Gian Pietro Di Sansebastiano

The Abscisic Acid–Related SNARE Homolog NtSyr1 Contributes to Secretion and Growth: Evidence from Competition with Its Cytosolic Domain

Syntaxins and other SNARE proteins are crucial for intracellular vesicle trafficking, fusion, and secretion. Previously, we isolated the syntaxin-related protein NtSyr1 (NtSyp121) from tobacco in a screen for abscisic acid–related signaling elements, demonstrating its role in determining the abscisic acid sensitivity of K+ and Cl− channels in stomatal guard cells. NtSyr1 is localized to the plasma membrane and is expressed normally throughout the plant, especially in root tissues, suggesting that it might contribute to cellular homeostasis as well as to signaling. To explore its functions in vivo further, we examined stably transformed lines of tobacco that expressed various constructs of NtSyr1, including the full-length protein and a truncated fragment, Sp2, corresponding to the cytosolic domain shown previously to be active in suppressing ion channel response to abscisic acid. Constitutively overexpressing NtSyr1 yielded uniformly high levels of protein (>10 times the wild-type levels) and was associated with a significant enhancement of root growth in seedlings but not with any obvious phenotype in mature, well-watered plants. Similar transformations with constructs encoding the Sp2 fragment of NtSyr1 showed altered leaf morphology but gave only low levels of Sp2 fragment, suggesting a strong selective pressure against plants expressing this protein. High expression of the Sp2 fragment was achieved in stable transformants under the control of a dexamethasone-inducible promoter. Sp2 expression was correlated positively with altered cellular and tissue morphology in leaves and roots and with a cessation of growth in seedlings. Overexpression of the full-length NtSyr1 protein rescued the wild-type phenotype, even in plants expressing high levels of the Sp2 fragment, supporting the idea that the Sp2 fragment interfered specifically with NtSyr1 function by competing with NtSyr1 for its binding partners. To explore NtSyr1 function in secretion, we used a green fluorescent protein (GFP)–based section assay. When a secreted GFP marker was coexpressed with Sp2 in tobacco leaves, GFP fluorescence was retained in cytosolic reticulate and punctate structures. In contrast, in plants coexpressing secreted GFP and NtSyr1 or secreted GFP alone, no GFP fluorescence accumulated within the cells. A new yellow fluorescent protein–based secretion marker was used to show that the punctate structures labeled in the presence of Sp2 colocalized with a Golgi marker. These structures were not labeled in the presence of a dominant Rab1 mutant that inhibited transport from the endoplasmic reticulum to the Golgi. We propose that NtSyr1 functions as an element in SNARE-mediated vesicle trafficking to the plasma membrane and is required for cellular growth and homeostasis.

An H+ P-ATPase on the tonoplast determines vacuolar pH and flower colour

The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H+-ATPase (V-ATPase), which, in plants, act together with H+-pyrophosphatases (PPase), whereas distinct P-type H+-ATPases in the cell membrane control the pH in the cytoplasm and energize the plasma membrane. Flower colour mutants have proved useful in identifying genes controlling the pH of vacuoles where anthocyanin pigments accumulate. Here we show that PH5 of petunia encodes a P3A-ATPase proton pump that, unlike other P-type H+-ATPases, resides in the vacuolar membrane. Mutation of PH5 reduces vacuolar acidification in petals, resulting in a blue flower colour and abolishes the accumulation of proanthocyanindins (condensed tannins) in seeds. Expression of PH5 is directly activated by transcription regulators of the anthocyanin pathway, in conjunction with PH3 and PH4. Thus, flower coloration, a key-factor in plant reproduction, involves the coordinated activation of pigment synthesis and a specific pathway for vacuolar acidification.

One protoplast is not the other

Protoplasts are widely used to study a variety of cellular processes because they are easy to transform, manipulate and image at high resolution. Protoplasts are thought to loose their original cell identity and therefore their origin is of minor unimportance. Hence, leaf mesophyll protoplasts are generally used to study processes active in other cell-types. However, there is no consensus whether data from protoplasts hold true for cells in intact tissues. We developed a procedure to isolate protoplasts from petals and compared their features with those of leaf protoplasts and intact tissues. Petal protoplasts derived from epidermis or mesophyll can be easily distinguished by their color and accurately reproduce the cell-specific differences in gene regulation and protein sorting observed in the intact tissue. We show that protoplasts maintain the features of the original cells in the intact tissue and, depending on their origin may produce completely different results in gene expression or protein localization assays. Consequently, analyses in leaf mesophyll protoplasts of genes or proteins that are expressed in other cell-types should be interpreted with caution. We conclude that with a few simple adjustments, to distinguish different cell-types, protoplasts can provide an excellent tool to reliably analyze highly cell-specific processes.

Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways

The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (secGFP) and secreted rat preputial β-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries.

Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco

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.

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 beta-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 events.

Asymmetric localization of Arabidopsis SYP124 syntaxin at the pollen tube apical and sub-apical zones is involved in tip growth

BackgroundThe 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.ResultsUsing 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.ConclusionsThe 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.

AtSYP51/52 Functions Diverge in the Post-Golgi Traffic and Differently Affect Vacuolar Sorting

Plant sensitive factor attachment protein receptors (SNAREs) encoded by genes of the same sub-family 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 sub-family 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 overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs (syntaxin of plants) 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 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.

Subcellular localisation of Medicago truncatula 9/13-hydroperoxide lyase reveals a new localisation pattern and activation mechanism for CYP74C enzymes

BackgroundHydroperoxide lyase (HPL) is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX) to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathogen attack. Together with their direct defence role towards different pathogens, these compounds are believed to play an important role in signalling within and between plants, and in the molecular cross-talk between plants and other organisms surrounding them. We have recently described the targeting of a seed 9-HPL to microsomes and putative lipid bodies and were interested to compare the localisation patterns of both a 13-HPL and a 9/13-HPL from Medicago truncatula, which were known to be expressed in leaves and roots, respectively.ResultsTo study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two M. truncatula HPL cDNAs and the localisation of the corresponding chimeras were verified by confocal microscopy in tobacco protoplasts and leaves. Results reported here indicated a distribution of M.truncatula 9/13-HPL (HPLF) between cytosol and lipid droplets (LD) whereas, as expected, M.truncatula 13-HPL (HPLE) was targeted to plastids. Notably, such endocellular localisation has not yet been reported previously for any 9/13-HPL. To verify a possible physiological significance of such association, purified recombinant HPLF was used in activation experiments with purified seed lipid bodies. Our results showed that lipid bodies can fully activate HPLF.ConclusionWe provide evidence for the first CYP74C enzyme, to be targeted to cytosol and LD. We also showed by sedimentation and kinetic analyses that the association with LD or lipid bodies can result in the protein conformational changes required for full activation of the enzyme. This activation mechanism, which supports previous in vitro work with synthetic detergent micelle, fits well with a mechanism for regulating the rate of release of volatile aldehydes that is observed soon after wounding or tissue disruption.

Localization of seed oil body proteins in tobacco protoplasts reveals specific mechanisms of protein targeting to leaf lipid droplets.

Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets (LD), whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum (ER). Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin-green fluorescent protein (GFP) peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein (BiP), were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.