Azeddine Driouich

Plant N-Glycan Processing Enzymes Employ Different Targeting Mechanisms for Their Spatial Arrangement along the Secretory Pathway

The processing of N-linked oligosaccharides in the secretory pathway requires the sequential action of a number of glycosidases and glycosyltransferases. We studied the spatial distribution of several type II membrane-bound enzymes from Glycine max, Arabidopsis thaliana, and Nicotiana tabacum. Glucosidase I (GCSI) localized to the endoplasmic reticulum (ER), α-1,2 mannosidase I (ManI) and N-acetylglucosaminyltransferase I (GNTI) both targeted to the ER and Golgi, and β-1,2 xylosyltransferase localized exclusively to Golgi stacks, corresponding to the order of expected function. ManI deletion constructs revealed that the ManI transmembrane domain (TMD) contains all necessary targeting information. Likewise, GNTI truncations showed that this could apply to other type II enzymes. A green fluorescent protein chimera with ManI TMD, lengthened by duplicating its last seven amino acids, localized exclusively to the Golgi and colocalized with a trans-Golgi marker (ST52-mRFP), suggesting roles for protein–lipid interactions in ManI targeting. However, the TMD lengths of other plant glycosylation enzymes indicate that this mechanism cannot apply to all enzymes in the pathway. In fact, removal of the first 11 amino acids of the GCSI cytoplasmic tail resulted in relocalization from the ER to the Golgi, suggesting a targeting mechanism relying on protein–protein interactions. We conclude that the localization of N-glycan processing enzymes corresponds to an assembly line in the early secretory pathway and depends on both TMD length and signals in the cytoplasmic tail.

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN-LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)1

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan‐proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β‐d‐glycosyl‐Yariv (β‐GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Epitopes of alkali‐soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti‐xyloglucan (anti‐XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC‐M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti‐XG antibody at the trans‐sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)‐β‐glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)‐β‐glucan (callose) could not be detected. The analytical results revealed that alkali‐soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)‐β‐d‐glucan.

Pollen tube cell walls of wild and domesticated tomatoes contain arabinosylated and fucosylated xyloglucan

BACKGROUND AND AIMSnIn flowering plants, fertilization relies on the delivery of the sperm cells carried by the pollen tube to the ovule. During the tip growth of the pollen tube, proper assembly of the cell wall polymers is required to maintain the mechanical properties of the cell wall. Xyloglucan (XyG) is a cell wall polymer known for maintaining the wall integrity and thus allowing cell expansion. In most angiosperms, the XyG of somatic cells is fucosylated, except in the Asterid clade (including the Solanaceae), where the fucosyl residues are replaced by arabinose, presumably due to an adaptive and/or selective diversification. However, it has been shown recently that XyG of Nicotiana alata pollen tubes is mostly fucosylated. The objective of the present work was to determine whether such structural differences between somatic and gametophytic cells are a common feature of Nicotiana and Solanum (more precisely tomato) genera.nnnMETHODSnXyGs of pollen tubes of domesticated (Solanum lycopersicum var. cerasiforme and var. Saint-Pierre) and wild (S. pimpinellifolium and S. peruvianum) tomatoes and tobacco (Nicotiana tabacum) were analysed by immunolabelling, oligosaccharide mass profiling and GC-MS analyses.nnnKEY RESULTSnPollen tubes from all the species were labelled with the mAb CCRC-M1, a monoclonal antibody that recognizes epitopes associated with fucosylated XyG motifs. Analyses of the cell wall did not highlight major structural differences between previously studied N. alata and N. tabacum XyG. In contrast, XyG of tomato pollen tubes contained fucosylated and arabinosylated motifs. The highest levels of fucosylated XyG were found in pollen tubes from the wild species.nnnCONCLUSIONSnThe results clearly indicate that the male gametophyte (pollen tube) and the sporophyte have structurally different XyG. This suggests that fucosylated XyG may have an important role in the tip growth of pollen tubes, and that they must have a specific set of functional XyG fucosyltransferases, which are yet to be characterized.

Sorption, diffusion and permeation properties of oxygen and water in copolymer of ethylene and polar monomers

Asymmetric membranes, based on Poly[ethylene-co-(vinyl acetate)] (EVA) containing 70 wt.% of vinyl acetate, were prepared by a treatment of unilateral hydrolysis using solutions of sodium hydroxide dissolved in a mixture of water and methanol. The depth of hydrolyzed layers and the concentration of hydroxyl groups in the membranes were controlled by the reaction time. The oxygen permeability, P O2 , of these membranes decrease with the reaction time while the water permeability, P H2O , reaches maximum at 30 min. The ideal separation factors of P H2O to P O2 of the EVA membranes treated for 1h to 4h are in the range of 3840 to 13500, and are greater than that of the EVA membranes. The plasticization effect of the membrane depends on the depth and concentration of hydroxyl groups and the concentration gradient of water in the membranes.

Perspectives on Structural, Physiological, Cellular, and Molecular Responses to Desiccation in Resurrection Plants

Resurrection plants possess a unique ability to counteract desiccation stress. Desiccation tolerance (DT) is a very complex multigenic and multifactorial process comprising a combination of physiological, morphological, cellular, genomic, transcriptomic, proteomic, and metabolic processes. Modification in the sugar composition of the hemicellulosic fraction of the cell wall is detected during dehydration. An important change is a decrease of glucose in the hemicellulosic fraction during dehydration that can reflect a modification of the xyloglucan structure. The expansins might also be involved in cell wall flexibility during drying and disrupt hydrogen bonds between polymers during rehydration of the cell wall. Cleavages by xyloglucan-modifying enzymes release the tightly bound xyloglucan-cellulose network, thus increasing cell wall flexibility required for cell wall folding upon desiccation. Changes in hydroxyproline-rich glycoproteins (HRGPs) such as arabinogalactan proteins (AGPs) are also observed during desiccation and rehydration processes. It has also been observed that significant alterations in the process of photosynthesis and photosystem (PS) II activity along with changes in the antioxidant enzyme system also increased the cell wall and membrane fluidity resulting in DT. Similarly, recent data show a major role of ABA, LEA proteins, and small regulatory RNA in regulating DT responses. Current progress in “-omic” technologies has enabled quantitative monitoring of the plethora of biological molecules in a high throughput routine, making it possible to compare their levels between desiccation-sensitive and DT species. In this review, we present a comprehensive overview of structural, physiological, cellular, molecular, and global responses involved in desiccation tolerance.

Arabinogalactan Proteins From Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes In Vitro

Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However, little is currently known regarding their potential activity toward skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability. Moreover, real‐time quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD‐2, TLR‐5, and IL1‐α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. J. Cell. Physiol. 232: 2558–2568, 2017.