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12 páginas., Thioredoxins (TRXs) are small ubiquitous oxidoreductases involved in disulfide bond reduction of a large panel of target proteins. The most complex cluster in the family of plant TRXs is formed by h-type TRXs. In Arabidopsis (Arabidopsis thaliana), nine members of this subgroup were described, which are less well known than their plastidial counterparts. The functional study of type-h TRXs is difficult because of the high number of isoforms and their similar biochemical characteristics, thus raising the question whether they have specific or redundant functions. Type-h TRXs are involved in seed germination and self incompatibility in pollen-pistil interaction. Their function as antioxidants has recently been proposed, but further work is needed to clarify this function in plants. In this study, we describe two new h-type TRXs from pea (Pisum sativum; stated PsTRXh1 and PsTRXh2). By functional complementation of a yeast (Saccharomyces cerevisiae) trx1D trx2D double mutant, we demonstrate that PsTRXh1 is involved in the redox-imbalance control, possibly through its interaction with peroxiredoxins. In contrast, PsTRXh2 provokes a phenotype of hypersensitivity to hydrogen peroxide in the yeast mutant. Furthermore, we show differential gene expression and protein accumulation of the two isoforms, PsTRXh1 protein being abundantly detected in vascular tissue and flowers, whereas PsTRXh2 gene expression was hardly detectable. By comparison with previous data of additional PsTRXh isoforms, our results indicate specific functions for the pea h-type TRXs so far described., This work was supported by the Dirección General de Investigación Científica y Te´cnica, Spain (grant nos. PB98–0474 and BF12002–00401), by the Junta de Andalucía, Spain (grant no. CVI 154), by CSIC (Acción Integrada grant no. HF2001–0136), and by the Spanish government (fellowship FPI98 to J.A.T.).

International audience; Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 β-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the β-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the β-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 β-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility.

International audience; PA1b (pea albumin 1, subunit b) is a small and compact 37-amino acid protein, isolated from pea seeds (Pisum sativum), that adopts a cystine knot fold. It acts as a potent insecticidal agent against major pests in stored crops and vegetables, making it a promising bioinsecticide. Here, we investigate the influence of individual residues on the structure and bioactivity of PA1b. A collection of 13 PA1b mutants was successfully chemically synthesized in which the residues involved in the definition of PA1b amphiphilic and electrostatic characteristics were individually replaced with an alanine. The three-dimensional structure of PA1b was outstandingly tolerant of modifications. Remarkably, receptor binding and insecticidal activities were both dependent on common well defined clusters of residues located on one single face of the toxin, with Phe-10, Arg-21, Ile-23, and Leu-27 being key residues of the binding interaction. The inactivity of the mutants is clearly due to a change in the nature of the side chain rather than to a side effect, such as misfolding or degradation of the peptide, in the insect digestive tract. We have shown that a hydrophobic patch is the putative site of the interaction of PA1b with its binding site. Overall, the mutagenesis data provide major insights into the functional elements responsible for PA1b entomotoxic properties and give some clues toward a better understanding of the PA1b mode of action.

International audience; The pea aphid genome includes 66 genes contributing to amino acid biosynthesis and 93 genes to amino acid degradation. In several respects, the pea aphid gene inventory complements that of its symbiotic bacterium, Buchnera aphidicola (Buchnera APS). Unlike other insects with completely sequenced genomes, the pea aphid lacks the capacity to synthesize arginine, which is produced by Buchnera APS. However, consistent with other insects, it has genes coding for individual reactions in essential amino acid biosynthesis, including threonine dehydratase and branched-chain amino acid aminotransferase, which are not coded in the Buchnera APS genome. Overall the genome data suggest that the biosynthesis of certain essential amino acids is shared between the pea aphid and Buchnera APS, providing the opportunity for precise aphid control over Buchnera metabolism.

International audience; Highly purified, intact chloroplasts were prepared from pea (Pisum sativum L.) and spinach (Spinacia oleracea L.) following an identical procedure, and were used to investigate the cupric cation inhibition on the photosynthetic activity. In both species, copper inhibition showed a similar inhibitor concentration that decreases the enzyme activity by 50% (IC(50) approximately 1.8 microM) and did not depend on the internal or external phosphate (Pi) concentration, indicating that copper did not interact with the Pi translocator. Fluorescence analysis suggested that the presence of copper did not facilitate photoinhibition, because there were no changes in maximal fluorescence (F(m)) nor in basal fluorescence (F(o)) of copper-treated samples. The electron transport through the photosystem II (PSII) was also not affected (operating efficiency of PSII-F'v/F'm similar in all conditions). Yet, under Cu(2+) stress, the proportion of open PSII reaction centers was dramatically decreased, and the first quinone acceptor (Q(A)) reoxidation was fully inhibited, as demonstrated by the constant photochemical quenching (q(P)) along experiment time. The quantum yield of PSII electron transport (Phi(PSII)) was also clearly affected by copper, and therefore reduced the photochemistry efficiency. Manganese, when added simultaneously with copper, delayed the inhibition, as measured by oxygen evolution and chlorophyll fluorescence, but neither reversed the copper effect when added to copper-inhibited plastids, nor prevented the inhibition of the Hill activity of isolated copper-treated thylakoids. Our results suggest that manganese competed with copper to penetrate the chloroplast envelope. This competition seems to be specific because other divalent cations e.g. magnesium and calcium, did not interfere with the copper action in intact chloroplasts. All results do suggest that, under these conditions, the stroma proteins, such as the Calvin-Benson cycle enzymes or others are the most probable first target for the Cu(2+) action, resulting in the total inhibition of chloroplast photosynthesis and in the consequent unbalanced rate of production and consumption of the reducing power.

International audience; Pea albumin 1b (PA1b) is a small sulphur-rich peptide from pea seeds, also named leginsulin because of the binding characteristics of its soybean orthologue. Its insecticidal properties were discovered more recently. By using a combination of molecular, biochemical and specific insect bioassays on seed extracts, we characterised genes from numerous Papilionoideae, but not from Caesalpinioideae or Mimosoideae, although the last group harboured species with partially positive cues (homologous biological activities). The A1b defence peptide family, therefore, appears to have evolved relatively late in the legume lineage, maybe from the sophoroid group (e.g. Styphnolobium japonicum). However, unambiguous sequence information is restricted to a group of tribes within the subfamily Papilionoideae (Psoraleae, Millettieae, Desmodieae, Hedysareae, Phaseoleae, Vicieae, and the now clearly polyphyletic "Trifolieae" and "Galegeae"). Recent diversification by gene duplications has occurred in many species, or longer ago in some lineages (Medicago truncatula), as well as probable gene or expression losses at different taxonomic levels (Loteae, Vigna subterranea).

Azospirillum strains have been used as plant-growth-promoting rhizobacteria (PGPR) of cereal crops, but their adaptation to the root remains poorly understood. Here, we used a global approach based on differential fluorescence induction (DFI) promoter trapping to identify genes of the wheat isolate Azospirillum brasilense Sp245 that are induced in the presence of spring wheat seed extracts. Fluorescence-based flow cytometry sorting of Sp245 cells was validated using PlacZ, PsbpA and PnifH promoters and egfp. A random promoter library was constructed by cloning 1–3 kb Sp245 fragments upstream of a promoterless version of egfp in the promoter-trap plasmid pOT1e (genome coverage estimated at threefold). Exposure to spring wheat seed extracts obtained using a methanol solution led to the detection of 300 induced DFI clones, and upregulation by seed extracts was confirmed in vitro for 46 clones. Sequencing of 21 clones enabled identification of seven promoter regions. Five of them displayed upregulation once inoculated onto spring wheat seedlings. Their downstream sequence was similar to (i) a predicted transcriptional regulator, (ii) a serine/threonine protein kinase, (iii) two conserved hypothetical proteins, or (iv) the copper-containing dissimilatory nitrite reductase NirK. Two of them were also upregulated when inoculated on winter wheat and pea but not on maize, whereas the three others (including PnirK) were upregulated on the three hosts. The amounts of nitrate and/or nitrite present in spring wheat seed extracts were sufficient for PnirK upregulation. Overall, DFI promoter trapping was useful to reveal Azospirillum genes involved in the interaction with the plant.

Late-embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to a high level in desiccation-tolerant tissues and are thus prominent in seeds. They are expected to play a protective role during dehydration; however, functional evidence is scarce. We identified a LEA protein of group 3 (PsLEAm) that was localized within the matrix space of pea (Pisum sativum) seed mitochondria. PsLEAm revealed typical LEA features such as high hydrophilicity and repeated motifs, except for the N-terminal transit peptide. Most of the highly charged protein was predicted to fold into amphiphilic α-helixes. PsLEAm was expressed during late seed development and remained in the dry seed and throughout germination. Application of the stress hormone abscisic acid was found to reinduce the expression of PsLEAm transcripts during germination. PsLEAm could not be detected in vegetative tissues; however, its expression could be reinduced in leaves by severe water stress. The recombinant PsLEAm was shown to protect two mitochondrial matrix enzymes, fumarase and rhodanese, during drying in an in vitro assay. The overall results constitute, to our knowledge, the first characterization of a LEA protein in mitochondria and experimental evidence for a beneficial role of a LEA protein with respect to proteins during desiccation.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; The toxicity of the pea albumin 1b (PA1b), a 37 amino-acid peptide extracted from pea seeds, for cereal weevils (Sitophilus oryzae, Sitophilus granarius and Sitophilus zeamais) was recently discovered. The mechanism of action of this new entomotoxin is still unknown and potentially involves a target protein in the insect tissues. This work describes the characterization of a high-affinity binding site for PA1b in a microsomal fraction of Sitophilus spp. extracts. Purified PA1b was labeled to a high specific radioactivity (c. 900 Ci.mmol-1) using 125I, and the iodinated ligand was found to be biologically active. Binding of this ligand to the microsomal fraction of S. oryzae extract was found to be saturable and reversible, with an affinity (Kd) of 2.6 nm, and a high maximal binding capacity (Bmax) of 40 pmol.mg-1 of protein. A binding site displaying similar characteristics was detectable in the five susceptible weevils strains tested, as well as in the pea aphid or in the fruit fly. However, no binding activity was detectable in extracts from four S. oryzae strains previously shown to be resistant to the toxin through a recessive monogenic mechanism. Therefore, we suggest that this binding site might be involved in the mechanism of action of PA1b.

International audience; Aphids feed on a protein-poor diet and are insensitive to several serine protease inhibitors. However, among the Bowman-Birk family of plant trypsin inhibitors (BBI), some members display significant toxicity to the pea aphid Acyrthosiphon pisum. A BBI isoform purified from pea seeds (PsTI-2) displays an IC50 of 41 microM and a LC50 of 48 microM at 7 days. Our data show that the chymotrypsin-directed active site from these bifunctional inhibitors is responsible for this activity, and that artificial cyclic peptides bearing the Bowman-Birk anti-chymotrypsin head induce much greater toxicity and growth inhibition than their anti-trypsin counterparts. The toxic syndrome included a rapid behavioural response of aphids on diets containing the toxic peptides, with induced restlessness after only 1 h of exposure to the chymotrypsin inhibitor. Nevertheless, chymotrypsin activity was not detected in aphid guts, using two chromogenic chymotrypsin substrates, and the physiological target of the chymotrypsin inhibitor remains unknown. These data show for the first time that plant chymotrypsin inhibitors, still widely unexplored, may act as paradoxical toxicants to aphids and serve as defensive metabolites for phloem-feeding insects.

International audience; Transmission electron micrographs of the pea aphid midgut revealed that its anterior region has cells with an apical complex network of lamellae (apical lamellae) instead of the usual regularly-arranged microvilli. These apical lamellae are linked to one another by trabeculae. Modified perimicrovillar membranes (MPM) are associated with the lamellae and project into the lumen. Trabeculae and MPM become less conspicuous along the midgut. The most active A. pisum digestive enzymes are membrane-bound. An aminopeptidase (APN) is described elsewhere. An alpha-glucosidase (alpha-Glu) has a molecular mass of 72 kDa, pH optimum 6.0 and catalyzes in vitro transglycosylations in the presence of an excess of the substrate sucrose. There is a major cysteine proteinase activity (CP) on protein substrates that has a molecular mass of 40 kDa, pH optimum 5.5, is inhibited by E-64 and chymostatin and is activated by EDTA+cysteine. The enzyme is more active against carbobenzoxy-Phe-Arg-4-methylcoumarin-7-amide (ZFRMCA) than against ZRRMCA. These features identify the purified CP as a cathepsin-L-like cysteine proteinase. Most CP is found in the anterior midgut, whereas alpha-Glu and APN predominate in the posterior midgut. With the aid of antibodies, alpha-Glu and CP were immunolocalized in cell vesicles and MPM, whereas APN was localized in vesicles, apical lamellae and MPM. The data suggest that the anterior midgut is structurally reinforced to resist osmotic pressures and that the transglycosylating alpha-Glu, together with CP and APN are bound to MPM, thus being both distributed over a large surface and prevented from excretion with honeydew. alpha-Glu frees glucose from sucrose without increasing the osmolarity, and CP and APN may process toxins or other proteins occasionally present in phloem.

Nod factors (NFs) are rhizobial lipo-chitooligosaccharide signals that trigger root nodule development in legumes. Modifications of NF structures influence their biological activity and affect their degradation by plant chitinases. Nodulation of certain pea cultivars by Rhizobium leguminosarum bv. viciae requires modification of NFs at the reducing end by either an O-acetyl or a fucosyl group. Fucosylated NFs were produced by an in vitro reaction with NodZ fucosyltransferase and purified. Their biological activity on pea was tested by measuring their capacity to stimulate the activity of a hydrolase that cleaves NFs. Non-modified and fucosylated NFs displayed this activity at nano- to picomolar concentrations, while a sulfated NF from Sinorhizobium meliloti was inactive. In an additional series of experiments, the stability of non-modified and fucosylated NFs in the presence of purified tobacco chitinases was compared. The presence of the fucosyl group affected the degradation rates and the accessibility of specific cleavage sites on the chitooligosaccharide backbone. These results suggest that the fucosyl group in NFs also weakens the interaction of NFs with certain chitinases or chitinase-related proteins in pea roots.