Anna Montefusco

Two glycosylated vacuolar GFPs are new markers for ER-to-vacuole sorting.

Vacuolar Sorting Determinants (VSDs) have been extensively studied in plants but the mechanisms for the accumulation of storage proteins in somatic tissues are not yet fully understood. In this work we used two mutated versions of well-documented vacuolar fluorescent reporters, a GFP fusion in frame with the C-terminal VSD of tobacco chitinase (GFPChi) and an N-terminal fusion in frame with the sequence-specific VSD of the barley cysteine protease aleurain (AleuGFP). The GFP sequence was mutated to present an N-glycosylation site at the amino-acid position 133. The reporters were transiently expressed in Nicotiana tabacum protoplasts and agroinfiltrated in Nicotiana benthamiana leaves and their distribution was identical to that of the non-glycosylated versions. With the glycosylated GFPs we could highlight a differential ENDO-H sensitivity and therefore differential glycan modifications. This finding suggests two different and independent routes to the vacuole for the two reporters. BFA also had a differential effect on the two markers and further, inhibition of COPII trafficking by a specific dominant-negative mutant (NtSar1h74l) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus.

Antioxidants in Varieties of Chicory (Cichorium intybus L.) and Wild Poppy (Papaver rhoeas L.) of Southern Italy

We report the hydrophilic and lipophilic antioxidant activities, as well as the total phenol, flavonoid, tocochromanol (tocopherol and tocotrienol), and carotenoid contents in the edible portion of wild and cultivated varieties of chicory (Cichorium intybus L.) and in the basal rosette leaves of the wild species of poppy (Papaver rhoeas L.), known by natives as “paparina,” collected in the countryside of Salento (South Apulia, Italy). We analyzed (1) two cultivars of chicory, the “Catalogna” harvested in the area between S. Pietro Vernotico and Tuturano (Brindisi) and the “Otrantina” harvested in Otranto (Lecce); (2) two wild chicory ecotypes harvested in S. Pietro Vernotico (Brindisi) and Statte (Taranto), respectively; (3) the basal leaves of wild poppy harvested in Sternatia (Lecce). In all samples, our results showed that the hydrophilic antioxidant activity is, generally, higher than the lipophilic activity. Poppy leaves exhibited the highest hydrophilic and lipophilic antioxidant activities and the highest concentration of total phenols and flavonoids. Tocopherols were detected only as traces. Among the extracted carotenoids, lutein and β-carotene were the most abundant in all analyzed samples. Total carotenoid content was greater in wild than in cultivated plants.

Brefeldin A: a specific inhibitor of cell wall polysaccharide biosynthesis in oat coleoptile segments

Abstract The effect of brefeldin A (BFA) on the synthesis and incorporation of polysaccharides, proteins and glycoproteins into the cell wall of subapical coleoptile segments isolated from etiolated oat seedlings ( Avena sativa L. cv. Angelica) has been investigated. In the presence of D-[U- 14 C]-glucose, the incorporation of radioactive glycosyl residues into buffer-soluble, membrane (matrix polysaccharides) and cell wall polysaccharides was drastically inhibited by increasing concentrations of BFA up to 10 μ·mL −1 . BFA also altered the pattern of these polysaccharides suggesting a different sensitivity of glycosyltransferases toward the action of the drug. The incorporation of [U- 14 C]-glycine or L-[U- 14 C]-leucine into non-covalently- and covalently-bound cell wall proteins as well as the incorporation of radioactive N-acetylglucosamine residues into the newly synthesised oligosaccharidic chains of cytosolic, membrane and cell wall glycoproteins remained unchanged in the presence of 10 μg·mL −1 BFA. The data demonstrate that, in oat coleoptile segments, BFA specifically inhibits the synthesis of cellulose and matrix polysaccharides without altering the synthesis and incorporation of proteins and glycoproteins into the cell wall. In addition, it is demonstrated that BFA does not affect the in vivo activity of glycosyltransferases involved in the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the oligosaccharidic chains of glycoproteins.

Cellular localization and biochemical characterization of a chimeric fluorescent protein fusion of Arabidopsis cellulose synthase-like A2 inserted into Golgi membrane.

Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of β-mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a 14C-mannan starting from GDP-d-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen.

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: Engineering attempts using a modified SNARE protein

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

Synthesis of cell-wall glycoproteins and their characterization in oat coleoptiles

D-[U-14C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP-N-acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N-glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N-glycosidase F and N-acetyl-beta-D-glucosaminidase on cell-wall glycoproteins indicated the presence of N-acetylglucosamine residues beta-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20 degrees, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50 degrees. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N-glycosidase F, N-acetyl-beta-D-glucosaminidase and NaOH at 50 degrees indicated that most N-acetylglucosamine residues were incorporated into N-linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N-linked glycoproteins.

When Color Really Matters: Horticultural Performance and Functional Quality of High-Lycopene Tomatoes

ABSTRACT Introgression of spontaneous or induced mutations has been used to increase the levels and diversify the profile of antioxidants in many fruits including tomato. The high-pigment (hp) and old-gold (og) alleles exemplify this approach as attractive genetic resources suitable to inbred elite high-lycopene (HLY) tomato lines with improved color and nutritional attributes. Although several studies have been published on HLY tomatoes, a systematic analysis of the information on their agronomic performances, processing features, and functional quality is lacking, leaving room for the assumption of their poor competitiveness with conventional tomato cultivars and limiting their agricultural diffusion. Therefore, the aim of this study is to critically review the most important agronomic, horticultural, and functional traits of HLY tomatoes, as well as the advances in some emerging (pre)industrial applications. Field experiments performed in different countries showed that most available HLY lines are productive, vigorous, with excellent foliage cover and with morphologically acceptable fruit. Tomato yield of HLY genotypes ranged from ∼30 to ∼178 t/ha exceeding, in some trials, that of highly productive cultivars. Red-ripe fruits of most HLY lines showed commercially suitable soluble solids and titratable acidity, in addition to increased levels of lycopene (up to 440 mg/kg fw) and other bioactive phytochemicals (mainly flavonoids and vitamin C) compared to their near isogenic conventional counterparts. Innovative (pre)industrial uses of HLY tomato include the following: (1) production of HLY sauces, juices, and powders; (2) supercritical-CO2 extraction of lycopene containing oleoresins; and (3) preparation of lycopene rich micro- and nano-carriers with improved stability and specific tissue delivery. In turn, the use of these innovative high-quality ingredients in the formulation of lycopene fortified foods, cosmetic products, nutraceuticals, and pharmaceuticals has been proposed as the basis of a novel highly profitable tomato product chain.

Assessment of sweet potato [Ipomoea batatas (L.) Lam] for bioethanol production in southern Italy.

The potential of sweet potato as an alternative crop for bioethanol production has been assessed. We evaluated the amount of soluble sugars, starch and cell wall polysaccharides in tubers of three sweet potato cultivars characterized by different pulp and peel colouration: “Yellow yam” with yellow flesh and brown peel, “White yam” with white flesh and white peel and “Orange yam” with orange flesh and brown peel. The results confirm the high concentration of carbohydrates in sweet potato tubers, especially “Yellow yam”, mainly in the form of starch (67%) and soluble sugars (26%). “Yellow yam”, which is the most widespread cultivar in Salento, appeared the best choice as biomass for bioethanol production. It is characterized by high productivity (20–40 tons/ha year). Results also suggest that “Yellow yam” cultivar has great potential as a bioethanol source in southern Italy with an estimated agroindustrial production yield higher than 2032 l/ha year.

Methodological approach for the study of glycoconjugates in Leptolyngbya VRUC 135

Abstract 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‐14C]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. Abbreviation: RPs, released polymers