Raoul Ranjeva

The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates.

Arbuscular mycorrhizal (AM) symbiosis is an association between obligate biotrophic fungi and more than 80% of land plants. During the pre-symbiotic phase, the host plant releases critical metabolites necessary to trigger fungal growth and root colonization. We describe the isolation of a semipurified fraction from exudates of carrot hairy roots, highly active on germinating spores of Gigaspora gigantea, G. rosea, and G. margarita. This fraction, isolated on the basis of its activity on hyphal branching, contains a root factor (one or several molecules) that stimulates, directly or indirectly, G. gigantea nuclear division. We demonstrate the presence of this active factor in root exudates of all mycotrophic plant species tested (eight species) but not in those of nonhost plant species (four species). We negatively tested the hypothesis that it was a flavonoid or a compound synthesized via the flavonoid pathway. We propose that this root factor, yet to be chemically characterized, is a key plant signal for the development of AM fungi.

Inositol trisphosphate stimulates the release of calcium from intact vacuoles isolated from Acer cells

On addition of inositol trisphosphate, intact vacuoles isolated from Acer pseudoplatanus cell suspension cultures release part of their calcium content. The process was specific, dose‐dependent (IC50 = 0.2μM) and was inhibited by an intracellular calcium antagonist. The calcium efflux elicited by inositol trisphosphate increased with the age of the cell suspension cultures, the maximum effect being obtained when the cultures reached the stationary phase. It is suggested that vacuoles play a role as an endocellular calcium store that is responsive to inositol trisphosphate in plants.

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Activation of plasma membrane voltage-dependent calcium-permeable channels by disruption of microtubules in carrot cells

Plasma membrane‐bound voltage‐dependent calcium channels may couple the perception of an initial stimulus to a regulated pathway for calcium influx. The activities of these channels have been shown to be very low and highly unstable but may be recruited by large‐predepolarizing pulses, according to a process referred to as recruitment. By combining pharmacological and electrophysiological approaches, we demonstrate in the present paper that the cytoskeleton plays an important role in the regulation of the activity and stability of voltage‐dependent calcium channels during whole‐cell patch‐clamp experiments on carrot protoplasts. Whereas drugs affecting the organization of the microfilament network have no measurable effect, the manipulation of the microtubule network elicits important changes. Thus, the addition of colchicine or oryzalin, which are known to disrupt microtubule organization, leads to a 6–10‐fold increase in calcium channel activities and half‐life. In contrast, stabilization of the microtubules by taxol has no effect on any of these parameters. The data obtained suggest that interactions of microtubules and voltage‐dependent calcium channels by either direct or indirect mechanisms inhibit channel activities and decrease their half‐life. In contrast, the disruption of the network overcomes such an inhibitory effect and allows the activation of calcium channels. It is speculated that under normal physiological conditions these protein‐protein interactions may work in a reversible manner and contribute to signal transduction in higher plants.

Phenolic metabolism in petunia tissues: IV. - Properties of p-coumarate: coenzyme A ligase isoenzymes

Three p-coumarate: CoA ligases were separated from Petunia leaves. There was no interconversion from one form to another. The isoenzymes had a number of common properties: optimum pH, instability in the absence of polyols, action on p-coumaric acid as the common substrate. These enzymes differed significantly with respect to: --their substrate specificity towards the other C6-C3 units of Petunia. Form Ia (caffeate: CoA ligase) acted on caffeic acid, form Ib (sinapate: CoA ligase) on sinapic acid form II (ferulate: CoA ligase) on ferulic acid. --their thermal stability. --their sensitivity to phenolics: (a) caffeate: CoA ligase was inhibited by p-coumaroyl and caffeoyl quinic esters. It was insensitive to p-coumaroyl-glucose, on one hand and to a number of flavonoids on the other. (b) ferulate: CoA ligase was specifically inhibited by naringenin. (c) sinapate: CoA ligase was not inhibited by the selected compounds. In all cases, the inhibition was of the non competitive type and the enzymes were desensized to the modifier action by thermal treatment independently from the enzyme activity. These results suggest the occurrence of distinct sites of reception for the substrate and the inhibitor on the enzyme molecule. All these data are consistent with the hypothesis of the possible participation of each individual form in a limited number of pathways. This would be of physiological interest since the metabolic fate of the different cinnamic acids could be independently controlled at the p-coumarate: CoA ligase level.

Identification of a High Affinity Binding Site for Lipo- oligosaccharidic NodRm Factors in the Microsomal Fraction of Medicago Cell Suspension Cultures

Protease-sensitive binding sites for a 35S-labeled ligand corresponding to the major lipo-oligosaccharidic symbiotic signal of Rhizobium meliloti (NodRm factor), have been identified in the microsomal fraction of Medicago varia cell suspension culture extracts. Binding was reversible and saturable and tetra-N-acetyl chitotetraose was a poor competitor of NodRm binding. Scatchard analysis suggests the presence of a high affinity binding site, termed Nod factor binding site two (NFBS2), with a Kd of 1.9 nM, and perhaps a second site with an affinity (Kd of 70 nM) similar to that of a site (NFBS1) previously characterized in Medicago truncatula root extracts.

Recruitment of plasma membrane voltage-dependent calcium-permeable channels in carrot cells.

Numerous biological assays and pharmacological studies have led to the suggestion that depolarization‐activated plasma membrane Ca2+ channels play prominent roles in signal perception and transduction processes during growth and development of higher plants. The recent application of patch‐clamp techniques to isolated carrot protoplasts has led to direct voltage‐clamp evidence for the existence of Ca2+ channels activated by physiological depolarizations in the plasma membrane of higher plant cells. However, these voltage‐dependent Ca2+ channels were not stable and their activities decreased following the establishment of whole‐cell recordings. We show here that large pre‐depolarizing pulses positive to 0 mV induced not only the recovery of Ca2+ channel activities, but also the activation of initially quiescent voltage‐dependent Ca2+ channels in the plasma membrane (recruitment). This recruitment was dependent on the intensity and duration of membrane depolarizations, i.e. the higher and longer the pre‐depolarization, the greater the recruitment. Pre‐depolarizing pulses to +118 mV during 30 s increased the initial calcium currents 5‐ to 10‐fold. The recruited channels were permeable to Ba2+ and Sr2+ ions. The data suggested that voltage‐dependent Ca(2+)‐permeable channels are regulated by biological mechanisms which might be induced by large pre‐depolarizations of the plasma membrane. In addition, this study provides evidence for the existence in the plasma membrane of higher plant cells of a large number of voltage‐dependent Ca2+ channels of which a major part are inactive and quiescent. It is suggested that quiescent Ca2+ channels can be rapidly recruited for Ca(2+)‐dependent signal transduction.

Modulation of quinate: NAD(+) oxidoreductase activity through reversible phosphorylation in carrot cell suspensions.

Quinate: NAD+ oxidoreductase (EC 1.1.1.24) from carrot cells was deactivated by incubating partially purified extract with MgCl2 at 30°C. The deactivation process was prevented by adding fluoride, a phosphatase inhibitor. Once inactivated, the enzyme could recover its initial activity on incubation with ATP-Mg either in combination with or not in combination with an exogenous protein kinase. 32PO4 was incorporated into the purified enzyme when the cell cultures were supplemented with labeled phosphate in vivo. Moreover, 32P from [γ-32P]ATP was incorporated into the reductase when the enzyme was reactivated in the presence of protein kinase. From these results, it is concluded that the activation-inactivation process is due to phosphorylation-dephosphorylation of quinate:NAD+ oxidoreductase.

A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein

Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.

Recherches sur les enzymes catalysant la formation des acides phénoliques chez Quercus pedunculata (EHRH.) II. Localisation intracellulaire de la phenylalanine ammoniaque-lyase, de la cinnamate 4-hydroxylase, et de la “benzoate synthase”

Abstract This paper deals with some properties of enzymes involved in the synthesis of phenolic acids, with special reference to their intracellular location. The results obtained can be summarized as follows: 1. 1. Two phenylalanine ammonia-lyase isoenzymes coexist in Quercus roots as has been previously described for oak leaves. The cinnamate-sensitive isoenzyme is associated with the microsomal fraction whereas the benzoate-sensitive isoenzyme is located in the “F 10 000” fraction including mitochondria and microbodies. 2. 2. The cinnamate 4-hydroxylase, catalyzing p-coumarate formation, is tightly bound to microsomes and the “benzoate synthase”, an emzyme involved in the benzoic acid formation from t-cinnamate, is confined to the “F 10 000” fraction, presumably in microbodies. These data show that phenolic acids biosynthesis pathways are closely compartmented in organelles so that phenylpropanoid compounds are mainly formed in microsomes whereas C6−C1 acids are formed in microbodies. The regulation of these pathways seems to involve sophisticated mechanisms, associating isoenzymes with tight compartments.