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Caractérisation des rôles des gènes PHO1 dans l'homéostasie du phosphate et les voies de transductions de signaux

English title characterization of the roles of PHO1 genes in Pi homeostasis and signal transduction pathways
Applicant Poirier Yves
Number 122493
Funding scheme Project funding (Div. I-III)
Research institution Dépt de Biologie Moléculaire Végétale Faculté de Biologie et de Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Molecular Biology
Start/End 01.01.2009 - 31.12.2011
Approved amount 466'207.00
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All Disciplines (2)

Discipline
Molecular Biology
Botany

Keywords (9)

phosphate; Arabidopsis; rice; Physcomitrella patens; signal transduction; abscissic acid; gibberellin; phosphate deficiency; transport

Lay Summary (French)

Lead
Lay summary
Le phosphate est un élément essentiel et limitant pour la croissance des plantes. Ce projet a pour but d'étudier le rôle des protéines PHO1 impliquées dans le transport et l'homéostasie du phosphate chez la plante modèle Arabidopsis thaliana.Résumé :Le phosphate est un élément essentiel à la croissance de tout organisme vivant. Chez les plantes, il est nécessaire non seulement d'acquérir le phosphate du sol via les racines, mais aussi de le transporter et distribuer vers les autres tissues, par exemple les feuilles et les fleurs. Notre laboratoire a isolé un gène, appelé PHO1, qui est impliqué dans le transport du phosphate de la racine vers les feuilles. L'étude du génome de la plante modèle Arabidopsis thaliana nous révèle qu'il existe 10 autres gènes similaires à PHO1. Notre but à long terme est de découvrir le rôle de cette famille de gène dans le transport et l'homéostasie du phosphate. Nous avons récemment découvert que PHO1 joue aussi un rôle important dans la réponse et l'adaptation de la plante à une carence en phosphate. But : Notre recherche a plusieurs axes. Premièrement, nous désirons comprendre comment PHO1 peut agir en même temps comme transporteur et régulateur de la carence en phosphate. Une des approches choisies est d'analyser l'interaction de PHO1 avec d'autres protéines qui pourraient moduler son activité. Nous désirons aussi comprendre le rôle de certains homologues de PHO1 dans le transport et l'homéostasie du phosphate. Ceci est principalement étudié via la caractérisation des plantes mutantes ayant une inactivation de ces gènesSignification :Le phosphate est un élément important des fertilisants utilisés en agriculture et est nécessaire à la croissance optimale des plantes. Malheureusement, le phosphate représente une ressource non-renouvelable et est une source de pollution des eaux, causant le phénomène d'eutrophication, i.e. une sur-croissance des algues dans les lacs et rivières. Ile est donc nécessaire de pouvoir développer de nouvelles plantes agricoles capables de pousser d'une manière optimale avec un apport réduit en fertilisant. Cette recherche à donc pour but de mieux comprendre comment la plante acquiert et distribue le phosphate dans ses tissues et comment elle peut mieux s'adapter à une carence de cet élément essentiel.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux
Stefanovic A, Arpat AB, Bligny R, Gout E, Vidoudez C, Bensimon M, Poirier Y (2011), Over-expression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux, in PLANT JOURNAL, 66(4), 689-699.
The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis
Rouached H, Secco D, Arpat B, Poirier Y (2011), The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis, in BMC PLANT BIOLOGY, 11, e19-e25.
Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis
Rouached H, Stefanovic A, Secco D, Arpat AB, Gout E, Bligny R, Poirier Y (2011), Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis, in PLANT JOURNAL, 65(4), 557-570.
Characterization of the Rice PHO1 Gene Family Reveals a Key Role for OsPHO1;2 in Phosphate Homeostasis and the Evolution of a Distinct Clade in Dicotyledons
Secco D, Baumann A, Poirier Y (2010), Characterization of the Rice PHO1 Gene Family Reveals a Key Role for OsPHO1;2 in Phosphate Homeostasis and the Evolution of a Distinct Clade in Dicotyledons, in PLANT PHYSIOLOGY, 152(3), 1693-1704.
Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis
Thibaud MC, Arrighi JF, Bayle V, Chiarenza S, Creff A, Bustos R, Paz-Ares J, Poirier Y, Nussaume L (2010), Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis, in PLANT JOURNAL, 64(5), 775-789.
Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis
Thibaud Marie-Christine, Arrighi Jean-Francois, Bayle Vincent, Chiarenza Serge, Creff Audrey, Bustos Regla, Paz-Ares Javier, Poirier Yves, Nussaume Laurent (2010), Dissection of local and systemic transcriptional responses to phosphate starvation in Arabidopsis, in Plant Journal, 64, 775-789.
Regulation of Phosphate Starvation Responses in Plants: Signaling Players and Cross-Talks
Rouached H, Arpat AB, Poirier Y (2010), Regulation of Phosphate Starvation Responses in Plants: Signaling Players and Cross-Talks, in MOLECULAR PLANT, 3(2), 288-299.

Collaboration

Group / person Country
Types of collaboration
CEA-Cadarache France (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Associated projects

Number Title Start Funding scheme
105874 Characterization of the PHO1 phosphate transporter family of Arabidopsis 01.01.2005 Project funding (Div. I-III)
138339 Dissection of the mode of action of the Arabidopsis PHO1 in phosphate homeostasis 01.07.2012 Project funding (Div. I-III)
145002 Microscale thermophoresis for the Faculty of Bology and Medicine in Lausanne 01.12.2012 R'EQUIP

Abstract

PHO1 is a gene identified and cloned in Arabidopsis thaliana that is involved in the loading of inorganic phosphate (Pi) to the xylem vessels in root. The gene is distinct from any other Pi transporter identified to date, including the family of H+-Pi co-transporters in yeast and plants. The PHO1 gene family comprises 11 members in Arabidopsis that have been characterized for their expression profile. With few exceptions, homologues of PHO1 are found in all eukaryotes, but not in prokaryotes, indicating an important role of this gene in eukaryotic cells. However, no specific functions have been assigned to any homologues of PHO1 in eukaryotes, with the exception of the some members of the PHO1gene family in Arabidopsis. We have recently shown that out of the 11 members of the PHO1 gene family in Arabidopsis, only 2 genes, i.e. PHO1 and its closest homologue PHO1;H1, are involved in the loading of Pi into the xylem for its long-distance transport to the shoot. The main question that thus arises is “what are the roles of the other members of the PHO1 gene family in Arabidopsis”? We have recently obtained evidence that PHO1 and its homologues are involved in mediating a number of responses of plants to phytohormones, as well as in the responses of plants to Pi deficiency, implicating their role in various signal transduction pathways. A significant part of this grant is thus aimed at further understanding this aspect of PHO1 biology. PHO1 and its homologue PHO1;H10 are strongly expressed in guard cells, and PHO1;H10 is strongly up-regulated by the phytohormone abscissic acid (ABA), a key hormone affecting the activity of guard cells. The pho1 mutant appears deficient in the response of stomata opening following ABA addition. We wish to firmly establish the role of PHO1, and potentially also of PHO1;H10, in the regulation of stomata opening through the detailed analysis of the pho1 and pho1;h10 single and double mutants. For this purpose, we will make use of grafts between pho1 shoots and wild type roots, where the shoot and guard cells are genetically pho1 but are Pi-sufficient because of the WT root, in order to separate the more indirect effect of leaf Pi deficiency from the more specific role of PHO1 and PHO1;H10 on stomata. These experiments involve measuring the response of guard cells to a number of agents affecting stomata opening, such as ABA, jasmonic acid and calcium. The electrophysiological signature of mutant and WT guard cells will be compared in collaboration with the group of Alain Vavasseur.We have recently discovered that mutants of the PHO1;H5 gene show abnormalities in the growth of the roots, with epidermal cells files being twisted instead of being straight. This phenotype, which is often associated with the orientation of microtubules, was corrected by the addition of the phytohormone gibberellic acid (GA). Furthermore, the PHO1;H5 gene is up-regulated by GA. The link between GA, PHO1;H5 and cell growth will be explored through a detailed analysis of the pho1;h5 mutant for other phenotypes typically associated with GA. Double mutants between pho1;h5 and mutants affected in components of the GA signal transduction cascade (e.g. mutants in GAI, RGL, SPY, G protein) will be analyzed in order to determine potential epistatic interactions. Furthermore, the PHO1;H5 gene has a very close homologue, PHO1;H3, and the double mutant pho1;h5 pho1:h3 will be generated and analyzed for GA responses. Comparison of the global gene expression analysis between the pho1;h5 mutant and WT, in the presence or absence of GA, will be performed in order to identify potential proximal effectors of PHO1;H5 action in the GA signal transduction pathway. Furthermore, complementation of the pho1;h5 mutant with other genes of the PHO1 gene family will be done to identify the homologues which could have the same role.Analysis of transgenic plants under-expressing the PHO1 gene revealed a truly remarkable phenotype, in that despite the shoot being severely Pi-deficient, shoot growth remained unaffected and the shoot did not respond to the Pi-deficiency by the expected up-regulation of numerous genes associated with Pi deficiency. These data imply that PHO1 participates in the transmission of a signal that is important in the response of the shoot to Pi deficiency, and that the reduced growth normally associated with Pi deficiency is not directly caused by the reduced intracellular Pi concentration but rather largely caused by the genetic response of the plant to the Pi deficiency. Based on these results, we hypothesize that it should be possible to isolate mutants that grow better than wild type under Pi-deficiency. The Pi-deficient mutant pho1 will be mutagenised by EMS, and secondary mutants growing better than the pho1 parent will be isolated and analyzed for Pi transport dynamic, and the genetic response to Pi deficiency. Genes form the most interesting mutants will be identified by map-base cloning.In order to understand the function and regulation of PHO1 in various pathways, we have previously aimed at identifying proteins interacting with PHO1. Extensive screening using yeast two-hydrid and split-ubiquitin has identified seven candidate proteins interacting in yeast with PHO1. Among the most interesting and promising proteins we find calnexin, a molecular chaperone involved in sorting glycoproteins through the ER, CIPK, a protein kinase interacting with calcineurin B-like proteins (CIPK), and the ß-subunit of the heterotrimeric G protein. Mutants in the identified candidate genes will be analyzed for potential defect in PHO1 function, including Pi transport to the xylem, the response of plants to Pi deficiency, as well as the response of stomata to ABA. Some candidates have close homologues, and in this case, double mutants will be created and analyzed. Furthermore, interaction of selected candidate proteins with PHO1 will be confirmed through various approaches, including co-immunoprecipitation and bi-molecular fluorescence complementation (BiFC) in planta.Finally, our research on understanding the roles the PHO1 genes in plants includes two other model plants, namely rice and the moss Physcomitrella patens. The PHO1 family in rice contains only 3 genes that are all more closely related to the original Arabidopsis PHO1 than to other members of the Arabidopsis PHO1 family, such as PHO1;H5. Furthermore, all three rice PHO1 genes have a cis-natural antisense transcripts associated with the sense transcript, a situation that is not found in Arabidopsis. These data suggest a potentially unique mode of regulation of the PHO1 genes in rice. The regulation of the three sense and antisense transcripts of the PHO1 genes in rice will be analyzed in detail to understand the potential implication of these antisense transcripts. Furthermore, insertional mutants affecting the sense and/or antisense transcripts of the three rice genes will be identified and the single and double mutants plants analyzed for defects in the Pi transport, Pi homeostasis, the response of plant to Pi deficiency, and other potential phenotypes associated with ABA or GA. The Physcomitrella patens genome contains seven PHO1 genes. It is speculated that moss could be a good model to discover roles of PHO1 in plant biology other than long-distance Pi transport, since mosses do not have an elaborate vascular cylinder with xylem vessels, and do not need to transfer Pi over long-distances like higher plants. Our analysis of the PHO1 gene in moss will principally focus on the two genes we have recently identified which are specifically upregulated by Pi deficiency. Single and double mutants in these genes will be created through homologous recombination and the resulting plants analyzed for phenotypes relating to Pi homeostasis or the response to environmental stress and phytohormones.
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