Channel characteristics of VDAC-3 from Arabidopsis thaliana.

Four different isoforms of the Voltage-Dependent Anion Channel (VDAC) have been identified in Arabidopsis plant cells. The electrophysiological characteristics of several VDAC channels from animal as well as plant cells are well documented, but those of this model plant are unknown. One isoform, AtVDAC-3 was obtained either directly by cell-free synthesis or produced in Escherichia coli, as inclusion bodies, and re-natured.

Differential targeting of VDAC3 mRNA isoforms influences mitochondria morphology.

Intracellular targeting of mRNAs has recently emerged as a prevalent mechanism to control protein localization. For mitochondria, a cotranslational model of protein import is now proposed in parallel to the conventional posttranslational model, and mitochondrial targeting of mRNAs has been demonstrated in various organisms. Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane and the major transport pathway for numerous metabolites.

Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria.

In mammals, the Voltage-dependent anion channels (VDACs) are predominant proteins of the outer mitochondrial membrane (OMM) where they contribute to the exchange of small metabolites essential for respiration. They were shown to be as well associated with the plasma membrane (PM) and act as redox enzyme or are involved in ATP release for example. In Arabidopsis, we show that four out of six genomic sequences encode AtVDAC proteins.

Plant organelle proteomics: collaborating for optimal cell function.

Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function.

The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the regulation of stomatal movements and contributes to salt tolerance.

In plant cells, anion channels and transporters are essential for key functions such as nutrition, resistance to biotic or abiotic stresses, and ion homeostasis. In Arabidopsis, members of the chloride channel (CLC) family located in intracellular organelles have been shown to be required for nitrate homeostasis or pH adjustment, and previous results indicated that AtCLCc is involved in nitrate accumulation. We investigated new physiological functions of this CLC member in Arabidopsis.

ATP binding to the C terminus of the Arabidopsis thaliana nitrate/proton antiporter, AtCLCa, regulates nitrate transport into plant vacuoles.

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP.

Two anion transporters AtClCa and AtClCe fulfil interconnecting but not redundant roles in nitrate assimilation pathways.

* In plants, the knowledge of the molecular identity and functions of anion channels are still very limited, and are almost restricted to the large ChLoride Channel (CLC) family. In Arabidopsis thaliana, some genetic evidence has suggested a role for certain AtCLC protein members in the control of plant nitrate levels. In this context, AtClCa has been demonstrated to be involved in nitrate transport into the vacuole, thereby participating in cell nitrate homeostasis.

Extent of N-terminal modifications in cytosolic proteins from eukaryotes.

Most proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software.

A novel pathway of cytochrome c biogenesis is involved in the assembly of the cytochrome b6f complex in arabidopsis chloroplasts.

We recently characterized a novel heme biogenesis pathway required for heme c(i)' covalent binding to cytochrome b6 in Chlamydomonas named system IV or CCB (cofactor assembly, complex C (b6f), subunit B (PetB)). To find out whether this CCB pathway also operates in higher plants and extend the knowledge of the c-type cytochrome biogenesis, we studied Arabidopsis insertion mutants in the orthologs of the CCB genes. The ccb1, ccb2, and ccb4 mutants show a phenotype characterized by a deficiency in the accumulation of the subunits of the cytochrome b6f complex and lack covalent heme binding to cytochrome b6.

Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively.

Though numerous pieces of evidence point to major physiological roles for anion channels in plants, progress in the understanding of their biological functions is limited by the small number of genes identified so far. Seven chloride channel (CLC) members could be identified in the Arabidopsis genome, amongst which AtCLCe and AtCLCf are both more closely related to bacterial CLCs than the other plant CLCs. It is shown here that AtCLCe is targeted to the thylakoid membranes in chloroplasts and, in agreement with this subcellular localization, that the clce mutants display a phenotype related to photosynthesis activity.

A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome.

The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS.

Anion channels and transporters in plant cell membranes.

Anion channels/transporters appear as key players in signaling pathways leading to the adaptation of plant cells to abiotic and biotic environmental stresses, in the control of metabolism and in the maintenance of electrochemical gradients. Focusing on the most recent advances, this review aims at providing a description of the role of these channels in various physiological functions such as control of stomatal movements, plant-pathogen interaction, xylem loading, compartmentalization of metabolites and coupling with proton gradients. These functions have been demonstrated by a combination of electrophysiology, pharmacology and genetics approaches, the key issue being to identify the corresponding proteins and genes.

The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles.

Nitrate, the major nitrogen source for most plants, is widely used as a fertilizer and as a result has become a predominant freshwater pollutant. Plants need nitrate for growth and store most of it in the central vacuole. Some members of the chloride channel (CLC) protein family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels, whereas the bacterial ClC-ec1 and mammalian ClC-4 and ClC-5 have recently been characterized as Cl-/H+ exchangers with unknown cellular functions.

A versatile method for deciphering plant membrane proteomes.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like that of Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. This article summarizes the different steps of a versatile strategy that has been developed to decipher plant membrane proteomes. Initiated with envelope membranes from spinach chloroplasts, this strategy has been adapted to thylakoids, and further extended to a series of membranes from the model plant Arabidopsis: chloroplast envelope membranes, plasma membrane, and mitochondrial membranes.

Plant membrane proteomics.

Plant membrane proteins are involved in many different functions according to their location in the cell. For instance, the chloroplast has two membrane systems, thylakoids and envelope, with specialized membrane proteins for photosynthesis and metabolite and ion transporters, respectively. Although recent advances in sample preparation and analytical techniques have been achieved for the study of membrane proteins, the characterization of these proteins, especially the hydrophobic ones, is still challenging.

Identification of new intrinsic proteins in Arabidopsis plasma membrane proteome.

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported.

Anion channels in higher plants: functional characterization, molecular structure and physiological role.

Anion channels are well documented in various tissues, cell types and membranes of algae and higher plants, and current evidence supports their central role in cell signaling, osmoregulation, plant nutrition and metabolism. It is the aim of this review to illustrate through a few selected examples the variety of anion channels operating in plant cells and some of their regulation properties and unique physiological functions. In contrast, information on the molecular structure of plant anion channels has only recently started to emerge.

The sax1 mutation defines a new locus involved in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

In this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J.

The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid.

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin-resistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (x 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1.

Impermeant auxin analogues have auxin activity.

Protein conjugates of 5-aminonaphthalene-1-acetic acid and of 5-azido-naphthalene-1-acetic acid have been prepared and evaluated for auxin activity in two types of assay. In standard elongation tests with pea (Pisum sativum L.) epicotyl sections the conjugates are inactive.

Use of mutants and transformed plants to study the action of auxins.

We describe here the results of a comparison of the properties of several plant genotypes differing in their reaction to auxins. The hormonal response used to compare the genotypes is the auxin-induced variation of the transplasmalemma electrical potential difference (delta Em) exhibited by protoplasts isolated from leaves or root tips. Using this membrane response, we have shown that large variations in the sensitivity to auxins can be induced either by mutagenesis in tobacco or by transformation of various materials by Agrobacterium rhizogenes.

Functional evidence for an auxin receptor at the plasmalemma of tobacco mesophyll protoplasts.

Tobacco mesophyll protoplasts were previously shown to respond to naphthaleneacetic acid by modifying their transmembrane potential difference. In the present work, evacuolated protoplasts were used to show that this response resides only at the plasmalemma. This electrical response was investigated by using polyclonal antibodies directed against plasma membrane antigens presumably involved in the reception and transduction of the auxin signal.