Brown S.C.,Institute des science du Vegetal |
Gaudin M.,Institute des science du Vegetal |
Pereira C.,University of Porto |
Marion J.,Institute des science du Vegetal |
Satiat-Jeunemaitre B.,Institute des science du Vegetal
Plant Journal | Year: 2010
Photoactivatable and photoconvertible fluorescent proteins capable of pronounced light-induced spectral changes are a powerful addition to the fluorescent protein toolbox of the cell biologist. They permit specific tracking of one subcellular structure (organelle or cell subdomain) within a differentially labelled population. They also enable pulse-chase analysis of protein traffic. The Kaede gene codes for a tetrameric protein found in the stony coral Trachyphyllia geoffroyi, which emits green fluorescence that irreversibly shifts to red following radiation with UV or violet light. We report here the use of Kaede to explore the plant secretory pathway. Kaede versions of the Golgi marker sialyl-transferase (ST-Kaede) and of the vacuolar pathway marker cardosin A (cardA-Kaede) were engineered. Several optical devices enabling photoconversion and observation of Kaede using these two constructs were assessed to optimize Kaede-based imaging protocols. Photoconverted ST-Kaede red-labelled organelles can be followed within neighbouring populations of non-converted green Golgi stacks, by their gradual development of orange/yellow coloration from de novo synthesis of Golgi proteins (green). Results highlight some aspects on the dynamics of the plant Golgi. For plant bio-imaging, the photoconvertible Kaede offers a powerful tool to track the dynamic behaviour of designated subpopulations of Golgi within living cells, while visualizing the de novo formation of proteins and structures, such as a Golgi stack. © 2010 Blackwell Publishing Ltd.
Planamente S.,Institute des science du Vegetal |
Planamente S.,French National Center for Scientific Research |
Vigouroux A.,French National Center for Scientific Research |
Mondy S.,Institute des science du Vegetal |
And 3 more authors.
Journal of Biological Chemistry | Year: 2010
Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe77 and Tyr275, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe77 restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr275 specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Heyno E.,CEA Saclay Nuclear Research Center |
Heyno E.,Institute des science du Vegetal |
Innocenti G.,University Paris - Sud |
Lemaire S.D.,University Pierre and Marie Curie |
And 2 more authors.
Philosophical Transactions of the Royal Society B: Biological Sciences | Year: 2014
In photosynthetic organisms, sudden changes in light intensity perturb the photosynthetic electron flow and lead to an increased production of reactive oxygen species. At the same time, thioredoxins can sense the redox state of the chloroplast. According to our hypothesis, thioredoxins and related thiol reactive molecules downregulate the activity of H2O2-detoxifying enzymes, and thereby allow a transient oxidative burst that triggers the expression of H2O2 responsive genes. It has been shown recently that upon light stress, catalase activity was reversibly inhibited in Chlamydomonas reinhardtii in correlation with a transient increase in the level of H2O2. Here, it is shown that Arabidopsis thaliana mutants lacking the NADP-malate dehydrogenase have lost the reversible inactivation of catalase activity and the increase in H2O2 levels when exposed to high light. The mutants were slightly affected in growth and accumulated higher levels of NADPH in the chloroplast than the wild-type. We propose that the malate valve plays an essential role in the regulation of catalase activity and the accumulation of a H2O2 signal by transmitting the redox state of the chloroplast to other cell compartments. © 2014 The Author(s) Published by the Royal Society. All rights reserved.
Janski N.,University of Strasbourg |
Masoud K.,University of Strasbourg |
Batzenschlager M.,University of Strasbourg |
Herzog E.,University of Strasbourg |
And 5 more authors.
Plant Cell | Year: 2012
Microtubules (MTs) are crucial for both the establishment of cellular polarity and the progression of all mitotic phases leading to karyokinesis and cytokinesis. MT organization and spindle formation rely on the activity of g-tubulin and associated proteins throughout the cell cycle. To date, the molecular mechanisms modulating γ-tubulin complex location remain largely unknown. In this work, two Arabidopsis thaliana proteins interacting with GAMMA-TUBULIN COMPLEX PROTEIN3 (GCP3), GCP3-INTERACTING PROTEIN1 (GIP1) and GIP2, have been characterized. Both GIP genes are ubiquitously expressed in all tissues analyzed. Immunolocalization studies combined with the expression of GIP-green fluorescent protein fusions have shown that GIPs colocalize with γ-tubulin, GCP3, and/or GCP4 and reorganize from the nucleus to the prospindle and the preprophase band in late G2. After nuclear envelope breakdown, they localize on spindle and phragmoplast MTs and on the reforming nuclear envelope of daughter cells. The gip1 gip2 double mutants exhibit severe growth defects and sterility. At the cellular level, they are characterized by MT misorganization and abnormal spindle polarity, resulting in ploidy defects. Altogether, our data show that during mitosis GIPs play a role in γ-tubulin complex localization, spindle stability and chromosomal segregation. © 2012 American Society of Plant Biologists. All rights reserved.
Majeran W.,Cornell University |
Majeran W.,Institute des science du Vegetal |
Friso G.,Cornell University |
Ponnal L.,Cornell University |
And 9 more authors.
Plant Cell | Year: 2010
C4 grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations.C4photosynthesis isestablishedalongthedevelopmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics ofmaize leafdevelopment and C4 differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C4 specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. Anonlineprotein expressionviewer is providedthrough the Plant Proteome Database. © 2010 American Society of Plant Biologists.
Chen X.,AM Technology |
Chen X.,Ghent University |
Grandont L.,Institute des science du Vegetal |
Li H.,AM Technology |
And 11 more authors.
Nature | Year: 2014
The prominent and evolutionarily ancient role of the plant hormone auxin is the regulation of cell expansion. Cell expansion requires ordered arrangement of the cytoskeleton but molecular mechanisms underlying its regulation by signalling molecules including auxin are unknown. Here we show in the model plant Arabidopsis thaliana that in elongating cells exogenous application of auxin or redistribution of endogenous auxin induces very rapid microtubule re-orientation from transverse to longitudinal, coherent with the inhibition of cell expansion. This fast auxin effect requires auxin binding protein 1 (ABP1) and involves a contribution of downstream signalling components such as ROP6 GTPase, ROP-interactive protein RIC1 and the microtubule-severing protein katanin. These components are required for rapid auxin-and ABP1-mediated re-orientation of microtubules to regulate cell elongation in roots and dark-grown hypocotyls as well as asymmetric growth during gravitropic responses. © 2014 Macmillan Publishers Limited. All rights reserved.
Jaffre T.,Institute Of Recherche Pour Le Developpement |
Pillon Y.,University of Hawaii at Hilo |
Thomine S.,Institute des science du Vegetal |
Merlot S.,Institute des science du Vegetal
Frontiers in Plant Science | Year: 2013
While an excess of metals such as zinc, cadmium or nickel (Ni) is toxic for most plants, about 500 plant species called hyperaccumulators are able to accumulate high amounts of these metals. These plants and the underlying mechanisms are receiving an increasing interest because of their potential use in sustainable biotechnologies such as biofortification, phytoremediation, and phytomining. Among hyperaccumulators, about 400 species scattered in 40 families accumulate Ni. Despite this wide diversity, our current knowledge of the mechanisms involved in Ni accumulation is still limited and mostly restricted to temperate herbaceous Brassicaceae. New Caledonia is an archipelago of the tropical southwest pacific with a third of its surface (5500 km2) covered by Ni-rich soils originating from ultramafic rocks. The rich New Caledonia flora contains 2145 species adapted to these soils, among which 65 are Ni hyperaccumulators, including lianas, shrubs or trees, mostly belonging to the orders Celastrales, Oxalidales, Malpighiales, and Gentianales. We present here our current knowledge on Ni hyperaccumulators from New Caledonia and the latest molecular studies developed to better understand the mechanisms of Ni accumulation in these plants. © 2013 Jaffré, Pillon, Thomine and Merlot.
Ramos M.S.,Institute des science du Vegetal |
Ramos M.S.,University of Udine |
Khodja H.,CEA Saclay Nuclear Research Center |
Mary V.,Institute des science du Vegetal |
Thomine S.,Institute des science du Vegetal
Frontiers in Plant Science | Year: 2013
Seeds are a crucial stage in plant life. They contain the nutrients necessary to initiate the development of a new organism. Seeds also represent an important source of nutrient for human beings. Iron (Fe) and zinc (Zn) deficiencies affect over a billion people worldwide. It is therefore important to understand how these essential metals are stored in seeds. In this work, Particle-Induced X-ray Emission with the use of a focused ion beam (μPIXE) has been used to map and quantify essential metals in Arabidopsis seeds. In agreement with Synchrotron radiation X-ray fluorescence (SXRF) imaging and Perls/DAB staining, μPIXE maps confirmed the specific pattern of Fe and Mn localization in the endodermal and subepidermal cell layers in dry seeds, respectively. Moreover, μPIXE allows absolute quantification revealing that the Fe concentration in the endodermal cell layer reaches ~800 μg·g-1 dry weight. Nevertheless, this cell layer accounts only for about half of Fe stores in dry seeds. Comparison between Arabidopsis wild type (WT) and mutant seeds impaired in Fe vacuolar storage (vit1-1) or release (nramp3nramp4) confirmed the strongly altered Fe localization pattern in vit1-1, whereas no alteration could be detected in nramp3nramp4 dry seeds. Imaging of imbibed seeds indicates a dynamic localization of metals as Fe and Zn concentrations increase in the subepidermal cell layer of cotyledons after imbibition. The complementarities between μPIXE and other approaches as well as the importance of being able to quantify the patterns for the interpretation of mutant phenotypes are discussed. © 2013 Schnell Ramos, Khodja, Mary and Thomine.
Lang J.,Institute des science du Vegetal |
Faure D.,Institute des science du Vegetal
Frontiers in Plant Science | Year: 2014
In Agrobacterium tumefaciens, horizontal transfer and vegetative replication of oncogenic Ti plasmids involve a cell-to-cell communication process called quorum-sensing (QS). The determinants of the QS-system belong to the LuxR/LuxI class. The LuxI-like protein TraI synthesizes N-acyl-homoserine lactone molecules which act as diffusible QS-signals. Beyond a threshold concentration, these molecules bind and activate the LuxR-like transcriptional regulator TraR, thereby initiating the QS-regulatory pathway. For the last 20 years, A. tumefaciens has stood as a prominent model in the understanding of the LuxR/LuxI type of QS systems. A number of studies also unveiled features which are unique to A. tumefaciens QS, some of them being directly related to the phytopathogenic lifestyle of the bacteria. In this review, we will present the current knowledge of QS in A. tumefaciens at both the genetic and molecular levels. We will also describe how interactions with plant host modulate the QS pathway of A. tumefaciens, and discuss what could be the advantages for the agrobacteria to use such a tightly regulated QS-system to disseminate the Ti plasmids. © 2014 Lang and Faure.
Bourcy M.,Institute des science du Vegetal
Plant signaling & behavior | Year: 2013
Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. In this organ, symbiotic cells contain large numbers of bacteroids. Remarkably, this chronic infection does not trigger visible defense reactions. Despite the importance of this phenomenon for potential transfer of the symbiotic capacity to non-legume plants, the molecular mechanisms underlying this tolerance are not understood. We have characterized the dnf2 M. truncatula mutant blocked in the symbiotic process after bacterial infection of the symbiotic cells. Nodules formed by the mutant contain only few layers of infected cells. Furthermore, they exhibit defense-like reactions which clearly contrast with premature senescence frequently observed during inefficient symbioses. This atypical phenotype raises DNF2 as an exciting starting point to investigate the molecular basis of symbiotic repression of plant defenses.