New Insights on Plant Cell Elongation: A Role for Acetylcholine

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

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

Endomembrane reorganization induced by heavy metals

Plant cells maintain plasmatic concentrations of essential heavy metal ions, such as iron, zinc, and copper, within the optimal functional range. To do so, several molecular mechanisms have to be committed to maintain concentrations of non-essential heavy metals and metalloids, such as cadmium, mercury and arsenic below their toxicity threshold levels. Compartmentalization is central to heavy metals homeostasis and secretory compartments, finely interconnected by traffic mechanisms, are determinant. Endomembrane reorganization can have unexpected effects on heavy metals tolerance altering in a complex way membrane permeability, storage, and detoxification ability beyond gene\u2019s expression regulation. The full understanding of endomembrane role is propaedeutic to the comprehension of translocation and hyper-accumulation mechanisms and their applicative employment. It is evident that further studies on dynamic localization of these and many more proteins may significantly contribute to the understanding of heavy metals tolerance mechanisms. The aim of this review is to provide an overview about the endomembrane alterations involved in heavy metals compartmentalization and tolerance in plants

Actin and microtubules differently contribute to vacuolar targeting specificity during the export from the er

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

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

Physico-chemical properties of inorganic nps influence the absorption rate of aquatic mosses reducing cytotoxicity on intestinal epithelial barrier model

Noble metals nanoparticles (NPs) and metal oxide NPs are widely used in different fields of application and commercial products, exposing living organisms to their potential adverse effects. Recent evidences suggest their presence in the aquifers water and consequently in drinking water. In this work, we have carefully synthesized four types of NPs, namely, silver and gold NPs (Ag NPs and Au NPs) and silica and titanium dioxide NPs (SiO2 NPs and TiO2 NPs) having a similar size and negatively charged surfaces. The synthesis of Ag NPs and Au NPs was carried out by colloidal route using silver nitrate (AgNO3 ) and tetrachloroauric (III) acid (HAuCl4 ) while SiO2 NPs and TiO2 NPs were achieved by ternary microemulsion and sol-gel routes, respectively. Once the characterization of NPs was carried out in order to assess their physico-chemical properties, their impact on living cells was studied. We used the human colorectal adenocarcinoma cells (Caco-2), known as the best representative intestinal epithelial barrier model to understand the effects triggered by NPs through ingestion. Then, we moved to explore how water contamination caused by NPs can be lowered by the ability of three species of aquatic moss, namely, Leptodictyum riparium, Vesicularia ferriei, and Taxiphyllum barbieri, to absorb them. The experiments were conducted using two concentrations of NPs (100 µM and 500 Mm as metal content) and two time points (24 h and 48 h), showing a capture rate dependent on the moss species and NPs type. Then, the selected moss species, able to actively capture NPs, appear as a powerful tool capable to purify water from nanostructured materials, and then, to reduce the toxicity associated to the ingestion of contaminated drinking water

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

Ipotesi sulla presenza di Glaucium flavum nel barocco leccese

Riassunto - Il barocco leccese si caratterizza per la ricca presenza di decorazioni vegetali, foglie, fusti e frutti. La tipica decorazione del capitello corinzio ha come elemento distintivo le foglie d’acanto. Per analogia con le decorazioni corinzie, molte altre foglie vengono definite “foglia d’acanto” pur non corrispondendo a tale pianta da un punto di vista anatomico. Sfruttando alcune caratteristiche botaniche, abbiamo identificato in molte decorazioni i tratti che riteniamo caratteristici della pianta tipica delle coste pugliesi Glaucium flavum Crantz comunemente noto come ‘papavero cornuto’. Se l’identificazione sarà confermata da valutazioni indipendenti, questa pianta potrebbe rappresentare un motivo decorativo estremamente ricorrente poiché parzialmente riprodotta in quasi ogni decorazione barocca del ‘700. L’importanza del riscontro di una pianta selvatica tipica degli ambienti salentini viene qui discussa

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