Tomato Rab11a Characterization Evidenced a Difference Between SYP121-Dependent and SYP122-Dependent exocytosis

The regulatory functions of Rab proteins in membrane trafficking lie in their ability to perform as molecular switches that oscillate between a GTP- and a GDP-bound conformation. The role of tomato LeRab11a in secretion was analyzed in tobacco protoplasts. Green fluorescent protein (GFP)/red fluorescent protein (RFP)-tagged LeRab11a was localized at the trans-Golgi network (TGN) in vivo. Two serines in the GTP-binding site of the protein were mutagenized, giving rise to the three mutants Rab11S22N, Rab11S27N and Rab11S22/27N. The double mutation reduced secretion of a marker protein, secRGUS (secreted rat β-glucuronidase), by half, whereas each of the single mutations alone had a much smaller effect, showing that both serines have to be mutated to obtain a dominant negative effect on LeRab11a function. The dominant negative mutant was used to determine whether Rab11 is involved in the pathway(s) regulated by the plasma membrane syntaxins SYP121 and SYP122. Co-expression of either of these GFP-tagged syntaxins with the dominant negative Rab11S22/27N mutant led to the appearance of endosomes, but co-expression of GFP-tagged SYP122 also labeled the endoplasmic reticulum and dotted structures. However, co-expression of Rab11S22/27N with SYP121 dominant negative mutants decreased secretion of secRGUS further compared with the expression of Rab11S22/27N alone, whereas co-expression of Rab11S22/27N with SYP122 had no synergistic effect. With the same essay, the difference between SYP121- and SYP122-dependent secretion was then evidenced. The results suggest that Rab11 regulates anterograde transport from the TGN to the plasma membrane and strongly implicate SYP122, rather than SYP121. The differential effect of LeRab11a supports the possibility that SYP121 and SYP122 drive independent secretory event

optimization of enzymatic synthesis of l arabinose ferulate catalyzed by feruloyl esterases from myceliophthora thermophila in detergentless microemulsions and assessment of its antioxidant and cytotoxicity activities

The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferula ..

New evidences on efficacy of boronic acid-based derivatization method to identify sugars in plant material by gas chromatography-mass spectrometry

This work presents an analytical procedure based on gas chromatography-mass spectrometry which allows the determination of aldoses (glucose, mannose, galactose, arabinose, xylose, fucose, rhamnose) and chetoses (fructose) in plant material. One peak for each target carbohydrate was obtained by using an efficient derivatization employing methylboronic acid and acetic anhydride sequentially, whereas the baseline separation of the analytes was accomplished using an ionic liquid capillary column. First, the proposed method was optimized and validated. Successively, it was applied to identify the carbohydrates present in plant material. Finally, the procedure was successfully applied to samples from a XVII century painting, thus highlighting the occurrence of starch glue and fruit tree gum as polysaccharide materials

Expression of a glycosylated GFP as a bivalent reporter in exocytosis

The complex-type N-linked glycans of plants differ markedly in structure from those of animals. Like those of insects and mollusks they lack terminal sialic acid(s) and may contain an α-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a β-(1,2)-xylose (Xyl) residue attached to the proximal mannose (Man) of the glycan core. N-glycosylated GFPs were used in previous studies showing their effective use to report on membrane traffic between the ER and the Golgi apparatus in plant cells. In all these cases glycosylated tags were added at the GFP termini. Because of the position of the tag and depending on the sorting and accumulation site of these modified GFP, there is always a risk of processing and degradation, and this protein design cannot be considered ideal. Here, we describe the development of three different GFPs in which the glycosylation site is internally localized at positions 80, 133, or 172 in the internal sequence. The best glycosylation site was at position 133. This glycosylated GFPgl133 appears to be protected from undesired processing of the glycosylation site and represents a bivalent reporter for biochemical and microscopic studies. After experimental validation, we can conclude that amino acid 133 is an effective glycosylation site and that the GFPgl133 is a powerful tool for in vivo investigations in plant cell biology

SPECIFICITA’ FUNZIONALE DELLE Qc-SNARE SYP51 e SYP52

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

Elucidation of Rituals and Food in the 'Cult Area' of Roca Through Chemical Analyses

The application of analytical chemistry to archaeological research has increased substantially over the last half-century and today represents a major methodological subfield within archaeological science. The paper reports the results obtained onto a selection of Final Bronze Age ceramic materials from Bronze Age site of Roca. Two pithoi in fine fabric -figulina- and five vessels in coarse fabric -impasto- (one olla, one dipper, three small cups) have been selected and analyzed. Regarding the archaeological contexts of provenience, one pithos comes from the so called Capanna-Magazzino located in SAS VI and all the other materials come from the South-East sector of the so called Capanna-Tempio located in SAS IX. Using high temperature gas chromatography and compound specific isotopic ratio, it has been possible to identify different types of lipids absorbed by the ceramic core. The pithos from the Capanna-Tempio shows residues of lipids clearly identified as olive oil. The olla and the dipper show residues of lipids referable to non-ruminant animals. One of the three small cups does not contain any traces of lipids but quantities of beeswax and some evidence of conifer's resins. Two cups contain residues of animal lipids that can be attributed to ruminant animals, particularly to sheep. The pithos from the Capanna-Tempio demonstrates processes of accumulation of huge quantities of olive oil in a ritual context characterized by precise comparisons with the Bronze Age Aegean. The traces of resin in the pithos from the Capanna-Magazzino could be interpreted both as results of surface treatment or as residues of various contents different from olive oil, such as flavored wine. The animal lipids found in the olla seem to confirm the use of this shape in food-preparing activities

Bitumen from archaeological pots of two Apulian Bronze Age settlements, Monopoli and Torre Santa Sabina: provenance and processing

Evidences of bitumen use were found in several Apulian Bronze Age coastal sites such as Otranto, Roca, Le Pazze (LE), Scoglio del Tonno (TA), Monopoli (BA) and Torre Santa Sabina (BR), but there are no archaeometric data about its source origin or use. Bitumen from archaeological potsherds collected from two Apulia excavations, Monopoli and Torre Santa Sabina, and geological samples from three different areas, Majella, Selenicë and Ragusa, were analysed by GC-MS/MS to assess the geographic origin of the archaeological bitumen. The distribution pattern of steranes and terpanes obtained from archaeological sample is very similar to those ofgeological samples collected from Selenicë.Archeological and Selenicë samples (SBI and SB2) are dominatedby C29 steranes (53–54%) followed by C27 steranes(26–33 %) and only minor proportions of C28 steranes(21%). This bitumen compositional family distinguished by having lower C27 regular steranes and higher C29 regular steranes is inferred to have a non-marine, possibly lacustrine source. Other biomarkers such as gammacerane and oleanane as well as carbon isotopic data on δ13C on asphaltene fractionalso suggested that the bitumen used for pottery came from the same source area in Selenicë. GC-MS was also employed to show the deliberate addition of fats to the archaeological bitumen, probably to lower the quite high softening point (about 120 °C) of Selenicë bitumen. Further, the presence of benzothiophene in selected samples suggests the use at temperatures below 220 °C (benzothiophene boiling point). The processing in such narrow temperature range, exclude direct contact of vessels with flame and points to indirect heating systems (e.g. water-bath heating). Since no direct evidences of use for bitumen were found, and taking into account its occurrence in Apulian coastal sites, its main utilization for caulking is supposed. Only in few cases, bitumen was used as adhesive to repair pottery

Bitumen in potsherds from two Apulian Bronze Age settlements, Monopoli and Torre Santa Sabina: Composition and origin

Bitumen was found to occur on archaeological potsherds collected from two Apulia Middle Bronze Age sites, Monopoli and Torre Santa Sabina (Italy). Bitumen from two different areas, Majella (Italy) and Selenicë (Albania), were analyzed as potential reference samples to assess the geographic origin of the archaeological bitumen using geochemical analytical techniques. Analysis of the archaeological samples from different layers at both sites showed that the bitumen possesses the same gross composition and biomarker distribution patterns. Sterane and terpane profiles from the archaeological samples were very similar to some of the geological samples collected from Selenicë. In both archeological and selected Selenicë samples, sterane distributions were dominated by the C29 homologues (46-54%), followed by the C27 (26-33%) and C28 homologues (ca. 21%). Other biomarkers, such as gammacerane and oleanane, as well as the stable carbon isotopic composition of the asphaltene fraction, also suggest that the bitumen from the two archaeological sites was imported from Albania during Middle Bronze Age

The genesis of vacuolinos, a membrane compartment on the route to the vacuole

In plants that rely on animals for the transfer of pollen, the cells of the epidermis of flower petals are specialized to attract pollinators. The differentiation program of these cells includes the synthesis of anthocyanin pigments, their accumulation in the central vacuole of these cells, the display of the color via modulation of the pH in the lumen of the vacuole, the building of a “papillary” cell shape that provides a handy landing surface to the pollinator and contributes to the final color of the petal by affecting the refraction angle of the light. All these mechanisms are controlled by a group of transcription factors that activate different sets of target genes (1). A set of genes has been shown to encode for the biosynthetic enzymes of the anthocyanin pathway, while two other target genes encode for two different P-ATPases (PH1 and PH5) that together acidify the lumen of the central vacuole and some 10 other target genes still have not been assigned a function (2); (3). Localization studies for the tonoplast proteins PH1 and PH5 has brought to the discovery that in petal epidermal cells, the sorting of proteins to the vacuole includes an intermediate organelle on which proteins reside before reaching the central vacuole. As this organelle looks like a small vacuole and is marked by vacuolar proteins (including vacuolar SNAREs), we called it “vacuolino”. In transient expression assays, the membrane of vacuolinos is marked by the PH5-GFP fusion 24 hours after cell transformation, while the tonoplast of the central vacuole shows fluorescence only ~48 hrs after transformation. In different cell types (like leaf cells or unpigmented petal mesophyll cells) vacuolinos are absent and PH5-GFP appears on the tonoplast already 24 hrs after transformation. All other vacuolar proteins we have observed in their sorting pathway to the vacuole in petal epidermal cells, reach the final destination after a short permanence on the vacuolinos. Mutants for any of the above mentioned transcription factors do not show vacuolinos implying that a set of their target genes is involved in the genesis of these compartments. We have used different methods to compare the transcriptomics of petals mutant for each of the transcription factors involved in the presence of vacuolinos (AN1, PH3 and PH4) and we have isolated a number of target genes of these regulators to isolate candidate genes involved in vacuolinos biogenesis. We are now isolating mutants for each of these genes by the screening of petunia BLASTABLE collections of transposon insertions and by RNAi technology. The first three genes for which we could see disappearance of vacuolinos in the mutants are involved in different steps of the vacuolino pathway to the vacuole: - in mutants for one of this gene vacuolar proteins directly go to the vacuole without passing through the vacuolino (like in mesophyll cells), - in another they get stuck in small vesicle-like structures, - in the third the markers remain on the vacuolinos as vacuolinos seem not to be able to fuse to the central vacuole. We are at the moment also studying when the vacuolinos appear during bud development as this could give some clue of their function. Analysis of the localization of vacuolar GFPs in epidermal petal cells of buds at different developmental stages shows that: - in young buds, the petal epidermal cells are rather small (compared to open flowers) and have small vacuoles with huge folded tonoplast - during development the cells enlarge and the tonoplast unfold - -vacuolinos only show up after flower opening, when petals are approaching senescence All three these genes encoded unexpected players in the genesis, physiology and fusion of membrane compartments. 1. Koes R, Verweij CW, & Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci. 5:236-242. 2. Faraco M, et al. (2014) Hyperacidification of Vacuoles by the Combined Action of Two Different P-ATPases in the Tonoplast Determines Flower Color. Cell reports 6(1):32-43. 3. Verweij W, et al. (2008) An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour. Nature cell biology 10(12):1456-1462