Evidence for intra- and extra-protoplasmic feruloylation and cross-linking in wheat seedlings roots.

The sub-cellular feruloylation and oxidative coupling sites of cell wall polysaccharides were investigated in planta by monitoring the kinetics of appearance of arabinosyl- and feruloyl-radiolabelled polysaccharides in the protoplasmic compartment and their secretion in the wall either in the presence or absence of brefeldin A (BFA). By using root apical segments excised from wheat seedlings (Triticum durum Desf.), incubated with trans-[U- 14C]cinnamic acid, we demonstrated that [14C]ferulate, likely [14C]diferulate, as well as trimers and larger products of ferulate are incorporated into the protoplasmic polysaccharides very rapidly within 1–3 min of [14C]cinnamate feeding. This agrees with the assumption that (glucurono) arabinoxylans [(G)AX] feruloylation and oxidative coupling occur intracellularly, likely in the Golgi apparatus. Simultaneously, polymer bound radioactive hydroxycinnamic acids appeared to be incorporated into the cell wall of root apical segments as early as 2 min after trans-[U- 14C]cinnamic acid feeding. On the contrary, starting from L-[1-14C]arabinose as tracer, the secretion of the pentose- containing polymers into the wall was between 5 to 10 min. These results indicated that (G)AX feruloylation and oxidative coupling occur both intra-protoplasmically and in muro. The occurrence of in muro feruloylation and oxidative coupling was conWrmed by the use of BFA a well known inhibitor of secretion. The drug caused a strong inhibition of the synthesis and secretion into the wall of the 14C-pentosyl-labelled polymers as well as of 14C-feruloylpolymers. In spite of this, the total amount of 14C-feruloylpolymers incorporated into the wall was only slightly aVected by BFA. This indicates the existence of a mechanism involved into secretion of the activated hydroxycinnamoyl precursors to the wall, alternative to that involved in polysaccharide secretion

Methodological approach for the study of glycoconjugates in Leptolyngbya VRUC 135

In this paper we report the metabolism of hexosamines and the cellular compartmentalization of glycoconjugates in the cyanobacterium Leptolyngbya VRUC 135 by using D -[U- 14 C]glucosamine as tracer. Glycoproteins as well as lipopolysaccharides were detected in the cell wall, membrane and buffer-soluble polymers. Evidence is also reported on the presence of lipopolysaccharides as released polymers

Fungal chitin induces trained immunity in human monocytes during cross-talk with Saccharomyces cerevisiae

Objectives: Candida albicans, a common colonizer of the human mucosa, is able to train innate immune cells in a process mediated by the β-glucans present in the cell wall1. Saccaromyces cerevisiae is another constant but harmful colonizer of humans and recently it has been demonstrated to be even more abundant than C. albicans2. The aim of this study was to investigate the in vitro training properties of S.cerevisiae on innate immune cells and the relation occurring between the cell wall composition and the observed effect. Methods: S. cerevisiae isolates from diverse ecological niches have been tested for their ability to induce trained immunity on human monocytes. The cell wall sugar composition of the S. cerevisiae strains have been investigated through ion-exchange chromatography. Glucan, mannan and chitin have been extracted from the yeast cell wall and tested independently for their capabilities to induce immune response. Results: The S. cerevisiae clinical isolates were able to strongly enhance pro-inflammatory cytokine production when monocytes were exposed to a secondary bacterial or fungal stimulus. Among cell wall components a higher content of chitin was detected in S. cerevisiae clinical isolates. Chitin was able alone to induce a potent immune response in terms of IL-6 and TNFα and to enhance the phagocytic killing by monocytes. Conclusions: The study gives novel prespective on how S.cerevisiae, a passenger microorganism present from centuries in our foods, could help priming organismal protection against pathogens. The chitin-induced training immunity suggests the importance of yeast cell wall modulation in protecting from dysbiosis

Protein trafficking to the cell wall occurs through different mechanisms distinguishable from the default sorting in tobacco

The secretory pathway in plants involves sustained traffic to the cell wall, as matrix components, polysaccharides and proteins reach the cell wall through the endomembrane system. We studied the secretion pattern of cell-wall proteins in tobacco protoplasts and leaf epidermal cells using fluorescent forms of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2). The two most representative protein fusions, secGFP-PMEI1 and PGIP2-GFP, reached the cell wall by passing through ER and Golgi stacks but using distinct mechanisms. secGFP-PMEI1 was linked to a glycosylphosphatidylinositol (GPI) anchor and stably accumulated in the cell wall, regulating the activity of the endogenous pectin methylesterases (PMEs) that are constitutively present in this compartment. A mannosamine-induced non-GPI-anchored form of PMEI1 as well as a form (PMEI1-GFP) that was unable to bind membranes failed to reach the cell wall, and accumulated in the Golgi stacks. In contrast, PGIP2-GFP moved as a soluble cargo protein along the secretory pathway, but was not stably retained in the cell wall, due to internalization to an endosomal compartment and eventually the vacuole. Stable localization of PGIP2 in the wall was observed only in the presence of a specific fungal endopolygalacturonase ligand in the cell wall. Both secGFP-PMEI1 and PGIP2-GFP sorting were distinguishable from that of a secreted GFP, suggesting that rigorous and more complex controls than the simple mechanism of bulk flow are the basis of cell-wall growth and differentiation