Marcello Salvatore Lenucci

Protein trafficking to the cell wall occurs through mechanisms distinguishable from 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.

Enzyme-aided extraction of lycopene from high-pigment tomato cultivars by supercritical carbon dioxide

This work reports a novel enzyme-assisted process for lycopene concentration into a freeze-dried tomato matrix and describes the results of laboratory scale lycopene supercritical CO2 (SC-CO2) extractions carried out with untreated (control) and enzyme-digested matrices. The combined use of food-grade commercial plant cell-wall glycosidases (Celluclast/Novozyme plus Viscozyme) allows to increase lycopene (∼153%) and lipid (∼137%) concentration in the matrix and rises substrate load onto the extraction vessel (∼46%) compared to the control. The addition of an oleaginous co-matrix (hazelnut seeds) to the tomato matrix (1:1 by weight) increases CO2 diffusion through the highly dense enzyme-treated matrix bed and provides lipids that are co-extracted increasing lycopene yield. Under the same operative conditions (50 MPa, 86 °C, 4 mL min(-1) SC-CO2 flow) extraction yield from control and Celluclast/Novozyme+Viscozyme-treated tomato matrix/co-matrix mixtures was similar, exceeding 75% after 4.5h of extraction. However, the total extracted lycopene was ∼3 times higher in enzyme-treated matrix than control.

Optimisation of biological and physical parameters for lycopene supercritical CO2 extraction from ordinary and high-pigment tomato cultivars

BACKGROUND Lycopene is used for several industrial applications. Supercritical CO(2) (SC-CO(2)) extraction from red-ripe tomato fruits is an excellent technique to replace the use of harmful solvents. In this study, starting from red-ripe tomatoes of ordinary and high-lycopene cultivars, the effect of different agronomical and technical aspects on lycopene content, stability and yield was evaluated throughout the production process from fresh tomatoes to the final SC-CO(2)-extracted oleoresin containing lycopene. RESULTS Red-ripe tomato cultivars differed in their lycopene content. Irrigation excess or deficit caused an increase in the amount of lycopene in the fruits. Fresh tomatoes were processed into a lyophilised matrix suitable for SC-CO(2) extraction, which could be stored for more than 6 months at -20 degrees C without lycopene loss. Under the optimal extraction conditions, efficiencies of up to 80% were achieved, but the recovery of lycopene in the extracted oleoresin was very low (approximately 24%). Co-extraction of the tomato matrix mixed with a lipid co-matrix allowed the recovery of approximately 90% of lycopene in the oleoresin. Using the high-lycopene cultivars, the yield of total extracted lycopene increased by approximately 60% with respect to the ordinary cultivars. Lipids and other biologically active molecules were present in the oleoresin. CONCLUSION A method for extracting, from a tomato matrix, a natural and solvent-free oleoresin containing lycopene dissolved in a highly unsaturated vegetable oil has been described. The oleoresin represents an excellent product for testing on cancer and cardiovascular disease prevention.

Supercritical carbon dioxide extraction of carotenoids from pumpkin (Cucurbita spp.): a review.

Carotenoids are well known for their nutritional properties and health promoting effects representing attractive ingredients to develop innovative functional foods, nutraceutical and pharmaceutical preparations. Pumpkin (Cucurbita spp.) flesh has an intense yellow/orange color owing to the high level of carotenoids, mainly α-carotene, β-carotene, β-cryptoxanthin, lutein and zeaxanthin. There is considerable interest in extracting carotenoids and other bioactives from pumpkin flesh. Extraction procedures able to preserve nutritional and pharmacological properties of carotenoids are essential. Conventional extraction methods, such as organic solvent extraction (CSE), have been used to extract carotenoids from plant material for a long time. In recent years, supercritical carbon dioxide (SC-CO2) extraction has received a great deal of attention because it is a green technology suitable for the extraction of lipophylic molecules and is able to give extracts of high quality and totally free from potentially toxic chemical solvents. Here, we review the results obtained so far on SC-CO2 extraction efficiency and quali-quantitative composition of carotenoids from pumpkin flesh. In particular, we consider the effects of (1) dehydration pre-treatments; (2) extraction parameters (temperature and pressure); the use of water, ethanol and olive oil singularly or in combination as entrainers or pumpkin seeds as co-matrix.

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

The immune system is essential to maintain the mutualistic homeostatic interaction between the host and its micro- and mycobiota. Living as a commensal, Saccharomyces cerevisiae could potentially shape the immune response in a significant way. We observed that S. cerevisiae cells induce trained immunity in monocytes in a strain-dependent manner through enhanced TNFα and IL-6 production upon secondary stimulation with TLR ligands, as well as bacterial and fungal commensals. Differential chitin content accounts for the differences in training properties observed among strains, driving induction of trained immunity by increasing cytokine production and direct antimicrobial activity both in vitro and in vivo. These chitin-induced protective properties are intimately associated with its internalization, identifying a critical role of phagosome acidification to facilitate microbial digestion. This study reveals how commensal and passenger microorganisms could be important in promoting health and preventing mucosal diseases by modulating host defense toward pathogens and thus influencing the host microbiota-immune system interactions.

Functional, textural and sensory properties of dry pasta supplemented with lyophilized tomato matrix or with durum wheat bran extracts produced by supercritical carbon dioxide or ultrasound

A study was carried out to produce functional pasta by adding bran aqueous extract (BW) and bran oleoresin (BO) obtained using ultrasound and supercritical CO2, respectively, or a powdery lyophilized tomato matrix (LT). The bioactive compounds, hydrophilic and lipophilic antioxidant activity (HAA and LAA) in vitro, were evaluated. BW supplementation did not improve antioxidant activity, whilst LT pasta showed unconventional taste and odor. BO pasta had good levels of tocochromanols (2551μg/100g pasta f.w.) and carotenoids (40.2μg/100g pasta f.w.), and the highest HAA and LAA. The oleoresin altered starch swelling and gluten network, as evidenced by scanning electron microscopy, therefore BO pasta had structural characteristics poor compared with the control (4.8% vs. 3.2% cooking loss), although this difference did not affect significantly overall sensory judgment (74 vs. 79 for BO and control, respectively). BO supplementation was most effective for increasing antioxidant activity without jeopardizing pasta quality.

Effects of sodium alginate bead encapsulation on the storage stability of durum wheat (Triticum durum Desf.) bran oil extracted by supercritical CO2.

The aim of this study was to investigate the influence of encapsulation on the storage stability of oil extracted by supercritical carbon dioxide from a micronized durum wheat bran fraction. Wheat bran oil was encapsulated in 2% (w/v) sodium alginate beads. Encapsulated and unencapsulated oil samples were stored at 4 or 25 °C, in daylight or darkness, over 90 days, and, at defined time points, subjected to stability evaluation based on fatty acid hydroperoxide production and tocopherol (α, β, and γ forms), tocotrienol (α, β, and γ forms) and carotenoid (lutein, zeaxanthin, and β-carotene) degradation. The encapsulation of the oil into alginate beads significantly increased stability, optimally when stored at 4 °C, maintaining high levels of isoprenoids and low content of fatty acid hydroperoxides over 30 days of storage.

Possible Use of the Carbohydrates Present in Tomato Pomace and in Byproducts of the Supercritical Carbon Dioxide Lycopene Extraction Process as Biomass for Bioethanol Production

This study provides information about the carbohydrate present in tomato pomace (skins, seeds, and vascular tissues) as well as in the byproducts of the lycopene supercritical carbon dioxide extraction (SC-CO₂) such as tomato serum and exhausted matrix and reports their conversion into bioethanol. The pomace, constituting approximately 4% of the tomato fruit fresh weight, and the SC-CO₂-exhausted matrix were enzyme saccharified with 0.1% Driselase leading to sugar yields of ~383 and ~301 mg/g dw, respectively. Aliquots of the hydrolysates and of the serum (80% tomato sauce fw) were fermented by Saccharomyces cerevisiae . The bioethanol produced from each waste was usually >50% of the calculated theoretical amount, with the exception of the exhausted matrix hydolysate, where a sugar concentration >52.8 g/L inhibited the fermentation process. Furthermore, no differences in the chemical solubility of cell wall polysaccharides were evidenced between the SC-CO₂-lycopene extracted and unextracted matrices. The deduced glycosyl linkage composition and the calculated amount of cell wall polysaccharides remained similar in both matrices, indicating that the SC-CO₂ extraction technology does not affect their structure. Therefore, tomato wastes may well be considered as potential alternatives and low-cost feedstock for bioethanol production.

Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets.

Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD), whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin-green fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP), were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.

Sphingomonas cynarae sp. nov., a proteobacterium that produces an unusual type of sphingan.

Strain SPC-1(T) was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered to be the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-stain-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide, formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30 °C in the presence of 0-0.5 % NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1(T) clustered together with species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, 2-hydroxymyristic acid (C(14 : 0) 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomic position. 16S rRNA gene sequence analysis showed that SPC-1(T) was most closely related to Sphingomonas hankookensis ODN7(T), Sphingomonas insulae DS-28(T) and Sphingomonas panni C52(T) (98.19, 97.91 and 97.11 % sequence similarities, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed; the type strain is SPC-1(T) ( = JCM 17498(T) = ITEM 13494(T)). A component analysis of the exopolysaccharide suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.