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

<p>Abstract</p> <p>Background</p> <p>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 <it>Arabidopsis thaliana </it>syntaxins are involved in this process and contribute to spatially define exocytosis and membrane recycling.</p> <p>Results</p> <p>Using GFP-fusion constructs, we imaged the distribution of pollen-specific (AtSYP124) and non-pollen syntaxins (AtSYP121 and AtSYP122) in transiently transformed <it>Nicotiana tabacum </it>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.</p> <p>Conclusions</p> <p>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.</p

AQUA1 is a mercury sensitive poplar aquaporin regulated at transcriptional and post-translational levels by Zn stress

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 re-localization 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 well-known 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

Vacuolar system distribution in Arabidopsis tissues, visualized using GFP fusion proteins

Green fluorescent protein (GFP) allows the direct visualization of gene expression and the subcellular localization of fusion proteins in living cells. The localization of different GFP fusion proteins in the secretory system was studied in stably transformed Arabidopsis plants cv. Wassilewskaja. Secreted GFP (SGFP) and GFP retained in the ER (GFP‐KDEL) confirmed patterns already known, but two vacuolar GFPs (GFP‐Chi and Aleu‐GFP) labelled the Arabidopsis vacuolar system for the first time, the organization of which appears to depend on cell differentiation. GFP stability in the vacuoles may depend on pH or degradation, but these vacuolar markers can, nevertheless, be used as a tool for physiological studies making these plants suitable for mutagenesis and gene‐tagging experiment

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

<p>Abstract</p> <p>Background</p> <p>Hydroperoxide 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 <it>Medicago truncatula</it>, which were known to be expressed in leaves and roots, respectively.</p> <p>Results</p> <p>To study the subcellular localisation of plant 9/13-HPLs, a set of YFP-tagged chimeric constructs were prepared using two <it>M. truncatula </it>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 <it>M</it>.<it>truncatula </it>9/13-HPL (HPLF) between cytosol and lipid droplets (LD) whereas, as expected, <it>M</it>.<it>truncatula </it>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.</p> <p>Conclusion</p> <p>We 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 <it>in vitro </it>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.</p

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

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

vacuolar sorting mechanisms are differently influenced by detoxification processes

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

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology

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.The UPMT committee is grateful to the sponsors supporting the meeting: The Company of Biologists, Fondazione Puglia, the American Society of Plant Biologists, the Biochemical Society, University of Salento, CNR-Nanotec, MDPI-International Journal of Molecular Science and Merck

The study and manipulation of compartmentalization and traffic in plant cells

Compartmentalization and traffic of endomembranes is at the base of eukaryotic cell organization involving very different molecular mechanisms: coated vesicles traffic, tubular traffic, biogenesis and turn-over of membranous compartments. Each compartment can be seen in a maturation process dependent on crosstalk between different traffic mechanisms (a). It is particularly true for vacuoles, compartments usually classified according to their functions, as protein storage vacuoles (PSV), lytic vacuoles (LVs), Senescence Associated Vacuoles (SAV), Vacuolinos or NaCl accumulating vacuoles. We explore the crosstalk of traffic routes of different vacuolar proteins. Comparing the traffic of soluble markers partially able to bypass the Golgi, and the interaction of different vacuolar transmembrane proteins, some following direct, other Golgi-mediated traffic from ER to tonoplast (b), we provide new insight for the endomembrane traffic comprehension