Development of a Whole-Cell System Based on the Use of Genetically Modified Protoplasts to Detect Nickel Ions in Food Matrices.

Heavy metals are dangerous contaminants that constitute a threat to human health because they persist in soils and are easily transferred into the food chain, causing damage to human health. Among heavy metals, nickel appears to be one of the most dangerous, being responsible for different disorders. Public health protection requires nickel detection in the environment and food chains. Biosensors represent simple, rapid, and sensitive methods for detecting nickel contamination. In this paper, we report on the setting up a whole-cell-based system, in which protoplasts, obtained from Nicotiana tabacum leaves, were used as transducers to detect the presence of heavy metal ions and, in particular, nickel ions. Protoplasts were genetically modified with a plasmid containing the Green Fluorescent Protein reporter gene (GFP) under control of the promoter region of a sunflower gene coding for a small Heat Shock Protein (HSP). Using this device, the presence of heavy metal ions was detected. Thus, the possibility of using this whole-cell system as a novel tool to detect the presence of nickel ions in food matrices was assessed.

Arabidopsis subtilases promote defense-related pectin methylesterase activity and robust immune responses to botrytis infection.

Plants involve a fine modulation of pectin methylesterase (PME) activity against microbes. PME activity can promote the cell wall stiffening and the production of damage signals able to induce defense responses and plant resistance to pathogens. However, the molecular mechanisms underlying PME activation during disease remain largely unknown. In this study, we explored the role of subtilases (SBTs) as PME activators in Arabidopsis immunity. By using biochemical and reverse genetic approaches, we found that the expression of SBT3.3 and SBT3.5 influences the induction of defense-related PME activity and resistance to the fungus Botrytis cinerea. Arabidopsis sbt3.3 and sbt3.5 knockout mutants showed decreased induction of PME activity and increased susceptibility to the fungus. SBT3.3 expression was stimulated by oligogalacturonides. Overexpression of SBT3.3 overactivated PME activity during fungal infection and enhanced resistance to B. cinerea. A negative correlation was observed between SBT3.3 expression and cell wall methyl ester content in the genotypes analyzed after B. cinerea infection. Increased expression of defense-related genes, including PAD3, CYP81F2 and WAK2, was also revealed in SBT3.3 overexpressing lines. We also demonstrated that SBT3.3 and pro-PME17 are both secreted into the cell wall using distinct protein secretion pathways and different kinetics. Our results propose SBT3.3 and SBT3.5 as modulators of PME activity in Arabidopsis against Botrytis to promptly boost immunity limiting the growth-defense trade-off.

CiXTH29 and CiLEA4 Role in Water Stress Tolerance in Cichorium intybus Varieties.

Drought causes massive crop quality and yield losses. Limiting the adverse effects of water deficits on crop yield is an urgent goal for a more sustainable agriculture. With this aim, six chicory varieties were subjected to drought conditions during seed germination and at the six week-old plant growth stage, in order to identify some morphological and/or molecular markers of drought resistance. Selvatica, Zuccherina di Trieste and Galatina varieties, with a high vegetative development, showed a major germination index, greater seedling development (6 days of growth) and a greater dehydration resistance (6 weeks of growth plus 10 days without water) than the other ones (Brindisina, Esportazione and Rossa Italiana). Due to the reported involvement, in the abiotic stress response, of xyloglucan endotransglucosylase/hydrolases (XTHs) and late embryogenesis abundant (LEA) multigene families, XTH29 and LEA4 expression profiles were investigated under stress conditions for all analyzed chicory varieties. We showed evidence that chicory varieties with high CiXTH29 and CiLEA4 basal expression and vegetative development levels better tolerate drought stress conditions than varieties that show overexpression of the two genes only in response to drought. Other specific morphological traits characterized almost all chicory varieties during dehydration, i.e., the appearance of lysigen cavities and a general increase of the amount of xyloglucans in the cell walls of bundle xylem vessels. Our results highlighted that high CiXTH29 and CiLEA4 basal expression, associated with a high level of vegetative growth, is a potential marker for drought stress tolerance.

Expression of Exogenous GFP-CesA6 in Tobacco Enhances Cell Wall Biosynthesis and Biomass Production.

Improved cellulose biosynthesis and plant biomass represent important economic targets for several biotechnological applications including bioenergy and biofuel production. The attempts to increase the biosynthesis of cellulose by overexpressing CesAs proteins, components of the cellulose synthase complex, has not always produced consistent results. Analyses of morphological and molecular data and of the chemical composition of cell walls showed that tobacco plants (F31 line), stably expressing the Arabidopsis CesA6 fused to GFP, exhibits a "giant" phenotype with no apparent other morphological aberrations. In the F31 line, all evaluated growth parameters, such as stem and root length, leaf size, and lignified secondary xylem, were significantly higher than in wt. Furthermore, F31 line exhibited increased flower and seed number, and an advance of about 20 days in the anthesis. In the leaves of F31 seedlings, the expression of primary CesAs (NtCesA1, NtCesA3, and NtCesA6) was enhanced, as well as of proteins involved in the biosynthesis of non-cellulosic polysaccharides (xyloglucans and galacturonans, NtXyl4, NtGal10), cell wall remodeling (NtExp11 and XTHs), and cell expansion (NtPIP1.1 and NtPIP2.7). While in leaves the expression level of all secondary cell wall CesAs (NtCesA4, NtCesA7, and NtCesA8) did not change significantly, both primary and secondary CesAs were differentially expressed in the stem. The amount of cellulose and matrix polysaccharides significantly increased in the F31 seedlings with no differences in pectin and hemicellulose glycosyl composition. Our results highlight the potentiality to overexpress primary CesAs in tobacco plants to enhance cellulose synthesis and biomass production.

Evaluation of Dittrichia viscosa Aquaporin Nip1.1 Gene as Marker for Arsenic-Tolerant Plant Selection.

Dittrichia viscosa (L.) Greuter is gaining attention for its high genetic plasticity and ability to adapt to adverse environmental conditions, including heavy metal and metalloid pollution. Uptake and translocation of cadmium, copper, iron, nickel, lead, and zinc to the shoots have been characterized, but its performance with arsenic is less known and sometimes contradictory. Tolerance to As is not related to a reduced uptake, but the null mutation of the aquaporin Nip1.1 gene in Arabidopsis makes the plant completely resistant to the metalloid. This aquaporin, localized in the endoplasmic reticulum, is responsible for arsenite and antimony (Sb) membrane permeation, but the uptake of arsenite occurs also in the null mutant, suggesting a more sophisticated action mechanism than direct uptake. In this study, the DvNip1 gene homologue is cloned and its expression profile in roots and shoots is characterized in different arsenic stress conditions. The use of clonal lines allowed to evidence that DvNip1.1 expression level is influenced by arsenic stress. The proportion of gene expression in roots and shoots can be used to generate an index that appears to be a promising putative selection marker to predict arsenic-resistant lines of Dittrichia viscosa plants.

Drought Stress Pre-Treatment Triggers Thermotolerance Acquisition in Durum Wheat.

Durum wheat is strongly affected by climatic constraints such as high temperatures and drought, which frequently lead to yield reduction. Damages due to high temperatures are related to plant thermotolerance, a trait determined by two components: basal and acquired thermotolerance. In this study, the effect of drought and heat stress imposed singularly or sequentially was investigated in ten durum wheat cultivars (cvs) at the physiological and molecular level. The traits analyzed were cell membrane stability, relative water content, proline content, and expression level of several genes for heat shock proteins (HSPs). Our results indicate that drought priming can induce the acquisition of thermotolerance in most cultivars already classified as able to acquire thermotolerance by heat pre-treatment. Proline accumulation was correlated to cell membrane stability, meaning that the most thermotolerant cvs were able to accumulate higher levels of proline. Acquired thermotolerance is also due to the activation of HSP gene expression; similarly, pre-treatment with water stress was able to activate HSPs expression. The results reported indicate that water stress plays an important role in inducing thermotolerance, comparable to mild heat stress pre-treatment. This is the first report on the effect of drought stress on the acquisition of thermotolerance.

Analysis of the Phytochemical Composition of Pomegranate Fruit Juices, Peels and Kernels: A Comparative Study on Four Cultivars Grown in Southern Italy.

The increasing popularity of pomegranate (Punica granatum L.), driven by the awareness of its nutraceutical properties and excellent environmental adaptability, is promoting a global expansion of its production area. This investigation reports the variability in the weight, moisture, pH, total soluble solids, carbohydrates, organic acids, phenolic compounds, fatty acids, antioxidant activities, and element composition of different fruit parts (juices, peels, and kernels) from four (Ako, Emek, Kamel, and Wonderful One) of the most widely cultivated Israeli pomegranate varieties in Salento (South Italy). To the best of our knowledge, this is the first systematic characterization of different fruit parts from pomegranate cultivars grown simultaneously in the same orchard and subjected to identical agronomic and environmental conditions. Significant genotype-dependent variability was observed for many of the investigated parameters, though without any correlation among fruit parts. The levels of phenols, flavonoids, anthocyanins, and ascorbic and dehydroascorbic acids of all samples were higher than the literature-reported data, as was the antioxidant activity. This is likely due to positive interactions among genotypes, the environment, and good agricultural practices. This study also confirms that pomegranate kernels and peels are, respectively, rich sources of punicic acid and phenols together, with several other bioactive molecules. However, the variability in their levels emphasizes the need for further research to better exploit their agro-industrial potential and thereby increase juice-production chain sustainability. This study will help to assist breeders and growers to respond to consumer and industrial preferences and encourage the development of biorefinery strategies for the utilization of pomegranate by-products as nutraceuticals or value-added ingredients for custom-tailored supplemented foods.

Progress towards Sustainable Control of Xylella fastidiosa subsp. pauca in Olive Groves of Salento (Apulia, Italy).

Xylella fastidiosa subsp. pauca is the causal agent of "olive quick decline syndrome" in Salento (Apulia, Italy). On April 2015, we started interdisciplinary studies to provide a sustainable control strategy for this pathogen that threatens the multi-millennial olive agroecosystem of Salento. Confocal laser scanning microscopy and fluorescence quantification showed that a zinc-copper-citric acid biocomplex-Dentamet®-reached the olive xylem tissue either after the spraying of the canopy or injection into the trunk, demonstrating its effective systemicity. The biocomplex showed in vitro bactericidal activity towards all X. fastidiosa subspecies. A mid-term evaluation of the control strategy performed in some olive groves of Salento indicated that this biocomplex significantly reduced both the symptoms and X. f. subsp. pauca cell concentration within the leaves of the local cultivars Ogliarola salentina and Cellina di Nardò. The treated trees started again to yield. A 1H-NMR metabolomic approach revealed, upon the treatments, a consistent increase in malic acid and γ-aminobutyrate for Ogliarola salentina and Cellina di Nardò trees, respectively. A novel endotherapy technique allowed injection of Dentamet® at low pressure directly into the vascular system of the tree and is currently under study for the promotion of resprouting in severely attacked trees. There are currently more than 700 ha of olive groves in Salento where this strategy is being applied to control X. f. subsp. pauca. These results collectively demonstrate an efficient, simple, low-cost, and environmentally sustainable strategy to control this pathogen in Salento.

Ride to cell wall: Arabidopsis XTH11, XTH29 and XTH33 exhibit different secretion pathways and responses to heat and drought stress.

The xyloglucan endotransglucosylase/hydrolases (XTHs) are enzymes involved in cell wall assembly and growth regulation, cleaving and re-joining hemicellulose chains in the xyloglucan-cellulose network. Here, in a homologous system, we compare the secretion patterns of XTH11, XTH33 and XTH29, three members of the Arabidopsis thaliana XTH family, selected for the presence (XTH11 and XTH33) or absence (XTH29) of a signal peptide, and the presence of a transmembrane domain (XTH33). We show that XTH11 and XTH33 reached, respectively, the cell wall and plasma membrane through a conventional protein secretion (CPS) pathway, whereas XTH29 moves towards the apoplast following an unconventional protein secretion (UPS) mediated by exocyst-positive organelles (EXPOs). All XTHs share a common C-terminal functional domain (XET-C) that, for XTH29 and a restricted number of other XTHs (27, 28 and 30), continues with an extraterminal region (ETR) of 45 amino acids. We suggest that this region is necessary for the correct cell wall targeting of XTH29, as the ETR-truncated protein never reaches its final destination and is not recruited by EXPOs. Furthermore, quantitative real-time polymerase chain reaction analyses performed on 4-week-old Arabidopsis seedlings exposed to drought and heat stress suggest a different involvement of the three XTHs in cell wall remodeling under abiotic stress, evidencing stress-, organ- and time-dependent variations in the expression levels. Significantly, XTH29, codifying the only XTH that follows a UPS pathway, is highly upregulated with respect to XTH11 and XTH33, which code for CPS-secreted proteins.

Actin and Microtubules Differently Contribute to Vacuolar Targeting Specificity during the Export from the ER.

Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins' functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton.

CesA6 and PGIP2 Endocytosis Involves Different Subpopulations of TGN-Related Endosomes.

Endocytosis is an essential process for the internalization of plasma membrane proteins, lipids and extracellular molecules into the cells. The mechanisms underlying endocytosis in plant cells involve several endosomal organelles whose origins and specific role needs still to be clarified. In this study we compare the internalization events of a GFP-tagged polygalacturonase-inhibiting protein of Phaseolus vulgaris (PGIP2-GFP) to that of a GFP-tagged subunit of cellulose synthase complex of Arabidopsis thaliana (secGFP-CesA6). Through the use of endocytic traffic chemical inhibitors (tyrphostin A23, salicylic acid, wortmannin, concanamycin A, Sortin 2, Endosidin 5 and BFA) it was evidenced that the two protein fusions were endocytosed through distinct endosomes with different mechanisms. PGIP2-GFP endocytosis is specifically sensitive to tyrphostin A23, salicylic acid and Sortin 2; furthermore, SYP51, a tSNARE with interfering effect on late steps of vacuolar traffic, affects its arrival in the central vacuole. SecGFP-CesA6, specifically sensitive to Endosidin 5, likely reaches the plasma membrane passing through the trans Golgi network (TGN), since the BFA treatment leads to the formation of BFA bodies, compatible with the aggregation of TGNs. BFA treatments determine the accumulation and tethering of the intracellular compartments labeled by both proteins, but PGIP2-GFP aggregated compartments overlap with those labeled by the endocytic dye FM4-64 while secGFP-CesA6 fills different compartments. Furthermore, secGFP-CesA6 co-localization with RFP-NIP1.1, marker of the direct ER-to-Vacuole traffic, in small compartments separated from ER suggests that secGFP-CesA6 is sorted through TGNs in which the direct contribution from the ER plays an important role. All together the data indicate the existence of a heterogeneous population of Golgi-independent TGNs.

Endomembrane Reorganization Induced by Heavy Metals.

Plant cells maintain plasmatic concentrations of essential heavy metal ions, such as iron, zinc, and copper, within the optimal functional range. To do so, several molecular mechanisms have to be committed to maintain concentrations of non-essential heavy metals and metalloids, such as cadmium, mercury and arsenic below their toxicity threshold levels. Compartmentalization is central to heavy metals homeostasis and secretory compartments, finely interconnected by traffic mechanisms, are determinant. Endomembrane reorganization can have unexpected effects on heavy metals tolerance altering in a complex way membrane permeability, storage, and detoxification ability beyond gene's expression regulation. The full understanding of endomembrane role is propaedeutic to the comprehension of translocation and hyper-accumulation mechanisms and their applicative employment. It is evident that further studies on dynamic localization of these and many more proteins may significantly contribute to the understanding of heavy metals tolerance mechanisms. The aim of this review is to provide an overview about the endomembrane alterations involved in heavy metals compartmentalization and tolerance in plants.

Bioactive composition and sensory evaluation of innovative spaghetti supplemented with free or α-cyclodextrin chlatrated pumpkin oil extracted by supercritical CO2.

The feasibility of producing durum wheat pasta enriched with a lipophilic phytocomplex, extracted using supercritical carbon dioxide (SC-CO2), from ripe pumpkin, as free oil or as ready-to-mix oil/α-cyclodextrins (α-CDs) powder, was explored. Four types of pasta were prepared: (i) control spaghetti (S-CTRL); (ii) spaghetti supplemented with α-CDs (S-α-CD); (iii) spaghetti supplemented with pumpkin oil (S-Oil) and (iv) spaghetti supplemented with the pumpkin oil/α-CD powder (S-Oil/α-CD). The chemical, antioxidant, textural and sensory attributes of the different pasta were evaluated and compared. S-Oil and S-Oil/α-CD spaghetti were significantly enriched with phytosterols, squalene, carotenoids, tocochromanols and unsaturated fatty acids. Spaghetti containing α-CDs were slightly improved in terms of fiber content. Oil chlatration increased the stability of some bioactives during pasta production and ameliorated poor textural and sensory characteristics of the cooked spaghetti compared with S-Oil sample. S-Oil/α-CD spaghetti might be accepted by customers, if the potential health benefits were also explained.

Td4IN2: A drought-responsive durum wheat (Triticum durum Desf.) gene coding for a resistance like protein with serine/threonine protein kinase, nucleotide binding site and leucine rich domains.

Wheat, the main food source for a third of world population, appears strongly under threat because of predicted increasing temperatures coupled to drought. Plant complex molecular response to drought stress relies on the gene network controlling cell reactions to abiotic stress. In the natural environment, plants are subjected to the combination of abiotic and biotic stresses. Also the response of plants to biotic stress, to cope with pathogens, involves the activation of a molecular network. Investigations on combination of abiotic and biotic stresses indicate the existence of cross-talk between the two networks and a kind of overlapping can be hypothesized. In this work we describe the isolation and characterization of a drought-related durum wheat (Triticum durum Desf.) gene, identified in a previous study, coding for a protein combining features of NBS-LRR type resistance protein with a S/TPK domain, involved in drought stress response. This is one of the few examples reported where all three domains are present in a single protein and, to our knowledge, it is the first report on a gene specifically induced by drought stress and drought-related conditions, with this particular structure.

Three Pectin Methylesterase Inhibitors Protect Cell Wall Integrity for Arabidopsis Immunity to Botrytis.

Infection by necrotrophs is a complex process that starts with the breakdown of the cell wall (CW) matrix initiated by CW-degrading enzymes and results in an extensive tissue maceration. Plants exploit induced defense mechanisms based on biochemical modification of the CW components to protect themselves from enzymatic degradation. The pectin matrix is the main CW target of Botrytis cinerea, and pectin methylesterification status is strongly altered in response to infection. The methylesterification of pectin is controlled mainly by pectin methylesterases (PMEs), whose activity is posttranscriptionally regulated by endogenous protein inhibitors (PMEIs). Here, AtPMEI10, AtPMEI11, and AtPMEI12 are identified as functional PMEIs induced in Arabidopsis (Arabidopsis thaliana) during B. cinerea infection. AtPMEI expression is strictly regulated by jasmonic acid and ethylene signaling, while only AtPMEI11 expression is controlled by PME-related damage-associated molecular patterns, such as oligogalacturonides and methanol. The decrease of pectin methylesterification during infection is higher and the immunity to B. cinerea is compromised in pmei10, pmei11, and pmei12 mutants with respect to the control plants. A higher stimulation of the fungal oxalic acid biosynthetic pathway also can contribute to the higher susceptibility of pmei mutants. The lack of PMEI expression does not affect hemicellulose strengthening, callose deposition, and the synthesis of structural defense proteins, proposed as CW-remodeling mechanisms exploited by Arabidopsis to resist CW degradation upon B. cinerea infection. We show that PME activity and pectin methylesterification are dynamically modulated by PMEIs during B. cinerea infection. Our findings point to AtPMEI10, AtPMEI11, and AtPMEI12 as mediators of CW integrity maintenance in plant immunity.

Drought and Heat Differentially Affect XTH Expression and XET Activity and Action in 3-Day-Old Seedlings of Durum Wheat Cultivars with Different Stress Susceptibility.

Heat and drought stress have emerged as major constraints for durum wheat production. In the Mediterranean area, their negative effect on crop productivity is expected to be exacerbated by the occurring climate change. Xyloglucan endotransglucosylase/hydrolases (XTHs) are chief enzymes in cell wall remodeling, whose relevance in cell expansion and morphogenesis suggests a central role in stress responses. In this work the potential role of XTHs in abiotic stress tolerance was investigated in durum wheat. The separate effects of dehydration and heat exposure on XTH expression and its endotransglucosylase (XET) in vitro activity and in vivo action have been monitored, up to 24 h, in the apical and sub-apical root regions and shoots excised from 3-day-old seedlings of durum wheat cultivars differing in stress susceptibility/tolerance. Dehydration and heat stress differentially influence the XTH expression profiles and the activity and action of XET in the wheat seedlings, depending on the degree of susceptibility/tolerance of the cultivars, the organ, the topological region of the root and, within the root, on the gradient of cell differentiation. The root apical region was the zone mainly affected by both treatments in all assayed cultivars, while no change in XET activity was observed at shoot level, irrespective of susceptibility/tolerance, confirming the pivotal role of the root in stress perception, signaling, and response. Conflicting effects were observed depending on stress type: dehydration evoked an overall increase, at least in the apical region of the root, of XET activity and action, while a significant inhibition was caused by heat treatment in most cultivars. The data suggest that differential changes in XET action in defined portions of the root of young durum wheat seedlings may have a role as a response to drought and heat stress, thus contributing to seedling survival and crop establishment. A thorough understanding of the mechanisms underlying these variations could represent the theoretical basis for implementing breeding strategies to develop new highly productive hybrids adapted to future climate scenarios.

Α-Cyclodextrin encapsulation of supercritical CO2 extracted oleoresins from different plant matrices: A stability study.

Here we describe the encapsulation in α-cyclodextrins (α-CDs) of wheat bran, pumpkin and tomato oleoresins, extracted by supercritical carbon dioxide, to obtain freeze-dried powders useful as ready-to-mix ingredients for novel functional food formulation. The stability of tocochromanols, carotenoids and fatty acids in the oleoresin/α-CD complexes, compared to the corresponding free oleoresins, was also monitored over time in different combinations of storage conditions. Regardless of light, storage at 25°C of free oleoresins determined a rapid decrease in carotenoids, tocochromanols and PUFAs. α-CD encapsulation improved the stability of most bioactive compounds. Storage at 4°C synergized with encapsulation in preventing degradation of bioactives. Unlike all other antioxidants, lycopene in tomato oleoresin/α-CD complex resulted to be more susceptible to oxidation than in free oleoresin, likely due to its selective sequestration from the interaction with other lipophilic molecules of the oleoresin.

Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1-10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal.

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.

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 ß -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.