Jean Pierre Renou

Jean Pierre Renou Portrait  

With June 2010, Jean-Pierre Renou left the URGV to take up a position as group leader at the Genhort in Angers. During his stay at the URGV, Jean-Pierre sucessfully establised the Microarray facility and set up many fruitful collaborations. The microarray group now merged with the group on Arabidopsis Functional Genomics by Claire Lurin who will coordinate the future activites of microarrays and deep sequencing transcriptomics at URGV.

Future address of Jean-Pierre Renou:
UMR Génétique et Horticulture (GenHort)
INRA / Agrocampus-ouest / Université d'Angers
Tel 33 02 41 22 57 74

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Scientific Career

1992 PhD thesis, University of Angers, France
1993 Engineer, UMR 1259 INRA, Genetics & Horticulture - Angers, France
2000 Research Assistant, UR 1196 INRA, Genomics & Physiology of mammary gland,
Jouy en Josas, France
2003 Senior Scientist (CR1), leader of the ADT group, Plant Genomics Research (URGV)
UMR INRA1165-CNRS8114-UEVE, Evry, France,
2007 Habilitation for PhD Supervision (HDR) University of Paris XI, France
2008 Director of Research, leader of the FGA group, Plant Genomics Research (URGV)
UMR INRA1165-CNRS8114-UEVE, Evry, France


Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes.

BMC Genomics. 2010 Nov 12;11(1):630. PMID: 21073700
Cohen D, Bogeat-Triboulot MB, Tisserant E, Balzergue S, Martin-Magniette ML, Lelandais G, Ningre N, Renou JP, Tamby JP, Le Thiec D, Hummel I.

BACKGROUND: Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance. Here we assess the genotype-dependent component of the drought-induced transcriptome response in two poplar genotypes differing in drought tolerance. Drought-induced responses were analysed in leaves and root apices and were compared with available transcriptome data from other Populus species.
RESULTS: Using a multi-species designed microarray, a genomic DNA-based selection of probesets provided an unambiguous between-genotype comparison. Analyses of functional group enrichment enabled the extraction of processes physiologically relevant to drought response. The drought-driven changes in gene expression occurring in root apices were consistent across treatments and genotypes. For mature leaves, the transcriptome response varied weakly but in accordance with the duration of water deficit. A differential clustering algorithm revealed similar and divergent gene co-expression patterns among the two genotypes. Since moderate stress levels induced similar physiological responses in both genotypes, the genotype-dependent transcriptional responses could be considered as intrinsic divergences in genome functioning. Our meta-analysis detected several candidate genes and processes that are differentially regulated in root and leaf, potentially under developmental control, and preferentially involved in early and long-term responses to drought.
CONCLUSIONS: In poplar, the well-known drought-induced activation of sensing and signalling cascades was specific to the early response in leaves but was found to be general in root apices. Comparing our results to what is known in arabidopsis, we found that transcriptional remodelling included signalling and a response to energy deficit in roots in parallel with transcriptional indices of hampered assimilation in leaves, particularly in the drought-sensitive poplar genotype.

A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.

New Phytol. 2010 Sep 3. PMID: 20819179
Pelletier S, Van Orden J, Wolf S, Vissenberg K, Delacourt J, Ndong YA, Pelloux J, Bischoff V, Urbain A, Mouille G, Lemonnier G, Renou JP, Höfte H.
Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, Route de St-Cyr (RD10), F-78026 Versailles Cedex, France.

Summary *We focused on a developmentally regulated growth acceleration in the dark-grown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. *To this end, precise transcriptome analysis on dissected dark-grown hypocotyls, Fourier transform infrared (FT-IR) microspectroscopy and kinematic analysis were used. *Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT-IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. *This study shows that the acceleration of cell elongation marks a developmental transition in dark-grown hypocotyl cells and supports a role for pectin de-methylesterification in the timing of this event.

Brain transcriptional stability upon prion protein-encoding gene invalidation in zygotic or adult mouse.

BMC Genomics. 2010 Jul 22;11(1):448. PMID: 20649983
Chadi S, Young R, Le Guillou S, Tilly G, Bitton F, Martin-Magniette ML, Soubigou-Taconnat L, Balzergue S, Vilotte M, Peyre C, Passet B, Beringue V, Renou JP, Le Provost F, Laude H, Vilotte JL.

BACKGROUND: The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages. RESULTS: Only subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences. CONCLUSIONS: These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

Expression of the Beet necrotic yellow vein virus p25 protein induces hormonal changes and a root branching phenotype in Arabidopsis thaliana.

Transgenic Res. 2010 Jul 3. PMID: 20602166
Peltier C, Schmidlin L, Klein E, Taconnat L, Prinsen E, Erhardt M, Heintz D, Weyens G, Lefebvre M, Renou JP, Gilmer D.

The RNA-3-encoded p25 protein was previously characterized as one of the major symptom determinants of the Beet necrotic yellow vein virus. Previous analyses reported the influence of the p25 protein in root proliferation phenotype observed in rhizomania disease on infected sugar beets (Beta vulgaris). A transgenic approach was developed, in which the p25 protein was constitutively expressed in Arabidopsis thaliana Columbia (Col-0) ecotype in order to provide new clues as to how the p25 protein might promote alone disease development and symptom expression. Transgenic plants were characterized by Southern blot and independent lines carrying single and multiple copies of the transgene were selected. Mapping of the T-DNA insertion was performed on the monocopy homozygote lines. P25 protein was localized both in the nucleus and in the cytoplasm of epidermal and root cells of transgenic plants. Although A. thaliana was not described as a susceptible host for BNYVV infection, abnormal root branching was observed on p25 protein-expressing A. thaliana plants. Moreover, these transgenic plants were more susceptible than wild-type plants to auxin analog treatment (2,4-D) but more resistant to methyl jasmonate (MeJA), abscisic acid (ABA) and to lesser extend to salicylic acid (SA). Hormonal content assays measuring plant levels of auxin (IAA), jasmonate (JA) and ethylene precursor (ACC) revealed major hormonal changes. Global transcript profiling analyses on roots displayed differential gene expressions that could corroborate root branching phenotype and stress signaling modifications.

Walls Are Thin1 (WAT1), an Arabidopsis homolog of Medicago truncatula NODULIN21, is a tonoplast-localized protein required for secondary wall formation in fibers.

Plant J. 2010 May 20, PMID: 20497379
Ranocha P, Denancé N, Vanholme R, Freydier A, Martinez Y, Hoffmann L, Köhler L, Pouzet C, Renou JP, Sundberg B, Boerjan W, Goffner D.

Summary By combining Zinnia elegans in vitro tracheary element genomics with reverse genetics in Arabidopsis, we have identified a new upstream component of secondary wall formation in xylary and interfascicular fibers. WAT1 (Walls Are Thin1), an Arabidopsis thaliana homolog of Medicago truncatula NODULIN21 (MtN21), encodes a plant-specific, predicted integral membrane protein and is a member of the Plant-Drug/Metabolite Exporter (P-DME) family (Transporter Classification number: TC 2.A.7.3). Although WAT1 is ubiquitously expressed throughout the plant, its expression is preferentially associated with vascular tissues including developing xylem vessels and fibers. WAT1:GFP fusion protein analysis demonstrated that WAT1 is localized on the tonoplast. Analysis of wat1 mutants revealed two cell wall-related phenotypes in stems: a defect in cell elongation resulting in a dwarfed habit and little to no secondary cell walls in fibers. Secondary walls of vessel elements were unaffected by the mutation. The secondary wall phenotype was supported by comparative transcriptomic and metabolomic analyses of wat1 and wild type stems, as many transcripts and metabolites involved in secondary wall formation were reduced in abundance. Unexpectedly, these experiments also revealed a modification in tryptophan (Trp) and auxin metabolism that might contribute to the wat1 phenotype. Together, our data demonstrate an essential role for the WAT1 tonoplast protein in the control of secondary cell wall formation in fibers.
Plant J. 2010, May 20

Arabidopsis GLUTATHIONE REDUCTASE 1 plays a crucial role in leaf responses to intracellular H2O2 and in ensuring appropriate gene expression through both salicylic acid and jasmonic acid signaling pathways.

Plant Physiol. 2010 May 20, PMID: 20488891
Mhamdi A, Hager J, Chaouch S, Queval G, Han Y, Taconnat L, Saindrenan P, Gouia H, Issakidis-Bourguet E, Renou JP, Noctor G.

Fig 8

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductases (GR), which is encoded by two genes in Arabidopsis thaliana (GR1 and GR2). This study addressed the role of GR1 in H2O2 responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role of changes in glutathione status in H2O2 signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione (GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven by H2O2. Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines were dependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicating glutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H2O2-glutathione interactions in cat2 gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H2O2 availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes, and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSG accumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but little or no evidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays a crucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis glutathione reductase gene or by thiol systems such as the thioredoxin system.

Cell wall biogenesis of Arabidopsis thaliana elongating cells: transcriptomics complements proteomics.

BMC Genomics. 2009 Oct 31;10(1):505. PMID: 19878582
Jamet E, Roujol D, San Clemente H, Irshad M, Soubigou-Taconnat L, Renou JP, Pont-Lezica R.

ABSTRACT: BACKGROUND: Plant growth is a complex process involving cell division and elongation. Arabidopsis thaliana hypocotyls undergo a 100-fold length increase mainly by cell elongation. Cell enlargement implicates significant changes in the composition and structure of the cell wall. In order to understand cell wall biogenesis during cell elongation, mRNA profiling was made on half- (active elongation) and fully-grown (after growth arrest) etiolated hypocotyls. RESULTS: Transcriptomic analysis was focused on two sets of genes. The first set of 856 genes named cell wall genes (CWGs) included genes known to be involved in cell wall biogenesis. A significant proportion of them has detectable levels of transcripts (55.5%), suggesting that these processes are important throughout hypocotyl elongation and after growth arrest. Genes encoding proteins involved in substrate generation or in synthesis of polysaccharides, and extracellular proteins were found to have high transcript levels. A second set of 2927 genes labeled secretory pathway genes (SPGs) was studied to search for new genes encoding secreted proteins possibly involved in wall expansion. Based on transcript level, 433 genes were selected. Genes not known to be involved in cell elongation were found to have high levels of transcripts. Encoded proteins were proteases, protease inhibitors, proteins with interacting domains, and proteins involved in lipid metabolism. In addition, 125 of them encoded proteins of unknown function. Finally, comparison with results of a cell wall proteomic study on the same material revealed that 48 out of the 137 identified proteins were products of the genes having high or moderate level of transcripts. About 15% of the genes encoding proteins identified by proteomics showed levels of transcripts below background. CONCLUSIONS: Members of known multigenic families involved in cell wall biogenesis, and new genes that might participate in cell elongation were identified. Significant differences were shown in the expression of such genes in half- and fully-grown hypocotyls. No clear correlation was found between the abundance of transcripts (transcriptomic data) and the presence of the proteins (proteomic data) demonstrating (i) the importance of post-transcriptional events for the regulation of genes during cell elongation and (ii) that transcriptomic and proteomic data are complementary.

Accumulation of Flavonoids in an ntra ntrb Mutant Leads to Tolerance to UV-C.

Mol Plant. 2009 Mar;2(2):249-58. Epub 2008 Oct 29. PMID: 19825611
Bashandy T, Taconnat L, Renou JP, Meyer Y, Reichheld JP.
Laboratoire Génome et Développement des Plantes, Université de Perpignan, UMR CNRS 5096, 52 avenue Paul Alduy, 66860 Perpignan, France.

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of thioredoxins. There are two genes encoding NTRs (NTRA and NTRB) in the Arabidopsis genome, each encoding a cytosolic and a mitochondrial isoform. A double ntra ntrb mutant has recently been characterized and shows slower plant growth, slightly wrinkled seeds and a remarkable hypersensitivity to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis. In this paper, we demonstrate that this mutant also accumulates higher level of flavonoids. Analysis of transcriptome data showed that several genes of the flavonoid pathway are overexpressed in the ntra ntrb mutant. Accumulation of flavonoids is generally considered a hallmark of plant stress. Nevertheless, no elevation of the expression of genes encoding ROS-detoxification enzymes was observed, suggesting that the ntra ntrb plants do not suffer from oxidative disease. Another hypothesis suggests that flavonoids are specifically synthesized in the ntra ntrb mutant in order to rescue the inactivation of NTR. To test this, the ntra ntrb mutant was crossed with transparent testa 4 (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis. As ntra ntrb plants are more resistant to UV-C treatment than wild-type plants, this higher resistance was abolished in the ntra ntrb tt4 mutant, suggesting that accumulation of flavonoids in the ntra ntrb mutant protects plants against UV-light.

Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis.

PLoS One. 2009 Oct 8;4(10):e7364. PMID: 19812700
Ping Hong Meng (1#), Cécile Raynaud (1#*), Guillaume Tcherkez (2), Sophie Blanchet (1), Kamal Massoud (1), Séverine Domenichini (1), Yves Henry (1), Ludivine Soubigou-Taconnat (3), Caroline Lelarge-Trouverie (2), Patrick Saindrenan1, Jean Pierre Renou (3), Catherine Bergounioux (1)
(1) Institut de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris-Sud XI, bât 630, Plateau de Moulon, Orsay, France
(2) Plateforme Métabolisme Métabolome IFR87, Institut de Biotechnologie des Plantes, Université Paris-Sud XI, bât 630, Plateau du Moulon, Orsay, France
(3) Unité de Recherche en Génomique Végétale, 2, CP5708, Evry, France

Figure 6. The transcriptome of atips1-1 is similar to that of several LMM mutants or plants infected by pathogens. Hierarchical clustering was performed using 150 transcripts across the different SD/LD conditions. Each vertical line displays the expression data for one gene. List of genetic backgrounds or treatment are displayed horizontally. Red and green indicate up- and down-regulation in mutants (A) or treated plants (B) compared to wild-type or untreated plants, respectively. Intensity of the colours is proportional to the absolute value of the fold difference. Images presented here correspond to a representative region of the global image which was too wide to be reproduced integrally. doi:10.1371/journal.pone.0007364.g006

BACKGROUND: Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI) biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. METHODOLOGY/PRINCIPAL FINDINGS: - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. CONCLUSION/SIGNIFICANCE: Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established.

Biotic Stress-Associated microRNAs: Identification, Detection, Regulation, and Functional Analysis.

Methods Mol Biol. 2010;592:183-202. PMID: 19802597
Florence Jay (1,*), Jean-Pierre Renou (2), Olivier Voinnet (1) and Lionel Navarro (1)
(1) Institut de Biologie Moléculaire des Plantes, CNRS UPR2353 – Université Louis Pasteur, Strasbourg Cedex, France
(2) UMR Génomique Végétale INRA-CNRS-UEVE, 2 rue G.Crémieux, Evry, France

The methods described herein first highlight the strategies that were used to discover a biotic stress-associated miRNA. This involved (1) the selection of transcripts that were more abundant in transgenic plants expressing viral-derived suppressors of RNA silencing and transcripts that were repressed in wild-type seedlings treated with a biotic stress, (2) a 5' RACE-derived assay to map miRNA target sites, and (3) a bioinformatic analysis to retrieve specific miRNA loci from the Arabidopsis genome. We then describe methods used to monitor (1) the levels of primary miRNA transcripts (pri-miRNAs)/mature miRNAs and (2) the transcriptional activity of miRNAs in response to a biotic stress and bacterial challenge. Furthermore, we present a strategy to identify additional biotic stress-responsive miRNA genes and get insight into their regulation. This involves (1) a microarray approach that allows detection of pri-miRNAs, coupled with (2) a promoter analysis of co-regulated miRNA genes. Finally, we describe strategies that can be used to functionally characterize individual biotic stress-associated miRNAs, or the miRNA pathway, in disease resistance.
Plant MicroRNAs

Uracil Salvage is Necessary for Early Arabidopsis Development.

Plant J. 2009 Jun 29. PMID: 19563437
Mainguet SE, Gakière B, Majira A, Pelletier S, Bringel F, Guérard F, Caboche M, Berthomé R, Renou JP.
- URGV, UMR INRA 1165 - CNRS 8114 - UEVE, 2, rue Gaston Crémieux, CP5708, 91057 Evry cedex, France.

Abstract Uridine nucleotides can be formed by energy-consuming de novo synthesis or by the energy-saving recycling of nucleobases coming from nucleotide catabolism. Uracil phosphoribosyltransferases (UPRTs; EC are involved in the salvage of pyrimidines by catalyzing the formation of UMP from uracil and phosphoribosyl pyrophosphate. To date, UPRTs are described as unessential, energy saving enzymes. In the present work, the six genes annotated as UPRTs in the Arabidopsis genome are examined through phylogenetic and functional complementation approaches, and the available T-DNA insertion mutants are characterized. We show that a single nuclear gene encoding a protein targeted to plastids, UPP, is responsible for almost all UPRT activity in Arabidopsis. The inability to salvage uracil caused a light-dependent dramatic pale-green to albino phenotype, dwarfism, and the inability to produce viable progeny in loss-of-function mutants. Plastid biogenesis and starch accumulation was affected in all analyzed tissues, with the exception of stomata. Therefore we propose that uracil salvage is of major importance for plant development.
Plant Journal

Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis thaliana cell suspensions.

Plant Physiol. 2009 Mar 20. PMID:19304931
Krinke O, Flemr M, Vergnolle C, Collin S, Renou JP, Taconnat L, Yu A, Burketova L, Valentova O, Zachowski A, Ruelland E.
UPMC Univ Paris 06, Unite de Recherche 5;
Centre National de la Recherche Scientifique, Equipe d'Accueil Conventionnee 7180, Laboratoire de Physiologie Cellulaire et Moleculaire des Plantes, Ivry-sur-Seine, F-94200 France;
Institute of Chemical Technology, Prague, Department of Biochemistry and Microbiology, Prague, 166 28 Czech Republic;
Unite Mixte de Recherche Institut National de la Recherche Agronomique 1165;
Centre National de la Recherche Scientifique 8114, Unite de Recherche en Genomique Vegetale, Evry, F-91057 France;
Academy of Sciences of the Czech Republic, Institute of Experimental Botany, v.v.i., Prague, 160 00 Czech Republic.

Salicylic acid (SA) plays a central role in defense against pathogen attack, as well as in germination, flowering, senescence and the acquisition of thermotolerance. In this report we investigate the involvement of phospholipase D (PLD) in the SA signaling pathway. In presence of exogenous primary alcohols, the production of phosphatidic acid (PA) by PLD is diverted towards the formation of phosphatidylalcohols through a reaction called transphosphatidylation. By in vivo metabolic phospholipid labeling with 33Pi, PLD activity was found to be induced 45 min after addition of SA. We show that incubation of Arabidopsis thaliana cell suspensions with primary alcohols inhibited the induction of two SA-responsive genes, PR1 and WRKY38, in a dose dependent manner. This inhibitory effect was more pronounced when the primary alcohols were more hydrophobic. Secondary or tertiary alcohols had no inhibitory effect. These results provide compelling arguments for PLD activity being upstream of the induction of these genes by SA. A subsequent study of n-butanol effects on the SA responsive transcriptome identified 1327 genes differentially expressed upon SA treatment. Strikingly, the SA-response of 380 of these genes was inhibited by n-butanol but not by tert-butanol. A detailed analysis of the regulation of these genes showed that PLD could act both positively and negatively, either on gene induction or gene repression. The overlap with the previously described Phosphatidylinositol-4-Kinase pathway is discussed.

A cadmium induced nitrite oxide production in Arabidopsis thaliana triggers upregulation of genes related to iron uptake and sets up resistance mechanisms through induction of a NAS4 dependant nicotianamide production.

[Nitric Oxide Contributes to Cadmium Toxicity in Arabidopsis by Promoting Cadmium Accumulation in Roots and by Up-Regulating Genes Related to Iron Uptake1,[W]] PMID: 19168643

Besson-Bard A., Gravot A., Richaud P., Auroy P., Gaymard F., Taconnat L., Renou J.P., Pugin A. and Wendehenne D. 2009.
Plant Physiology 149:1302-1315, 2009 Jan 23. [First published online January 23, 2009; 10.1104/pp.108.133348]
UMR INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, 21065 Dijon cedex, France (A.B.-B., A.P., D.W.); UMR 118 Amélioration des Plantes et Biotechnologies Végétales, INRA/Agrocampus Rennes/Université Rennes 1, 35653 Le Rheu cedex, France (A.G.); Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, SBVME, IBEB, DSV, CEA, CNRS, Université Aix Marseille, 13108 Saint Paul lez Durance, France (P.R., P.A.); Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, UMR 5004 Agro-M/CNRS/INRA/UMII, 34060 Montpellier cedex 1, France (C.D., F.G.); and Unité de Recherche en Génomique Végétale, UMR 8114 CNRS/INRA/Université d'Evry-Val d'Essonne, 91057 Evry, France (L.T., J.-P.R.)

Nitric oxide (NO) functions as a cell-signaling molecule in plants. In particular, a role for NO in the regulation of iron homeostasis and in the plant response to toxic metals has been proposed. Here, we investigated the synthesis and the role of NO in plants exposed to cadmium (Cd2+), a nonessential and toxic metal. We demonstrate that Cd2+ induces NO synthesis in roots and leaves of Arabidopsis (Arabidopsis thaliana) seedlings. This production, which is sensitive to NO synthase inhibitors, does not involve nitrate reductase and AtNOA1 but requires IRT1, encoding a major plasma membrane transporter for iron but also Cd2+. By analyzing the incidence of NO scavenging or inhibition of its synthesis during Cd2+ treatment, we demonstrated that NO contributes to Cd2+-triggered inhibition of root growth. To understand the mechanisms underlying this process, a microarray analysis was performed in order to identify NO-modulated root genes up- and down-regulated during Cd2+ treatment. Forty-three genes were identified encoding proteins related to iron homeostasis, proteolysis, nitrogen assimilation/metabolism, and root growth. These genes include IRT1. Investigation of the metal and ion contents in Cd2+-treated roots in which NO synthesis was impaired indicates that IRT1 up-regulation by NO was consistently correlated to NO's ability to promote Cd2+ accumulation in roots. This analysis also highlights that NO is responsible for Cd2+-induced inhibition of root Ca2+ accumulation. Taken together, our results suggest that NO contributes to Cd2+ toxicity by favoring Cd2+ versus Ca2+ uptake and by initiating a cellular pathway resembling those activated upon iron deprivation.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes.

BMC Plant Biol. 2009 Jan 16;9(1):6. [Epub ahead of print] PMID: 19149885
Minic Z, Jamet E, San-Clemente H, Pelletier S, Renou JP, Rihouey C, Okinyo DP, Proux C, Lerouge P, Jouanin L.

ABSTRACT: BACKGROUND: Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. RESULTS: Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. CONCLUSION: Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins.

An integrated genomics approach to define niche establishment by Rhodococcus fascians.

Plant Physiol. 2008 Dec 31. [Epub ahead of print] PMID: 19118125
Depuydt S, Trenkamp S, Fernie AR, Elftieh S, Renou JP, Vuylsteke M, Holsters M, Vereecke D.
Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Gent, Belgium;
Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium;
Max-Planck Institute of Molecular Plant Physiology, University of Potsdam, 14476 Potsdam-Golm, Germany;
Unite Mixte de Recherche en Genomique Vegetale, Institut National de la Recherche Agronomique, F-91057, Evry, France.

Rhodococcus fascians is a Gram-positive phytopathogen that induces shooty hyperplasia on its host through the secretion of cytokinins. Global transcriptomics using microarrays combined with profiling of primary metabolites on infected Arabidopsis thaliana plants revealed that this Actinomycete modulated pathways to convert its host into a niche. The transcript data demonstrated that R. fascians leaves a very characteristic mark on Arabidopsis with an outspoken cytokinin response illustrated by the activation of cytokinin perception, signal transduction, and homeostasis. The microarray data further suggested active suppression of an oxidative burst during the R. fascians pathology and comparison with publicly available transcript datasets implied a central role for auxin in the prevention of plant defense activation. Gene ontology categorization of the differentially expressed genes hinted at a significant impact of infection on the primary metabolism of the host, which was confirmed by subsequent metabolite profiling. The much higher levels of sugars and amino acids in infected plants are presumably accessed by the bacteria as carbon and nitrogen sources to support epiphytic and endophytic colonization. Hexoses, accumulating from a significantly increased invertase activity, assumingly inhibited expression of photosynthesis genes and photosynthetic activity in infected leaves. Altogether these changes are indicative of sink development in symptomatic tissues. The metabolomics data furthermore point to the possible occurrence of secondary signaling during the interaction that might contribute to symptom development. The data are placed in the context of regulation of bacterial virulence gene expression, suppression of defense, infection phenotype, and niche establishment.

The Arabidopsis Abscisic Acid Catabolic Gene CYP707A2 Plays a Key Role in Nitrate Control of Seed Dormancy.

Plant Physiol. 2008 Dec 12. PMID: 19074630
Matakiadis T, Alboresi A, Jikumaru Y, Tatematsu K, Pichon O, Renou JP, Kamiya Y, Nambara E, Truong HN.
Unite de la Nutrition Azotee des Plantes, IJPB, INRA, 78026 Versailles cedex, France;
Growth Regulation Research Group, RIKEN Plant Science Center,
1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Japan 230-0045;
URGV, INRA, CNRS, 2, rue Gaston Cremieux, CP5708, 91057 Evry cedex

Nitrate releases seed dormancy in Arabidopsis thaliana Col-0 seeds in part by reducing abscisic acid (ABA) levels. Nitrate led to lower levels of ABA in imbibed seeds when included in the germination media (exogenous nitrate). Nitrate also reduced ABA levels in dry seeds when provided to the mother plant during seed development (endogenous nitrate). Transcript profiling of imbibed seeds treated with or without nitrate revealed that exogenous nitrate led to a higher expression of nitrate responsive genes whereas endogenous nitrate led to a profile similar to stratified or after-ripened seeds. Profiling experiments indicated that the expression of the ABA catabolic gene CYP707A2 was regulated by exogenous nitrate. The cyp707a2-1 mutant failed to reduce seed ABA levels in response to both endogenous and exogenous nitrate. In contrast both endogenous and exogenous nitrate reduced ABA levels of the wild type and cyp707a1-1 mutant seeds. The CYP707A2 mRNA levels in developing siliques were positively correlated with different nitrate doses applied to the mother plants. This was consistent with a role of the CYP707A2 gene in controlling seed ABA levels in response to endogenous nitrate. The cyp707a2-1 mutant was less sensitive to exogenous nitrate for breaking seed dormancy. Altogether our data underline the central role of the CYP707A2 gene in the nitrate-mediated control of ABA levels during seed development and germination.

A genome-wide and metabolic analysis determined the adaptive response of Arabidopsis cells to folate depletion induced by methotrexate.

Plant Physiol. 2008 Oct 17. PMID: 18931140
Loizeau K, De Brouwer V, Gambonnet B, Yu A, Renou JP, Van Der Straeten D, Lambert WE, Rebeille F, Ravanel S.
Laboratoire de Physiologie Cellulaire Vegetale, UMR5168 CNRS-CEA-INRA-Universite Joseph Fourier Grenoble I, Institut de Recherches en Technologies et Sciences pour le Vivant, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France,
Laboratory of Toxicology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium,
UMR INRA1165 CNRS8114 UEVE, Unite de Recherche en Genomique Vegetale, 2 rue Gaston Cremieux, CP5708, F-91057 Evry, France,
Unit Plant Hormone Signaling and Bio-imaging, Department of Molecular Genetics, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.

Control of folate homeostasis is essential to sustain the demand for one-carbon (C1) units that are necessary for major biological functions, including nucleotide synthesis and methylation reactions. In this study we analyzed the genome-wide and metabolic adaptive response of Arabidopsis thaliana cells to folate depletion induced by the antifolate methotrexate. Drug treatment induced a response typical to xenobiotic stress and important changes in folate content and composition. This resulted in a reduction of cell division and primary energy metabolism that was likely associated with perturbation of nucleotide homeostasis. Through a modification of serine metabolism, folate depletion also induced O-acetylserine accumulation and mimicked sulfur-deficiency response. The major adaptive response to folate limitation concerned the composition of the folate pool rather than the intracellular level of cofactors. Thus, no significant change in the expression of genes involved in cofactor synthesis, degradation or trafficking was observed. However, changes in the distribution of C1-derivatives pools and increased expression levels for transcripts coding enzymes manipulating C1-moieties in plastids suggested a re-orientation of C1-units towards the synthesis of purine and thymidylate. Also, no genomic or metabolic adaptation was built up to counterbalance the major impairment of the methyl index, which controls the efficiency of methylation reactions in the cell. Together, these data suggested that the metabolic priority of Arabidopsis cells in response to folate limitation was to shuttle the available folate derivatives to the synthesis of nucleotides at the expense of methylation reactions.

Accumulation of Flavonoids in an ntra ntrb Mutant Leads to Tolerance to UV-C.

Talaat Bashandy (a), Ludivine Taconnat (b), Jean-Pierre Renou (b), Yves Meyer (a) and Jean-Philippe Reichheld (a,1,2)
Molecular Plant, doi:10.1093/mp/ssn065 Oct 29, 2008
(a)Laboratoire Génome et Développement des Plantes, Université de Perpignan, UMR CNRS 5096, 52 avenue Paul Alduy, 66860 Perpignan, France
(b)Unité de Recherche en Génomique Végétale, 91057 Evry cedex, France
(1)To whom correspondence should be addressed. E-mail, fax (+33) 4 68 66 84 99.
(2)The author responsible for distribution of materials integral to the findings presented in this article.

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of thioredoxins. There are two genes encoding NTRs (NTRA and NTRB) in the Arabidopsis genome, each encoding a cytosolic and a mitochondrial isoform. A double ntra ntrb mutant has recently been characterized and shows slower plant growth, slightly wrinkled seeds and a remarkable hypersensitivity to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis. In this paper, we demonstrate that this mutant also accumulates higher level of flavonoids. Analysis of transcriptome data showed that several genes of the flavonoid pathway are overexpressed in the ntra ntrb mutant. Accumulation of flavonoids is generally considered a hallmark of plant stress. Nevertheless, no elevation of the expression of genes encoding ROS-detoxification enzymes was observed, suggesting that the ntra ntrb plants do not suffer from oxidative disease. Another hypothesis suggests that flavonoids are specifically synthesized in the ntra ntrb mutant in order to rescue the inactivation of NTR. To test this, the ntra ntrb mutant was crossed with transparent testa 4 (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis. As ntra ntrb plants are more resistant to UV-C treatment than wild-type plants, this higher resistance was abolished in the ntra ntrb tt4 mutant, suggesting that accumulation of flavonoids in the ntra ntrb mutant protects plants against UV-light.

The nodule inception-like protein 7 modulates nitrate sensing and metabolism in Arabidopsis.

Plant J. 2008 Sep 26. PMID 18826430
Castaings L, Camargo A, Pocholle D, Gaudon V, Texier Y, Boutet-Mercey S, Taconnat L, Renou JP, Daniel-Vedele F, Fernandez E, Meyer C, Krapp A.
IJPB, Unité de Nutrition Azotée des Plantes, INRA, route de St. Cyr, F-78026 Versailles Cedex, France.

coverimage Plant Journal

Abstract Nitrate is an essential nutrient, and it is involved in many adaptive responses of plants, such as localized proliferation of roots, flowering or stomatal movements. How such nitrate specific mechanisms are regulated at the molecular level is poorly understood. Although the Arabidopsis ANR1 transcription factor seems to control the stimulation of lateral root elongation in response to nitrate, no regulators of nitrate assimilation have so far been identified in higher plants. Legume-specific symbiotic nitrogen fixation is under the control of the putative transcription factor, NIN, in Lotus japonicus. Recently the algal homolog NIT2 was found to regulate nitrate assimilation. Here we report that Arabidopsis thaliana NIN Like Protein 7 (NLP7) knockout mutants constitutively display several traits of nitrogen starved plants and that they are tolerant to drought stress. We show that nlp7 mutants are impaired in the transduction of the nitrate signal and that NLP7 expression pattern is consistent with a function of NLP7 in the sensing of N. Translational fusions with the green fluorescent protein (GFP) show a nuclear localization for the NLP7 putative transcription factor. Altogether, we propose NLP7 as an important element of the nitrate signal transduction pathway and as a new regulatory protein specific for N assimilation in non-nodulating plants.

Genome-scale Arabidopsis promoter array identifies targets of the histone acetyltransferase GCN5.

Plant J. 2008 Jul 4. PMID: 18644002
Benhamed M, Martin-Magniette ML, Taconnat L, Bitton F, Servet C, De Clercq R, De Meyer B, Buysschaert C, Rombauts S, Villarroel R, Aubourg S, Beynon J, Bhalerao RP, Coupland G, Gruissem W, Menke FL, Weisshaar B, Renou JP, Zhou DX, Hilson P.
Institut de Biotechnologie des Plantes, UMR 8618, Centre National de la Recherche Scientifique, Université de Paris Sud 11, 91405 Orsay, France.

We have built a repertoire of approximately 20,000 Arabidopsis thaliana promoter regions, compatible with functional studies that require cloning and with microarray applications. The promoter fragments can be captured as modular entry clones (MultiSite Gateway format) via site-specific recombinational cloning and be transferred into vectors of choice to investigate transcriptional networks. The fragments can also be amplified by PCR and printed on glass arrays. In combination with the immunoprecipitation of protein-DNA complexes (ChIP-chip), these arrays enable the characterization of the binding sites of chromatin-associated proteins or the extent of chromatin modifications at genome scale. The Arabidopsis histone acetyltransferase GCN5 associated with 40% of the tested promoters. At most sites, binding did not depend on the integrity of the GCN5 bromodomain. However, the presence of the bromodomain was necessary for binding to 11% of the promoter regions and correlated with the acetylation of histone H3 lysine 14 in these promoters. Combined analysis of ChIP-chip and transcriptomic data indicated that the binding of GCN5 does not strictly correlate with gene activation. As GCN5 had previously been shown to be required for light-regulated gene expression and growth, we found that GCN5 targets were enriched in the early light-responsive genes. Thus, besides its transcriptional activation function GCN5 might play an important role in priming activation of inducible genes under non-induced conditions.

Physiological and transcriptomic aspects of urea uptake and assimilation in Arabidopsis plants.

Plant Physiology Preview, Published on May 28, 2008; 10.1104/pp.108.119339, PMID: 18508958
Patricia Merigout , Maud Lelandais , Frédérique Bitton , Jean-Pierre Renou , Xavier Briand , Christian Meyer , and Francoise Daniel-Vedele
INRA, IJPB, Unité de Nutrition Azotee des Plantes, F-78000 Versailles, France; INRA, Unité Mixte de Recherche en Génomique Végétale, F-91057 Evry, France; BiotechMarine BP 65, 22260 Pontrieux, France

Urea is the major nitrogen (N) form supplied as fertilizer in agriculture but also an important N metabolite in plants. Urea transport and assimilation were investigated in Arabidopsis. Uptake studies using (15)N-labelled urea demonstrated the capacity of Arabidopsis to absorb urea, and that the urea uptake was regulated by the initial N status of the plants. Urea uptake was stimulated by urea, but was reduced by the presence of ammonium nitrate in the growth medium. N deficiency in plants did not affect urea uptake. Urea exerted a repressive effect on nitrate influx whereas urea enhanced ammonium uptake. The use of [(15)N]urea and [(15)N]ammonium tracers allowed us to show that urea and ammonium assimilation pathways were similar. Finally, urea uptake was less efficient than nitrate uptake, and urea grown-plants presented signs of N starvation. We also report the first analysis of Arabidopsis gene expression profiling in response to urea. Our transcriptomic approach revealed that nitrate and ammonium transporters were transcriptionally regulated by urea, as well as key enzymes of the GS-GOGAT pathway. AtDUR3, a high-affinity urea transporter in Arabidopsis, was strongly up-regulated by urea. Moreover, our transcriptomic data suggest that other genes are also involved in urea influx.
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Plant DELLAs Restrain Growth and Promote Survival of Adversity by Reducing the Levels of Reactive Oxygen Species.


Curr Biol. 2008 Apr 30 PMID: 18450450
Achard P, Jean-Pierre Renou, Berthomé R, Harberd NP, Genschik P.
Institut de Biologie Moléculaire des Plantes, Conventionné avec l'Université Louis Pasteur, 67084 Strasbourg, France.
Unité de Recherche en Génomique Végétale (URGV), 91057 Evry cedex, France
University of Oxford, Department of Plant Sciences, South Parks Road, Oxford OX1 3RB, United Kingdom

Plant growth is adaptively modulated in response to environmental change. The phytohormone gibberellin (GA) promotes growth by stimulating destruction of the nuclear growth-repressing DELLA proteins [1-7], thus providing a mechanism for environmentally responsive growth regulation [8, 9]. Furthermore, DELLAs promote survival of adverse environments [8]. However, the relationship between these survival and growth-regulatory mechanisms was previously unknown. Here, we show that both mechanisms are dependent upon control of the accumulation of reactive oxygen species (ROS). ROS are small molecules generated during development and in response to stress that play diverse roles as eukaryotic intracellular second messengers [10]. We show that Arabidopsis DELLAs cause ROS levels to remain low after either biotic or abiotic stress, thus delaying cell death and promoting tolerance. In essence, stress-induced DELLA accumulation elevates the expression of genes encoding ROS-detoxification enzymes, thus reducing ROS levels. In accord with recent demonstrations that ROS control root cell expansion [11, 12], we also show that DELLAs regulate root-hair growth via a ROS-dependent mechanism. We therefore propose that environmental variability regulates DELLA activity [8] and that DELLAs in turn couple the downstream regulation of plant growth and stress tolerance through modulation of ROS levels.

Normalization for triple-target microarray experiments

BMC Bioinformatics. 2008 Apr 28;9(1):216 PMID: 18442385
Marie-Laure Martin-Magniette*¹², Julie Aubert¹, Avner Bar-Hen (4), Samira Elftieh² , Frederic Magniette³, Jean-Pierre Renou² and Jean-Jacques Daudin¹
¹   UMR AgroParisTech-INRA MIA 518, 75231 Paris Cedex05, France
²   UMR INRA 1165-CNRS 8114-UEVE URGV, 91057 Evry Cedex, France
³   Unit´e MOY300, D´el´egation CNRSˆIle de France Est, 94532 Thiais Cedex, France
(4)   Universite Paris Descartes, MAP 5, PARIS cedex 06, France
*   Corresponding author

Most microarray studies are made using labelling with one or two dyes which allows the hybridization of one or two samples on the same slide. In such experiments, the most frequently used dyes are Cy3 and Cy5. Recent improvements in the technology (dye-labelling, scanner and, image analysis) allow hybridization up to four samples simultaneously. The two additional dyes are Alexa 488 and Alexa 494. The triple-target or four-target technology is very promising, since it allows us more flexibility in the design of experiments, an increase in the statistical power when comparing gene expressions induced by different conditions and a scaled down number of slides. However, there have been few methods proposed for statistical analysis of such data. Moreover the lowess correction of the global dye effect is available for only two-color experiments, and even if its application can be derived, it does not allow simultaneous correction of the raw data.
RESULTS: We propose a two-step normalization procedure for triple-target experiments. First the dye bleeding is evaluated and corrected if necessary. Then the signal in each channel is normalized using a generalized lowess procedure to correct a global dye bias. The normalization procedure is validated using triple-self experiments and by comparing the results of triple-target and two-color experiments. Although the focus is on triple-target microarrays, the proposed method can be used to normalize p differently labelled targets co-hybridized on a same array, for any value of p greater than 2.
CONCLUSIONS: The proposed normalization procedure is effective: the technical biases are reduced, the number of false positives is under control in the analysis of differentially expressed genes, and the triple-target experiments are more powerful than the corresponding two-color experiments. There is room for improving the microarray experiments by simultaneously hybridizing more than two samples.

Cell wall modifications in Arabidopsis thaliana plants with altered {alpha}-Larabinofuranosidase activity.

Plant Physiol. 2008 Mar 14 PMID: 18344421
Chavez Montes RA, Ranocha P, Martinez Y, Minic Z, Jouanin L, Marquis M, Saulnier L, Fulton LM, Cobbett CS, Bitton F, Renou JP, Jauneau A, Goffner D.
UMR 5546 CNRS-UPS "Surfaces Cellulaires et Signalisation chez les Vegetaux", 24 chemin de Borde Rouge, BP 42617 Auzeville, 31326 Castanet-Tolosan, France;
Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Route de St-Cyr, 78026 Versailles Cedex, France;
Biopolymeres Interactions Assemblages, Unite de Recherche sur les Polysaccharides leurs Organisations et Interactions, Institut National de la Recherche Agronomique, BP 71627, 44316 Nantes Cedex 03, France;
Department of Genetics, University of Melbourne, Victoria 3010, Australia;
Unité de Recherche en Génomique Végétale INRA-CNRS, 2 rue Gaston Crémieux, CP 5708, 91057 Evry cedex, France.

Although cell wall remodelling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis thaliana plants with altered expression of ARAF1, a bifunctional alpha-L-arabinofuranosidase / beta-D-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalisation experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labelling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalisation studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.
Plant Physiology
Supplemental Data 1 (xls)
Supplemental Data 2 (xls)

Simultaneous Interaction of Arabidopsis thaliana with Bradyrhizobium Sp. Strain ORS278 and Pseudomonas syringae pv. tomato DC3000 Leads to Complex Transcriptome Changes.

Molecular Plant Microbe Interactions 2008 Feb;21(2):244-59. PMID: 18184068
Fabienne Cartieaux (1), Céline Contesto (1), Adrien Gallou (1), Guilhem Desbrosses (1), Joachim Kopka (2), Ludivine Taconnat (3), Jean-Pierre Renou (3), and Bruno Touraine (1)
(1) Laboratoire des Symbioses Tropicales et Méditerranéennes (UMR113, Université Montpellier, Institut de Recherche pour le Développement, Cirad, Ecole Nationale Supérieure d'Agronomie de Montpellier, INRA
(2) Max Planck Institute of Molecular Plant Physiology, Am Muhlenberg 1, 14476 Golm, Germany;
(3) URGV (UMR 8114 CNRS, INRA, Université d'Evry-Val d'Essonne), Evry, France

Induced systemic resistance (ISR) is a process elicited by telluric microbes, referred to as plant growth-promoting rhizobacteria (PGPR), that protect the host plant against pathogen attacks. ISR has been defined from studies using Pseudomonas strains as the biocontrol agent. Here, we show for the first time that a photosynthetic Bradyrhizobium sp. strain, ORS278, also exhibits the ability to promote ISR in Arabidopsis thaliana, indicating that the ISR effect may be a widespread ability. To investigate the molecular bases of this response, we performed a transcriptome analysis designed to reveal the changes in gene expression induced by the PGPR, the pathogen alone, or by both. The results confirm the priming pattern of ISR described previously, meaning that a set of genes, of which the majority was predicted to be influenced by jasmonic acid or ethylene, was induced upon pathogen attack when plants were previously colonized by PGPR. The analysis and interpretation of transcriptome data revealed that 12-oxo-phytodienoic acid, an intermediate of the jasmonic acid biosynthesis pathway, is likely to be an actor in the signaling cascade involved in ISR. In addition, we show that the PGPR counterbalanced the pathogen-induced changes in expression of a series of genes.
Supplementary Table 1 Download xls file
Supplementary Table 2 Genes that were identified as down-regulated in Arabidopsis plants colonized by Bradyrhizobium ORS278. (pdf file)

Di- and tri- but not mono-methylation on histone H3 lysine 36 marks active transcription of genes involved in flowering time regulation and other processes in Arabidopsis thaliana

Mol Cell Biol. 2007 Dec 10 PMID: 18070919
Lin Xu, Zhong Zhao, Aiwu Dong, Ludivine Soubigou-Taconnat, Jean-Pierre Renou, Andre Steinmetz, and Wen-Hui Shen
Institut de Biologie Moléculaire des Plantes (IBMP), Centre National de la Recherche Scientifique (CNRS), Université Louis Pasteur de Strasbourg (ULP), Strasbourg, France; Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai, China; URGV, UMR INRA 1165 - CNRS 8114 – UEVE, France; CRP-Santé, Luxembourg;

Histone lysines can be mono-, di- or tri-methylated, providing an ample magnitude of epigenetic information for transcription regulation. In fungi, SET2 is the sole methyltransferase responsible for mono-, di- and tri-methylation of H3K36. Here we show that in Arabidopsis the degree of H3K36 methylation is regulated by distinct methyltransferases. The SET2-homologs SDG8 and SDG26 each can methylate oligonucleosomes in vitro and both proteins are localized in the nucleus. While the previously reported loss-of-function sdg8 mutants have an early-flowering phenotype, the loss-of-function sdg26 mutants show a late-flowering phenotype. Consistently, several MADS-box flowering repressors are down-regulated by sdg8 but up-regulated by sdg26. The sdg8 but not the sdg26 mutant plants show a dramatically reduced level of both di- and tri-methyl-H3K36 and an increased level of mono-methyl-H3K36. SDG8 is thus specifically required for di- and tri-methylation of H3K36. Our results further establish that H3K36 di- and tri- but not mono-methylation correlates with transcription activation. Finally, we show that SDG8 and VIP4, which encodes a component of the PAF1 complex, act independently and synergistically in transcription regulation. Together our results reveal that the deposition of H3K36 methylation is finely regulated, possibly to cope with the complex regulation of growth and development in higher eukaryotes.

Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana: novel insights into atrazine toxicity and sucrose-induced tolerance.

[Transcriptome profiling reveals the apoptotic effects of the herbicide atrazine and large-scale protective effects of sucrose signaling in Arabidopsis thaliana plantlets.]

BMC Genomics. 2007 Dec 5;8(1):450 PMID: 18053238
Ramel F (1), Sulmon C (1) Cabello-Hurtado F (1), Taconnat L (2), Martin-Magniette ML (2, 3), Jean-Pierre Renou (2), Elamrani A (1), Couee I (1), Gouesbet G. (1)
(1) CNRS, Université de Rennes 1, UMR 6553 ECOBIO, France
(2) UMR INRA 1165-CNRS 8114-UEVE, Unité de Recherche en Génomique Végétale (URGV),Evry, France
(3) UMR AgroParisTech-INRA, Mathématique et Informatique Appliquées 518, Paris, France

ABSTRACT: BACKGROUND: Soluble sugars, which play a central role in plant structure and metabolism, are also involved in the responses to a number of stresses, and act as metabolite signalling molecules that activate specific or hormone-crosstalk transduction pathways. The different roles of exogenous sucrose in the tolerance of Arabidopsis thaliana plantlets to the herbicide atrazine and oxidative stress were studied by a transcriptomic approach using CATMA arrays. RESULTS: Parallel situations of xenobiotic stress and sucrose-induced tolerance in the presence of atrazine, of sucrose, and of sucrose plus atrazine were compared. These approaches revealed that atrazine affected gene expression and therefore seedling physiology at a much larger scale than previously described, with potential impairment of protein translation and of reactive-oxygen-species (ROS) defence mechanisms. Correlatively, sucrose-induced protection against atrazine injury was associated with important modifications of gene expression related to ROS defence mechanisms and repair mechanisms. These protection-related changes of gene expression did not result only from the effects of sucrose itself, but from combined effects of sucrose and atrazine, thus strongly suggesting important interactions of sucrose and xenobiotic signalling or of sucrose and ROS signalling. CONCLUSIONS: These interactions resulted in characteristic differential expression of gene families such as ascorbate peroxidases, glutathione-S-transferases and cytochrome P450s, and in the early induction of an original set of transcription factors. These genes used as molecular markers will eventually be of great importance in the context of xenobiotic tolerance and phytoremediation.

Analysis of CATMA transcriptome data identifies hundreds of novel functional genes and improves gene models in the Arabidopsis genome.

BMC Genomics. 2007 Nov 2;8(1):401 PMID: 17980019
Sébastien Aubourg,  Martin-Magniette ML, Brunaud V, Taconnat L, Bitton F, Balzergue S, Jullien PE, Ingouff M, Thareau V, Schiex T,  Alain Lecharny,  Jean-Pierre Renou

Figure 7: Expression intensity and expression range of the novel genes

ABSTRACT: BACKGROUND: Since the finishing of the sequencing of the Arabidopsis thaliana genome, the Arabidopsis community and the annotator centers have been working on the improvement of gene annotation at the structural and functional levels. In this context, we have used the large CATMA resource on the Arabidopsis transcriptome to search for genes missed by different annotation processes. Probes on the CATMA microarrays are specific gene sequence tags (GSTs) based on the CDS models predicted by the Eugene software. Among the 24 576 CATMA v2 GSTs, 677 are in regions considered as intergenic by the TAIR annotation. We analyzed the cognate transcriptome data in the CATMA resource and carried out data-mining to characterize novel genes and improve gene models.
The statistical analysis of the results of more than 500 hybridized samples distributed among 12 organs provides an experimental validation for 465 novel genes. The hybridization evidence was confirmed by RT-PCR approaches for 88% of the 465 novel genes. Comparisons with the current annotation show that these novel genes often encode small proteins, with an average size of 137 aa. Our approach has also led to the improvement of pre-existing gene models through both the extension of 16 CDS and the identification of 13 gene models erroneously constituted of two merged CDS.
This work is a noticeable step forward in the improvement of the Arabidopsis genome annotation. We increased the number of Arabidopsis validated genes by 465 novel transcribed genes to which we associated several functional annotations such as expression profiles, sequence conservation in plants, cognate transcripts and protein motifs.

CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes.

BMC Bioinformatics. 2007 Oct 18;8:400. PMID: 17945016
Sclep G, Allemeersch J, Liechti R, De Meyer B, Beynon J, Bhalerao R, Moreau Y, Nietfeld W,  Jean-Pierre Renou,  Reymond P, Kuiper MT, Hilson P.

Figure 1: Overview of the GST classification and design process yielding the CATMAv3 repertoire.

The Complete Arabidopsis Transcript MicroArray (CATMA) initiative combines the efforts of laboratories in eight European countries [1] to deliver gene-specific sequence tags (GSTs) for the Arabidopsis research community. The CATMA initiative offers the power and flexibility to regularly update the GST collection according to evolving knowledge about the gene repertoire. These GST amplicons can easily be reamplified and shared, subsets can be picked at will to print dedicated arrays, and the GSTs can be cloned and used for other functional studies. This ongoing initiative has already produced approximately 24,000 GSTs that have been made publicly available for spotted microarray printing and RNA interference.
GSTs from the CATMA version 2 repertoire (CATMAv2, created in 2002) were mapped onto the gene models from two independent Arabidopsis nuclear genome annotation efforts, TIGR5 and PSB-EuGene, to consolidate a list of genes that were targeted by previously designed CATMA tags. A total of 9,027 gene models were not tagged by any amplified CATMAv2 GST, and 2,533 amplified GSTs were no longer predicted to tag an updated gene model. To validate the efficacy of GST mapping criteria and design rules, the predicted and experimentally observed hybridization characteristics associated to GST features were correlated in transcript profiling datasets obtained with the CATMAv2 microarray, confirming the reliability of this platform. To complete the CATMA repertoire, all 9,027 gene models for which no GST had yet been designed were processed with an improved version of the Specific Primer and Amplicon Design Software (SPADS). A total of 5,756 novel GSTs were designed and amplified by PCR from genomic DNA. Together with the pre-existing GST collection, this new addition constitutes the CATMAv3 repertoire. It comprises 30,343 unique amplified sequences that tag 24,202 and 23,009 protein-encoding nuclear gene models in the TAIR6 and EuGene genome annotations, respectively. To cover the remaining untagged genes, we identified 543 additional GSTs using less stringent design criteria and designed 990 sequence tags matching multiple members of gene families (Gene Family Tags or GFTs) to cover any remaining untagged genes. These latter 1,533 features constitute the CATMAv4 addition.
To update the CATMA GST repertoire, we designed 7,289 additional sequence tags, bringing the total number of tagged TAIR6-annotated Arabidopsis nuclear protein-coding genes to 26,173. This resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin RNA silencing vectors. All information about the resulting updated CATMA repertoire is available through the CATMA database .

CATdb: a public access to Arabidopsis transcriptome data from the URGV-CATMA platform.

Figure 1: Results of a query of CATdb for an experiment called Circadian cycle

Nucleic Acids Res. 2007 Oct 16 PMID: 17940091
Gagnot S, Tamby JP, Martin-Magniette ML, Bitton F, Taconnat L, Balzergue S, Sébastien Aubourg, Alain Lecharny, Jean-Pierre Renou,  Brunaud V.
URGV - UMR INRA 1165-CNRS 8114-UEVE, Laboratoire de Biologie Cellulaire - Institut J.P. Bourgin - INRA Centre de Versailles-Grignon, Versailles, France,
Unité de Mathématiques et Informatique Appliquées (MIA) - UMR 518 AgroParisTech-INRA, Paris
Université Paris-Sud, Institut de Biotechnologie des Plantes (IBP) - UMR CNRS UPS, Orsay, France.

CATdb is a free resource available at that provides public access to a large collection of transcriptome data for Arabidopsis thaliana produced by a single Complete Arabidopsis Transcriptome Micro Array (CATMA) platform. CATMA probes consist of gene-specific sequence tags (GSTs) of 150-500 bp. The v2 version of CATMA contains 24 576 GST probes representing most of the predicted A. thaliana genes, and 615 probes tiling the chloroplastic and mitochondrial genomes. Data in CATdb are entirely processed with the same standardized protocol, from microarray printing to data analyses. CATdb contains the results of 53 projects including 1724 hybridized samples distributed between 13 different organs, 49 different developmental conditions, 45 mutants and 63 environmental conditions. All the data contained in CATdb can be downloaded from the web site and subsets of data can be sorted out and displayed either by keywords, by experiments, genes or lists of genes up to 100. CATdb gives an easy access to the complete description of experiments with a picture of the experiment design.

A Receptor-like Kinase Mediates the Response of Arabidopsis Cells to the Inhibition of Cellulose Synthesis

Current Biology, Vol 17, 922-931, 05 June 2007 PMID: 17540573
Kian Hématy, Pierre-Etienne Sado, Ageeth Van Tuinen, Soizic Rochange, Thierry Desnos, Sandrine Balzergue, Sandra Pelletier, Jean-Pierre Renou, and Herman Höfte
Laboratoire de Biologie Cellulaire, UR501, Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique Centre de Versailles, Versailles, France, URGV

A major challenge is to understand how the walls of expanding plant cells are correctly assembled and remodeled, often in the presence of wall-degrading micro-organisms. Plant cells, like yeast, react to cell-wall perturbations as shown by changes in gene expression, accumulation of ectopic lignin, and growth arrest caused by the inhibition of cellulose synthesis.
We have identified a plasma-membrane-bound receptor-like kinase (THESEUS1), which is present in elongating cells. Mutations in THE1 and overexpression of a functional THE1-GFP fusion protein did not affect wild-type (WT) plants but respectively attenuated and enhanced growth inhibition and ectopic lignification in seedlings mutated in cellulose synthase CESA6 without influencing the cellulose deficiency. A T-DNA insertion mutant for THE1 also attenuated the growth defect and ectopic-lignin production in other but not all cellulose-deficient mutants. The deregulation of a small number of genes in cesA6 mutants depended on the presence of THE1. Some of these genes are involved in pathogen defense, in wall crosslinking, or in protecting the cell against reactive oxygen species.
The results show that THE1 mediates the response of growing plant cells to the perturbation of cellulose synthesis and may act as a cell-wall-integrity sensor.

Phosphatidylinositol 4-kinase Activation Is an Early Response to Salicylic Acid in Arabidopsis Suspension Cells

Plant Physiol. 2007 Jul;144(3):1347-59. Epub 2007 May 11. PMID: 17496105
Ondrej Krinke, Eric Ruelland, Olga Valentová, Chantal Vergnolle, Jean-Pierre Renou, Ludivine Taconnat, Matyás Flemr, Lenka Burketová, and Alain Zachowski, 2007
Université Pierre et Marie Curie-Paris 6 and Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7180, Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Ivry-sur-Seine, France.

Salicylic acid (SA) has a central role in defence against pathogen attack. Besides, its role in such diverse processes as germination, flowering, senescence and thermotolerance acquisition has been documented. However, little is known about the early signalling events triggered by SA. Using Arabidopsis thaliana suspension cells as a model, it was possible to show by in vivo metabolic phospholipid labelling with 33Pi that SA addition induced a rapid and early (in few minutes) decrease in a pool of phosphatidylinositol (PI). This decrease paralleled with an increase in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. These changes could be inhibited by two different inhibitors of type III PI 4-kinases, phenylarsine oxide and 30 µM wortmannin; no inhibitory effect was seen with 1 µM wortmannin, a concentration inhibiting PI 3-kinases but not PI 4-kinases. We therefore undertook a study of wortmannin effects on SA responsive transcriptome. Using the Complete Arabidopsis Transcriptome MicroArray (CATMA) chip, we could identify 774 genes differentially expressed upon SA treatment. Strikingly, amongst these genes, the response to SA of 112 of them was inhibited by 30 µM wortmannin but not by 1 µM wortmannin.

ATM-Mediated Transcriptional and Developmental Responses to gamma-rays in Arabidopsis.

Figure 1, Root tip morphology and expression of fluorescent markers of WT seedlings after IR.

PLoS ONE. 2007 May 9;2(5):e430. PMID: 17487278
Ricaud L, Proux C, Renou JP, Pichon O, Fochesato S, Ortet P, Montane MH. May 2007
CEA, DSV, Institut de Biologie Environnementale et de Biotechnologie (iBEB), Service de biologie végétale et de microbiologie environnementales (SBVME), Cadarache, Saint Paul-lez-Durance, France.

ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of gamma-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post-IR were compiled in a single table, which was used to import gene information and extract gene sets. Sequence and function homology searches; import of spatio-temporal, cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes in all functional classes. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA replication, repair, and recombination; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints.

Figure 2. CLSM optical longitudinal sections of WT and atm stem cells and QC post-IR.

Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. The transcription burst was almost exclusively AtATM-dependent or weakly AtATR-dependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways after IR are also discussed.

Arabidopsis NRP1 and NRP2 Encode Histone Chaperones and Are Required for Maintaining Postembryonic Root Growth.

Root tip and stem-root junction region, respectively, from a transgenic Arabidopsis plant expressing YFP:NRP1.

Plant Cell. 2006 Nov;18(11):2879-92. Epub 2006 Nov 22. PMID: 17122067
Zhu Y, Dong A, Meyer D, Pichon O, Renou JP, Cao K, Shen WH.
Institut de Biologie Moléculaire des Plantes, Laboratoire Propre du Centre National de la Recherche Scientifique, Unité Propre de Recherche 2357, Conventioné avec l'Université Louis Pasteur, Strasbourg, France.

NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) is conserved from yeast to human and was proposed to act as a histone chaperone. While budding yeast contains a single NAP1 gene, multicellular organisms, including plants and animals, contain several NAP1 and NAP1-RELATED PROTEIN (NRP) genes. However, the biological role of these genes has been largely unexamined. Here, we show that, in Arabidopsis thaliana, simultaneous knockout of the two NRP genes, NRP1 and NRP2, impaired postembryonic root growth. In the nrp1-1 nrp2-1 double mutant, arrest of cell cycle progression at G2/M and disordered cellular organization occurred in root tips. The mutant seedlings exhibit perturbed expression of ~100 genes, including some genes involved in root proliferation and patterning. The mutant plants are highly sensitive to genotoxic stress and show increased levels of DNA damage and the release of transcriptional gene silencing. NRP1 and NRP2 are localized in the nucleus and can form homomeric and heteromeric protein complexes. Both proteins specifically bind histones H2A and H2B and associate with chromatin in vivo. We propose that NRP1 and NRP2 act as H2A/H2B chaperones in the maintenance of dynamic chromatin in epigenetic inheritance.

Genetic variation suggests an interaction between cold acclimation and the metabolic regulation of leaf senescence.

Figure 3. Function of genes affected by Glc independent of the genetic background

Plant Physiol. 2007 Jan;143(1):434-46. Epub 2006 Nov 10. PMID: 17098848
Masclaux-Daubresse C, Purdy S, Lemaitre T, Pourtau N, Taconnat L, Renou JP and Wingler A. 2007.
Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, Versailles, France.

The extent to which leaf senescence is induced by nitrogen deficiency or by sugar accumulation varies between natural accessions of Arabidopsis (Arabidopsis thaliana). Analysis of senescence in plants of the Bay-0 x Shahdara recombinant inbred line (RIL) population revealed a large variation in developmental senescence of the whole leaf rosette, which was in agreement with the extent to which glucose (Glc) induced senescence in the different lines. To determine the regulatory basis of genetic differences in the Glc response, we investigated changes in gene expression using Complete Arabidopsis Transcriptome MicroArray (CATMA) analysis. Genes whose regulation did not depend on the genetic background, as well as genes whose regulation was specific to individual RILs, were identified. In RIL 310, a line that does not show the typical senescence response to Glc, stress response genes, especially those responding to cold stress, were induced by Glc. We therefore tested whether cold acclimation delays senescence by reducing sugar sensitivity. In cold-acclimated plants, leaf senescence was severely delayed and Glc did not induce the typical senescence response. Together, our results suggest that cold acclimation extends rosette longevity by affecting metabolic regulation of senescence, thereby allowing vernalization-dependent plants to survive the winter period. The role of functional chloroplasts and of nitrogen and phosphate availability in this regulation is discussed.

Cross-talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb-4 transcription factor.

[Analysis of transcriptional networks regulated by the sunflower Hahb-4 transcription factor in Arabidopsis revealed a conserved role in ethylene signaling.]

Plant J. 2006 Oct;48(1):125-37. PMID: 16972869
Manavella PA, Arce AL, Dezar CA, Bitton F, Renou JP, Crespi M, Chan RL.
Cátedra de Biología Celular y Molecular, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, CONICET, Santa Fe, Argentina.

Figure 2. Transgenic plants enter into the senescence program later and are insensitive to ethylene.

Hahb-4 is a member of the Helianthusannuus (sunflower) subfamily I of HD-Zip proteins that is transcriptionally regulated by water availability and abscisic acid. Transgenic Arabidopsis thaliana plants overexpressing this transcription factor (TF) exhibit a characteristic phenotype that includes a strong tolerance to water stress. Here we show that this TF is a new component of ethylene signalling pathways, and that it induces a marked delay in senescence. Plants overexpressing Hahb-4 are less sensitive to external ethylene, enter the senescence pathway later and do not show the typical triple response. Furthermore, transgenic plants expressing this gene under the control of its own inducible promoter showed an inverse correlation between ethylene sensitivity and Hahb-4 levels. Potential targets of Hahb-4 were identified by comparing the transcriptome of Hahb-4-transformed and wild-type plants using microarrays and quantitative RT-PCR. Expression of this TF has a major repressive effect on genes related to ethylene synthesis, such as ACO and SAM, and on genes related to ethylene signalling, such as ERF2 and ERF5. Expression studies in sunflower indicate that Hahb-4 transcript levels are elevated in mature/senescent leaves. Expression of Hahb-4 is induced by ethylene, concomitantly with several genes homologous to the targets identified in the transcriptome analysis (HA-ACOa and HA-ACOb). Transient transformation of sunflower leaves demonstrated the action of Hahb-4 in the regulation of ethylene-related genes. We propose that Hahb-4 is involved in a novel conserved mechanism related to ethylene-mediated senescence that functions to improve desiccation tolerance.

Genome-wide transcriptome profiling of the early cadmium response of Arabidopsis roots and shoots.

Biochimie. 2006 Nov;88(11):1751-65. Epub 2006 Jun 6. PMID: 16797112
Herbette S., Taconnat L., Hugouvieux V, Martin-Magniette M.L., Cuine S., Auroy P., Richaud P., Forestier C., Bourguignon J. Renou JP , Vavasseur A. and Leonhart N.2006.
CEA Cadarache, DSV/DEVM/Laboratoire des Echanges Membranaires et Signalisation, UMR 6191 CNRS-CEA-Aix-Marseille-II, 13108 Saint-Paul-les-Durance cedex, France.

Transcriptional regulation in response to cadmium treatment was investigated in both roots and leaves of Arabidopsis, using the whole genome CATMA microarray containing at least 24,576 independent probe sets. Arabidopsis plants were hydroponically treated with low (5 microM) or high (50 microM) cadmium concentrations during 2, 6, and 30 hours. At each time point, Cd level was determined using ICP-AES showing that both plant tissues are able to accumulate the heavy metal. RT-PCR of eight randomly selected genes confirmed the reliability of our microarray results. Analyses of response profiles demonstrate the existence of a regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to cadmium. One of the main response observed in roots was the induction of genes involved in sulfur assimilation-reduction and glutathione (GSH) metabolism. In addition, HPLC analysis of GSH and phytochelatin (PC) content shows a transient decrease of GSH after 2 and 6 h of metal treatment in roots correlated with an increase of PC contents. Altogether, our results suggest that to cope with cadmium, plants activate the sulfur assimilation pathway by increasing transcription of related genes to provide an enhanced supply of GSH for PC biosynthesis. Interestingly, in leaves an early induction of several genes encoding enzymes involved in the biosynthesis of phenylpropanoids was observed. Finally, our results provide new insights to understand the molecular mechanisms involved in transcriptional regulation in response to cadmium exposure in plants.

MGOUN3: evidence for chromatin-mediated regulation of FLC expression.

J Exp Bot. 2006;57(9):2111-9. Epub 2006 May 25. PMID: 16728410
Guyomarch's S, Benhamed M, Lemonnier G, Renou JP, Zhou DX and Delarue M. 2006.
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Bât. 630. Université Paris XI, Orsay, France.

Journal of Experimental Botany

The MGOUN3(MGO3)/BRUSHY1(BRU1)/TONSOKU(TSK) gene of Arabidopsis thaliana encodes a nuclear leucine–glycine–asparagine (LGN) domain protein that may be implicated in chromatin dynamics and genome maintenance. Mutants with defects in MGO3 display a fasciated stem and disorganized meristem structures. The transition to flowering was examined in mgo3 mutants and it was found that, under short days, the mutants flowered significantly earlier than the wild-type plants. Study of flowering-time associated gene expression showed that the floral transition inhibitor gene FLC was under-expressed in the mutant background. Ectopic expression of the flower-specific genes AGAMOUS (AG), PISTILLATA (PI), and SEPALLATA3 (SEP3) in mgo3 vegetative organs was also detected. Western blot and chromatin immunoprecipitation experiments suggested that histone H3 acetylation may be altered in the mgo3 background. Together, these data suggest that MGO3 is required for the correct transition to flowering and that this may be mediated by histone acetylation and associated changes in FLC expression.

A coumaroyl-ester-3- hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth.

Figure 4. Ectopic lignification phenotypes of cyp98A3 insertion and cosuppressed plants.

Plant Physiol. 2006 Jan;140(1):30-48. Epub 2005 Dec 23. PMID: 16377748
Abdulrazzak N, Pollet B, Ehlting J, Larsen K, Asnaghi C, Ronseau S, Proux C, Erhardt M, Seltzer V, Renou JP, Ullmann P, Pauly M, Lapierre C & Werck-Reichhart D. 2006.
Department of Plant Metabolic Responses , Institute of Plant Molecular Biology Centre National de la Recherche Scientifique-Unité Propre de Recherche 2357, Université Louis Pasteur, 67000 Strasbourg, France.

Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.

The cold-induced early activation of phospholipase C and D pathways determines the response of two distinct clusters of genes in Arabidopsis cell suspensions.

Plant Physiol. 2005 Nov;139(3):1217-33. Epub 2005 Oct 28. PMID: 16258011
Vergnolle C, Vaultier MN, Taconnat L, Renou JP, Kader JC, Zachowski A, Ruelland E.
Laboratoire de Physiologie Cellulaire et Moléculaire des Plantes, Formation de Recherche en Evolution 2846, Centre National de la Recherche Scientifique/Université Pierre et Marie Curie, F-94200 Ivry-sur-Seine, France.

Figure 2. Functional categories of the cold-induced genes or the coldrepressed genes.

In plants, a temperature downshift represents a major stress that will lead to the induction or repression of many genes. Therefore, the cold signal has to be perceived and transmitted to the nucleus. In response to a cold exposure, we have shown that the phospholipase D (PLD) and the phospholipase C (PLC)/diacylglycerol kinase pathways are simultaneously activated. The role of these pathways in the cold response has been investigated by analyzing the transcriptome of cold-treated Arabidopsis (Arabidopsis thaliana) suspension cells in the presence of U73122 or ethanol, inhibitors of the PLC/diacylglycerol kinase pathway and of the phosphatidic acid produced by PLD, respectively. This approach showed that the expression of many genes was modified by the cold response in the presence of such agents. The cold responses of most of the genes were repressed, thus correlating with the inhibitory effect of U73122 or ethanol. We were thus able to identify 58 genes that were regulated by temperature downshift via PLC activity and 87 genes regulated by temperature downshift via PLD-produced phosphatidic acid. Interestingly, each inhibitor appeared to affect different cold response genes. These results support the idea that both the PLC and PLD pathways are upstream of two different signaling pathways that lead to the activation of the cold response. The connection of these pathways with the CBF pathway, currently the most understood genetic system playing a role in cold acclimation, is discussed.

Genome-wide expression profiling of the host response to root-knot nematode infection in Arabidopsis.

Figure 5. In vivo validation by promoter GUS fusion of downregulation of a trypsin protease inhibitor TPI and upregulation of the formin AtFH10.

Plant J. 2005 Nov;44(3):447-58. PMID: 16236154

Jammes F, Lecomte P, de Almeida-Engler J, Bitton F, Martin-Magniette ML, Renou JP, Abad P, Favery B.
UMR INRA 1064-UNSA-CNRS 6192, Interactions Plantes-Microorganismes et Santé Végétale, 400 route des Chappes, BP 167, 06903 Sophia Antipolis, France.

During a compatible interaction, root-knot nematodes (Meloidogyne spp.) induce the redifferentiation of root cells into multinucleate nematode feeding cells (giant cells). Hyperplasia and hypertrophy of the surrounding cells leads to the formation of a root gall. We investigated the plant response to root-knot nematodes by carrying out a global analysis of gene expression during gall formation in Arabidopsis, using giant cell-enriched root tissues. Among 22 089 genes monitored with the complete Arabidopsis transcriptome microarray gene-specific tag, we identified 3373 genes that display significant differential expression between uninfected root tissues and galls at different developmental stages. Quantitative PCR analysis and the use of promoter GUS fusions confirmed the changes in mRNA levels observed in our microarray analysis. We showed that a comparable number of genes were found to be up- and downregulated, indicating that gene downregulation might be essential to allow proper gall formation. Moreover, many genes belonging to the same family are differently regulated in feeding cells. This genome-wide overview of gene expression during plant-nematode interaction provides new insights into nematode feeding-cell formation, and highlights that the suppression of plant defence is associated with nematode feeding-site development.

hca: An Arabidopsis mutant exhibiting unusual cambial activity and altered vascular patterning.

Plant J. 2005 Oct;44(2):271-89. PMID: 16212606
Pineau C, Freydier A, Ranocha P, Jauneau A, Turner S, Lemonnier G, Renou JP, Tarkowski P, Sandberg G, Jouanin L, Sundberg B, Boudet AM, Goffner D, Pichon M.
Surfaces Cellulaires et Signalisation chez les Végétaux, Unité Mixte de Recherche, Centre National de la Recherche Scientifique - Université Paul Sabatier 5546, Pôle de Biotechnologie Végétale, 31326 Castanet Tolosan, France.

Figure 5. hca/cov1 morphology and vasculature organization.

By screening a T-DNA population of Arabidopsis mutants for alterations in inflorescence stem vasculature, we have isolated a mutant with a dramatic increase in vascular tissue development, characterized by a continuous ring of xylem/phloem. This phenotype is the consequence of premature and numerous cambial cell divisions in both the fascicular and interfascicular regions that result in the loss of the alternate vascular bundle/fiber organization typically observed in Arabidopsis stems. The mutant was therefore designated high cambial activity (hca). The hca mutation also resulted in pleiotropic effects including stunting and a delay in developmental events such as flowering and senescence. The physiological characterization of hca seedlings in vitro revealed an altered auxin and cytokinin response and, most strikingly, an enhanced sensitivity to cytokinin. These results were substantiated by comparative microarray analysis between hca and wild-type plants. The genetic analysis of hca indicated that the mutant phenotype was not tagged by the T-DNA and that the hca mutation segregated as a single recessive locus, mapping to the long arm of chromosome 4. We propose that hca is involved in mechanisms controlling the arrangement of vascular bundles throughout the plant by regulating the auxin-cytokinin sensitivity of vascular cambial cells. Thus, the hca mutant is a useful model for examining the genetic and hormonal control of cambial growth and differentiation.

Molecular and functional characterization of Arabidopsis Cullin 3A

Plant J. 2005 Feb;41(3):386-99. PMID: 15659098
Dieterle M, Thomann A, Renou JP, Parmentier Y, Cognat V, Lemonnier G, Muller R, Shen WH, Kretsch T, Genschik P.
Institut de Biologie Moléculaire des Plantes du CNRS, Strasbourg, France.

Cullin proteins, which belong to multigenic families in all eukaryotes, associate with other proteins to form ubiquitin protein ligases (E3s) that target substrates for proteolysis by the 26S proteasome. Here, we present the molecular and genetic characterization of a plant Cullin3. In contrast to fungi and animals, the genome of the model plant Arabidopsis thaliana contains two related CUL3 genes, called CUL3A and CUL3B. We found that CUL3A is ubiquitously expressed in plants and is able to interact with the ring-finger protein RBX1. A genomic search revealed the existence of at least 76 BTB-domain proteins in Arabidopsis belonging to 11 major families. Yeast two-hybrid experiments indicate that representative members of certain families are able to physically interact with both CUL3A and CUL3B, suggesting that Arabidopsis CUL3 forms E3 protein complexes with certain BTB domain proteins. In order to determine the function of CUL3A, we used a reverse genetic approach. The cul3a null mutant flowers slightly later than the control plants. Furthermore, this mutant exhibits a reduced sensitivity of the inhibition of hypocotyl growth in far-red light and miss-expresses COP1. The viability of the mutant plants suggests functional redundancy between the two CUL3 genes in Arabidopsis.

Arabidopsis HAF2 gene encoding TATA-binding protein (TBP)-associated factor TAF1, is required to integrate light signals to regulate gene expression and growth.

J Biol Chem. 2005 Jan 14;280(2):1465-73. Epub 2004 Nov 3. PMID: 15525647
Bertrand C, Benhamed M, Li YF, Ayadi M, Lemonnier G, Renou JP, Delarue M, Zhou DX.
Institut de Biotechnologie des Plantes, UMR8618, Université Paris-sud XI, 91405 Orsay, France.

Plant growth and development are sensitive to light. Light-responsive DNA-binding transcription factors have been functionally identified. However, how transcription initiation complex integrates light signals from enhancer-bound transcription factors remains unknown. In this work, we characterized mutations within the Arabidopsis HAF2 gene encoding TATA-binding protein-associated factor TAF1 (or TAF(II)250). The mutation of HAF2 induced decreases on chlorophyll accumulation, light-induced mRNA levels, and promoter activity. Genetic analysis indicated that HAF2 is involved in the pathways of both red/far-red and blue light signals. Double mutants between haf2-1 and hy5-1, a mutation of a light signaling positive DNA-binding transcription factor gene, had a synergistic effect on photomorphogenic traits and light-activated gene expression under different light wavelengths, suggesting that HAF2 is required for interaction with additional light-responsive DNA-binding transcription factors to fully respond to light induction. Chromatin immunoprecipitation assays showed that the mutation of HAF2 reduced acetylation of histone H3 in light-responsive promoters. In addition, transcriptome analysis showed that the mutation altered the expression of about 9% of genes in young leaves. These data indicate that TAF1 encoded by the Arabidopsis HAF2 gene functions as a coactivator capable of integrating light signals and acetylating histones to activate light-induced gene transcription.

Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis.

Figure 3. Motif Structure of Arabidopsis PPR Proteins

Plant Cell. 2004 Aug;16(8):2089-103. Epub 2004 Jul 21. PMID: 15269332
Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, Caboche M, Debast C, Gualberto J, Hoffmann B, Lecharny A, Le Ret M, Martin-Magniette ML, Mireau H, Peeters N, Renou JP, Szurek B, Taconnat L, Small I.
URGV, INRA, CNRS, Université d'Evry Val d'Essone

The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

Loss of protein phosphatase 2A expression correlates with phosphorylation of DP-1 and reversal of dysplasia through differentiation in a conditional mouse model of cancer progression.

Cancer Res. 2003 Nov 15;63(22):7668-73. PMID: 14633688
Tilli MT, Hudgins SL, Frech MS, Halama ED, Renou JP, Furth PA. 2003.
Graduate Program in Human Genetics, University of Maryland at Baltimore, Baltimore, MD, USA.

A conditional mouse model of time-dependent dysplasia reversal demonstrated that reversal and differentiation of dysplastic salivary gland tissue at the 4-month reversible stage was characterized by the appearance of a phosphorylated slower mobility form of Differentiation Related Transcription Factor 1-polypeptide-1 that was correlated with cellular differentiation. The phosphorylated form of DP-1 was not found at the 7-month irreversible stage or in adenocarcinomas. At the 4-month reversible stage, protein phosphatase 2A expression was down-regulated coincident with loss of oncogene expression, whereas PP2A expression persisted at the 7-month irreversible stage. Results are consistent with the hypothesis that persistent PP2A expression prevented the appearance of the phosphorylated form of DP-1 required for cellular differentiation and reversal of dysplasia after loss of oncogene expression.

Identification of genes differentially expressed in mouse mammary epithelium transformed by an activated beta-catenin.

Oncogene. 2003 Jul 17;22(29):4594-610. PMID: 12881717
Renou JP, Bierie B, Miyoshi K, Cui Y, Djiane J, Reichenstein M, Shani M and Hennighausen L. 2003.
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Beta-catenin is an executor of Wnt signaling and it can control cell fate and specification. Deletion of exon 3 from the endogenous beta-catenin gene in differentiating mammary alveolar epithelium of the mouse results in the generation of an activated protein that lacks amino acids 5-80. This is accompanied by a loss of mammary epithelial differentiation and a transdifferentiation process to squamous metaplasias. To further understand the molecular process of transdifferentiation, the expression of genes in mammary tissue was profiled in the absence and presence of activated of beta-catenin. Microarrays were generated that carry about 8500 cDNA clones with approximately 6000 obtained from mammary tissue. Mutant tissues, which had undergone either partial (TD1) or complete (TD2) squamous transdifferentiation, were compared with wild-type mammary tissue. Four groups of genes were identified. Group 1 contained genes whose expression was induced in both mutant tissues. Groups 2 and 3 contained genes that were active preferentially in TD2 and TD1, respectively. Group 4 contained genes suppressed in both samples. Using this approach, known and unknown genes activated in the transdifferentiation process were identified. A new 20 kDa protein (PANE1) induced upon transdifferentiation was nuclear in nonconfluent cells and cytoplasmic in confluent or dividing cells. Lastly, stabilization of beta-catenin resulted in the retention of differentiated epithelium upon involution and altered activities of several proteases in transdifferentiated mammary epithelium.

Activation of beta-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation.

[Stabilization of ß-catenin in prostate epithelium induces hyperplasias and squamous transdifferentiation.]

Oncogene. 2003 Jun 19;22(25):3875-87. PMID: 12813461
Bierie B, Nozawa M, Renou JP, Shillingford JM, Morgan F, Oka T, Taketo M, Cardiff RD, Miyoshi K, Wagner KU, Robinson GW and Hennighausen L. 2003.
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

The Wnt/beta-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to beta-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the beta-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of beta-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of high-grade prostate intraepithelial neoplasia were detected. Since beta-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of beta-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of beta-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to beta-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

PTEN overexpression suppresses proliferation and differentiation and enhances apoptosis of the mouse mammary epithelium.

[Overexpression of PTEN in mous mammary epithelium suppresses epithelial proliferation and differentiation and enhances apoptosis.]

J Clin Invest. 2002 Sep;110(6):815-25. PMID: 12235113
Dupont J, Renou JP, Shani M, Hennighausen L and Leroith D. 2002.
Section on Molecular and Cellular Physiology, Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892, USA.

The phosphatase PTEN regulates growth, adhesion, and apoptosis, among many other cell processes. To investigate its role during mouse mammary gland development, we generated MK-PTEN, a transgenic mouse model in which human PTEN is overexpressed in ductal and alveolar mammary epithelium during puberty, pregnancy, lactation, and involution. No obvious phenotype was observed in mammary tissue of pubescent virgin mice. However, MK-PTEN females could not lactate normally, and approximately 30% of pups died, with survivors exhibiting growth retardation. Transgenic offspring nursed by wild-type foster mothers, conversely, developed normally. This phenotype is consistent with a reduced number of alveolar epithelial cells due to a decrease in cell proliferation and an increase in apoptosis. Using mammary-enriched cDNA microarrays, we identified several genes that were preferentially expressed in MK-PTEN mammary tissue, including the IGF-binding protein-5 (Igfbp5) gene, and others whose expression was reduced, including the genes for c-Jun amino-terminal kinase. Secretory epithelial cell differentiation was impaired, as measured by the expression of specific milk protein genes. MK-PTEN mice also exhibited a 50% decrease in the phosphorylation state of Akt. Taken together, these results suggest that PTEN controls mammary gland development and, consequently, lactation.

Mammary epithelial cells are not able to undergo pregnancy-dependent differentiation in the absence of the helix-loop-helix inhibitor Id2.

Mol Endocrinol. 2002 Dec;16(12):2892-901. PMID: 12456807
Miyoshi K, Meyer B, Gruss P, Cui Y, Renou JP, Morgan F, Smith GH, Reichenstien M Shani M, Hennighausen L and Robinson G.W. 2002.
Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases/NIH, Building 8, Room 101, 8 Center Drive, Bethesda, MD 20892, USA.

Mammary alveolar development during pregnancy is triggered by hormone signals. The prolactin receptor/Jak2/signal transducer and activator of transcription (Stat) 5 signal transduction pathway is the principal mediator of these cues and alveolar development is abrogated in its absence. The loss of the basic helix-loop-helix protein inhibitor of differentiation (Id)2 results in a similar defect. To investigate the role of Id2 in mammary epithelium, we performed structural and molecular analyses. Id2-null mammary epithelial cells were unable to form alveoli; the epithelial architecture was disorganized and dissimilar from early stages of alveologenesis in wild-type glands. The epithelial cells retained the ductal marker Na-K-Cl cotransporter (NKCC)1. Nuclear localization of Stat5a and down-regulation of NKCC1 was observed in some areas, indicating a limited response to pregnancy signals. The differentiation status of Id2-null tissue at term was further characterized with cDNA microarrays enriched in mammary specific sequences (mammochip). Some of the early differentiation markers for mammary epithelium were expressed in the Id2-null tissue, whereas genes that are expressed at later stages of pregnancy were not induced. From these results, we conclude that, in the absence of Id2, mammary epithelial development is arrested at an early stage of pregnancy.

Expression of a yeast RNase III gene in transgenic tobacco silences host nitrite reductase genes.

Plant Mol Biol. 2000 Sep;44(1):53-60. PMID: 11094979
Berthomé R, Techeney PY, Renou JP, Okada Y and Tepfer M., 2000.

When a gene encoding the Schizosaccharomyces pombe dsRNA-specific RNase III, pac1, was expressed in transgenic tobacco plants, six out of thirteen transformed plants gave progeny among which were individuals displaying a distinctive chlorotic phenotype. These chlorotic plants strongly resemble those transformed with a 35S-Nii (nitrite reductase) transgene, in which both Nii host genes and the 35S-Nii transgene are silenced by co-suppression. RNA blots showed that the host Nii genes were silenced in chlorotic 35S-pac1 plants but not in individuals with a normal green phenotype. Neither the transcript levels of the other cellular genes tested nor the transcription of Nii genes was significantly affected by the expression of pac1. This is the first observation of post-transcriptional silencing of host genes by a transgene with no apparent sequence similarity to the target gene.

Microsatellites for cultivar identification in Pelargonium

Theoretical and Applied Genetics (Theor. Appl. Genet.) ISSN 0040-5752 CODEN THAGA6 2000, vol. 101, no4, pp. 643-651 (33 ref.)
Becher SA, Steinmetz K, Weising K, Boury S, Peltier D, Renou JP, Kahl G, and Wolff K. 2000.

We have isolated and characterised microsatellite loci from Pelargonium sp. to explore the potential of these markers for cultivar identification. Small-insert libraries from a zonal (Pelargonium x hortorum cv. Isabell) and an ivy-leaved variety (P. peltatum cv. Guenievre gergue) were enriched for d(AG), d(AC), d(CAA), d(GAA) and d(GATA) repeats. Of 141 positive clones sequenced, 133 contained a microsatellite. Primers for PCR amplification were designed to the flanking regions of 57 microsatellites, resulting in interpretable amplification products of the expected size for 29 loci. Seventeen primer pairs amplifying 18 loci were used to fingerprint 44 di- and tetra-ploid Pelargonium accessions representative of commercially available varieties. Multilocus genotypes obtained at 3 loci distinguished among all accessions, except for three known flower colour sports and a fourth, phenotypically very similar, variety. Allelic composition was also identical within two other sport families' typed at the same 18 loci. UPGMA and principal co-ordinate analysis of pairwise distance matrices derived from PCR amplification patterns revealed four distinct assemblages. The first group consisted of tetraploid P. x hortorum varieties; a second group contained diploid P. x hortorum, a third, tetraploid P. peltatum accessions, while a fourth, very distinct, group consisted solely of diploid P. peltatum varieties. Polymorphism in P. peltatum was equal or greater than in P. x hortorum at 17 of the 18 loci, indicating that the analysed P. peltatum varieties form a genetically more variable array.

Relationship of the pelargonium flower break carmovirus (PFBV) coat protein gene with that of other carmoviruses

Archives of virology (Arch. virol.) ISSN 0304-8608 1998, vol. 143, no9, pp. 1823-1829 (38 ref.)
Berthomé R, Kusiak C, Renou JP, Albouy J, Freire MA and Dinant S. 1998.

The 3'-terminal 1500 nucleotides of the genome of pelargonium flower break carmovirus (PFBV) were sequenced from RT-PCR amplification products. One large ORF was found, encoding a 345 amino acid protein of Mr 37 kDa, which corresponds to the coat protein, as confirmed by immunoprecipitation of products of in vitro transcription and translation. The sequence also included the putative promoter of the coat protein gene subgenomic RNA, as well as its 5' and 3' untranslated regions. The PFBV coat protein was more similar to that of saguaro cactus virus and carnation mottle virus than to that of other carmoviruses. Despite the lower level of similarity of CP gene sequences compared to the RNA dependent RNA polymerase (RdRp) gene sequences of small icosahedral viruses used in taxonomic studies, PFBV CP sequence comparisons and alignments confirmed that PFBV is related to carmoviruses, tombusviruses and a dianthovirus, as previously concluded from the analysis of a PFBV RdRp gene fragment.

Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia × intermedia

Plant Molecular Biology, Volume 35, Number 3 / Oktober 1997, Pages 303-311 PMID: 9349254
Rosati C, Cadic A, Duron M, Renou JP and Simoneau P. 1997.

The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia × intermedia cv. lsquoSpring Gloryrsquo. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals.
Plant Molecular Biology

Interspecific crosses between Pelargonium X hortorum and P. quinquelobatum using embryo reçue and molecular characterization of hybrids by an endogenous chs probe.

Plant Breeding, (1997) 116, 177-180.
Denis-Peixoto L, Cadic A and Renou JP, 1997.

Evaluation of the genetic variability in the genus Pelargonium using RAPD markers.

Journal of Horticultural Science, (1997) 72 (2), 229-237.
Renou JP, Aubry C, Serveau M and Jalouzot M., 1997

Regeneration and Agrobacterium mediated transformation of Forsythia x intermedia "Spring glory".

Plant Cell Reports, Volume 16, Numbers 1-2 / November 1996 Pages 114-117
Rosati C, Cadic A, Renou JP and Duron M. 1996.

Internode explants ofin vitro plants of Forsythia x intermedia "Spring Glory" were transformed with thegus andnpt II genes after inoculation with theA. tumefaciens strain EHA 101 harbouring the plasmid pFAJ3000. Shoot organogenesis took place from callused edges of explants. The first transformed buds were detected 4 to 6 weeks after transfer on regeneration medium, containing 25 mg/l kanamycin as selective agent. An average of 1% of explants regenerated transgenic shoots. ß-glucuronidase assays and culture on kanamycin-containing medium provided the first indication of integration and expression of introduced genes in transformants. Southern blot and polymerase chain reaction amplification analyses gave molecular confirmation of genetic transformation. Transgenic plants were acclimatized in the greenhouse. Enzymatic assays on several organs of mature plants still showed ß-glucuronidase activity, thus confirming stable integration of T-DNA in the plant genome.
Plant Cell Reports

Genetic transformation of Pelargonium X hortorum.

Plant Cell Reports, Volume 15, Numbers 1-2 / Januar 1995 Pages 63-67
Robichon MP, Renou JP, and Jalouzot R. 1995.

TransgenicPelargonium X hortorum have been producedvia Agrobacterium tumefaciens-mediated transformation. The regeneration protocol used provided a regeneration frequency approximately to 95 percent. Clumps of regenerants, from cotyledons and hypocotyls ofPelargonium X hortorum seedlings, were inoculated with the disarmed strain EHA101 ofAgrobacterium tumefaciens. This strain contains a binary vector carrying neomycin phosphotransferase II, hygromycin B phosphotransferase and ß-glucuronidase genes. Selection on the regeneration medium supplemented with hygromycin allowed production of transgenic plants in up to 20% of the inoculated explants. The insertion of foreign DNA was demonstrated by Southern and polymerase chain reaction analysis: these experiments indicated that the inserted T-DNA is not full length for most of the plants. All RO transgenic plants exhibited a normal phenotype and are fertile.
Plant Cell Reports

Recovery of transgenic chrysanthemum (Dendranthema grandiflora Tzvelev) after hygromycin resistance selection.

Plant Science, 89, 185-197.
Renou JP, Brochard P and Jalouzot R. 1993.