Functional Genomics of Arabidopsis

Claire Lurin Portratit

Claire Lurin
Group Leader DR2
lurin@evry.inra.fr
+33 1 60 87 45 13

Table of Contents
The Team
Goals
The PPR Family
The Transcriptomic Platform
The ORFeome Platform
Publications
Research Activity of the Team
Platform Activity of the Team
Former Members

Richard Berthome

Richard Berthomé
CR1
berthome@evry.inra.fr
+33 1 60 87 45 17

Etienne Delannoy

Etienne Delannoy
CR1
delannoy@evry.inra.fr
+33 1 60 87 45 17

Alexandra Avon Portrait

Alexandra Avon
Technician (TR)
avon@evry.inra.fr
+33 1 60 87 45 25

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Sandrine Balzergue

Sandrine Balzergue
Engineer (IR)
balzerg@evry.inra.fr
+33 1 60 87 45 28

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Caius Jose

José Caius
Assistant technician (AJT) half-time
caius@evry.inra.fr
+33 1 60 87 45 28

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Laure Heurtevin Portrait

Laure Heurtevin
Technician (TR)
heurtevin@evry.inra.fr
+33 1 60 87 45 25

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Stephanie Huguet Portrait

Stéphanie Huguet
Technician (TR)
huguet@evry.inra.fr
+33 1 60 87 45 28

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Stephanie Pateyron

Stéphanie Pateyron
Technician (TR) half-time
pateyron@evry.inra.fr
+33 1 60 87 45 20

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Ludivine Soubigou-Taconnat Portrait

Ludivine
Soubigou-Taconnat

Research Assistant (AI)
soubigou@evry.inra.fr
+33 1 60 87 45 20

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Clement Boussardon Portrait

Clément Boussardon
PhD student
(Univ. Evry, Fr/ Univ. Perth, AUS)
boussardon@evry.inra.fr
+33 1 60 87 45 25

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Obando

Mauricio Lopez Obando
PhD Student
(Univ. Evry, Fr)
obando@evry.inra.fr
+33 1 60 87 45 25

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Eddy Blondet

Eddy Blondet
CDD Research Assistant
blondet@evry.inra.fr
+33 1 60 87 45 20

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Delphine Gey Portrait

Delphine Gey
Engineer Génoplante (IE)
gey@evry.inra.fr
+33 1 60 87 45 20

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The Team

Team Functional Genomics of Arabidopsis

 

Goals

The goal of the team is to combine functional genomics approaches to identify gene and protein functions in Arabidopsis. Using the advanced technology of the platforms and the expertise available in the team (meta-analyses of transcriptome data, forward genetics and protein-protein interactions), we are aiming to accelerate the study of the Arabidopsis Orphan genes for which no function has so far been attributed. In particular, we pioneered the functional analysis of the PPR family, one of the largest protein families in Arabidopsis and rice and we work on the characterisation and the engineering of the pyrimidin salvage pathway in Arabidopsis for tissue-specific transcription profiling.

The previous Arabidopsis biggest family of unknown proteins encodes post-transcriptional regulation factors.

The PPR (pentatricopeptide repeat) family was until recently the plant biggest multigene family (more than 450 members in Arabidopsis) without any known function. Our team was heavily involved in its discovery and pioneered its analysis using functional genomics. PPR proteins are mostly localised in both plastids and mitochondria, and appear to have similar roles in both cases. They have been recently shown to be involved in almost all stages of gene expression, including mRNA transcription, splicing, processing, editing, translation and stability.

ppr

Currently, the main projects of the team concerning the PPR family are:

  1. To investigate putative non-organelar localization of Arabidopsis PPR proteins and to use reverse genetics to elucidate their functions,

  2. To further elucidate the function of PPR proteins in RNA editing,

  3. To characterize PPR containing protein complexes

transcriptomic platform

The transcriptomic platform, performs profiling experiments as a microarray facility in collaboration with partner laboratories. Thousands of data, accessible through the CATdb public database, have been produced using CATMA microarrays, Affymetrix and NimbleGen plateforms. Our major current methodological development is transcriptomic analysis by High-Throughput Sequencing (HTS).

 

 

 

atome-orfeome

The ORFeome platform has generated thousands of Arabidopsis ORF clones in Gateway™ entry vectors (projects funded by Genoplante, ANR and EU). The ATOME collections are distributed by the CNRGV (Toulouse, France) and can be searched and ordered online.

The ORF clones constructed by the team have been included in a systematic yeast 2-hybrid screening (collaboration with the Arabidopsis interactome project funded by the US National Science Foundation) which has generated thousands of interactions.

Several Gateway destination vectors have been constructed and can be ordered by sending an email to Alexandra Avon: avon@evry.inra.fr

  • pRedGate, DsREd2 C-term fusion, 2X35S promotor, transient expression
  • pGreenGate, GFP C-term fusion, 2X35S promotor, transient expression
  • p0229-RFP2, DsREd2 C-term fusion, 2X35S promotor, binary vector (pGreen 0229)
  • p0229-GFP, GFP C-term fusion, 2X35S promotor, binary vector (pGreen 0229)
  • pAGRIKOLA , RNAi vector

Because the silencing cassette in pAGRIKOLA was extracted from the pHELLSGATE12 vector developed by the group of Peter Waterhouse at CSIRO, you first need to file with CSIRO the MTA for that vector. You will find all the necessary information about the M T A here.

 

Publications

Research activity of the team         2004         2007         2008         2009         2010

2010

Dreze M., Monachello D., Lurin C., Cusick M.E., Hill D.E., Vidal M. and Braun P. 2010.

High quality binary interactome mapping.

Meth. Enzymol. 470: 281- 315. DOI: 10.1016/S0076-6879(10)70012-4


Falcon de Longevialle A., Small I. and Lurin C. 2010.

Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

Mol Plant. 2010 Jul;3(4):691-705. Epub 2010 Jul 5. ABSTRACT


2009

Mainguet SE, Gakière B, Majira A, Pelletier S, Bringel F, Guérard F, Caboche M, Berthomé R, Renou JP.

Uracil Salvage is Necessary for Early Arabidopsis Development.

Plant J. 2009 Jun 29. PMID: 19563437
- 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 2.4.2.9) 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


2008

Chateigner-Boutin AL, Ramos-Vega M, Guevara-García A, Andrés C, de la Luz Gutiérrez-Nava M, Cantero A, Delannoy E, Jiménez LF, Lurin C, Small I, León P.

CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts.

Plant J. 2008 Nov;56(4):590-602. Epub 2008 Aug 23. PMID: 18657233
ARC Centre of Excellence in Plant Energy Biology, Molecular and Chemical Sciences Building (M316), University of Western Australia, 35 Stirling Highway, Crawley, Perth 6009 WA, Australia.

RNA editing changes the sequence of many transcripts in plant organelles, but little is known about the molecular mechanisms determining the specificity of the process. Here, we present the characterization of CLB19 (also known as PDE247), a gene required for editing of two distinct chloroplast transcripts, rpoA and clpP. Loss of function clb19 mutants presents a yellow phenotype with impaired chloroplast development and early seedling lethality under greenhouse growing conditions. Transcript patterns are profoundly affected in the mutant plants, with a pattern entirely consistent with a defect in activity of the plastid-encoded RNA polymerase. CLB19 encodes a PPR protein similar to the editing specificity factors CRR4 and CRR21, but unlike them is implicated in editing of two target sites.


de Longevialle AF, Hendrickson L, Taylor NL, Delannoy E, Lurin C, Badger M, Harvey Millar A, Small I.

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana.

Plant J. 2008 Jun 13. PMID: 18557832
Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 WA, Australia.

The Arabidopsis thaliana chloroplast contains 20 group II introns in its genome and seven known splicing factors are required for splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2 and also influences splicing of several other group IIa introns. Loss of OTP51 has far-reaching consequences for photosystem I and II assembly and the photosynthetic fluorescence characteristics of mutant plants.


Marie-Laure Martin-Magniette*¹², Julie Aubert¹, Avner Bar-Hen (4), Samira Elftieh² , Frederic Magniette³, Jean-Pierre Renou² and Jean-Jacques Daudin¹

Normalization for triple-target microarray experiments

BMC Bioinformatics. 2008 Apr 28;9(1):216 PMID: 18442385
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

ABSTRACT: BACKGROUND:
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.


Nicholas O, Hattori M, Andres C, Iida K, Lurin C, Schmitz-Linneweber C, Sugita M, Small I.

On the expansion of the pentatricopeptide repeat gene family in plants.

Mol Biol Evol. 2008 Mar 14 [Epub ahead of print] PMID: 18343892
Centre for Computational Systems Biology, University of Western Australia, Perth, Australia.

Pentatricopeptide repeat (PPR) proteins form a huge family in plants (450 members in Arabidopsis and 477 in rice) defined by tandem repetitions of characteristic sequence motifs. Some of these proteins have been shown to play a role in post-transcriptional processes within organelles and they are thought to be sequence-specific RNA binding proteins. The origins of this family are obscure as they are lacking from almost all prokaryotes, and the spectacular expansion of the family in land plants is equally enigmatic. In this study we investigate the growth of the family in plants by undertaking a genome-wide identification and comparison of the PPR genes of three organisms: the flowering plants Arabidopsis thaliana and Oryza sativa, and the moss Physcomitrella patens. A large majority of the PPR genes in each of the flowering plants are intron-less. In contrast, most of the 103 PPR genes in Physcomitrella are intron-rich. A phylogenetic comparison of the PPR genes in all three species shows similarities between the intron-rich PPR genes in Physcomitrella and the few intron-rich PPR genes in higher plants. Intron-poor PPR genes in all three species also display a bias towards a position of their introns at their 5' ends. These results provide compelling evidence that one or more waves of retrotransposition were responsible for the expansion of the PPR gene family in flowering plants. The differing numbers of PPR proteins are highly correlated with differences in organellar RNA editing between the three species.

Molecular Biology and Evolution


2007

Andrés C, Lurin C, Small ID (2007) URGV

The multifarious roles of PPR proteins in plant mitochondrial gene expression.

Physiologia Plantarum, Volume 129, Number 1, January 2007 , pp. 14-22(9)

With a few rare exceptions, genes encoding pentatricopeptide (PPR) proteins are present in all sequenced eukaryotic genomes but absent from prokaryotic and archaeal genomes. The family has greatly expanded in plants, to more than 400 genes in each species. So far, the evidence indicates that PPR proteins are generally involved in regulation of organelle genome expression, in other words they are eukaryotic proteins selected for the control of genomes of prokaryotic origin. PPR proteins are localised in both plastids and mitochondria, and appear to have similar roles in both cases. They have been implicated in almost all stages of gene expression, including messenger RNA (mRNA) transcription, splicing, processing, editing, translation and stability. The most probable hypothesis for explaining these diverse roles is that PPR proteins are sequence-specific RNA-binding adaptors capable of directing effector enzymes to defined sites on mRNAs. Much of the recent interest in the role of PPR proteins in mitochondria has been driven by the discovery that most cytoplasmic male sterility systems comprise fertility restorer genes that are members of this fascinating family.
IngentaConnect


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

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

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.
RESULTS:
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.
CONCLUSIONS:
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.


de Longevialle AF, Meyer EH, Andrés C, Taylor NL, Lurin C, Millar AH, Small ID.

The Pentatricopeptide Repeat Gene OTP43 Is Required for trans-Splicing of the Mitochondrial nad1 Intron 1 in Arabidopsis thaliana.

Plant Cell. 2007 Oct;19(10):3256-65. Epub 2007 Oct 26. PMID: 17965268
Unité Mixte de Recherche Génomique Végétale (Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Université d'Evry/Val d'Essone), 91057 Evry, France.

The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a large protein complex formed from both nuclearly and mitochondrially encoded subunits. Subunit ND1 is encoded by a mitochondrial gene comprising five exons, and the mature transcript requires four RNA splicing events, two of which involve trans-splicing independently transcribed RNAs. We have identified a nuclear gene (OTP43) absolutely required for trans-splicing of intron 1 (and only intron 1) of Arabidopsis thaliana nad1 transcripts. This gene encodes a previously uncharacterized pentatricopeptide repeat protein. Mutant Arabidopsis plants with a disrupted OTP43 gene do not present detectable mitochondrial Complex I activity and show severe defects in seed development, germination, and to a lesser extent in plant growth. The alternative respiratory pathway involving alternative oxidase is significantly induced in the mutant.


Gagnot S, Tamby JP, Martin-Magniette ML, Bitton F, Taconnat L, Balzergue S, Sébastien Aubourg, Alain Lecharny, Jean-Pierre Renou,  Brunaud V.

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
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 http://urgv.evry.inra.fr/CATdb 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.


Véronique Salonea,b, Mareike Rüdingerc, Monika Polsakiewiczc, Beate Hoffmanna, Milena Groth-Malonekc, Boris Szureka, Ian Smalla,b, Volker Knoopc, Claire Lurina,* 2007

A hypothesis on the identification of the editing enzyme in plant organelles

FEBS Lett. 2007 Sep 4;581(22):4132-8. Epub 2007 Aug 10. PMID: 17707818
a URGV, 2 Rue Gaston Crémieux, F-91057 Evry Cedex, France
b ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, Australia
c IZMB, Abt. Molekulare Evolution, Kirschallee 1, D-53115 Bonn, Germany

RNA editing in plant organelles is an enigmatic process leading to conversion of cytidines into uridines. Editing specificity is determined by proteins; both those known so far are pentatricopeptide repeat (PPR) proteins. The enzyme catalysing RNA editing in plants is still totally unknown. We propose that the DYW domain found in many higher plant PPR proteins is the missing catalytic domain. This hypothesis is based on two compelling observations: (i) the DYW domain contains invariant residues that match the active site of cytidine deaminases; (ii) the phylogenetic distribution of the DYW domain is strictly correlated with RNA editing.


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.

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

BMC Bioinformatics. 2007 Oct 18;8:400. PMID: 17945016

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

ABSTRACT: BACKGROUND:
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.
RESULTS:
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.
CONCLUSIONS:
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 .


2004

Hilson P, Allemeersch J, Altmann T, Aubourg S, Avon A, Beynon J, Bhalerao RP, Bitton F, Caboche M, Cannoot B, Chardakov V, Cognet-Holliger C, Colot V, Crowe M, Darimont C, Durinck S, Eickhoff H, de Longevialle AF, Farmer EE, Grant M, Kuiper MT, Lehrach H, Leon C, Leyva A, Lundeberg J, Lurin C, Moreau Y, Nietfeld W, Paz-Ares J, Reymond P, Rouze P, Sandberg G, Segura MD, Serizet C, Tabrett A, Taconnat L, Thareau V, Van Hummelen P, Vercruysse S, Vuylsteke M, Weingartner M, Weisbeek PJ, Wirta V, Wittink FR, Zabeau M, Small I.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Versatile gene-specific sequence tags

Genome Res. 2004 Oct;14(10B):2176-89. PMID: 15489341
Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, B-9052 Gent, Belgium. pierre.hilson@psb.ugent.be

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.


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.

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
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.


Small I, Peeters N, Legeai F and Lurin C (2004)

Predotar: A tool for rapidly screening proteomes for N-terminal targeting sequences.

Proteomics. 2004 Jun;4(6):1581-90. PMID: 15174128
Station de Génétique et Amélioration des Plantes, INRA, Versailles, France. small@evry.inra.fr

Probably more than 25% of the proteins encoded by the nuclear genomes of multicellular eukaryotes are targeted to membrane-bound compartments by N-terminal targeting signals. The major signals are those for the endoplasmic reticulum, the mitochondria, and in plants, plastids. The most abundant of these targeted proteins are well-known and well-studied, but a large proportion remain unknown, including most of those involved in regulation of organellar gene expression or regulation of biochemical pathways. The discovery and characterization of these proteins by biochemical means will be long and difficult. An alternative method is to identify candidate organellar proteins via their characteristic N-terminal targeting sequences. We have developed a neural network-based approach (Predotar--Prediction of Organelle Targeting sequences) for identifying genes encoding these proteins amongst eukaryotic genome sequences. The power of this approach for identifying and annotating novel gene families has been illustrated by the discovery of the pentatricopeptide repeat family.


 

Platform Activity of the Team         2005         2006         2007         2008         2009         2010

2010

Krugman T, Chagué V, Peleg Z, Balzergue S, Just J, Korol AB, Nevo E, Saranga Y, Chalhoub B, Fahima T.

Multilevel regulation and signalling processes associated with adaptation to terminal drought in wild emmer wheat.

Funct Integr Genomics. 2010 Mar 24. PMID: 20333536

Low water availability is the major environmental factor limiting crop productivity. Transcriptome analysis was used to study terminal drought response in wild emmer wheat, Triticum dicoccoides, genotypes contrasting in their productivity and yield stability under drought stress. A total of 5,892 differentially regulated transcripts were identified between drought and well-watered control and/or between drought resistant (R) and drought susceptible (S) genotypes. Functional enrichment analyses revealed that multilevel regulatory and signalling processes were significantly enriched among the drought-induced transcripts, in particular in the R genotype. Therefore, further analyses were focused on selected 221 uniquely expressed or highly abundant transcripts in the R genotype, as potential candidates for drought resistance genes. Annotation of the 221 genes revealed that 26% of them are involved in multilevel regulation, including: transcriptional regulation, RNA binding, kinase activity and calcium and abscisic acid signalling implicated in stomatal closure. Differential expression patterns were also identified in genes known to be involved in drought adaptation pathways, such as: cell wall adjustment, cuticular wax deposition, lignification, osmoregulation, redox homeostasis, dehydration protection and drought-induced senescence. These results demonstrate the potential of wild emmer wheat as a source for candidate genes for improving drought resistance.


2009

Bashandy T, Taconnat L, Renou JP, Meyer Y, Reichheld JP.

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
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.


Besson-Bard A., Gravot A., Richaud P., Auroy P., Gaymard F., Taconnat L., Renou J.P., Pugin A. and Wendehenne D. 2009.

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

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.


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.

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

Plant J. 2008 Sep 26. PMID 18826430
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.


Elis S, Blesbois E, Couty I, Balzergue S, Martin-Magniette ML, Batellier F, Govoroun MS.

Identification of germinal disk region derived genes potentially involved in hen fertility.

Mol Reprod Dev. 2009 Nov;76(11):1043-55. PMID: 19484757

Despite the regular decrease in fertility observed in hens, especially in "meat" lines, little is known about genes affecting fertility. We used the Affymetrix microarray to search for oocyte genes whose expression would vary in relation to fertility rate in both "laying" and "meat" line hens. We focused on oocyte genes because several of them have been found to be involved in fertility in other species. Based on microarray analysis, 54 and 84 genes were differentially expressed between germinal disc regions (GDR) of F1 maturation stage oocytes from hens exhibiting either high (100%) or low (from 22% to 80%) fertility rate from laying and meat lines respectively. Most of these differentially expressed genes were distributed between "laying" and "meat" lines indicating that mechanisms involved in the decrease in fertility rates in these two cases were independent. Real time RT-PCR performed on the same samples which were used for microarray confirmed in several cases differences in gene expression levels detected by microarray. Moreover the correlations between gene expression levels and fertility rates were evaluated for the 10 most interesting genes at different stages of follicular maturation and early embryo development on individual GDR samples from hens exhibiting different fertility rates. In total, we identified five genes whose expression levels correlated with fertility rate in accordance with findings of microarray analysis and real time RT-PCR: VWC2, CR407412, TAPA, FGL2, and TRAP6. The biological significance of these genes sheds light on potential mechanisms influencing fertility and could provide candidates for fertility markers in the hen.


Matakiadis T, Alboresi A, Jikumaru Y, Tatematsu K, Pichon O, Renou JP, Kamiya Y, Nambara E, Truong HN.

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

Plant Physiol. 2008 Dec 12. PMID: 19074630
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.


Minic Z, Jamet E, San-Clemente H, Pelletier S, Renou JP, Rihouey C, Okinyo DP, Proux C, Lerouge P, Jouanin L.

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

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.


Andrea Pitzschke (a), Armin Djamei (a,b,) Frédérique Bitton (c) and Heribert Hirt (a,c,1)

A Major Role of the MEKK1–MKK1/2–MPK4 Pathway in ROS Signalling

Molecular Plant Advance Access published January 6, 2009 | Molecular Plant • Pages 1–18, 2008
(a) Department of Plant Molecular Biology, Max F. Perutz Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030 Vienna, Austria
(b) Present address: Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany
(c) URGV Plant Genomics Laboratory, 2 Rue Gaston Cre´ mieux, 91057 Evry, France
(1) To whom correspondence should be addressed. E-mail hirt@evry.inra.fr

molecular plant

ABSTRACT Over the last few years, it has become evident that reactive oxygen species (ROS) signalling plays an important role in various physiological responses, including pathogen defense and stomatal opening/closure. On the other hand, ROS overproduction is detrimental for proper plant growth and development, indicating that the regulation of an appropriate redox balance is essential for plants. ROS homeostasis in plants involves the mitogen-activated protein kinase (MAPK) pathway consisting of the MAPK kinase kinase MEKK1 and the MAPK MPK4. Phenotypic and molecular analysis revealed that the MAPK kinases MKK1 and MKK2 are part of a cascade, regulating ROS and salicylic acid (SA) accumulation. Gene expression analysis shows that of 32 transcription factors reported to be highly responsive to multiple ROS-inducing conditions, 20 are regulated by the MEKK1, predominantly via the MEKK1–MKK1/2–MPK4 pathway. However, MEKK1 also functions on other as yet unknown pathways and part of the MEKK1-dependent MPK4 responses are regulated independently of MKK1 and MKK2. Overall, this analysis emphasizes the central role of this MAPK cascade in oxidative stress signalling, but also indicates the high level of complexity revealed by this signalling network.


2008

Achard P, Jean-Pierre Renou, Berthomé R, Harberd NP, Genschik P.

Plant DELLAs Restrain Growth and Promote Survival of Adversity by Reducing the Levels of Reactive Oxygen Species.

xx

Curr Biol. 2008 Apr 30 PMID: 18450450
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.


Talaat Bashandy (a), Ludivine Taconnat (b), Jean-Pierre Renou (b), Yves Meyer (a) and Jean-Philippe Reichheld (a,1,2)

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

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 jpr@univ-perp.fr, 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.


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.

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

Plant J. 2008 Jul 4. PMID: 18644002
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.


Bouchabke-Coussa O, Quashie ML, Seoane-Redondo J, Fortabat MN, Gery C, Yu A, Linderme D, Trouverie J, Granier F, Téoulé E, Durand-Tardif M.

ESKIMO1 is a key gene involved in water economy as well as cold acclimation and salt tolerance.

BMC Plant Biol. 2008 Dec 7;8:125. PMID: 19061521
Cell Biology Laboratory, IJPB, INRA-CIRAD, UR0501, Route de St Cyr, F-78026 Versailles, France. bouchabk@versailles.inra.fr

BACKGROUND: Drought is a major social and economic problem resulting in huge yield reduction in the field. Today's challenge is to develop plants with reduced water requirements and stable yields in fluctuating environmental conditions. Arabidopsis thaliana is an excellent model for identifying potential targets for plant breeding. Drought tolerance in the field was successfully conferred to crops by transferring genes from this model species. While involved in a plant genomics programme, which aims to identify new genes responsible for plant response to abiotic stress, we identified ESKIMO1 as a key gene involved in plant water economy as well as cold acclimation and salt tolerance. RESULTS: All esk1 mutants were more tolerant to freezing, after acclimation, than their wild type counterpart. esk1 mutants also showed increased tolerance to mild water deficit for all traits measured. The mutant's improved tolerance to reduced water supply may be explained by its lower transpiration rate and better water use efficiency (WUE), which was assessed by carbon isotope discrimination and gas exchange measurements. esk1 alleles were also shown to be more tolerant to salt stress. Transcriptomic analysis of one mutant line and its wild-type background was carried out. Under control watering conditions a number of genes were differentially expressed between the mutant and the wild type whereas under mild drought stress this list of genes was reduced. Among the genes that were differentially expressed between the wild type and mutant, two functional categories related to the response to stress or biotic and abiotic stimulus were over-represented. Under salt stress conditions, all gene functional categories were represented equally in both the mutant and wild type. Based on this transcriptome analysis we hypothesise that in control conditions the esk1 mutant behaves as if it was exposed to drought stress. CONCLUSION: Overall our findings suggest that the ESKIMO1 gene plays a major role in plant response to water shortage and in whole plant water economy. Further experiments are being undertaken to elucidate the function of the ESKIMO1 protein and the way it modulates plant water uptake.


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)

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
(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)


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.

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

Plant Physiol. 2008 Mar 14 PMID: 18344421
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)


Cossegal M, Chambrier P, Mbelo S, Balzergue S, Martin-Magniette ML, Moing A, Deborde C, Guyon V, Perez P, Rogowsky P.

Transcriptional and metabolic adjustments in AGPase deficient bt2 maize kernels.

Plant Physiol. 2008 Feb 20 PMID: 18287491
RDP, UMR 5667 INRA-CNRS-ENSL-UCBL, IFR128 BioSciences Lyon-Gerland, ENS-Lyon, 46 Allee d'Italie, F-69364 Lyon Cedex 07, France; URGV, UMR1165 INRA-CNRS-UEVE, 2 rue Gaston Cremieux, F-91057 Evry Cedex, France; Pole Metabolome de la Plateforme Genomique Fonctionnelle Bordeaux, IFR BVI, BP 81, F-33883 Villenave d'Ornon, France; Biogemma SAS, Laboratoire de Biologie Cellulaire et Moleculaire, 8 rue des freres Lumiere, F-63028 Clermont-Ferrand Cedex 2, France; INRA UMR AgroParisTech/INRA MIA 518, 16 rue Claude Bernard, F-75231 Paris Cedex 05, France.

During the cloning of monogenic recessive mutations responsible for defective kernel phenotypes in a Mutator induced maize mutant collection, we isolated a new mutant allele in Brittle2 (Bt2), which codes for the small subunit of ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch synthesis. RT-PCR experiments with gene-specific primers confirmed a predominant expression of Bt2 in endosperm, of Agpsemzm in embryo and of Agpslzm in leaf, but also revealed considerable additional expression in various tissues for all three genes. Bt2a, the classical transcript coding for a cytoplasmic isoform, was almost exclusively expressed in the developing endosperm, while Bt2b, an alternative transcript coding for a plastidial isoform, was expressed in almost all tissues tested with a pattern very similar to that of Agpslzm. The phenotypic analysis showed that at 30 days after pollination (DAP) mutant kernels were plumper than wildtype ones, that the onset of kernel collapse took place between 31 and 35 DAP and that the number of starch grains was greatly reduced in the mutant endosperm but not the mutant embryo. A comparative transcriptome analysis of wildtype and bt2-H2328 kernels at mid-development (35 DAP) with the 18K GeneChip(R) Maize Genome Array led the conclusion that the lack of Bt2 encoded AGPase triggers large scale changes on the transcriptional level that concern mainly genes involved in carbohydrate or amino acid metabolic pathways. Principal component analysis (PCA) of (1)H-NMR metabolic profiles confirmed the impact of the bt2-H2328 mutation on these pathways and revealed that the bt2-H2328 mutation did not affect exclusively the endosperm but also the embryo at the metabolic level. These data suggest that in the bt2-H2328 endosperms regulatory networks are activated that redirect excess carbon into alternative biosynthetic pathways (amino acid synthesis) or into other tissues (embryo).


Depuydt S, Trenkamp S, Fernie AR, Elftieh S, Renou JP, Vuylsteke M, Holsters M, Vereecke D.

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

Plant Physiol. 2008 Dec 31. [Epub ahead of print] PMID: 19118125
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.


Elis S, Batellier F, Couty I, Balzergue S, Martin-Magniette ML, Monget P, Blesbois E, Govoroun MS.

Search for the genes involved in oocyte maturation and early embryo development in the hen.

oocyte maturation

BMC Genomics. 2008 Feb 29;9(1):110 PMID: 18312645

ABSTRACT: BACKGROUND: The initial stages of development depend on mRNA and proteins accumulated in the oocyte, and during these stages, certain genes are essential for fertilization, first cleavage and embryonic genome activation. The aim of this study was first to search for avian oocyte-specific genes using an in silico and a microarray approaches, then to investigate the temporal and spatial dynamics of the expression of some of these genes during follicular maturation and early embryogenesis. RESULTS: The in silico approach allowed us to identify 18 chicken homologs of mouse potential oocyte genes found by digital differential display. Using the chicken Affymetrix microarray, we identified 461 genes overexpressed in granulosa cells (GCs) and 250 genes overexpressed in the germinal disc (GD) of the hen oocyte. Six genes were identified using both in silico and microarray approaches. Based on GO annotations, GC and GD genes were differentially involved in biological processes, reflecting different physiological destinations of these two cell layers. Finally we studied the spatial and temporal dynamics of the expression of 21 chicken genes. According to their expression patterns all these genes are involved in different stages of final follicular maturation and/or early embryogenesis in the chicken. Among them, 8 genes (btg4, chkmos, wee, zpA, dazL, cvh, zar1 and ktfn) were preferentially expressed in the maturing occyte and cvh, zar1 and ktfn were also highly expressed in the early embryo. CONCLUSIONS: We showed that in silico and Affymetrix microarray approaches were relevant and complementary in order to find new avian genes potentially involved in oocyte maturation and/or early embryo development, and allowed the discovery of new potential chicken mature oocyte and chicken granulosa cell markers for future studies. Moreover, detailed study of the expression of some of these genes revealed promising candidates for maternal effect genes in the chicken. Finally, the finding concerning the different state of rRNA compared to that of mRNA during the postovulatory period shed light on some mechanisms through which oocyte to embryo transition occurs in the hen.


Loizeau K, De Brouwer V, Gambonnet B, Yu A, Renou JP, Van Der Straeten D, Lambert WE, Rebeille F, Ravanel S.

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
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.


Patricia Merigout , Maud Lelandais , Frédérique Bitton , Jean-Pierre Renou , Xavier Briand , Christian Meyer , and Francoise Daniel-Vedele

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
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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Ribot C, Hirsch J, Balzergue S, Tharreau D, Nottéghem JL, Lebrun MH, Morel JB.

Susceptibility of rice to the blast fungus, Magnaporthe grisea.

J Plant Physiol. 2008 Jan;165(1):114-24. Epub 2007 Oct 1. PMID: 17905473

The interaction between rice and the blast fungus Magnaporthe grisea is the focus of extensive studies on rice disease resistance and fungal infection mechanisms. Here, we review the characteristics of susceptible rice blast infections in terms of physiology, cytology and both host and pathogen transcriptional responses. The success of the infection and the type of disease symptoms strongly depend on environmental and developmental cues. After its penetration into a host cell, the fungus differentiates invasive hyphae that fill up the plant cell lumen and are in direct contact with the membrane of the infected cell. The infected plant cell is alive, displaying considerable vesicle accumulation near the fungus, which is consistent with the establishment of a biotrophic phase at this stage of the infection. Colonization of host tissues by the fungus occurs through the perforation of cell walls from adjacent cells, likely using plasmodesmata as breaking points, or through hyphal growth in the apoplasm. After a few days of biotrophic growth within rice tissues, the fungus switches to a necrotrophic-like phase associated with the onset of sporulation, leading to visible lesions. Genome-wide transcriptomic studies have shown that classical plant defence responses are triggered during a susceptible infection, although the kinetics and amplitude of these responses are slower and lower than in resistant interactions. Infected rice cells are submitted to an intense transcriptional reprogramming, where responses to hormones such as auxins, abscissic acid and jasmonates are likely involved. Consistent with the extensive plant-fungal exchanges during the biotrophic phase, many rice genes expressed during infection encode plasma membrane proteins. At the onset of lesion formation (5 days after the start of infection), M. grisea is actively reprogramming its transcription towards active DNA, RNA and protein syntheses to sustain its rapid growth in infected tissues. A striking characteristic of M. grisea genes expressed at this stage of the infection is the over-representation of genes encoding secreted proteins, mainly of unknown function. However, some of these secreted proteins are enzymes involved in cell wall, protein and lipid degradation, suggesting that the fungus is starting to degrade host polymers and cell walls or is remodelling its own cell wall. The next challenge will be to decipher the role of these induced plant and fungal genes in the susceptible interaction.


Ruffel S, Freixes S, Balzergue S, Tillard P, Jeudy C, Martin-Magniette ML, van der Merwe MJ, Kakar K, Gouzy J, Fernie AR, Udvardi M, Salon C, Gojon A, Lepetit M.

Systemic signaling of the plant N status triggers specific transcriptome responses depending on the N source in Medicago truncatula.

Plant Physiol. 2008 Feb 20 PMID: 18287487
Biochimie et Physiologie Moleculaire des Plantes, UMR 5004, INRA-CNRS-Sup Agro-UM2, Institut de Biologie Integrative des Plantes, 2 Place Viala, F-34060 Montpellier, France; Unite de Genetique et Ecophysiologie des Legumineuses, UMR INRA, BP 86510, F-21065 Dijon, France; Unite de Recherche en Genomique Vegetale – UMR INRA 1165-CNRS 8114-UEVE, 2 Rue Gaston Cremieux, F-91057 Evry, France; Max-Planck-Institut fur Molekulare Pflanzenphysiologie, 14476 Potsdam-Golm, Germany; Laboratoire des Interactions Plantes Micro-organismes, UMR INRA/CNRS 441/2594, F-31326 Castanet Tolosan, France; UMR AgroParisTech/INRA MIA 518, 16 rue Claude Bernard, F-75231 Paris France.

Legumes can acquire nitrogen from NO3(-), NH4(+) and N2 (through symbiosis with rhizobium bacteria), however, the mechanisms by which uptake and assimilation of these N forms are coordinately regulated to match the N demand of the plant are currently unknown. Here we find, by use of the split-root approach in Medicago truncatula plants, that NO3(-) uptake, NH4(+) uptake and N2 fixation are under general control by systemic signaling of plant N status. Indeed, irrespective of the nature of the N source, N acquisition by one side of the root system is repressed by high N supply to the other side. Transcriptome analysis facilitated the identification of in excess of 3000 genes that were regulated by systemic signaling of the plant N status. However, detailed scrutiny of the data revealed that the observation of differential gene expression was highly dependent on the N source. Localized N starvation results, in the unstarved roots of the same plant, in a strong compensatory up-regulation of NO3(-) uptake, but not of either NH4(+) uptake or N2 fixation. This indicates that the three N acquisition pathways do not always respond similarly to a change in plant N status. When taken together, these data indicate that although systemic signals of N status control root N acquisition, the regulatory gene networks targeted by these signals, as well as the functional response of the N acquisition systems, are predominantly determined by the nature of the N source.


2007

Kian Hématy, Pierre-Etienne Sado, Ageeth Van Tuinen, Soizic Rochange, Thierry Desnos, Sandrine Balzergue, Sandra Pelletier, Jean-Pierre Renou, and Herman Höfte

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
Laboratoire de Biologie Cellulaire, UR501, Institut Jean-Pierre Bourgin, Institut National de la Recherche Agronomique Centre de Versailles, Versailles, France, URGV

Background
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.
Results
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.
Conclusions
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.


Ondrej Krinke, Eric Ruelland, Olga Valentová, Chantal Vergnolle, Jean-Pierre Renou, Ludivine Taconnat, Matyás Flemr, Lenka Burketová, and Alain Zachowski, 2007

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
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. ondrej.krinke@vscht.cz

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.


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)

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
(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.


Ribot C, Hirsch J, Balzergue S, Tharreau D, Nottéghem JL, Lebrun MH, Morel JB.

Susceptibility of rice to the blast fungus, Magnaporthe grisea.

Journal of Plant Physiology 2007 Sep 28 doi:10.1016/j.jplph.2007.06.013 PMID: 17905473
UMR 5240 CNRS-UCB-INSA-BCS, Bayer CropScience, 14-20 rue Pierre Baizet BP9163, 69263 Lyon Cedex 09, France.

The interaction between rice and the blast fungus Magnaporthe grisea is the focus of extensive studies on rice disease resistance and fungal infection mechanisms. Here, we review the characteristics of susceptible rice blast infections in terms of physiology, cytology and both host and pathogen transcriptional responses. The success of the infection and the type of disease symptoms strongly depend on environmental and developmental cues. After its penetration into a host cell, the fungus differentiates invasive hyphae that fill up the plant cell lumen and are in direct contact with the membrane of the infected cell. The infected plant cell is alive, displaying considerable vesicle accumulation near the fungus, which is consistent with the establishment of a biotrophic phase at this stage of the infection. Colonization of host tissues by the fungus occurs through the perforation of cell walls from adjacent cells, likely using plasmodesmata as breaking points, or through hyphal growth in the apoplasm. After a few days of biotrophic growth within rice tissues, the fungus switches to a necrotrophic-like phase associated with the onset of sporulation, leading to visible lesions. Genome-wide transcriptomic studies have shown that classical plant defence responses are triggered during a susceptible infection, although the kinetics and amplitude of these responses are slower and lower than in resistant interactions. Infected rice cells are submitted to an intense transcriptional reprogramming, where responses to hormones such as auxins, abscissic acid and jasmonates are likely involved. Consistent with the extensive plant-fungal exchanges during the biotrophic phase, many rice genes expressed during infection encode plasma membrane proteins. At the onset of lesion formation (5 days after the start of infection), M. grisea is actively reprogramming its transcription towards active DNA, RNA and protein syntheses to sustain its rapid growth in infected tissues. A striking characteristic of M. grisea genes expressed at this stage of the infection is the over-representation of genes encoding secreted proteins, mainly of unknown function. However, some of these secreted proteins are enzymes involved in cell wall, protein and lipid degradation, suggesting that the fungus is starting to degrade host polymers and cell walls or is remodelling its own cell wall. The next challenge will be to decipher the role of these induced plant and fungal genes in the susceptible interaction.


Ricaud L, Proux C, Renou JP, Pichon O, Fochesato S, Ortet P, Montane MH. May 2007

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
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.


Lin Xu, Zhong Zhao, Aiwu Dong, Ludivine Soubigou-Taconnat, Jean-Pierre Renou, Andre Steinmetz, and Wen-Hui Shen

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
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.


2006

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.

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
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.


Ferrario-Mery S, Besin E, Pichon O, Meyer C, Hodges M.

The regulatory PII protein controls arginine biosynthesis in Arabidopsis.

FEBS Lett. 2006 Apr 3;580(8):2015-20. Epub 2006 Mar 10. PMID: 16545809
Unité de Nutrition Azotée des Plantes, Institut National de la Recherche Agronomique, INRA, Versailles, France

In higher plants, PII is a nuclear-encoded plastid protein which is homologous to bacterial PII signalling proteins known to be involved in the regulation of nitrogen metabolism. A reduced ornithine, citrulline and arginine accumulation was observed in two Arabidopsis PII knock-out mutants in response to NH4+ resupply after N starvation. This difference could be explained by the regulation of a key enzyme of the arginine biosynthesis pathway, N-acetyl glutamate kinase (NAGK) by PII. In vitro assays using purified recombinant proteins showed the catalytic activation of Arabidopsis NAGK by PII giving the first evidence of a physiological role of the PII protein in higher plants. Using Arabidopsis transcriptome microarray (CATMA) and RT-PCR analyses, it was found that none of the genes involved in the arginine biosynthetic or catabolic pathways were differentially expressed in a PII knock-out mutant background. In conclusion, the observed changes in metabolite levels can be explained by the reduced activation of NAGK by PII.


Guyomarch's S, Benhamed M, Lemonnier G, Renou JP, Zhou DX & Delarue M. 2006.

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

J Exp Bot. 2006;57(9):2111-9. Epub 2006 May 25. PMID: 16728410
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.


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.

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
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.


Manavella PA, Arce AL, Dezar CA, Bitton F, Renou JP, Crespi M, Chan RL.

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
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.


Masclaux-Daubresse C, Purdy S, Lemaitre T, Pourtau N, Taconnat L, Renou JP and Wingler A. 2007.

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
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.


Zhu Y, Dong A, Meyer D, Pichon O, Renou JP, Cao K, Shen WH.

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
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.


2005

Bertrand C, Benhamed M, Li YF, Ayadi M, Lemonnier G, Renou JP, Delarue M, Zhou DX.

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
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.


Dieterle M, Thomann A, Renou JP, Parmentier Y, Cognat V, Lemonnier G, Muller R, Shen WH, Kretsch T, Genschik P.

Molecular and functional characterization of Arabidopsis Cullin 3A

Plant J. 2005 Feb;41(3):386-99. PMID: 15659098
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.


Jammes F, Lecomte P, de Almeida-Engler J, Bitton F, Martin-Magniette ML, Renou JP, Abad P, Favery B.

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
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.


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.

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

Plant J. 2005 Oct;44(2):271-89. PMID: 16212606
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.


Vergnolle C, Vaultier MN, Taconnat L, Renou JP, Kader JC, Zachowski A, Ruelland E.

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
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.


Former Members

Charles Andres PhD student 2003 - 2007 charles.andres@unine.ch
Guillaume Barbier Genopole Post-doc 2005 – 2007 gbarbier@metabolic-explorer.com
Salima Berkani Master Student 2004 berkanisalima@yahoo.fr
Clément Bernard Master Student 2007 clementbernard84@gmail.com
Frédérique Bitton Engineer 2000-2008 bitton@evry.inra.fr
Jean Colcombet INRA Research scientist 2005 – 2007 colcombet@evry.inra.fr
Nolwen Dautrevaux Master Student / CDD Engineer AGRIKOLA 2005 / 2005 - 2006 n.dautrevaux@gmail.com
Samira Elftieh CDD Engineer Génoplante 2006-2008  
Marina Ferrand Licence Student 2008 - 2009 ferrand@versailles.inra.fr
David Glissant ATER Univ. Evry 2008  
Beate Hoffmann INRA Technician 2000 – 2003 Hoffmann@versailles.inra.fr
Andéol Falcon de Longevialle Agrikola Resarch Assistant Jan 2003 – March 2006 falcon@cyllene.uwa.edu.au
Blandine Helvig Genoplante Resarch Assistant 2002 – 2003 jeromeseigneur@yahoo.fr
Samuel Mainguet PhD student 2006 - 2010 samuel.e.mainguet@gmail.com
Anne Marmagne Agrikola Post-doc 2004 – 2006 marmagne@versailles.inra.fr
Karine Martin Master Student Jan 2006 – Sept 2006 martin_ka91@hotmail.com
Stéphanie Pateyron INRA Technician 2005 – 2006 pateyron@evry.inra.fr
Sandra Pelletier INRA Technician 2007 - 2010 pelletier@angers.inra.fr
Jean-Pierre Renou INRA Senior Scientist 2003 - 2010 renou@angers.inra.fr
Christian Schmitz-Linneweber Post-doc 2004 – 2005 christian.schmitz-linneweber@rz.hu-berlin.de
Véronique Salone PhD student 2004 - 2008 v.salone@free.fr
Ian Small INRA Senior Scientist 2000 – 2006 iansmall@cyllene.uwa.edu.au
Boris Szurek Genopole Post-doc   boris.szurek@mpl.ird.fr
Jennifer Yansouri Master Student/CDD Engineer 2007 / 2007 - 2010 yansouri@versailles.inra.fr
A. Yu Engineer Génoplante 2006 - 2008