Plant SNAREs encoded by genes of the same subfamily are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless the application of innovative experimental approaches highlighted that members of the same gene subfamily often have different functional specificities. In this work two closely related Qc-SNAREs, the AtSYP51 and the AtSYP52 are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently over-expressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs (i-SNAREs) when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 role differ in the traffic to the vacuole. The findings are a novel contribution for the functional characterization of plant SNAREs that reveal additional non-fusogenic roles.

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 is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion (Krause et al., 2013) thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of protoplasts derived from Artemisia suspension cultures and explore a novel strategy based on compartmentalization engineering using a modified SNARE protein as molecular tools. We used several fluorescent markers to visualize the vacuolar organization in vivo and a truncated form of AtSYP51, 51H3, to induce a compartment generated by the contribution of endocytosis and ER-to-Vacuole traffic. 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 (De Benedictis et al. 2013), and new hits to engineer eukaryote endomembranes for future biotechnological applications. De Benedictis, M., Bleve, G., Faraco, M., Stigliano, E., Grieco, F., Piro, G., Dalessandro, G., Di Sansebastiano, G.P., 2013. AtSYP51/52 functions diverge in the Post-Golgi traffic and differently affect vacuolar sorting. Mol Plant. 6, 916–930. Krause, C., Richter, S., Knöll, C., Jürgens, G., 2013. Plant secretome - from cellular process to biological activity. Biochim Biophys Acta. 1834, 2429–2441.

The immediate visual comparison of platinum chemotherapeutics' effects in eukaryotic cells using accessible plant models of transgenic Arabidopsis thaliana is reported. The leading anticancer drug cisplatin, a third generation drug used for colon cancer, oxaliplatin and kiteplatin, promising Pt-based anticancer drugs effective against resistant lines, were administered to transgenic A. thaliana plants monitoring their effects on cells from different tissues. The transgenic plants' cell cytoskeletons were labelled by the green fluorescent protein (GFP)-tagged microtubule-protein TUA6 (TUA6-GFP), while the vacuolar organization was evidenced by two soluble chimerical GFPs (GFPChi and AleuGFP) and one transmembrane GFP-tagged tonoplast intrinsic protein 1-1 (TIP1.1-GFP). The three drugs showed easily recognizable effects on plant subcellular organization, thereby providing evidence for a differentiated drug targeting. Genetically modified A. thaliana are confirmed as a possible rapid and low-cost screening tool for better understanding the mechanism of action of human anticancer drugs.

SNAREs (N-ethylmaleimide-sensitive factor adaptor protein receptors) are small polypeptides characterized by a particular domain, the SNARE motif, that can form a coiled-coil structure. Via hetero-oligomeric interactions, these proteins form highly stable protein-protein interactions, the so called SNARE-complex, that allow membrane fusion. SNAREs also interact with several proteins acting as regulators of SNARE-complex formation. Stoichiometry of these proteins reveals that they are more abundant than required for membrane traffic. Indeed their function appears to be more diversified. It was shown (1) that they may assemble to form non-fusogenic complexes acting as interfering-SNAREs or iSNARE (2) as in the case of AtSYP51 and SYP52. It was also shown that plasma membrane SNAREs can be phosphorylated as part of the signaling cascade elicited by interaction with microorganisms or hormonal stimulation and that they influence turnover of channels. In particulat Grefen and co-workers (3; 4) provided direct evidence that SYP121 is part of a scaffold of proteins associated, by direct interaction with channel KAT1, with the membrane transport of K+. In fact, few SNARE proteins are known to interact with ion channels, notably mammalian Syntaxin 1A, which binds several different Ca2+ and K+ channels in nerves. Here we show that AtSYP51 interacts directly with a non-SNARE protein, AtNLM1, probably regulating autophagocytosis processes. Aknowledgement The authors thank the contribution of the Italian project ‘Reti di Laboratori Pubblici di Ricerca per la Selezione, Caratterizzazione e Conservazione di Germoplasma 2009’. 1) De Benedictis M, Bleve G, Faraco M, Stigliano S, Grieco F, Piro G, Dalessandro G, Di Sansebastiano GP. (2013) AtSYP51/52 Functions Diverge in the Post-Golgi Traffic and Differently Affect Vacuolar Sorting, Mol. Plant 6(3): 916-930. 2) Di Sansebastiano GP. (2013) Defining new SNARE functions: the i-SNARE. Front Plant Sci. 4: 99. 3) Grefen C, Blatt MR. (2008) SNAREs--molecular governors in signalling and development. Curr Opin Plant Biol. 11(6): 600-9. 4) Grefen C, Chen Z, Honsbein A, Donald N, Hills A, Blatt MR. (2010) A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis. Plant Cell. 22(9): 3076-92.

n this work a process for obtaining high vitamin E and carotenoid yields by supercritical carbon dioxide (SC-CO2) extraction from pumpkin (Cucurbita moschata Duch.) is described. The results show that the use of a vacuum oven-dried [residual moisture (∼8%)] and milled (70 mesh sieve) pumpkin flesh matrix increased SC-CO2 extraction yields of total vitamin E and carotenoids of ∼12.0- and ∼8.5-fold, respectively, with respect to the use of a freeze-dried and milled flesh matrix. The addition of milled (35 mesh) pumpkin seeds as co-matrix (1:1, w/w) allowed a further ∼1.6-fold increase in carotenoid yield, besides to a valuable enrichment of the extracted oil in vitamin E (274 mg/100 g oil) and polyunsaturated fatty acids. These findings encourage further studies in order to scale up the process for possible industrial production of high quality bioactive ingredients from pumpkin useful in functional food or cosmeceutical formulation.

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.

This study reports quali-quantitative analyses on isoprenoids, phospholipids, neutral lipids, phytosterols, and proteins in purified plastids isolated from fresh fruits of traditional (Donald and Incas) and high-pigment (Kalvert and HLY-18) tomato cultivars at four ripening stages. In all of the investigated cultivars, lycopene, beta-catotene, lutein, and total carotenoids varied significantly during ripening. Chromoplasts of red-ripe tomato fruits of high-pigment cultivars accumulated twice as much as lycopene (307.6 and 319.2 mu g/mg of plastid proteins in Kalvert and HLY-18, respectively) than ordinary cultivars (178.6 and 151.7 mu g/mg of plastid proteins in Donald and Incas, respectively); differences in chlorophyll and a-tocopherol contents were also evidenced. Phospholipids and phytosterols increased during ripening, whereas triglycerides showed a general decrease. Regardless of the stage of ripening, palmitic acid was the major fatty acid in all cultivars (ranging from 35 to 52% of the total fatty acids), followed by stearic, oleic, linoleic, linolenic, and myristic acids, but their relative percentage was affected by ripening. Most of the bands detected on the SDS-PAGEs of plastid proteins were constantly present during chloroplast-to-chromoplast conversion, some others disappeared, and only one, with a molecular weight of similar to 41.6 kDa, was found to increase in intensity.

ABSTRACT - The vacuolar sap epifluorescence as a quality parameter of fruits and vegetables.- Phenolic compounds play a major role in the interaction of plants with their environment. Tomatoes are an important part of the human diet because they supply essential nutrients such as vitamins and minerals and they are also considered important to human health and well-being because they contain other necessary compounds such as antioxidants. Consumers are concerned about the quality of the fruits they eat and the quality is linked to the richness in antioxidant compounds. In this work, phenolic antioxidant compounds localization has been studied by using S. lycopersicum fruits as experimental system. Metabolites accumulation in the vacuole has been revealed by using confocal laser scanning microscopy. In fact, CLSM provide the opportunity to study tissue localization of phenolic compounds thanks to their epifluorescence. Moreover, CLSM aids in the identification of chemical components using their specific fluorescence characteristics, on the basis of their absorbance and emission behavior.

This work demonstrates that lipid-detergent mixed micelles can be employed successfully in order to achieve and modulate the transfer of bio-active hydrophobic compounds into lipid carriers by means of a simple and bio-safe procedure. In our specific investigation, liposome preparations incorporating mixtures of natural carotenoids with high lycopene content were developed and characterized, aiming to obtain formulations of potential nutraceutical and pharmaceutical interest. The starting material was a solvent-free high-quality lycopene rich oleoresin (LRO) obtained by extracting a freeze-dried tomato matrix with supercritical carbon dioxide (SC-CO2). Mixed micelles containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholate were loaded with LRO antioxidants by means of two slightly different procedures, which surprisingly resulted in significant differences in both quality and quantity of incorporated carotenoids. In particular, the selective incorporation of (all-E)-lycopene was achieved by extracting the oleoresin with a pre-formed cholate/POPC micelle suspension whilst (Z)-isomers were preferentially integrated when treating a POPC/LRO mixed film with cholate. The micelle to vesicle transition (MVT) method was employed in order to produce vesicles of well-defined lamellarity and size. Visible and infrared (IR) spectroscopy as well as Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) measurements allowed the extensive characterization of LRO-loaded micelles and liposomes. The antioxidant potential of preparations was assessed by measuring the radical scavenging activity towards the coloured radical cation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS). Important information about the reliability of different approaches for antioxidant capacity evaluation of micelle and liposome preparations was gained and the successful incorporation of LRO antioxidant power in a bio-deliverable water-dispersed form was demonstrated.

NIP1;1 (Nod 26 like Intrinsic Protein 1;1) is an aquaglyceroporin involved in transport and tolerance to As(III) and regulated negatively by ABA, NaCl, dark and dry stress. It is a MIP (Major Intrinsic Proteins) as many others transmembrane proteins collectively named aquaporins, involved in regulation of the cell’s homeostasis. The cDNAs cluster into three groups. The tonoplast aquaporins (TIPs) and the plasma membrane aquaporins (PIPs) form large groups, whereas NIPs remain few, distinct from both TIPs and PIPs. Aquaporins have a predicted topology with six membrane-spanning domains connected by five loops and short N-terminal and C-terminal domains in the cytoplasm. NIP1;1 has a hydrophilic N-terminal tail with approximately the same charges found in PIPs but the C-terminal tail has two negative charges and is closer to plant TIPs. Unique to the NIP1;1 subgroup is a highly conserved Cys residue in, or close to, the end of the first putative transmembrane domain. It is interesting that the aminoacidic motif NPA (asparagine-proline-alanine), characteristic for the MIP family, is not fully conserved in NIP1;1. The NPA motif is replaced by NPG (Asn-Pro-Gly) in NIP1;1. Kamiya and co-workers reported to have localized GFP-NIP1;1 to the plasma membrane but their images could be questioned. In fact no co-localization experiments were performed and the distribution of fluorescence was not sharp and limited to the plasma membrane but distributed in the cell protoplasma (Kamiya et al., 2009). We described for AtNIP1;1 a more complex distribution using an identical GFP-tagged chimera. We observed GFP-NIP1;1 in the endoplasmic reticulum and in well defined donut-like structures that could be described as karmellae but of which we started the characterization. A bioinformatics analysis on network interaction data, evidenced a direct interaction between NIP1;1 and some SNAREs among which is found SYP51, a Qc SNARE involved in vacuolar transport, exocytosis and endocytosis (De Benedictis et al., 2013). The closely related Qc SNARE SYP52, with an high level of homology to SYP51, did not interact with NIP1;1. We have investigated in vivo if NIP 1.1 interact with SYP51/52 trough ratiometric bimolecular fluorescence complementation (rBiFC) (Grefen and Blatt, 2012) using appropriate controls. The experiments confirmed the specific interaction between NIP1;1 and SYP51. Essential bibliography: De Benedictis, M., Bleve, G., Faraco, M., Stigliano, E., Grieco, F., Piro, G., Dalessandro, G., and Di Sansebastiano, G. Pietro (2013). AtSYP51/52 functions diverge in the post-Golgi traffic and differently affect vacuolar sorting. Mol. Plant 6: 916–30. Grefen, C. and Blatt, M.R. (2012). A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC). Biotechniques 53: 311–14. Kamiya, T., Tanaka, M., Mitani, N., Ma, J.F., Maeshima, M., and Fujiwara, T. (2009). NIP1;1, an aquaporin homolog, determines the arsenite sensitivity of Arabidopsis thaliana. J. Biol. Chem. 284: 2114–20.

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.

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.

We investigated the effect of auxin and acetylcholine on the expression of the tomato expansin gene LeEXPA2, a specific expansin gene expressed in elongating tomato hypocotyl segments. Since auxin interferes with clathrin-mediated endocytosis, in order to regulate cellular and developmental responses we produced protoplasts from tomato elongating hypocotyls and followed the endocytotic marker, FM4-64, internalization in response to treatments. Tomato protoplasts were observed during auxin and acetylcholine treatments after transient expression of chimerical markers of volume-control related compartments such as vacuoles. Here we describe the contribution of auxin and acetylcholine to LeEXPA2 expression regulation and we support the hypothesis that a possible subcellular target of acetylcholine signal is the vesicular transport, shedding some light on the characterization of this small molecule as local mediator in the plant physiological response.

Lycopene is used for several industrial applications. Supercritical CO2 (SC-CO2) 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-CO2-extracted oleoresin containing lycopene.

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.

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.

Tribulus terrestris L. (Zygophyllaceae) is an annual plant commonly known as Puncture vine. It is dramatically gaining interest as a rich source of saponins. T. terrestris is a promising ingredient for many industries and recent patents on dermatological applications support the use of this plant for cosmetics and hygiene. Nonetheless problems arise in the selection of the material to be used. The extracts of different origins may differ substantially. Natural speciation processes normally influence ‘variations’ in wild-crafted medicinal plants. The genus Tribulus is emblematic. Taxonomic status of T. terrestris is complicated by the wide geographical distribution leading to high levels of genetic polymorphism. Being aware of such variability we selected 3 commercial Tribulus extracts and compared their biological effect on Candida albicans with the effect produced by an extract from local plants (South of Apulia, Italy). One of the commercial extracts with the best anti-candida performance was used to substitute triclosan in a detergent formulation and it proved to improve the product performance in the control of potentially pathogenic skin flora such as C. albicans.

Le proteine SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) sono i principali determinanti della specificità nei processi di fusione di una vescicola al suo compartimento accettore; la loro caratteristica è quella di possedere il cosiddetto dominio SNARE con cui interagiscono tra loro. Recentemente sono emersi dati che indicano un loro ruolo nella definizione dell’identità delle membrane, che va oltre l’evento di fusione. In particolare due Qc-SNARE della famiglia genica delle SYP5 di Arabidopsis thaliana (AtSYP51 e AtSYP52) hanno manifestato capacità di agire come i-SNARE (SNARE inibitorie). Queste SNARE rappresentano una nuova classe funzionale i cui componenti inibiscono la fusione mediante sostituzione o legame ad una subunità di un complesso SNARE incompleto, formando un complesso non-fusogenico (Di Sansebastiano, 2013). È stato dimostrato in precedenza che le due proteine della famiglia SYP5 localizzano sul tonoplasto e su endosomi/TGN svolgendo ruoli diversi nelle due localizzazioni; in particolare sembra abbiano un effetto i-SNARE sul tonoplasto (De Benedictis et al., 2013). In questo lavoro sono stati prodotti e caratterizzati diversi costrutti chimerici formati dai domini Qc-SNARE (H3) di AtSYP51 e AtSYP52 isolati o fusi ad un tag fluorescente (GFP o RFP). Tali costrutti espressi in cotiledoni di Arabidopsis thaliana e in foglie di Nicotiana tabacum, hanno mostrato una distribuzione caratteristica di ciascun gene. GFP51H3 viene deviata verso gli endosomi di riciclo, dove co-localizza con marcatori della membrana plasmatica; GFP52H3 si ferma nei Trans-Golgi-Network. Utilizzando marker di secrezione e di ritenzione al vacuolo di tipo enzimatico e fluorescente, è stata verificata la persistenza di un effetto i-SNARE anche dovuto alla sola presenza del dominio Qc. Approfondimenti sulle interazioni di queste proteine supportano l’idea che l’effetto i-SNARE si esplichi attraverso interazioni del dominio Qc-SNARE con partner proteici non-SNARE, aprendo interessanti prospettive riguardo il mantenimento della compartimentazione cellulare. 1) G.P. Di Sansebastiano (2013) Front Plant Sci., 4: 99. 2) M. De Benedictis, G. Bleve, M. Faraco, E. Stigliano, F. Grieco, G. Piro, G. Dalessandro, G.P. Di Sansebastiano (2013) Mol Plant. 6(3): 916-30.

Abstract: Strain SPC-1T was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide and 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-1T clustered together with species of the genus Sphingomonas sensu strictu. 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, and 2-hydroxymiristic acid (C14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomical position. 16S rRNA gene sequence analysis showed that SPC-1 was most closely related to Sphingomonas hankookensis ODN7T, Sphingomonas insulae DS-28T and Sphingomonas panni C52T (98.19%, 97.91% and 97.11% similarity, 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-1T (JCM 17498; ITEM 13494) represents a new species of Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed. A component analysis of the exopolysaccharide (named SPC-1T EPS) 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.

Writing supports are exceptional archeological finds. The interest in the written texts is such that usually it overshadows the nature of the object itself. Some well-diversified materials like parchment or papyrus, find their true place within their historical context but others, like wooden tablets, are more neglected and rarely analyzed in depth. In this work we have analyzed through LSM and ESEM microscopy samples from two wooden supports from Egypt, dated to the Roman Period and exposed at the Museo Papirologico of the University of Salento, in Lecce, directed by Mario Capasso, professor of Papyrology in the same University. Preliminary microscopic analysis revealed that these tablets are not made of wood, intended as a portion of secondary xylem, but are the result of complex fabrication processes. In one case the material consists of wood but it presents evidence of extended manipulations; in the second case the tablet apparently made of wood is in fact made of compressed fibers. In both cases a specific and unknown technology was used to produce these materials. The existence of dedicated technologies to produce woody writing supports from ligno-cellulosic materials is not well documented in the different historical periods. The need of specific technologies to produce large amounts of writing supports without the direct consumption of good quality wood was evidently stronger in those environmental conditions in which hard wood was imported and probably expensive. It was the case of the ancient Egypt, the area of recovery of the analyzed samples. More attention has to be paid to the reconstruction of the ancient technologies to produce ligno-cellulosic materials that appear striking modern and provide information on the value given to the written documentation and writing in general in the studied historical context.

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.

Support of confocal microscopy to the preliminary assessment of archaeological wood samples.- Each wooden artifact represents a specific case of study and the multi-disciplinary diagnostic analysis to be performed always vary. The first step usually consists in carrying out a macroscopic morphological investigation, possibly in situ, especially if the sample is very large. Then it is opportune to run the xilotomic analysis through microscopic examination of a small sample. For some samples, especially the archaeological remains, the limits to the availability of fragments to be analyzed induces to proceed directly to chemical and molecular expensive analysis even without a preliminary microscopic observeation, or the scanning electron microscope is used directly. To quickly determine whether the object of investigation is really wood allows programming the correct analysis, with significant benefits. This work demonstrates the effectiveness of a rapid microscopic investigation performed with the laser scanning confocal microscope. This tool allows a highly informative, easy and low cost microscopic examination.

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.

Due to the numerous roles plant vacuoles play in cell homeostasis, detoxification, and protein storage, the trafficking pathways to this organelle have been extensively studied. Recent evidence, however, suggests that our vision of transport to the vacuole is not as simple as previously imagined. Alternative routes have been identified and are being characterized. Intricate interconnections between routes seem to occur in various cases, complicating the interpretation of data. In this review, we aim to summarize the published evidence and link the emerging data with previous findings. We discuss the current state of information on alternative and classical trafficking routes to the plant vacuole.

In this work we explored the possibility of using genetically modified Arabidopsis thaliana plants as a rapid and low-cost screening tool for evaluating human anticancer drugs action and efficacy. Here, four different inhibitors with a validated anticancer effect in humans and distinct mechanism of action were screened in the plant model for their ability to interfere with the cytoskeletal and endomembrane networks. We used plants expressing a green fluorescent protein (GFP) tagged microtubule-protein (TUA6-GFP), and three soluble GFPs differently sorted to reside in the endoplasmic reticulum (GFPKDEL) or to accumulate in the vacuole through a COPII dependent (AleuGFP) or independent (GFPChi) mechanism. Our results demonstrated that drugs tested alone or in combination differentially influenced the monitored cellular processes including cytoskeletal organization and endomembrane trafficking. In conclusion, we demonstrated that A. thaliana plants are sensitive to the action of human chemotherapeutics and can be used for preliminary screening of drugs efficacy. The cost-effective subcellular imaging in plant cell may contribute to better clarify drugs subcellular targets and their anticancer effects.

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 (NtSar1h74I) confirmed that GFPChi transport from the ER to the vacuole is not fully dependent on the Golgi apparatus. (C) 2013 Elsevier Masson SAS. All rights reserved.

Glyphosate is a non-selective herbicide that inhibits the shikimate pathway’s enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) preventing the production of aromatic amino acids. This herbicide is largely used and appreciated because it controls a wide range of annual and perennial weeds but it has a minimal environmental impact when compared with other herbicides. Initially it was thought that resistance to glyphosate was not easy to evolve but the continuous applications, as it happened for other herbicides, have induced the development of several glyphosate-resistant weeds. Glyphosate resistance can be developed as target-site and non-target-site mechanisms. In the target-site mechanism of resistance, either a mutation on the EPSPS enzyme (enzyme modification) or the overexpression of the EPSPS enzyme have been found to confer resistance. In the non-target-site mechanism of glyphosate resistance, the herbicide translocation and neutralization is observed. Pumping glyphosate into vacuoles via membrane transporters has been suggested as a possible process involved in the restricted glyphosate translocation. As a consequence, a different vacuolar organization or plasticity could be an interesting character or marker to correlate to glyphosate resistance. Vacuolar markers AleuGFP (Sar1 dependent sorting) or GFPChi (Sar1 independent sorting) respectively can be used to monitor independent vacuolar sorting mechanisms during glyphosate induced stress. We observed that the adaptive reaction of tobacco protoplasts vacuolar system to the treatment with glyphosate, can be mimicked by the overexpression of a Triticum durum TdGST gene. Previous analysis evidenced that the herbicide glyphosate increased TdGST expression, confirming the role of GST in the protection against xenobiotics. Non-target-site glyphosate resistance mechanisms may correlate with an independent regulation of cell compartmentalization and herbicide induced genes may have a direct effect on it.

Xylella fastidiosa is a Gram-negative, xylem-limited, bacterium which is responsible, in Italy, for the olive quick decline syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce’s disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In this study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analysing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars “Ogliarola salentina” and “Cellina di Nardò,” and the tolerant cultivar “Leccino.” Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.

Xylella outbreak in Puglia is ravaging olive plants and research is trying different paths moving through serious social and practical difficulties. The laboratory of Botany in Lecce, in collaboration with CNR ISPA local section, University of Firenze and University of Neuchatel supports the scientific community effort investigating the Olea europea xylem lumen through microscopic analysis. This apoplastic space is the channel through which the pathogen may be targeted by drugs but also a battleground on which the plant itself deploys its natural defensive barriers. In particular woody plants are able to selectively block the traffic through vessels with an active process known as tylose. It is known that tylose impairs water transport during the development of Pierce's disease (PD), caused by Xylella fastidiosa in grape. The cavitation-induced embolisms of xylem due to the bacterial colonization appears to develop before tyloses (Perez-Donoso et al 2016), anyhow both embolisms and tyloses develop with the same pattern and correlate to symptoms. Scanning Electron Microscope observations performed in the xylem of olive plants allowed the identification of different forms of xylem defects, from tylose to biotic obstructions and allowed the correct interpretation of vessels obstructions visualized at low magnification by optical microscopy and confocal microscopy. A visual screening of sections from branches of the three most representative Olea europea cultivars present in the infected area of South Puglia, Cellina di Nardò, Leccino and Ogliarola salentina, was performed. The results will be discussed. Essential bibliography Perez-Donoso, A. G., Lenhof, J. J., Pinney, K., Labavitch, J. M. (2016) Vessel embolism and tyloses in early stages of Pierce's disease. AUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH Volume: 22 Issue: 1 Pages: 81-86