Papers by Gian Pietro Di Sansebastiano
Plant Direct, 2020
Abstract Flowering and fruiting are processes subject to complex control by environmental and end... more Abstract Flowering and fruiting are processes subject to complex control by environmental and endogenous signals. Endogenous signals comprise, besides classical phytohormones, also signaling peptides and miniproteins. Tomato cystine‐knot miniproteins (TCMPs), which belong to a Solanaceous‐specific group of Cys‐rich protein family, have been recently involved in fruit development. TCMP‐1 and TCMP‐2 display a highly modulated expression pattern during flower and fruit development. A previous study reported that a change in the ratio of the two TCMPs affects the timing of fruit production. In this work, to investigate TCMP‐2 mode of action, we searched for its interacting partners. One of the interactors identified by a yeast two hybrid screen, was the B‐box domain‐containing protein 16 (SlBBX16), whose closest homolog is the Arabidopsis microProtein 1b implicated in flowering time control. We demonstrated the possibility for the two proteins to interact in vivo in tobacco epidermal cells. Arabidopsis plants ectopically overexpressing the TCMP‐2 exhibited an increased level of FLOWERING LOCUS T (FT) mRNA and anticipated flowering. Similarly, in previously generated transgenic tomato plants with increased TCMP‐2 expression in flower buds, we observed an augmented expression of SINGLE‐FLOWER TRUSS gene, the tomato ortholog of FT, whereas the expression of the antiflorigen SELF‐PRUNING was unchanged. Consistently, these transgenic plants showed alterations in the flowering pattern, with an accelerated termination of the sympodial units. Overall, our study reveals a novel function for TCMP‐2 as regulatory factor that might integrate, thanks to its capacity to interact with SlBBX16, into the signaling pathways that control flowering, and converge toward florigen regulation.
Journal of experimental botany, Jan 18, 2017
Due to the numerous roles plant vacuoles play in cell homeostasis, detoxification, and protein st... more 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.
Journal of Experimental Botany, 2005
Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with ... more Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL) from almonds, to the endomembrane system and lipid bodies, both as enzyme activity in almond seeds and as GFP fusions transiently expressed in tobacco protoplasts, is described. Such association of a CYP74 with lipid bodies has not been reported previously. Also described are the properties of a 9-HPL gene, the developmental regulation of its expression, the production and characterization of recombinant 9-HPL in Escherichia coli, and the developmental correlation between gene expression, enzyme activity, and the appearance of volatile C 9 aldehydes from HPL action.
Abstract: In this work we explored the possibility of using genetically modified Arabidopsis thal... more Abstract: 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...
syntaxin at the pollen tube apical and sub-apical zones is involved in tip growth
We evaluated the ability of Dittrichia viscosa to extract cadmium (Cd) and arsenic (As) from agar... more We evaluated the ability of Dittrichia viscosa to extract cadmium (Cd) and arsenic (As) from agarized substrates with moderately low levels of contamination. This plant species spontaneously colonizes contaminated soils and has already been recognized as suitable for metal phytoremediation. D. viscosa retains As mainly in the roots and translocates it in limited amount to the aerial parts. We compared D. viscosa subpopulations to other plant species (Solalun lycopersicum L., Nicotiana tabacum L. and Daucus carota L.) and observed that their minor cultural needs not always accompanied higher phytoextraction performances. Different subpopulations exhibited different behavior. Based on the results obtained on the analyzed plants, we suggest that D. viscosa is a promising candidate for the phytoextraction of Cd and As from soils with relatively low contamination, cautioning that specific genetic improvements have to be pursued in order to prepare D. viscosa for widespread adoption.
Plant Physiology and Biochemistry
Membranes
Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targetin... more Plants rely on both actin and microtubule cytoskeletons to fine-tune sorting and spatial targeting of membranes during cell growth and stress adaptation. Considerable advances have been made in recent years in the comprehension of the relationship between the trans-Golgi network/early endosome (TGN/EE) and cytoskeletons, but studies have mainly focused on the transport to and from the plasma membrane. We address here the relationship of the cytoskeleton with different endoplasmic reticulum (ER) export mechanisms toward vacuoles. These emergent features of the plant endomembrane traffic are explored with an in vivo approach, providing clues on the traffic regulation at different levels beyond known proteins’ functions and interactions. We show how traffic of vacuolar markers, characterized by different vacuolar sorting determinants, diverges at the export from the ER, clearly involving different components of the cytoskeleton.
Plants
Artemisia annua L. is well-known as the plant source of artemisinin, a sesquiterpene lactone with... more Artemisia annua L. is well-known as the plant source of artemisinin, a sesquiterpene lactone with effective antimalarial activity. Here, a putative ortholog of the Arabidopsis thaliana WRKY40 transcription factor (TF) was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends in A. annua and named AaWRKY40. A putative nuclear localization domain was identified in silico and experimentally confirmed by using protoplasts of A. annua transiently transformed with AaWRKY40-GFP. A genome-wide analysis identified 122 WRKY genes in A. annua, and a manually curated database was obtained. The deduced proteins were categorized into the major WRKY groups, with group IIa containing eight WRKY members including AaWRKY40. Protein motifs, gene structure, and promoter regions of group IIa WRKY TFs of A. annua were characterized. The promoter region of AaWRKY group IIa genes contained several abiotic stress cis-acting regulatory elements, among which a highl...
Plants
The production of crocin, an uncommon and valuable apocarotenoid with strong biological activity,... more The production of crocin, an uncommon and valuable apocarotenoid with strong biological activity, was obtained in a cell suspension culture of saffron (Crocus sativus L.) established from style-derived calli to obtain an in-vitro system for metabolite production. Salycilic acid (SA) was used at different concentrations to elicit metabolite production, and its effect was analyzed after a 4 days of treatment. HPLC-DAD analysis was used for total crocin quantification while the Folin-Ciocâlteu method was applied for phenolic compounds (PC) content. Interestingly, despite cell growth inhibition, a considerable exudation was observed when the highest SA concentration was applied, leading to a 7-fold enhanced production of crocin and a 4-fold increase of phenolics compared to mock cells. The maximum antioxidant activity of cell extracts was evidenced after SA 0.1 mM elicitation. Water-soluble extracts of saffron cells at concentrations of 1, 0.5, and 0.1 µg mL−1 showed significant inhibit...
International Journal of Molecular Sciences
Heavy metals (HMs) are released into the environment by many human activities and persist in wate... more Heavy metals (HMs) are released into the environment by many human activities and persist in water even after remediation. The efficient filtration of solubilized HMs is extremely difficult. Phytoremediation appears a convenient tool to remove HMs from polluted water, but it is limited by the choice of plants able to adapt to filtration of polluted water in terms of space and physiological needs. Biomasses are often preferred. Aquatic moss biomasses, thanks to gametophyte characteristics, can act as live filtering material. The potential for phytoremediation of Hypnales aquatic mosses has been poorly investigated compared to aquatic macrophytes. Their potential is usually indicated as a tool for bioindication and environmental monitoring more than for pollutant removal. When phytoremediation has been considered, insufficient attention has been paid to the adaptability of biomasses to different needs. In this study the heavy metal uptake of moss Taxiphyllum barbieri grown in two diff...
Journal of Archaeological Science: Reports
Archaeological investigations carried out at Case Bastione (Enna, central Sicily) provide a key i... more Archaeological investigations carried out at Case Bastione (Enna, central Sicily) provide a key insight into the cultural and environmental changes that occurred during the transition from the Copper Age to the Bronze Age. Preliminary data of an ongoing paleoenvironmental reconstruction through archaeobotanical analyses are here presented. The selective exploitation of vegetation, the adaptation of lifestyle to local resources, and changing climatic conditions are analysed using different on-site and off-site environmental and archaeological proxies. The environment around the site was constituted by mixed oak woodland. Dietary preferences were reconstructed through the analysis of carpo-remains. Isotopic values provide new data on the 4.2 ka BP event and its effects on vegetation in central Sicily. In a whole, first results from Case Bastione give new light to human choices of vegetal resources exploitation. Comparison of the local results with the regional pollen data support the hypothesis that the growth in population and settlement in the inland part of the island since the Late Copper Age may reflect changing climatic conditions in coastal areas. environment since prehistoric times (Behre, Jacomet 1991; Mercuri, Sadori 2013). This combination of studies by complementary disciplines demonstrates that the full integration of analysed datasets focused on anthropogenic and natural processes provides the most powerful way to understand human activity and adaptation to landscapes and environments (
Frontiers in Plant Science
Endocytosis is an essential process for the internalization of plasma membrane proteins, lipids a... more Endocytosis is an essential process for the internalization of plasma membrane proteins, lipids and extracellular molecules into the cells. The mechanisms underlying endocytosis in plant cells involve several endosomal organelles whose origins and specific role needs still to be clarified. In this study we compare the internalization events of a GFP-tagged polygalacturonase-inhibiting protein of Phaseolus vulgaris (PGIP2-GFP) to that of a GFP-tagged subunit of cellulose synthase complex of Arabidopsis thaliana (secGFP-CesA6). Through the use of endocytic traffic chemical inhibitors (tyrphostin A23, salicylic acid, wortmannin, concanamycin A, Sortin 2, Endosidin 5 and BFA) it was evidenced that the two protein fusions were endocytosed through distinct endosomes with different mechanisms. PGIP2-GFP endocytosis is specifically sensitive to tyrphostin A23, salicylic acid and Sortin 2; furthermore, SYP51, a tSNARE with interfering effect on late steps of vacuolar traffic, affects its arrival in the central vacuole. SecGFP-CesA6, specifically sensitive to Endosidin 5, likely reaches the plasma membrane passing through the trans Golgi network (TGN), since the BFA treatment leads to the formation of BFA bodies, compatible with the aggregation of TGNs. BFA treatments determine the accumulation and tethering of the intracellular compartments labeled by both proteins, but PGIP2-GFP aggregated compartments overlap with those labeled by the endocytic dye FM4-64 while secGFP-CesA6 fills different compartments. Furthermore, secGFP-CesA6 co-localization with RFP-NIP1.1, marker of the direct ER-to-Vacuole traffic, in small compartments separated from ER suggests that secGFP-CesA6 is sorted through TGNs in which the direct contribution from the ER plays an important role. All together the data indicate the existence of a heterogeneous population of Golgi-independent TGNs.
Frontiers in Plant Science
SYP51 and 52 are the two members of the SYP5 Qc-SNARE gene family in Arabidopsis thaliana. These ... more SYP51 and 52 are the two members of the SYP5 Qc-SNARE gene family in Arabidopsis thaliana. These two proteins, besides their high level of sequence identity (85%), have shown to have differential functional specificity and possess a different interactome. Here we describe a unique and specific interaction of SYP51 with an ER aquaporin, AtNIP1;1 (also known as NLM1) indicated to be able to transport arsenite [As(III)] and previously localized on PM. In the present work we investigate in detail such localization in vivo and characterize the interaction with SYP51. We suggest that this interaction may reveal a new mechanism regulating tonoplast invagination and recycling. We propose this interaction to be part of a regulatory mechanism associated with direct membrane transport from ER to tonoplast and Golgi mediated vesicle trafficking. We also demonstrate that NIP1;1 is important for plant tolerance to arsenite but does not alter its uptake or translocation. To explain such phenomenon the hypothesis that SYP51/NIP1;1 interaction modifies ER and vacuole ability to accumulate arsenite is discussed.
Plant Physiology and Biochemistry
Aquaporins are water channel proteins that regulate plant development, growth, and response to en... more Aquaporins are water channel proteins that regulate plant development, growth, and response to environmental stresses. Populus trichocarpa is one of the plants with the highest number of aquaporins in its genome, but only few of them have been characterized at the whole plant functional level. Here we analyzed a putative aquaporin gene, aqua1, a gene that encodes for a protein of 257 amino acid with the typical NPA (Asp-Pro-Ala) signature motif of the aquaporin gene family. aqua1 was down-regulated of ∼10 fold under excess Zn in both leaves and roots, and conferred Zn tolerance when expressed in yeast Zn hypersensitive strain. In vivo localization of AQUA1-GFP in Arabidopsis protoplast showed a heterogeneous distribution of this protein on different membranes destined to form aggregates related to autophagic multivesicular bodies. Zn-dependent AQUA1-GFP relocalization was perturbed by phosphatases' and kinases' inhibitors that could affect both intracellular trafficking and aquaporins' activity. Exposed to high concentration of Zn, AQUA1 also co-localized with AtTIP1;1, a wellknown Arabidopsis vacuolar marker, probably in pro-vacuolar multivesicular bodies. These findings suggest that high concentration of Zn down-regulates aqua1 and causes its re-localization in new forming pro-vacuoles. This Zn-dependent re-localization appears to be mediated by mechanisms regulating intracellular trafficking and aquaporins' post-translational modifications. This functional characterization of a poplar aquaporin in response to excess Zn will be a useful reference for understanding aquaporins' roles and regulation in response to high concentration of Zn in poplar.
Journal of experimental botany, Jan 18, 2017
How many different routes for protein secretion? To enter the conventional secretory pathway, pro... more How many different routes for protein secretion? To enter the conventional secretory pathway, proteins have signal peptides that are recognized for translocation into the ER during synthesis. Once these proteins are exported to the Golgi apparatus from the ER, there are two potential routes to the plasma membrane: (1) cargo are loaded into secretory vesicles at the Golgi or (2) cargo are exported to the TGN and then loaded into secretory vesicles for transport to the plasma
Plant Cell Reports
Triticum durum Glutathione S-transferase Z1 is specifically responsive to glyphosate. Its express... more Triticum durum Glutathione S-transferase Z1 is specifically responsive to glyphosate. Its expression influences the receptor-mediated vacuolar sorting mechanisms involved in tolerance mechanisms. A zeta subfamily glutathione S-transferase gene from Triticum durum (cv Cappelli) (TdGSTZ1) was characterized as part of a complex detoxification mechanism. The effect of different abiotic stresses on TdGSTZ1 revealed that the gene is unexpectedly responsive to glyphosate (GLY) herbicide despite it should not be part of tolerance mechanisms. Its role in the non-target-site mechanism of GLY resistance was then investigated. To analyze the GLY and the TdGSTZ1 overexpression effects on vacuolar sorting mechanisms, we performed transient transformation experiments in Nicotiana tabacum protoplasts using two vacuolar markers, AleuGFPgl133 and GFPgl133Chi, labeling the Sar1 dependent or independent sorting, respectively. We observed that the adaptive reaction of tobacco protoplasts vacuolar system to the treatment with GLY could be partially mimicked by the overexpression of TdGSTZ1 gene. To confirm the influence of GLY on the two vacuolar markers accumulation and the potential involvement of the secretion pathway activity in detoxification events, Arabidopsis thaliana transgenic plants overexpressing the non-glycosylated versions of the two markers were analyzed. The results suggested that GLY treatment specifically altered different vacuolar sorting characteristics, suggesting an involvement of the receptor-mediated AleuGFP sorting mechanism in GLY resistance. Finally, the expression analysis of selected genes confirmed that the non-target-site GLY resistance mechanisms are related to vacuolar sorting.
Cell reports, Jun 20, 2017
It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of mu... more 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.
International Journal of Molecular Sciences, 2017
Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pat... more 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.
Uploads
Papers by Gian Pietro Di Sansebastiano