URGV - Plant Genomics Research
Unité de Recherche en Génomique Végétale

Plant Protein Interactomes.

Annu Rev Plant Biol. 2013 Jan 16. PMID: 23330791
Braun P, Aubourg S, Van Leene J, De Jaeger G, Lurin C.

Protein-protein interactions are a critical element of biological systems, and the analysis of interaction partners can provide valuable hints about unknown functions of a protein. In recent years, several large-scale protein interaction studies have begun to unravel the complex networks through which plant proteins exert their functions. Two major classes of experimental approaches are used for protein interaction mapping: analysis of direct interactions using binary methods such as yeast two-hybrid or split ubiquitin, and analysis of protein complexes through affinity purification followed by mass spectrometry. In addition, bioinformatics predictions can suggest interactions that have evaded detection by other methods or those of proteins that have not been investigated. Here we review the major approaches to construct, analyze, use, and carry out quality control on plant protein interactome networks. We present experimental and computational approaches for large-scale mapping, methods for validation or smaller-scale functional studies, important bioinformatics resources, and findings from recently published large-scale plant interactome network maps. Expected final online publication date for the Annual Review of Plant Biology Volume 64 is April 29, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Two Interacting Proteins Are Necessary for the Editing of the NdhD-1 Site in Arabidopsis Plastids.

Plant Cell. 2012 Sep 21 PMID: 23001034
Boussardon C, Salone V, Avon A, Berthomé R, Hammani K, Okuda K, Shikanai T, Small I, Lurin C.

After transcription, mRNA editing in angiosperm chloroplasts and mitochondria results in the conversion of cytidine to uridine by deamination. Analysis of Arabidopsis thaliana mutants affected in RNA editing have shown that many pentatricopeptide repeat proteins (PPRs) are required for specific cytidine deamination events. PPR proteins have been shown to be sequence-specific RNA binding proteins allowing the recognition of the C to be edited. The C-terminal DYW domain present in many editing factors has been proposed to catalyze C deamination, as it shows sequence similarities with cytidine deaminases in other organisms. However, many editing factors, such as the first to be discovered, CHLORORESPIRATORY REDUCTION4 (CRR4), lack this domain, so its importance has been unclear. Using a reverse genetic approach, we identified DYW1, an RNA editing factor acting specifically on the plastid ndhD-1 editing site recognized by CRR4. Unlike other known editing factors, DYW1 contains no identifiable PPR motifs but does contain a clear DYW domain. We were able to show interaction between CRR4 and DYW1 by bimolecular fluorescence complementation and to reconstitute a functional chimeric CRR4-DYW1 protein complementing the crr4 dyw1double mutant. We propose that CRR4 and DYW1 act together to edit the ndhD-1 site.

Evidence for network evolution in an Arabidopsis interactome map.

Science. 2011 Jul 29;333(6042):601-7. PMID: 21798944
Arabidopsis Interactome Mapping Consortium.
Braun P, Carvunis AR, Charloteaux B, Dreze M, Ecker JR, Hill DE, Roth FP, Vidal M, Galli M, Balumuri P, Bautista V, Chesnut JD, Kim RC, de los Reyes C, Gilles P, Kim CJ, Matrubutham U, Mirchandani J, Olivares E, Patnaik S, Quan R, Ramaswamy G, Shinn P, Swamilingiah GM, Wu S, Ecker JR, Dreze M, Byrdsong D, Dricot A, Duarte M, Gebreab F, Gutierrez BJ, MacWilliams A, Monachello D, Mukhtar MS, Poulin MM, Reichert P, Romero V, Tam S, Waaijers S, Weiner EM, Vidal M, Hill DE, Braun P, Galli M, Carvunis AR, Cusick ME, Dreze M, Romero V, Roth FP, Tasan M, Yazaki J, Braun P, Ecker JR, Carvunis AR, Ahn YY, Barabási AL, Charloteaux B, Chen H, Cusick ME, Dangl JL, Dreze M, Ecker JR, Fan C, Gai L, Galli M, Ghoshal G, Hao T, Hill DE, Lurin C, Milenkovic T, Moore J, Mukhtar MS, Pevzner SJ, Przulj N, Rabello S, Rietman EA, Rolland T, Roth FP, Santhanam B, Schmitz RJ, Spooner W, Stein J, Tasan M, Vandenhaute J, Ware D, Braun P, Vidal M.
Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA

Plants have unique features that evolved in response to their environments and ecosystems. A full account of the complex cellular networks that underlie plant-specific functions is still missing. We describe a proteome-wide binary protein-protein interaction map for the interactome network of the plant Arabidopsis thaliana containing about 6200 highly reliable interactions between about 2700 proteins. A global organization of plant biological processes emerges from community analyses of the resulting network, together with large numbers of novel hypothetical functional links between proteins and pathways. We observe a dynamic rewiring of interactions following gene duplication events, providing evidence for a model of evolution acting upon interactome networks. This and future plant interactome maps should facilitate systems approaches to better understand plant biology and improve crops.

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
Epub 2010 Mar 1. PMID: 20946815

Physical interactions mediated by proteins are critical for most cellular functions and altogether form a complex macromolecular “interactome” network. Systematic mapping of protein–protein, protein–DNA, protein–RNA, and protein–metabolite interactions at the scale of the whole proteome can advance understanding of interactome networks with applications ranging from single protein functional characterization to discoveries on local and global systems properties. Since the early efforts at mapping protein–protein interactome networks a decade ago, the field has progressed rapidly giving rise to a growing number of interactome maps produced using high-throughput implementations of either binary protein–protein interaction assays or co-complex protein association methods. Although high-throughput methods are often thought to necessarily produce lower quality information than low-throughput experiments, we have recently demonstrated that proteome-scale interactome datasets can be produced with equal or superior quality than that observed in literature-curated datasets derived from large numbers of small-scale experiments. In addition to performing all experimental steps thoroughly and including all necessary controls and quality standards, careful verification of all interacting pairs and validation tests using independent, orthogonal assays are crucial to ensure the release of interactome maps of the highest possible quality. This chapter describes a high-quality, high-throughput binary protein–protein interactome mapping pipeline that includes these features.

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. PMID: 20603383

Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

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
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.
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.

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

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

Mol Biol Evol. 2008 Mar 14 [Epub ahead of print] PMID: 18343892
Nicholas O, Hattori M, Andres C, Iida K, Lurin C, Schmitz-Linneweber C, Sugita M, Small I.
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

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

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
Véronique Salonea,^b, Mareike Rüdinger^c, Monika Polsakiewicz^c, Beate Hoffmann^a, Milena Groth-Malonek^c, Boris Szurek^a, Ian Small^a,b, Volker Knoop^c, Claire Lurin^a,* 2007
^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.

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

Physiologia Plantarum, Volume 129, Number 1, January 2007 , pp. 14-22(9)
Andrés C, Lurin C, Small ID (2007) URGV

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

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

Genome Res. 2004 Oct;14(10B):2176-89. PMID: 15489341
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.
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.

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

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

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

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

Proteomics. 2004 Jun;4(6):1581-90. PMID: 15174128
Small I, Peeters N, Legeai F and Lurin C (2004)
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.

Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein.

Nature. 2004 Apr 29;428(6986):945-50. PMID: 15118728
Wang KL, Yoshida H, Lurin C, Ecker JR.
Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

Ethylene gas is used as a hormone by plants, in which it acts as a critical growth regulator. Its synthesis is also rapidly evoked in response to a variety of biotic and abiotic stresses. The Arabidopsis ethylene-overproducer mutants eto2 and eto3 have previously been identified as having mutations in two genes, ACS5 and ACS9, respectively; these encode isozymes of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), which catalyse the rate-limiting step in ethylene biosynthesis. Here we report that another ethylene-overproducer mutation, eto1, is in a gene that negatively regulates ACS activity and ethylene production. The ETO1 protein directly interacts with and inhibits the enzyme activity of full-length ACS5 but not of a truncated form of the enzyme, resulting in a marked accumulation of ACS5 protein and ethylene. Overexpression of ETO1 inhibited induction of ethylene production by the plant growth regulator cytokinin, and promoted ACS5 degradation by a proteasome-dependent pathway. ETO1 also interacts with CUL3, a constituent of ubiquitin ligase complexes in which we propose that ETO1 serves as a substrate-specific adaptor protein. ETO1 thus has a dual mechanism, inhibiting ACS enzyme activity and targeting it for protein degradation. This permits rapid modulation of the concentration of ethylene.

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

Biochem J. 2000 Jun 1;348 Pt 2:291-5. PMID: 10816421
Lurin C, Güclü J, Cheniclet C, Carde JP, Barbier-Brygoo H, Maurel C.
Institut des Sciences Végétales, UPR 40, CNRS, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Disruption of putative anion channel gene AtCLC-a in Arabidopsis suggests a role in the regulation of nitrate content.

Plant J. 2000 Feb;21(3):259-67. PMID: 10758477
Geelen D, Lurin C, Bouchez D, Frachisse JM, Lelièvre F, Courtial B, Barbier-Brygoo H, Maurel C.
Institut des Sciences Végétales, Centre National de la Recherche Scientifique (CNRS), Bât 22, avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France.

In animals and yeast, voltage-dependent chloride channels of the CLC family play a role in basic cellular functions such as epithelial transport, plasma membrane excitability, and control of pH and membrane potential in intracellular compartments. To assess the function of CLCs in plants, we searched for CLC insertion mutants in a library of Arabidopsis lines transformed by Agrobacterium tumefaciens transferred DNA (T-DNA). Using a polymerase chain reaction-based screening procedure, an Arabidopsis line that carries a T-DNA insertion within the C-terminus of the AtCLC-a coding sequence was identified. Progeny from this plant line, clca-1, showed dramatically altered transcription of the AtCLC-a gene. Plants homozygous for the clca-1 mutation exhibited normal development and a morphology indistinguishable from the wild-type. However, their capacity to accumulate nitrate under conditions of nitrate excess was reduced in roots and shoots, by approximately 50%, while chloride, sulphate and phosphate levels were similar to the wild-type. In addition, the herbicide chlorate, an analogue of nitrate, induced a faster and more pronounced chlorosis in mutant plants. Hypersensitivity to chlorate as well as decreased nitrate levels co-segregated with the T-DNA insertion. They were found at various time points of the clca-1 life cycle, supporting the idea that AtCLC-a has a general role in the control of the nitrate status in Arabidopsis. Concordant with such a function, AtCLC-a mRNA was found in roots and shoots, and its levels rapidly increased in both tissues upon addition of nitrate but not ammonium to the culture medium. The specificity of AtCLC-a function with respect to nitrate is further supported by a similar free amino acid content in wild-type and clca-1 plants. Although the cellular localization of AtCLC-a remains unclear, our results suggest that AtCLC-a plays a role in controlling the intracellular nitrate status.

Cloning and functional expression of a plant voltage-dependent chloride channel.

Plant Cell. 1996 Apr;8(4):701-11. PMID: 8624442
Lurin C, Geelen D, Barbier-Brygoo H, Guern J, Maurel C. (1996)
Institut des Sciences Végétales, Centre National de la Recherche Scientifique (CNRS), Gif sur Yvette, France.

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants.