R. Esnault

Alfalfa Root Flavonoid Production Is Nitrogen Regulated

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.

Pathological and molecular characterizations of alfalfa interactions with compatible and incompatible bacteria, Xanthomonas campestris pv. alfalfae and Pseudomonas syringae pv. pisi

We report on the interactions of alfalfa with Xanthomonas campestris pv. alfalfae and Pseudomonas syringae pv. pisi. A hypersensitive response was observed when leaves were infiltrated with P. s. pv. pisi, which remained strictly limited to the injected zone. The compatible interaction with X. c. pv. alfalfae was characterized by water-soaking symptoms and the spreading of the bacterium into the leaf blade. Analyses of transcript accumulation were conducted with cDNAs encoding enzymes involved in phytoalexin synthesis: chalcone synthase (CHS), chalcone isomerase (CHI), and isoflavone reductase (IFR). In incompatible interactions the maximum accumulation of the CHS, CHI, and IFR transcripts was observed 6 hr postinfection. In the compatible interaction, the induction of these transcripts was delayed until 25-30 hr postinfection, and the level of their accumulation was considerably lower. Extending this molecular analysis to the root system showed that the reaction of roots during an incompatible interaction was quite comparable to that of leaves. To complete these analyses, expression of genes encoding pathogenesis-related (PR) proteins in leaves was also analyzed by polymerase chain reaction. High-level accumulation of a 0.8-kb transcript encoding a PR protein was observed 6 to 30 hr postinfection in the incompatible interaction.

Nucleotide Sequences of Three Chalcone Reductase Genes from Alfalfa

An important reaction in the biosynthesis of many phytoalexins in leguminous plants is the formation of 4,2‘,4‘trihydroxychalcone from 4-coumaroyl-COA and malonylCOA, first demonstrated by Ayabe et al. (1988). Welle and Grisebach (1988) purified a reductase from soybean cell cultures and demonstrated that the enzyme, named CHR, co-acts, with NADPH as cofactor, with CHS in the formation of the 4,2’,4’-trihydroxychalcone. They showed also that the two enzymes are induced coordinately after treatment of the cell cultures with elicitor-type molecules. Moreover, Welle et al. (1991) isolated the encoding cDNA. This reductase can play a fundamental role in planta in the balancing of the production of 5-deoxyflavonoids or 5-hydroxyflavonoids, the synthesis of which depends on the co-action of CHS and CHR or on CHS alone, respectively. Despite this pivotal role and except for the pioneering work already cited, the CHR has not been extensively studied, either at the enzymatic or gene expression levels. We have previously shown that during an incompatible interaction, Medicago sativalPseudomonas syringae pv pisi, severa1 genes involved in phytoalexin production (CHS and isoflavone reductase) were induced (Esnault et al., 1993). This led us to address the question of an involvement of CHR. Our strategy was based first on the isolation of cDNA encoding CHR in alfalfa as well as its genomic organization, and second on the analysis of its expression. In this communication, we report the isolation of three cDNAs encoding CHR. The soybean CHR cDNA clone (Welle et al., 1991) was used to screen a AgtlO cDNA library made from poly(A)+ RNA extracted from leaves harvested 6 h after infection with the incompatible bacterium P. syringae pv pisi. After three rounds of screening and subcloning in the pBS SKplasmid, seven clones were obtained and sequenced. Three of them, MsCHRla, MsCHRlc, MsCHRZa, were full length and their features are described in Table I. They contain an insert of 1202,1115, and 1134 bp, respectively, with an open reading frame of 936 nucleotides. The predicted size of the reductase is 312 amino acids with a molecular mass of around 35 kD. The deduced amino acid sequences are more than 90% homologous to the soybean CHR, and they are related to the aldo-keto reductase family. No signal or ~~

Nucleotide sequences of four pathogen-induced alfalfa peroxidase- encoding cDNAs

We constructed an alfalfa cDNA library from mRNA extracted from leaves after infection with Pseudomonas syringae (incompatible interaction). Screening with oligodeoxyribonucleotides designed from regions conserved in all known peroxidases allowed the isolation of four cDNAs (Msprx1A, 1B, 1C and 2). Sequence analysis revealed the presence of open reading frames of 351, 355, 358 and 323 amino acids, respectively, with the characteristic consensus sequences of plant peroxidases. Sequence comparison showed that the Msprx2 product is significantly different from the others and, particularly, lacks a C-terminal propeptide which might be required for vacuolar targeting.

Measurement and detection of peroxidase

Abstract The purity and the concentration based on the RZ value and the molar extinction coefficient was determined for the cationic peroxidase isolated from peanut (Arachis hypogaea L.) suspension culture medium. The maintenance of a concentrated sample and, if necessary, suspension in slightly alakline buffer was found to be the best condition for storage. As little as 3 ng of peroxidase could be immuno-precipitated, subjected to electrophoresis and immunoprobed and still be detected.

Effect of salinity stress on ribonucleolytic activities in glycophytic and halophytic plant species

Summary The ribonuclease activities (RNase, EC 2.7.7.17) of the halophyte, Suaeda maritima (L.) DUM var. macrocarpa MOQ, and the glycophytes, Phaseolus vulgaris (L.) and Vicia faba (L.), were studied in relation to the salinity (NaCl) of the nutrient medium. A reduction of growth was observed for the three species subjected to «salt stresso (i.e. hypersalinity for glycophytes or absence of salt for the halophyte). Both short-term (salt shock) and long-term treatments with NaCl induced a marked increase in RNase activity, and a close relationship was established between «salt stress» and a high RNase activity. The stimulation of RNase activity was independent of the species (halophyte or glycophyte), the analyzed organ (root or leaf) and the nature of the salt treatment (saline shock or steady state culture). Ribonuclease activities of S. maritima leaves and V. faba roots were studied in detail by ion-exchange chromatography. Under the influence of salt treatment it appears that both anionic and cationic RNase activities were increased. It has been demonstrated for the first time that an anionic RNase is stimulated to a greater extent than any other ribonucleolytic enzyme in both the halophyte and the glycophyte. The RNases, particularly the anionic enzymes, can thus be considered as good markers for conditions of saline contraint.

CLONING AND EXPRESSION OF A CDNA ENCODING A CYTOPLASMIC L5 RIBOSOMAL PROTEIN FROM ALFALFA (MEDICAGO SATIVA L.)

A cDNA encoding a putative cytoplasmic ribosomal protein L5 from alfalfa (MsRL5), the first sequence from higher plants, has been characterized. The derived amino acid sequence of 181 residues contains the L5 signature, is 72.2% identical to yeast ribosomal L5 and shares high identity with other RL5 peptides from eukaryotic origin. The sequence does not contain any signal or transit peptide and therefore might be cytoplasmic. In all alfalfa organs examined MsRL5 transcripts were detected at approximately equal levels.

Preliminary crystallographic study of peanut peroxidase.

The cationic isozyme of peroxidase isolated from suspension cultures of peanut cells is a heme-containing and calcium-dependent glycoprotein having four covalently attached oligosaccharide chains. Attempts were made to crystallize the glycoprotein for X-ray diffraction analysis, and these have met with some success. Crystals have now been grown that are suitable for a full three-dimensional structural analysis. The crystals are thin plates and we have shown them to be of the orthorhombic space group P2(1)2(1)2(1) with a = 48.1, b = 97.2, c = 146.2 A. The crystals diffract to beyond 2.8 A resolution, appear to be stable to lengthy X-ray exposure, and contain two molecules of 40,000 daltons each in the asymmetric unit.

A Highly Specific Monoclonal Antibody to a Catonic Peroxidase from Cultured Peanut Cells

Summary A hybridoma cell line (CP-4) producing a monoclonal antibody (IgG2a), against a cationic peroxidase isolated from cultured peanut cells is described. This IgG selectively recognized the peroxidase molecule from cultured peanut cells on western blots, as detected by antimouse- 125 IIgG.

Analysis of Peroxidase Gene Expression in an Hypersensitive Response Induced by Pathogenic Bacteria on Alfalfa

Peroxidases constitute an enzyme family that displays tremendous diversity in form, function and distribution throught the plant kingdom. They have been implicated in polysac-charide or extensin monomers cross-linking, lignification, wound-healing, phenol oxidation, pathogen defence, indole-3-acetic acid oxidation and the regulation of the cell elongation.