In vivo imaging of the tonoplast intrinsic protein family in Arabidopsis roots

Background: Tonoplast intrinsic proteins (TIPs) are widely used as markers for vacuolar compartments in higher plants. Ten TIP isoforms are encoded by the Arabidopsis genome. For several isoforms, the tissue and cell specific pattern of expression are not known. Results: We generated fluorescent protein fusions to the genomic sequences of all members of the Arabidopsis TIP family whose expression is predicted to occur in root tissues (TIP1;1 and 1;2; TIP2;1, 2;2 and 2;3; TIP4;1) and expressed these fusions, both individually and in selected pairwise combinations, in transgenic Arabidopsis. Analysis by confocal microscopy revealed that TIP distribution varied between different cell layers within the root axis, with extensive co-expression of some TIPs and more restricted expression patterns for other isoforms. TIP isoforms whose expression overlapped appeared to localise to the tonoplast of the central vacuole, vacuolar bulbs and smaller, uncharacterised structures. Conclusion: We have produced a comprehensive atlas of TIP expression in Arabidopsis roots, which reveals novel expression patterns for not previously studied TIPs

Development of an oligosaccharide library to characterise the structural variation in glucuronoarabinoxylan in the cell walls of vegetative tissues in grasses.

BACKGROUND: Grass glucuronoarabinoxylan (GAX) substitutions can inhibit enzymatic degradation and are involved in the interaction of xylan with cell wall cellulose and lignin, factors which contribute to the recalcitrance of biomass to saccharification. Therefore, identification of xylan characteristics central to biomass biorefining improvement is essential. However, the task of assessing biomass quality is complicated and is often hindered by the lack of a reference for a given crop. RESULTS: In this study, we created a reference library, expressed in glucose units, of Miscanthus sinensis GAX stem and leaf oligosaccharides, using DNA sequencer-Assisted Saccharide analysis in high throughput (DASH), supported by liquid chromatography (LC), nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Our analysis of a number of grass species highlighted variations in substitution type and frequency of stem and leaf GAX. In miscanthus, for example, the β-Xylp-(1 → 2)-α-Araf-(1 → 3) side chain is more abundant in leaf than stem. CONCLUSIONS: The reference library allows fast identification and comparison of GAX structures from different plants and tissues. Ultimately, this reference library can be used in directing biomass selection and improving biorefining

Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis.

All members of the DUF579 family characterized so far have been described to affect the integrity of the hemicellulosic cell wall component xylan: GXMs are glucuronoxylan methyltransferases catalyzing 4-O-methylation of glucuronic acid on xylan; IRX15 and IRX15L, although their enzymatic activity is unknown, are required for xylan biosynthesis and/or xylan deposition. Here we show that the DUF579 family members, AGM1 and AGM2, are required for 4-O-methylation of glucuronic acid of a different plant cell wall component, the highly glycosylated arabinogalactan proteins (AGPs)

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14.

Xylan is a crucial component of many plant primary and secondary cell walls. However, the structure and function of xylan in the dicotyledon primary cell wall is not well understood. Here, we characterized a xylan that is specific to tissues enriched in Arabidopsis primary cell walls. Unlike previously described xylans, this xylan carries a pentose linked 1-2 to the α-1,2-d-glucuronic acid (GlcA) side chains on the β-1,4-Xyl backbone. The frequent and precisely regular spacing of GlcA substitutions every six xylosyl residues along the backbone is also unlike that previously observed in secondary cell wall xylan. Molecular genetics, in vitro assays, and expression data suggest that IRX9L, IRX10L and IRX14 are required for xylan backbone synthesis in primary cell wall synthesising tissues. IRX9 and IRX10 are not involved in the primary cell wall xylan synthesis but are functionally exchangeable with IRX9L and IRX10L. GUX3 is the only glucuronyltransferase required for the addition of the GlcA decorations on the xylan. The differences in xylan structure in primary versus secondary cell walls might reflect the different roles in cross-linking and interaction with other cell wall components.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 grant BB/K005537/1. JCM’s work at the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE -AC02-05CH11231. NFB was supported by a PhD studentship from the Portuguese Foundation for Science and Technology. AN was supported by a summer studentship award from the Biochemical Society. The authors are grateful to the European Community’s Seventh Framework Programme SUNLIBB (FP7/2007-2013) under the grant agreement no 251132.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/tpj.1289

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

Localisation et dynamique sub-cellulaire des aquaporines d'Arabidopsis thaliana

Les aquaporines sont des canaux facilitant le passage de l'eau au travers des membranes biologiques. Chez les plantes, elles contribuent de façon majeure au transport de l'eau dans tous les organes. Le maintien de l'équilibre hydrique de la plante entière dépend ainsi, en grande partie, de la régulation des AQP. Chez Arabidopsis, nous nous sommes intéressés au contrôle de la dynamique et de la localisation sub-cellulaires des PIP (Plasma membrane Intrinsic Protein). Ce contrôle semble être un mécanisme original de modulation de la fonction de nutrition hydrique des organes, et en particulier de la racine. La première partie nous a permis de mettre en évidence une relation entre l'effet du traitement salin sur l'inhibition du transport d'eau dans la racine et l'internalisation des PIP de la membrane plasmique (MP) vers des endosomes. En effet, dans ses étapes précoces, le traitement salin provoque une endocytose accrue des membranes lipidiques qui s'accompagne de celle des PIP. Cette découverte a été rendue possible grâce à l'utilisation de la technique de Fluorescence Recovery After Photobleaching (FRAP) qui nous a permis de discriminer entre des fusions PIP-GFP présentes dans la MP de celles présentes dans les compartiments endosomiaux, malgré leur très grande proximité. Précisément, nous avons, dans un premier temps, confirmé que les aquaporines cyclent de manière constitutive via une voie d'endocytose dépendante de l'adaptateur AP-2 et de la clathrine et via une voie d'exocytose dépendante de GNOM. Dans les étapes précoces du traitement salin, le pool d'endosomes et peut-être le cyclage entre ce pool et la MP sont augmentés selon une voie indépendante d'AP-2, mais par contre dépendante d'une Phosphatidyl-inositol-3 kinase. Ces données nous suggèrent l'existence de deux populations d'endosomes cyclant avec des dynamiques différentes pourrait, en partie, expliquer l'inhibition du transport d'eau de la racine observée en traitement salin, mais nous proposons également un mécanisme concomitant d'inhibition de l'activité intrinsèque des PIP. Par des approches de mutagenèse dirigées et de fusions à la GFP, la seconde partie de ce travail a permis de mettre en évidence un motif di-acide DXE (Asp4Val5Glu6) impliqué dans l'export hors du RE et situé sur l'extrémité N-terminale de PIP2;1. La mutation de l'un et/ou l'autre de ses résidus acides conduit à une rétention de la PIP2;1 dans le RE. Nous avons également étudié le rôle sur l'export hors du RE de modification post-traductionelles (MPT) originales que sont la di- et la mono-méthylation portées, respectivement, par les résidus Lys3 et Glu6. La Lys3 et la di-méthylation qu'elle porte ne sont pas impliquées dans l'export hors du RE de PIP2;1 et la question du rôle de la méthylation du Glu6 reste en suspens. Nous avons également mis en évidence des interactions entre sites où des mutations d'un résidu conduisent à des changements de nature des MPT des résidus à proximité. Enfin, la sur-expression d'une PIP2;1-GFP mutée sur le motif di-acide et retenue dans le RE, perturbe l'adressage à la MP des PIP2 et des PIP1 endogènes. Ce résultat suggère des interactions entre ces deux sous-groupes dans leur adressage à la MP. Nos études montrent qu'il existe de nombreux moyens de réguler la localisation et la dynamique sub-cellulaires des PIP, dans une cellule végétale. Il nous semble la mise en place de différentes voies d'endocytoses en réponse à des stimuli de l'environnement est un mécanisme de régulation des aquaporines crucial à explorer

Localisation et dynamique subcellulaires des aquaporines d'Arabidopsis thaliana

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Processing and Properties of MDF Fibre-Reinforced Biopolyesters with Chain Extender Additives

Biopolyesters are a way to improve natural fibre composite sustainability. This study explores, for the first time, the potential of using medium density fibreboard (MDF) fibres to reinforce four biobased and biodegradable polyester matrices to create a fully “green composite.” Added at 30 wt %, MDF fibres did not improve the strength of the injection moulded NFCs and this deficiency was investigated by measuring fibre length, viscosity, and molecular weight of the matrices. Compared to other lignocellulosic fibres, the use of MDF fibres led to a molecular weight reduction of biopolyesters during processing. This effect was particularly striking for PLA. The addition of a chain extender enhanced the molecular weight of PLA and improved its processability. The tensile strength increase was correlated to a reduction of fibre pull-out, enabling the MDF fibre to fulfil its expected reinforcement role within the biopolyester composite

Mechanisms and effects of retention of over-expressed aquaporin AtPIP2;1 in the endoplasmic reticulum.

International audiencePlasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity