Anne Marmagne

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. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

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. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.

Arabidopsis Formin AtFH6 Is a Plasma Membrane-Associated Protein Upregulated in Giant Cells Induced by Parasitic Nematodes

Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Δ bnr1Δ mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.

Analysis of gene expression in resynthesized Brassica napus allotetraploids: transcriptional changes do not explain differential protein regulation.

Polyploidy, or whole genome duplication, is a major evolutionary process that has shaped eukaryotic genomes, notably those of flowering plants. The mechanisms underlying the regulation of, and sharing of functions between, the duplicated genes originating from polyploidy events, which lead to novel phenotypes, remain to be elucidated. A previous comparative proteomic study identified 360 proteins that were differentially regulated between the diploid Brassica progenitors and their synthetic allotetraploid derivatives. For 102 of these proteins, using the same resynthesized Brassica napus allotetraploids, we assayed the accumulation of the transcripts of the corresponding genes. We compared transcript levels quantified in the synthetic allotetraploids with the mid-parent expression values. Although all of the genes surveyed encoded nonadditive proteins, we found that two-thirds of them had additive transcript levels, indicating that most of the differential protein regulation is not explained by transcriptional changes. Our data suggest that differential protein regulation is mainly governed by post-transcriptional modifications. Summarizing available data from transcriptomic studies of other synthetic allopolyploid models, we describe the general trends of transcript regulation in an allopolyploid genome and discuss putative underlying molecular mechanisms, with particular emphasis on the small RNA pathway for the post-transcriptional control of gene expression.

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. All four AtVDACs are ubiquitously expressed in the plant but each of them displays a specific expression pattern in root cell types. Using two complementary approaches, we demonstrate conclusively that the four expressed AtVDACs are targeted to both mitochondria and plasma membrane but in differential abundance, AtVDAC3 being the most abundant in PM, and conversely, AtVDAC4 almost exclusively associated with mitochondria. These are the first plant proteins to be shown to reside in both these two membranes. To investigate a putative function of AtVDACs, we analyzed T-DNA insertion lines in each of the corresponding genes. Knock-out mutants for AtVDAC1, AtVDAC2 and AtVDAC4 present slow growth, reduced fertility and yellow spots in leaves when atvdac3 does not show any visible difference compared to wild-type plants. Analyses of atvdac1 and atvdac4 reveal that yellow areas correspond to necrosis and the mitochondria are swollen in these two mutants. All these results suggest that, in spite of a localization in plasma membrane for three of them, AtVDAC1, AtVDAC2 and AtVDAC4 have a main function in mitochondria.

Purification and fractionation of membranes for proteomic analyses.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, such as Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. However, membrane proteomics remains a challenge. One way to bring into view the complex mixture of proteins present in a membrane is to develop proteomic analyses based on (1) the use of highly purified membrane fractions and (2) fractionation of membrane proteins to retrieve as many proteins as possible (from the most to the less hydrophobic ones). To illustrate such strategies, we choose two types of membranes, the plasma membrane and the chloroplast envelope membranes. Both types of membranes can be prepared in a reasonable degree of purity from different types of tissues: the plasma membrane from cultured cells and the chloroplast envelope membrane from whole plants. This article is restricted to the description of methods for the preparation of highly purified and characterized plant membrane fractions and the subsequent fractionation of these membrane proteins according to simple physicochemical criteria (i.e., chloroform/methanol extraction, alkaline or saline treatments) for further analyses using modern proteomic methodologies.

ASN1-encoded asparagine synthetase in floral organs contributes to nitrogen filling in Arabidopsis seeds

Despite a general view that asparagine synthetase generates asparagine as an amino acid for long-distance transport of nitrogen to sink organs, its role in nitrogen metabolic pathways in floral organs during seed nitrogen filling has remained undefined. We demonstrate that the onset of pollination in Arabidopsis induces selected genes for asparagine metabolism, namely ASN1 (At3g47340), GLN2 (At5g35630), GLU1 (At5g04140), AapAT2 (At5g19950), ASPGA1 (At5g08100) and ASPGB1 (At3g16150), particularly at the ovule stage (stage 0), accompanied by enhanced asparagine synthetase protein, asparagine and total amino acids. Immunolocalization confined asparagine synthetase to the vascular cells of the silique cell wall and septum, but also to the outer and inner seed integuments, demonstrating the post-phloem transport of asparagine in these cells to developing embryos. In the asn1 mutant, aberrant embryo cell divisions in upper suspensor cell layers from globular to heart stages assign a role for nitrogen in differentiating embryos within the ovary. Induction of asparagine metabolic genes by light/dark and nitrate supports fine shifts of nitrogen metabolic pathways. In transgenic Arabidopsis expressing promoterCaMV35S ::ASN1 fusion, marked metabolomics changes at stage 0, including a several-fold increase in free asparagine, are correlated to enhanced seed nitrogen. However, specific promoterNapin2S ::ASN1 expression during seed formation and a six-fold increase in asparagine toward the desiccation stage result in wild-type seed nitrogen, underlining that delayed accumulation of asparagine impairs the timing of its use by releasing amide and amino nitrogen. Transcript and metabolite profiles in floral organs match the carbon and nitrogen partitioning to generate energy via the tricarboxylic acid cycle, GABA shunt and phosphorylated serine synthetic pathway.