1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plant... more 1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plants. MAS solid state NMR experiments were performed on a widebore Bruker (Karlsruhe, Germany) AVANCE III 850 MHz solid state NMR spectrometer operating at 20 T, corresponding to 1H and 13C Larmor frequencies of 850.2 and 213.8 MHz, respectively, using a 4 mm double-resonance MAS probe. Experiments were conducted at room temperature at a MAS frequency of 12.0 kHz ± 5 Hz. The 13C cross-polarization (CP) MAS spectra were acquired using a 1ms ramped contact time, a spectral window of 100 kHz (468 ppm), a recycle delay of 2 s and 28800 transients were co-added for each sample. The 1H 90° pulse duration was 3.4 s. Two-pulse phase-modulated (TPPM) decoupling was applied during acquisition at a 1H nutation frequency of 83 kHz58. The 13C chemical shift was determined using the carbonyl peak at 177.8 ppm of L-alanine as an external reference with respect to TMS. Two independent stem samples were analysed.This research data supports “Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis” which will be published in “Nature communications”.This work was supported by the BBSRC [grant number BB/G016240/1]
Xylan is a hemicellulose present in the cell walls of all land plants. Glycosyltransferases of th... more Xylan is a hemicellulose present in the cell walls of all land plants. Glycosyltransferases of the GT43 (IRX9/IRX9L and IRX14/IRX14L) and GT47 (IRX10/IRX10L) families are involved in the biosynthesis of its β-1,4-linked xylose backbone, which can be further modified by acetylation and sugar side chains. However, it remains unclear how the different enzymes work together to synthesize the xylan backbone. A xylan synthesis complex (XSC) has been described in the monocots wheat and asparagus, and co-expression of asparagus AoIRX9, AoIRX10 and AoIRX14A is required to form a catalytically active complex for secondary cell wall xylan biosynthesis. Here, we argue that an equivalent XSC exists for the synthesis of the primary cell wall of the eudicot Arabidopsis thaliana, consisting of IRX9L, IRX10L and IRX14. This would suggest the existence of distinct XSCs for primary and secondary cell wall xylan synthesis, reminiscent of the distinct cellulose synthesis complexes (CSCs) of the primary ...
1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plant... more 1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plants. MAS solid state NMR experiments were performed on a widebore Bruker (Karlsruhe, Germany) AVANCE III 850 MHz solid state NMR spectrometer operating at 20 T, corresponding to 1H and 13C Larmor frequencies of 850.2 and 213.8 MHz, respectively, using a 4 mm double-resonance MAS probe. Experiments were conducted at room temperature at a MAS frequency of 12.0 kHz ± 5 Hz. The 13C cross-polarization (CP) MAS spectra were acquired using a 1ms ramped contact time, a spectral window of 100 kHz (468 ppm), a recycle delay of 2 s and 28800 transients were co-added for each sample. The 1H 90° pulse duration was 3.4 s. Two-pulse phase-modulated (TPPM) decoupling was applied during acquisition at a 1H nutation frequency of 83 kHz58. The 13C chemical shift was determined using the carbonyl peak at 177.8 ppm of L-alanine as an external reference with respect to TMS. Two independent stem samples were analysed.This research data supports “Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis” which will be published in “Nature communications”.This work was supported by the BBSRC [grant number BB/G016240/1]
Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-c... more Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-consuming experimental procedures, and considerable effort is invested in the generation of such data. Multiple research groups have described a variety of approaches for establishing high-quality proteome-wide datasets. However, data analysis is as critical as data production for reliable and insightful biological interpretation, and no consistent and robust solutions have been offered to the community so far. Here, we introduce the requirements for rigorous spatial proteomics data analysis, as well as the statistical machine learning methodologies needed to address them, including supervised and semi-supervised machine learning, clustering, and novelty detection. We present freely available software solutions that implement innovative state-of-the-art analysis pipelines and illustrate the use of these tools through several case studies involving multiple organisms, experimental designs,...
The Plant journal : for cell and molecular biology, 2014
The interaction between xylan and cellulose microfibrils is important for secondary cell wall pro... more The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecul...
The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glyc... more The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains...
The molecular architecture of plant secondary cell walls is still not resolved. There are several... more The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.
Polysaccharide synthesis in the Golgi apparatus is considered a multistep reaction that requires ... more Polysaccharide synthesis in the Golgi apparatus is considered a multistep reaction that requires the cooperative and orchestrated action of several enzymes, mostly glycosyltransferases (GTs). The organisation of such biosynthetic machinery determines the properties of the polysaccharide made: its size, substitution degree and pattern, translating into specific rheological behaviour, defining the polysaccharide function. The mechanism of synthesis is poorly understood; not only is very little known about the specificity of the GTs, but even less about their interaction or the identity of other types of proteins involved. Studies towards determining all the machinery components and their abundances are therefore of high importance. In order to obtain such insight we explored three different quantitative proteomic approaches. Firstly, the LOPIT technique led to the localisation of nearly 200 proteins in the Golgi apparatus, 79 of which have not been previously reported [1]. We found, u...
1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plant... more 1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plants. MAS solid state NMR experiments were performed on a widebore Bruker (Karlsruhe, Germany) AVANCE III 850 MHz solid state NMR spectrometer operating at 20 T, corresponding to 1H and 13C Larmor frequencies of 850.2 and 213.8 MHz, respectively, using a 4 mm double-resonance MAS probe. Experiments were conducted at room temperature at a MAS frequency of 12.0 kHz ± 5 Hz. The 13C cross-polarization (CP) MAS spectra were acquired using a 1ms ramped contact time, a spectral window of 100 kHz (468 ppm), a recycle delay of 2 s and 28800 transients were co-added for each sample. The 1H 90° pulse duration was 3.4 s. Two-pulse phase-modulated (TPPM) decoupling was applied during acquisition at a 1H nutation frequency of 83 kHz58. The 13C chemical shift was determined using the carbonyl peak at 177.8 ppm of L-alanine as an external reference with respect to TMS. Two independent stem samples were analysed.This research data supports “Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis” which will be published in “Nature communications”.This work was supported by the BBSRC [grant number BB/G016240/1]
Xylan is a hemicellulose present in the cell walls of all land plants. Glycosyltransferases of th... more Xylan is a hemicellulose present in the cell walls of all land plants. Glycosyltransferases of the GT43 (IRX9/IRX9L and IRX14/IRX14L) and GT47 (IRX10/IRX10L) families are involved in the biosynthesis of its β-1,4-linked xylose backbone, which can be further modified by acetylation and sugar side chains. However, it remains unclear how the different enzymes work together to synthesize the xylan backbone. A xylan synthesis complex (XSC) has been described in the monocots wheat and asparagus, and co-expression of asparagus AoIRX9, AoIRX10 and AoIRX14A is required to form a catalytically active complex for secondary cell wall xylan biosynthesis. Here, we argue that an equivalent XSC exists for the synthesis of the primary cell wall of the eudicot Arabidopsis thaliana, consisting of IRX9L, IRX10L and IRX14. This would suggest the existence of distinct XSCs for primary and secondary cell wall xylan synthesis, reminiscent of the distinct cellulose synthesis complexes (CSCs) of the primary ...
1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plant... more 1D solid state NMR data for mature stems of stello cellulose synthesis mutant and wild type plants. MAS solid state NMR experiments were performed on a widebore Bruker (Karlsruhe, Germany) AVANCE III 850 MHz solid state NMR spectrometer operating at 20 T, corresponding to 1H and 13C Larmor frequencies of 850.2 and 213.8 MHz, respectively, using a 4 mm double-resonance MAS probe. Experiments were conducted at room temperature at a MAS frequency of 12.0 kHz ± 5 Hz. The 13C cross-polarization (CP) MAS spectra were acquired using a 1ms ramped contact time, a spectral window of 100 kHz (468 ppm), a recycle delay of 2 s and 28800 transients were co-added for each sample. The 1H 90° pulse duration was 3.4 s. Two-pulse phase-modulated (TPPM) decoupling was applied during acquisition at a 1H nutation frequency of 83 kHz58. The 13C chemical shift was determined using the carbonyl peak at 177.8 ppm of L-alanine as an external reference with respect to TMS. Two independent stem samples were analysed.This research data supports “Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis” which will be published in “Nature communications”.This work was supported by the BBSRC [grant number BB/G016240/1]
Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-c... more Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-consuming experimental procedures, and considerable effort is invested in the generation of such data. Multiple research groups have described a variety of approaches for establishing high-quality proteome-wide datasets. However, data analysis is as critical as data production for reliable and insightful biological interpretation, and no consistent and robust solutions have been offered to the community so far. Here, we introduce the requirements for rigorous spatial proteomics data analysis, as well as the statistical machine learning methodologies needed to address them, including supervised and semi-supervised machine learning, clustering, and novelty detection. We present freely available software solutions that implement innovative state-of-the-art analysis pipelines and illustrate the use of these tools through several case studies involving multiple organisms, experimental designs,...
The Plant journal : for cell and molecular biology, 2014
The interaction between xylan and cellulose microfibrils is important for secondary cell wall pro... more The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β-(1,4)-linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one-half of the xylosyl residues are O-acetylated at C-2 or C-3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecul...
The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glyc... more The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains...
The molecular architecture of plant secondary cell walls is still not resolved. There are several... more The molecular architecture of plant secondary cell walls is still not resolved. There are several proposed structures for cellulose fibrils, the main component of plant cell walls and the conformation of other molecules is even less well known. Glucuronic acid (GlcA) substitution of xylan (GUX) enzymes, in CAZy family glycosyl transferase (GT)8, decorate the xylan backbone with various specific patterns of GlcA. It was recently discovered that dicot xylan has a domain with the side chain decorations distributed on every second unit of the backbone (xylose). If the xylan backbone folds in a similar way to glucan chains in cellulose (2-fold helix), this kind of arrangement may allow the undecorated side of the xylan chain to hydrogen bond with the hydrophilic surface of cellulose microfibrils. MD simulations suggest that such interactions are energetically stable. We discuss the possible role of this xylan decoration pattern in building of the plant cell wall.
Polysaccharide synthesis in the Golgi apparatus is considered a multistep reaction that requires ... more Polysaccharide synthesis in the Golgi apparatus is considered a multistep reaction that requires the cooperative and orchestrated action of several enzymes, mostly glycosyltransferases (GTs). The organisation of such biosynthetic machinery determines the properties of the polysaccharide made: its size, substitution degree and pattern, translating into specific rheological behaviour, defining the polysaccharide function. The mechanism of synthesis is poorly understood; not only is very little known about the specificity of the GTs, but even less about their interaction or the identity of other types of proteins involved. Studies towards determining all the machinery components and their abundances are therefore of high importance. In order to obtain such insight we explored three different quantitative proteomic approaches. Firstly, the LOPIT technique led to the localisation of nearly 200 proteins in the Golgi apparatus, 79 of which have not been previously reported [1]. We found, u...
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