N-glycosylation is a major modification of proteins in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide into high-mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycans occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination. At the end of their maturation, some plant N-glycans have typical structures that differ from those found in their mammalian counterpart by the absence of sialic acid and the presence of β(1,2)-xylose and α(1,3)-fucose residues. Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion to form mature N-glycans. On the basis of this assembly line concept, fast progress is currently made by using N-linked glycan structures as milestones of the intracellular transport of proteins along the plant secretory pathway. Further developments of this approach will need to more precisely define the topological distribution of glycosyltransferases within a plant Golgi stack. In contrast with their acknowledged role in the targeting of lysosomal hydrolases in mammalian cells, N-glycans have no specific function in the transport of glycoproteins into the plant vacuole. However, the presence of N-glycans, regardless of their structures, is necessary for an efficient secretion of plant glycoproteins. In the biotechnology field, transgenic plants are rapidly emerging as an important system for the production of recombinant glycoproteins intended for therapeutic purposes, which is a strong motivation to speed up research in plant glycobiology. In this regard, the potential and limits of plant cells as a factory for the production of mammalian glycoproteins will be illustrated.

N-glycoprotein biosynthesis in plants : recent developments and future trends

INTROD UCTION . . . . . . . . . . . .. . . . . . . . . . .. ....... . . ... . . . . . . . . ..... .. . . . ........ . . . 137 DYNAMIC S O F VAC UOLAR FORM AND STR UCT URE....... . . . . . . . .... . . . . . . ...... . . . . 1 38 Ontogeny and Diff erentiation of Vacuoles... . . . . . . ........ . . . . . . . . . ....... . . . . .. ...... . . . . ... 1 38 Tonoplast and Membrane Flow ..... . . . .... . . . . . ....... . . . . . . . .. . . .... . .. . . . ...... . . ... . . . . .... 139 DYNAMIC S O F MACROMOLEC ULES IN THE VAC UOLAR COMPARTMENT . . . . 140 Vacuoles as Lysosomes ... . . . . . . . ........ . . . . . . . . ... . ... . . . .. . . . .. . ... ... . . . . . . . . .... ... . . . . . . ... 140 Import and Export of Vacuolar Proteins ..... . . . . . . ......... . . . . ...... . . .. .. . ... . .. 141 Digestion of Cytoplasmic Components in Vacuoles? . . . . . . . . . ..... . . . . . . . .. . . . . . . . . . . . . . . . . 142 Vacuoles and Heterophagy.... .. . . . . .. . . . . . . . . 143 DYNAMIC S OF METABOLITES AND IONS IN THE VAC UOLAR COMPARTMENT 144 Composition of the Vacuolar Sap . . .. . ...... 144 Energization of the Tonoplast...... . . . . . 145 Mechanisms of Transport and Accumulation in Vacuoles ........ ......... . .. ........ . . . .. 149 Homeostatic Functions of Vacuolar Compartmentation . . . ...... . . .. . . . . .... . . . . ... . . . . . . . . 152 Metabolic Processes Involving the Vacuoles . . . . . . . .. ........ .. . . . . .... ..... . . . . . . ...... . .... 155 CONCL USION .. .. . . . . ... . . . . .. ...... . . . .. ... . . . . . . .. . ..... . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . .. 156

Dynamics of Vacuolar Compartmentation

In plants, most proteins of the extracellular compartment and the endomembrane system are glycosylated by N-linked oligosaccharides. The N-glycosylation of proteins has a great impact both on their physicochemical properties and on their biological functions. Over the last ten years, a number of laboratories have contributed considerably to the understanding of the structure, the biosynthesis and the function of plant N-linked glycans. In this review, data on this domain will be summarized and the recent results on the N-glycosylation of a vacuolar lectin, the bean phytohaemagglutinin (PHA) will also be included. This PHA, used as a model glycoprotein, was expressed in different plant systems and the N-glycosylation patterns of different recombinant PHA were compared. In addition to this study on plant-specific glycosylation, the same model glycoprotein was used to investigate whether or not N-glycosylation and N-glycan maturation is organ-specific in plants.

The protein N-glycosylation in plants

Leaf and whole plant-level functional traits were studied in five dominant woody savannah species from Central Brazil (Cerrado) to determine whether reduction of nutrient limitations in oligotrophic Cerrado soils affects carbon allocation, water relations and hydraulic architecture. Four treatments were used: control, N additions, P additions and N plus P additions. Fertilizers were applied twice yearly, from October 1998 to March 2004. Sixty-three months after the first nutrient addition, the total leaf area increment was significantly greater across all species in the N- and the N + P-fertilized plots than in the control and in the P- fertilized plots. Nitrogen fertilization significantly altered several components of hydraulic architecture: specific conductivity of terminal stems increased with N addi- tions, whereas leaf-specific conductivity and wood density decreased in most cases. Average daily sap flow per individ- ual was consistently higher with N and N + P additions compared to the control, but its relative increase was not as great as that of leaf area. Long-term additions of N and N + P caused midday Y L to decline significantly by a mean of 0.6 MPa across all species because N-induced relative reductions in soil-to-leaf hydraulic conductance were greater than those of stomatal conductance and tran- spiration on a leaf area basis. Phosphorus-fertilized trees did not exhibit significant changes in midday Y L. Analysis of xylem vulnerability curves indicated that N-fertilized trees were significantly less vulnerable to embolism than trees in control and P-fertilized plots. Thus, N-induced decreases in midday Y L appeared to be almost entirely compensated by increases in resistance to embolism. Leaf tissue water relations characteristics also changed as a result of N-induced declines in minimum Y L : osmotic potential at full turgor decreased and symplastic solute con- tent on a dry matter basis increased linearly with declining midday Y L across species and treatments. Despite being adapted to chronic nutrient limitations, Cerrado woody species apparently have the capacity to exploit increases in nutrient availability by allocating resources to maximize carbon gain and enhance growth. The cost of increased allocation to leaf area relative to water transport capacity involved increased total water loss per plant and a decrease in minimum leaf water potentials. However, the risk of increased embolism and turgor loss was relatively low as xylem vulnerability to embolism and leaf osmotic charac- teristics changed in parallel with changes in plant water status induced by N fertilization.

https://fs.fed.us/pnw/pubs/journals/pnw_2006_bucci001.pdf

Nutrient availability constrains the hydraulic architecture and water relations of savannah trees.

In plants, glycoproteins with asparagine-linked glycans (oligosaccharides) are found in vacuoles, in the extracellular space or matrix, and associated with the endo-membrane system (endoplasmic reticulum, Golgi apparatus, plasma membrane, tonoplast). These glycans are of the high-mannose type, with a structure identical to that found in other organisms (mammals, yeast), or of the complex type with a β1–2 linked xylosyl residue not found in mammalian complex glycans. Asparagine-linked glycans play multiple roles by modifying the physicochemical properties of the polypeptides to which they are attached.

Structure, biosynthesis, and function of asparagine-linked glycans on plant glycoproteins

We have failed to detect the presence of mannose-6-phosphate in the oligosaccharide moiety of glycoproteins from pea ( Pisum sativum L. cv Burpeeana) cotyledons using an assay system sensitive to 10 picomoles of mannose-6-phosphate. We were also unable to demonstrate any retention of glycosidase activity from pea seedlings and pea cotyledons on Sepharose-coupled phosphomannosyl receptor proteins isolated from bovine liver which were, however, able to retain phosphomannosylated hexosaminidase purified from Dictyostelium discoideum secretions. Furthermore, although Sepharose-coupled phosphomannosylated hexosaminidase from Dictyostelium was able to bind phosphomannosyl receptors from bovine liver we were unable to detect the retention of any protein from acetone powder extracts of pea seedlings or from endoplasmic reticulum-associated proteins of pea cotyledons. Based on this collective evidence we conclude that mannose-6-phosphate does not appear to play a role in the targeting of hydrolytic enzymes from the endoplasmic reticulum to the protein bodies in pea cotyledons.

Protein bodies and vacuoles as lysosomes : investigations into the role of mannose-6-phosphate in intracellular transport of glycosidases in pea cotyledons.

Pierre-Richard Gaudreault

Papers

Protein bodies and vacuoles as lysosomes : investigations into the role of mannose-6-phosphate in intracellular transport of glycosidases in pea cotyledons.

The Association of Acid Hydrolase Activity with the Microsomal Fraction from Pea Cotyledons (Pisum sativum L.)

Glycoprotein Nature of Glycosidases from Leaves of Pisum sativum L