatorial searches by Boolean terms. The results output now includes interactive visualization of annotated fragmentation spectra and the ability to export spectra and peptide sequences as text files for use in other applications. We have also implemented dynamic links to other web resources thus augmenting PhosPhAt-specific information with external protein-related data. For experimental phosphorylation sites with information about dynamic behavior in response to external stimuli, we display simple time-resolved diagrams. We have included predictions for pT and pY sites and updated pS predictions. Access to prediction algorithm now allows 'on-the-fly' prediction of phosphorylation of any user-uploaded protein sequence. Protein Pfam domain structures are now mapped onto the protein sequence display next to experimental and predicted phosphorylation sites. Finally, we have implemented functional annotation of proteins using MAPMAN ontology. These new developments make the PhosPhAt resource a useful and powerful tool for the scientific community as a whole beyond the plant sciences.

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.

The PhosPhAt database provides a resource consolidating our current knowledge of mass spectrometry-based identified phosphorylation sites in Arabidopsis and combines it with phosphorylation site prediction specifically trained on experimentally identified Arabidopsis phosphorylation motifs. The database currently contains 1187 unique tryptic peptide sequences encompassing 1053 Arabidopsis proteins. Among the characterized phosphorylation sites, there are over 1000 with unambiguous site assignments, and nearly 500 for which the precise phosphorylation site could not be determined. The database is searchable by protein accession number, physical peptide characteristics, as well as by experimental conditions (tissue sampled, phosphopeptide enrichment method). For each protein, a phosphorylation site overview is presented in tabular form with detailed information on each identified phosphopeptide. We have utilized a set of 802 experimentally validated serine phosphorylation sites to develop a method for prediction of serine phosphorylation (pSer) in Arabidopsis. An analysis of the current annotated Arabidopsis proteome yielded in 27,782 predicted phosphoserine sites distributed across 17,035 proteins. These prediction results are summarized graphically in the database together with the experimental phosphorylation sites in a whole sequence context. The Arabidopsis Protein Phosphorylation Site Database (PhosPhAt) provides a valuable resource to the plant science community and can be accessed through the following link http://phosphat.mpimp-golm.mpg.de.

Knowledge of protein localisation contributes towards our understanding of protein function and of biological inter-relationships. A variety of experimental methods are currently being used to produce localisation data that need to be made accessible in an integrated manner. Chimeric fluorescent fusion proteins have been used to define subcellular localisations with at least 1100 related experiments completed in Arabidopsis. More recently, many studies have employed mass spectrometry to undertake proteomic surveys of subcellular components in Arabidopsis yielding localisation information for approximately 2600 proteins. Further protein localisation information may be obtained from other literature references to analysis of locations (AmiGO: approximately 900 proteins), location information from Swiss-Prot annotations (approximately 2000 proteins); and location inferred from gene descriptions (approximately 2700 proteins). Additionally, an increasing volume of available software provides location prediction information for proteins based on amino acid sequence. We have undertaken to bring these various data sources together to build SUBA, a SUBcellular location database for Arabidopsis proteins. The localisation data in SUBA encompasses 10 distinct subcellular locations, >6743 non-redundant proteins and represents the proteins encoded in the transcripts responsible for 51% of Arabidopsis expressed sequence tags. The SUBA database provides a powerful means by which to assess protein subcellular localisation in Arabidopsis (http://www.suba.bcs.uwa.edu.au).

The Arabidopsis Mitochondrial Protein Database is an Internet-accessible relational database containing information on the predicted and experimentally confirmed protein complement of mitochondria from the model plant Arabidopsis thaliana (http://www.ampdb.bcs.uwa.edu.au/). The database was formed using the total non-redundant nuclear and organelle encoded sets of protein sequences and allows relational searching of published proteomic analyses of Arabidopsis mitochondrial samples, a set of predictions from six independent subcellular-targeting prediction programs, and orthology predictions based on pairwise comparison of the Arabidopsis protein set with known yeast and human mitochondrial proteins and with the proteome of Rickettsia. A variety of precomputed physical-biochemical parameters are also searchable as well as a more detailed breakdown of mass spectral data produced from our proteomic analysis of Arabidopsis mitochondria. It contains hyperlinks to other Arabidopsis genomic resources (MIPS, TIGR and TAIR), which provide rapid access to changing gene models as well as hyperlinks to T-DNA insertion resources, Massively Parallel Signature Sequencing (MPSS) and Genome Tiling Array data and a variety of other Arabidopsis online resources. It also incorporates basic analysis tools built into the query structure such as a BLAST facility and tools for protein sequence alignments for convenient analysis of queried results.

Experimental analyses of the proteins found in the mitochondria of yeast, humans and Arabidopsis have confirmed some expectations but given some surprises and some insights into the evolutionary origins of mitochondrial proteins.