Marc Boutry

Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021

Sinorhizobium meliloti is an α-proteobacterium that forms agronomically important N2-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.

The Plasma Membrane H+-ATPase (A Highly Regulated Enzyme with Multiple Physiological Functions)

The proton-pump ATPase (H+-ATPase) of the plant plasma membrane acts as a primary transporter by pumping protons out of the cell, thereby creating pH and electrical potential differences across the plasmalemma (Fig. 1). Transport of many solutes (ions, metabolites, etc.) into and out of the cell involves secondary transporters whose ability to function is directly dependent on the proton-motive force created by the H+-ATPase. Depending on the electrical charge of the solute to be transported, the direction of its transport, and its concentration on either side of the membrane, it is possible to predict from Figure 1 the type of transport protein required. For instance, the uptake of a cation is energetically favorable because of the positive external electrical potential, and therefore requires only a diffusion facilitator, such as a channel protein or a uniport. Conversely, to be energetically favorable, the uptake of an anion must be accompanied by the uptake of one or more protons in a symport system. In addition to activating secondary transport, the H+-ATPase promotes more specialized physiological functions.

The plant plasma membrane H+-ATPase: structure, function and regulation.

The proton-pumping ATPase (H(+)-ATPase) of the plant plasma membrane generates the proton motive force across the plasma membrane that is necessary to activate most of the ion and metabolite transport. In recent years, important progress has been made concerning the identification and organization of H(+)-ATPase genes, their expression, and also the kinetics and regulation of individual H(+)-ATPase isoforms. At the gene level, it is now clear that H(+)-ATPase is encoded by a family of approximately 10 genes. Expression, monitored by in situ techniques, has revealed a specific distribution pattern for each gene; however, this seems to differ between species. In the near future, we can expect regulatory aspects of gene expression to be elucidated. Already the expression of individual plant H(+)-ATPases in yeast has shown them to have distinct enzymatic properties. It has also allowed regulatory aspects of this enzyme to be studied through random and site-directed mutagenesis, notably its carboxy-terminal region. Studies performed with both plant and yeast material have converged towards deciphering the way phosphorylation and binding of regulatory 14-3-3 proteins intervene in the modification of H(+)-ATPase activity. The production of high quantities of individual functional H(+)-ATPases in yeast constitutes an important step towards crystallization studies to derive structural information. Understanding the specific roles of H(+)-ATPase isoforms in whole plant physiology is another challenge that has been approached recently through the phenotypic analysis of the first transgenic plants in which the expression of single H(+)-ATPases has been up- or down-regulated. In conclusion, the progress made recently concerning the H(+)-ATPase family, at both the gene and protein level, has come to a point where we can now expect a more integrated investigation of the expression, function and regulation of individual H(+)-ATPases in the whole plant context.

Genomic Comparison of P-Type ATPase Ion Pumps in Arabidopsis and Rice

Members of the P-type ATPase ion pump superfamily are found in all three branches of life. Forty-six P-type ATPase genes were identified in Arabidopsis, the largest number yet identified in any organism. The recent completion of two draft sequences of the rice (Oryza sativa) genome allows for comparison of the full complement of P-type ATPases in two different plant species. Here, we identify a similar number (43) in rice, despite the rice genome being more than three times the size of Arabidopsis. The similarly large families suggest that both dicots and monocots have evolved with a large preexisting repertoire of P-type ATPases. Both Arabidopsis and rice have representative members in all five major subfamilies of P-type ATPases: heavy-metal ATPases (P1B), Ca2+-ATPases (endoplasmic reticulum-type Ca2+-ATPase and autoinhibited Ca2+-ATPase, P2A and P2B), H+-ATPases (autoinhibited H+-ATPase, P3A), putative aminophospholipid ATPases (ALA, P4), and a branch with unknown specificity (P5). The close pairing of similar isoforms in rice and Arabidopsis suggests potential orthologous relationships for all 43 rice P-type ATPases. A phylogenetic comparison of protein sequences and intron positions indicates that the common angiosperm ancestor had at least 23 P-type ATPases. Although little is known about unique and common features of related pumps, clear differences between some members of the calcium pumps indicate that evolutionarily conserved clusters may distinguish pumps with either different subcellular locations or biochemical functions.

A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense.

A nuclear gene encoding the beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia

The beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia is encoded by two nuclear genes, atp2-1 and atp2-2, which are both expressed. The complete nucleotide sequence of atp2-1 has been determined. It contains eight introns ranging from 88 to 1453 bp. The last intron contains a putative insertion element (Inp), of 812 bp bordered by 35-bp inverted repeats which share an 11-bp homology with Agrobacterium tumefaciens T-DNA borders. Sequences homologous to Inp are present in multiple copies in the N. plumbaginifolia and the N. tabacum genome but not in more distant species. The atp2-1 encoded polypeptide is highly homologous to beta subunits from other ATP synthases but it contains an extension at the N terminus which is probably involved in mitochondrial targeting. A sequence homology between exon 4 of atp2-1 and exon 1 of the human ras genes suggests a common ancestral origin for these exons.

The plasma membrane proton pump ATPase: the significance of gene subfamilies

Abstract. The plasma membrane proton pump ATPase (H+-ATPase) plays a central role in transport across the plasma membrane. As a primary transporter, it mediates ATP-dependent H+ extrusion to the extracellular space, thus creating pH and potential differences across the plasma membrane that activate a large set of secondary transporters. In several species, the H+-ATPase is encoded by a family of approximately 10 genes, classified into 5 gene subfamilies and we might ask what can this tell us about the concept, and the evolution, of gene families in plants. All the highly expressed H+-ATPase genes are classified into only two gene subfamilies, which diverged before the emergence of present plant species, raising the questions of the significance of the existence of these two well-conserved subfamilies and whether this is related to different kinetic or regulatory properties. Finally, what can we learn from experimental approaches that silence specific genes? In this review, we would like to discuss these questions in the light of recent data.

Production of antibodies in plants: status after twenty years.

Thanks to their potential to bind virtually all types of molecules; monoclonal antibodies are in increasing demand as therapeutics and diagnostics. To overcome the overloading of current production facilities, alternative expression systems have been developed, of which plants appear the most promising. In this review, we focus on the expression of monoclonal IgG or IgM in plant species. We analyse the data for 32 different antibodies expressed in various ways, differing in DNA construction, transformation method, signal peptide source, presence or absence of an endoplasmic reticulum retention sequence, host species and the organs tested, together resulting in 98 reported combinations. A large heterogeneity is found in the quantity and quality of the antibody produced. We discuss in more detail the strategy used to express both chains, the nature of the transcription promoters, subcellular localization and unintended proteolysis, when encountered.

The ATP-Binding Cassette Transporters: Structure, Function, and Gene Family Comparison between Rice and Arabidopsis

Cells are separated from their external environment by a membrane barrier, which ensures that certain ions, metabolic intermediates, and macromolecules, such as proteins, remain within the cell. However, life is not possible without the exchange of material and information with the external

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump‐ATPase (H(+)‐ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)‐ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)‐ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C‐terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild‐type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2‐ to 3‐fold increase of H(+)‐pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)‐ATPase improve the coupling between H(+)‐pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild‐type PMA2, indicating a relationship between H(+)‐ATPase activity and perturbations of the secretory pathway.