Xylan decoration patterns and the plant secondary cell wall molecular architecture.

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.We thank Nadine Anders for helpful comments on the manuscript. The work was supported by a Leverhulme Trust Programme Grant : The Centre for Natural Material Innovation and the Biotechnology and Biological Sciences Research Council grant numbers [BB/K005537/1] and [BB/G016240/1].This is the author accepted manuscript. The final version is available from Portland Press via http://dx.doi.org/10.1042/BST2015018

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana.

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 molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan-cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy.The work conducted by TT and NN was supported by a grant from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) to PD and DNB. The work of PD was supported by the European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement #251132. The NMR facility infrastructure was supported by the BBSRC and the Wellcome Trust. TCFG thanks CNPq (Brazil) for a graduate fellowship (grant # 140978/2009-7). MSS thanks CEPROBIO (grant # 490022/2009- 0) and FAPESP for funding (grant #2013/08293-7).This is the accepted version of the following article: "Busse-Wicher, M; Gomes, T.C.F; Tryfona, T; Nikolovski, N; Stott, K; Grantham, N.J; Bolam, D.N; Skaf, M.S; Dupree, P. (2014) "The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a two-fold helical screw in the secondary plant cell wall of Arabidopsis thaliana." The Plant Journal. Accepted article [electronic] 10.1111/tpj.12575", which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/tpj.12575/abstrac

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus

A foundation for reliable spatial proteomics data analysis.

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, mass spectrometry platforms, and quantitation techniques. We also propose sound analysis strategies for identifying dynamic changes in subcellular localization by comparing and contrasting data describing different biological conditions. We conclude by discussing future needs and developments in spatial proteomics data analysis..G., C.M.M., and M.F. were supported by the European Union 7th Framework Program (PRIME-XS Project, Grant No. 262067). L.M.B. was supported by a BBSRC Tools and Resources Development Fund (Award No. BB/K00137X/1). T.B. was supported by the Proteomics French Infrastructure (ProFI, ANR-10-INBS-08). A.C. was supported by BBSRC Grant No. BB/D526088/1. A.J.G. was supported by BBSRC Grant No. BB/E024777/ and a generous gift from King Abdullah University for Science and Technology, Saudi Arabia. D.J.N.H. was supported by a BBSRC CASE studentship (BB/I016147/1)

Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus.The work presented in this paper was supported by grants from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) and the European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement n° 251132 to PD. The UK 850 MHz solid-state NMR Facility was funded by EPSRC and BBSRC, as well as the University of Warwick including via part funding through Birmingham Science City Advanced Materials Projects 1 and 2 supported by Advantage West Midlands (AWM) and the European Regional Development Fund (ERDF); we thank Dinu Iuga for experimental assistance, and Chris Somerville for helpful discussions and suggesting the name STELLO. The authors acknowledge LNBio and LNLS for providing X-ray beam time (proposal GAR 15208), and the Sainsbury Laboratory Cambridge University for imaging facilities. TV was supported by an EMBO long-term fellowship (ALTF 711-2012) and by postdoctoral funding from the Philomathia Foundation. HEM was supported by an EMBO Long Term Fellowship (ALTF-1246-2013) and an NSERC Postdoctoral Fellowship (PDF-454454-2014). SP and YZ were supported by the Max-Planck Gesellschaft, and SP was also supported by a R@MAP Professor position at UoM. We thank the Biological Optical Microscopy Platform (BOMP) at University of Melbourne, and Tom Simmons and Rita Marques for assistance on sugar analyses.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms11656

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Potsdam 2009Published online at th

Research data supporting "Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis"

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]

β-1,4-Xylan backbone synthesis in higher plants: How complex can it be?

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 and secondary cell wall. In contrast to the CSC, in which each CESA protein has catalytic activity, the XSC seems to contain proteins with non-catalytic function with each component bearing potentially unique but crucial roles. Moreover, the core XSC formed by a combination of IRX9/IRX9L, IRX10/IRX10L and IRX14/IRX14L might not be stable in its composition during transit from the endoplasmic reticulum to the Golgi apparatus. Instead, potential dynamic changes of the XSC might be a means of regulating xylan biosynthesis to facilitate coordinated deposition of tailored polysaccharides in the plant cell wall

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of arabidopsis thaliana

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 molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan-cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy. © 2014 The Authors. 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Eng., 80, pp. 1008-1016Vranken, W.F., Boucher, W., Stevens, T.J., Fogh, R.H., Pajon, A., Llinas, P., Ulrich, E.L., Laue, E.D., The CCPN data model for NMR spectroscopy: Development of a software pipeline (2005) Proteins, 59, pp. 687-696Xiong, G., Cheng, K., Pauly, M., Xylan O-acetylation impacts xylem development and enzymatic recalcitrance as indicated by the Arabidopsis mutant tbl29 (2013) Mol. Plant, 6, pp. 1373-1375Zhang, Q., Brumer, H., Agren, H., Tu, Y.Q., The adsorption of xyloglucan on cellulose: Effects of explicit water and side chain variation (2011) Carbohydr. Res., 346, pp. 2595-2602The work conducted by TT and NN was supported by a grant from the BBSRC: BB/G016240/1 BBSRC Sustainable Energy Centre Cell Wall Sugars Programme (BSBEC) to PD and DNB. The work of PD was supported by the European Community's Seventh Framework Programme S