It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV) of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

Background: The continuous polarized vesicle secretion in pollen tubes is essential for tip growth but the location of endo- and exocytic sub-domains remains however controversial. In this report we aimed to show that Arabidopsis thaliana syntaxins are involved in this process and contribute to spatially define exocytosis and membrane recycling. Results: Using GFP-fusion constructs, we imaged the distribution of pollen-specific (AtSYP124) and non-pollen syntaxins (AtSYP121 and AtSYP122) in transiently transformed Nicotiana tabacum pollen tubes. All three proteins associate with the plasma membrane and with apical vesicles indicating a conserved action mechanism for all SYPs. However, the GFP tagged SYP124 showed a specific distribution with a higher labelling at the plasma membrane flanks, 10-25 μm behind the apex. This distribution is affected by Ca2+ fluxes as revealed by treatment with Gd3+ (an inhibitor of extracellular Ca2+ influx) and TMB-8 (an inhibitor of intracellular Ca2+ release). Both inhibitors decreased growth rate but the distribution of SYP124 at the plasma membrane was more strongly affected by Gd3+. Competition with a related dominant negative mutant affected the specific distribution of SYP124 but not tip growth. In contrast, co-expression of the phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4) or of the small GTPase Rab11 perturbed polarity and the normal distribution of GFP-SYP but did not inhibit the accumulation in vesicles or at the plasma membrane. Conclusions: The results presented suggest that in normal growing pollen tubes, a net exocytic flow occurs in the flanks of the tube apex mediated by SYP124. The specific distribution of SYP124 at the plasma membrane is affected by changes in Ca2+ levels in agreement with the importance of this ion for exocytosis. Apical growth and the specific localization of SYP124 were affected by regulators of membrane secretion (Ca2+, PIP5K4 and Rab11) but competition with a dominant negative mutant affected only SYP distribution. These data thus suggest that syntaxins alone do not provide the level of specificity that is required for apical growth and that additional signalling and functional mechanisms are required.

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.

Plant SNAREs coded by genes of the same family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless the application of innovative experimental approaches evidenced that members of the same gene family often have different functional specificities. The role of the two closely related Qc-SNAREs, the AtSYP51 and the AtSYP52 in the vesicle sorting to the central vacuole has been investigated. SYP5 proteins appeared to have functional specificity and to be part of a new functional class, the inhibitory SNAREs (i-SNAREs). 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 i-SNARE when they accumulate on the tonoplast. A higher level of complexity emerges for functional classification of plant SNAREs.

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.

Plant chitinases have been studied for their importance in the defense of crop plants from pathogen attacks and for their peculiar vacuolar sorting determinants. A peculiarity of the sequence of many family 19 chitinases is the presence of a C-terminal extension that seems to be important for their correct recognition by the vacuole sorting machinery. The 7 amino acids long C-terminal vacuolar sorting determinant (CtVSD) of tobacco chitinase A is necessary and sufficient for the transport to the vacuole. This VSD shares no homology with other CtVSDs such as the phaseolin's tetrapeptide AFVY (AlaPheValTyr) and it is also sorted by different mechanisms. While a receptor for this signal has not yet been convincingly identified, the research using the chitinase CtVSD has been very informative, leading to the observation of phenomena otherwise difficult to observe such as the presence of separate vacuoles in differentiating cells and the existence of a Golgi-independent route to the vacuole. Thanks to these new insights in the endoplasmic reticulum (ER)-to-vacuole transport, GFPChi (Green Fluorescent Protein carrying the chitinase A CtVSD) and other markers based on chitinase signals will continue to help the investigation of vacuolar biogenesis in plants.

SNAREs (N-ethylmaleimide-sensitive factor adaptor protein receptors) have been often seen to have a dishomogeneous distribution on membranes and are apparently present in excess of the amount required to assure correct vesicle traffic. It was also shown in few cases that SNARE on the target membrane (t-SNARE) with a fusogenic role, can become non-fusogenic when overexpressed. When SNAREs concentration is inversely proportional to the expected fusogenic activity, they can be reasonably defined as “inhibitory” or “interfering” (i-SNAREs). In fact i-SNAREs have been proposed to form a new functional class of SNAREs. In this manuscript I discuss data obtained in various eukaryotic models that leave open different possibilities for the action mechanism of the i-SNAREs in plants.

In Arabidopsis thaliana, different types of vacuolar receptors were discovered. The AtVSR (Vacuolar Sorting Receptor) receptors are well known to be involved in the traffic to lytic vacuole (LV), while few evidences demonstrate the involvement of the receptors from AtRMR family (Receptor Membrane RING-H2) in the traffic to the protein storage vacuole (PSV). In this study we focused on the localization of two members of AtRMR family, AtRMR1 and -2, and on the possible interaction between these two receptors in the plant secretory pathway. Our experiments with agroinfiltrated Nicotiana benthamiana leaves demonstrated that AtRMR1 was localized in the endoplasmic reticulum (ER), while AtRMR2 was targeted to the trans-Golgi network (TGN) due to the presence of a cytosolic 23-amino acid sequence linker. The fusion of this linker to an equivalent position in AtRMR1 targeted this receptor to the TGN, instead of the ER. By using a Bimolecular Fluorescent Complementation (BiFC) technique and experiments of co-localization, we demonstrated that AtRMR2 can make homodimers, and can also interact with AtRMR1 forming heterodimers that locate to the TGN. Such interaction studies strongly suggest that the transmembrane domain and the few amino acids surrounding it, including the sequence linker, are essential for dimerization. These results suggest a new model of AtRMR trafficking and dimerization in the plant secretory pathway.

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.

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 a-(1,3)-fucose (Fuc) linked to the proximal GlcNAc residue and/or a b-(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 evelopment 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.

Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils.

The acidification of endomembrane compartments is essential for enzyme activities, sorting, trafficking, and trans-membrane transport of various compounds. Vacuoles are mildly acidic inmost plant cells because of the action of V-ATPase and/or pyrophosphatase proton pumps but are hyperacidified in specific cells by mechanisms that remained unclear. Here, we show that the blue petal color of petunia ph mutants is due to a failure to hyperacidify vacuoles. We report that PH1 encodes a P-3B-ATPase, hitherto known as Mg2+ transporters in bacteria only, that resides in the vacuolar membrane (tonoplast). In vivo nuclear magnetic resonance and genetic data show that PH1 is required and, together with the tonoplast H+ P-3A-ATPase PH5, sufficient to hyperacidify vacuoles. PH1 has no H+ transport activity on its own but can physically interact with PH5 and boost PH5 H+ transport activity. Hence, the hyperacidification of vacuoles in petals, and possibly other tissues, relies on a heteromeric P-ATPase pump.

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.

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.

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.

An alternative study involving proteome analysis of the 24 hour Nicotiana tabacum protoplast culture medium was performed with the aim to confirm relations among regulatory elements of exocytotic processes. Protoplasts present many convenient features to study cellular processes during transient over-expression or suppression of specific gene's products. We performed a proteomic analysis of the culture medium fraction of protoplasts transiently expressing transgenes for 24 hours to characterize the effect of various regulatory proteins dominant negative mutants. A total number of 49 spots were found reproducible in the medium. 24 of these spots were identified with nano RP-HPLC-ESI-MS/MS. Only three and six spots were respectively identified as canonical and non-canonical secreted cell wall proteins. The low number of spots present in the culture medium fraction allowed us the ambitious experiment to analyze the influence of various SNAREs (SYP121, SYP122, SNAP33) and Rab (Rab11) dominant negative mutants. Missing a reasonable number of identified proteins the analyses gave rise to a similarity matrix statistically analyzed considering variation within the presence of 24 spots reproducible in presence of transient over-expression of SNAREs (SYP121 and SYP122) and Rab11 native cDNAs. The similarity confirmed the closer relation between the function of SYP122 and Rab11 as evidenced by the secRGUS based analysis. This analysis included the effect of SNAP33 DN mutant and showed that this Qb-c-SNARE influence both SYP121 and SYP122 SNARE complexes.

SYP51 and SYP52 are sorted through the Golgi apparatus and accumulate on TGN compartments and tonoplast. When overexpressed (a condition naturally occurring in pollen tubes) these proteins and especially SYP51, cause an effect that inhibit vacuolar sorting (1; 2). The interference causes an alteration of endocytic compartments that seem to receive material also from a direct ER-to-vacuole pathway. Bioinformatic analysis on network interaction data, allowed us to evidence direct interactions between SNARE proteins and aquaporins that may have a role in the interference mechanism. Aquaporins have an important role in the modification of membranes propriety (3). One interaction between SNARE and aquaporin have already been described involving the Qc-SNARE SYP61, the Qa-SNARE SYP121 and the aquaporin PIP2;7. These proteins physically interact on plasma membrane (4). In our work, we investigated by ratiometric bimolecular fluorescence complementation (rBiFC) approach (5) the in vivo interaction of the Qc SNAREs AtSYP51 and SYP52 with aquaporins apparently able to bypass the Golgi apparatus. These interactions may control Multi-Vesicular-Bodies formation and represents a regulatory mechanism for unconventional vacuolar transport, exocytosis and endocytosis. Essential bibliography: 1. De Benedictis et al. (2013). AtSYP51/52 functions diverge in the post-golgi traffic and differently affect vacuolar sorting. Mol. Plant 6: 916–930. 2. Di Sansebastiano GP (2013) Defining new SNARE functions: the i-SNARE. Front Plant Sci. 2013 Apr 16;4:99. 3. Maeshima M (2001). Tonoplast Transporters: Organization and Function. Annu. Rev. Plant Physiol. 52: 469–497. 4. Hachez et al. (2014). Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability. Plant Cell 26: 3132–47. 5. Grefen, C. and Blatt, M.R. (2012). A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC). Biotechniques 53: 311–314.

Plant protoplasts are often used as experimental material without paying attention to the tissue they are isolated from (a protoplast is a protoplast), whereas in other cases, they are considered not sufficiently able to reproduce the in planta situation. Here, we show that protoplasts are a very reliable experimental system, as long as we carefully chose their source.

In the eukaryotic systems the membrane trafficking inside the cells is indispensible. The membrane trafficking is a highly regulated process in which various molecular machineries are involved. It involves the vesicle formation, tethering, and finally fusion. According to the phylogenetic analysis, these processes are highly conserved among various organisms. This suggests the acquisition of common ancestral lineages by eukaryotes. In addition, to the similarity in components of trafficking in eukaryotes, each organism has also acquired various specific regulatory molecules which ascertain the diversification to membrane trafficking. In this review we summarize the progress in recent times about the plant-specific Rab GTPases in membrane trafficking events. Rab GTPases are a diverse group which are involved in various processes of membrane trafficking. Further, there are some reports which suggest Rab GTPases’ role in signalling pathways involving light, hormones, biotic, and abiotic stresses. Despite these there is still some inhibition among the scientific community to ascribe the latter roles to Rab GTPases with certainty even though the membrane trafficking events are integrated with signalling.

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.

SNAREs (N-ethylmaleimide-sensitive factor adaptor protein receptors) are small polypeptides (∼200–400 amino acid) which are characterized by a particular domain, the SNARE motif that can form a coiled-coil structure via hetero-oligomeric interactions. These protein interactions are highly stable leading to the formation of the so-called SNARE complex which allows the membrane fusion. SNAREs also interact with several proteins acting as regulators of SNARE complex formation. By regulating vesicle traffic, SNAREs have a clear influence on several signaling pathways. SNAREs take part to receptors turnover through endocytosis and exocytosis, but they can also directly gate channels and interact with membrane proteins potentially involved in signaling processes. Phosphorylation of SNAREs upon elicitation is known, and hormonal control confirms that SNAREs have a role in signaling processes.

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.

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.

In humans, the autosomal recessive Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2), caused by mutations in the BSCL2 gene encoding a protein known as seipin, is characterised by an almost completely absence of adipose tissue and low but detectable lipid content. The seipin protein family appears to widely spread in animals, plants and some microorganisms. In yeast, seipin is localised at the endoplasmic reticulum-lipid droplet (ER-LD) junction and its absence resulted in irregular LDs, clustered together. LDs are found in nearly all eukaryotic cells and in some prokaryotes and function as an energy-storage reservoir in the form of neutral lipids, predominantly triacylglycerols and steryl esters. LD consist of a neutral lipid core surrounded by a phospholipid monolayer with acyl chains deeply inserted into the core and are originated from the (ER), even though the precise mechanisms underlying their biogenesis remains largely unclear. In Arabidopsis thaliana, similarly to other plant species, homologues to human and yeast seipins have been identified and are encoded by a gene family. However their physiological significance is completely unknown. Here we report about subcellular localisation of the three Arabidopsis thaliana seipins by CLSM in tobacco leaves. These results together with the peculiar expression pattern of the seipin genes allowed us to speculate about possible physiological roles of this class of proteins in plants.

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.

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

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.

Despite a long case history, the use of protoplasts in cell biology research still divides scientists but their weaknesses can be exploited as strengths. Transient expression in protoplasts can saturate protein–protein interactions very efficiently, inhibiting the process of interest more efficiently than other approaches at gene expression level. The method described here consists of an assay providing a functional characterization of SNARE proteins in a heterogeneous population of cells, by the comparison of native and dominant negative mutant forms. In particular, it allows for discriminating between t-SNARE and i-SNARE functional classes.

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.

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on "Unconventional Protein and Membrane Traffic" (UPMT) during 4-7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes.

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