A 75-kDa polypeptide, located primarily at the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888.

Calcium channel blockers of the phenylalkylamine family bind specifically to membranes and inhibit calcium uptake in carrot protoplast. LU 49888, an azido derivative of phenylalkylamine, behaves as its unmodified homolog in terms of affinity and specificity and therefore allows us to probe the receptor by photoaffinity labeling. Upon UV irradiation, a 75-kDa peptide was specifically labeled. Incubation of microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent, led to the solubilization of the LU 49888-binding protein. Electrophoretic analysis under denaturing conditions and gel filtration of the solubilized "receptor-ligand" complex show a 75-kDa peptide mainly located at the plasma membrane. Consequently the LU 49888-binding protein in plants differs significantly from its animal counterpart by its size and may be a primary target for external signal molecules.

Recent advances in the regulation of plant calcium channels: evidence for regulation by G-proteins, the cytoskeleton and second messengers.

Important aspects of the regulatory properties of plant calcium channels have been discovered during the past few years. These include the control of plasma membrane-bound channels by regulatory proteins and the characterization of a plethora of intracellular calcium release channels. Deciphering the mechanisms of regulation of different Ca2+ channels and the probable co-operation of their activities in response to various stimuli is leading to a better understanding of Ca2+-signalling processes in higher plants.

Perception of lipo-chitooligosaccharidic Nod factors in legumes.

Lipo-chitooligosaccharides produced by rhizobia are a class of signalling molecules that mediate recognition and nodule organogenesis in the legume-rhizobia symbiosis. Their synthesis is specified by the nodulation genes of rhizobia and hence they are commonly known as Nod factors. They are amphiphilic molecules and induce a variety of responses in the roots of the legume hosts. Studies using plant and rhizobial mutants and purified molecules suggest that Nod factors are recognized by more than one receptor. In this article, we review evidence about the affinity, specificity and location of these putative receptors and describe recent studies with regard to their identification.

Phenolic metabolism in petunia tissues. I. Characteristic responses of enzymes involved in different steps of polyphenol synthesis to different hormonal influences.

Pronounced changes in enzymatic patterns occur in petunia tissues when calluses are subcultured on media containing different growth substances. As judged by variations of enzymes related to primary metabolism (6-phosphogluconate and malate dehydrogenases) there are individual responses for each metabolic pathway. Concerning the enzymes of aromatic metabolism: (a) Phenylalanine ammonia-lyase, cinnamate and p-coumarate hydroxylases and the enzyme(s) activating phenylpropanoid units vary in the same manner. (b) Chalcone-flavanone isomerase, a key enzyme in the synthesis of flavonoids, and coniferyl alcohol dehydrogenase, which leads to the monomers of lignins, have, on the other hand, an independent behaviour. These responses show that the enzymes involved in the synthesis and activation of phenylpropanoid units seem to act coordinately in plants. Moreover, the data suggest that the common pathway leading to the activated cinnamic acids and the specific metabolic steps of lignin and flavonoid synthesis are regulated in a different way.

The nucleus together with the cytosol generates patterns of specific cellular calcium signatures in tobacco suspension culture cells.

Plant cell suspension cultures respond to osmotic changes by alterations in levels of free cellular calcium. Using the aequorin recombinant method, we have measured the spatial and temporal characteristics of calcium signatures in the nucleus and the cytosol of BY-2 tobacco suspension cells challenged with hypo- or hyper-osmotic shock. We show here that the nuclear compartment contributes together with the cytosol to produce calcium signal patterns that discriminate hypo- from hyper-osmotic treatments, i.e. turgor from tension. We also demonstrate that calcium responses in the nucleus and the cytosol are differentially modulated by the strength and the nature of hyper-osmotic treatments. We conclude that qualitative and quantitative changes in the parameters of an external stimulus such as osmotic changes are converted into calcium signatures, distinctive in their temporal and subcellular characteristics, involving both the nucleus and the cytosol. Our results illustrate the versatility of calcium signaling in plant cells. In addition to the physiological 'address' of the cell, the compartmentation of the calcium signal is probably an important parameter in encoding response specificity.

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.

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.

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.

Evidence for the involvement of activated oxygen in fungal degradation of lignocellulose.

Oxygen has been shown to be necessary as a cosubstrate for the fungal degradation of lignins. In this work, the active forms of oxygen were tentatively identified in three ways: --effect of chemically generated active radicals and molecular species on lignocellulosic complexes, --use of activated oxygen scavengers in culture media of ligninolytic fungi, --characterization of active forms of oxygen by specific reactions. The data obtained strongly suggest that two main oxygen species are involved, namely OH radical and singlet oxygen (1O2). Chemical or enzymic scavengers inhibit the degradation of lignocelluloses by Phanerochaete chrysosporium. The fungus has been demonstrated to synthesize OH.

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.

Recent advances in the study of nod factor perception and signal transduction.

Rhizobial lipochitooligosaccharidic Nod factors mediate the specific recognition between leguminous plants and their prokaryotic symbionts. This review summarizes recent findings on the way plants could perceive and transduce these bacterial signals. It starts by summarizing knowledge about Nod factor binding sites, before moving to the potential implications in Nod factor signal transduction of G proteins, root-hair plasma membrane depolarisation, cytoplasmic and extracellular alkalinisation and finally variations in cytoplasmic calcium concentration.

Recherches sur les enzymes catalysant la biosynthese des acides phénoliques chez Quercus pedunculata (EHRH.): I - Formation des premiers termes des series cinnamique et benzöique.

Occurence of PAL, cinnamate-hydroxylase and p-coumarate-hydroxylase, is found in cell-free extracts from Quercus pedunculata roots; moreover, an enzyme system which catalyzes benzoïc acid formation from cinnamic acid is caracterized for the first time. Role of these enzymes and their interactions within the same organ are discussed.

A calcium-binding protein is a regulatory subunit of quinate:NAD+ oxidoreductase from dark-grown carrot cells.

On transfer of carrot cell-suspensions from light to dark conditions, quinate:NAD+ oxidoreductase binds, post-translationally, an additional subunit. As a consequence, the oligomeric enzyme becomes activatable by Ca++ and behaves as a Ca++ binding protein. The additional subunit has been shown to be the Ca++ binding moiety of the molecule that protects the phosphorylated amino acids from dephosphorylation. It is suggested that the regulatory subunit may be a new class of calciprotein.

Plant graviperception and gravitropism: a newcomer's view.

Gravitropism is an adaptable mechanism corresponding to the directed growth by which plants orient in response to the gravity vector. The overall process is generally divided into three distinct stages: graviperception, gravitransduction, and asymmetric growth response. The phenomenology of these different steps has been described by using refined cell biology approaches combined with formal and molecular genetics. To date, it clearly appears that the cellular organization plays crucial roles in gravisensing and that gravitropism is genetically different between organs. Moreover, while interfering with other physical or chemical stimuli and sharing probably some common intermediary steps in the transduction pathway, gravity has its own perception and transduction systems. The intimate mechanisms involved in these processes have to be unveiled at the molecular level and their biological relevance addressed at the cellular and whole plant levels under normal and microgravitational conditions. gravitropism: a newcomer's view.

External electric fields stimulate the electrogenic calcium/sodium exchange in plant protoplasts.

External electric fields of low intensity stimulated calcium influx in protoplasts isolated from carrot cell suspension cultures in field intensity dependent and frequency-dependent ways. The field-induced calcium uptake involved a temperature-dependent system that was saturable by external calcium. The induction process appeared mainly cumulative as long as the morphology of the protoplasts did not change (up to 10 min). The stimulation elicited by the electric fields was effective even after switching the field off; the influx increased for 5 min and then slowed down to its initial value 15 min later. During electrostimulation, an additional amount of ATP was accumulated; on removal of the stimulatory field, the extra amount of ATP was consumed, whereas the plasma membrane was hyperpolarized and sodium ions were expelled from the protoplasts. Inhibition of either ATP accumulation or consumption results in the inhibition of both calcium influx and sodium efflux, demonstrating that these processes are coupled. From the data obtained in this work, it may be concluded that the electric field stimulates an ATP synthase like activity; the consumption of the ATP thus formed elicits an electric potential (probably due to the efflux of cations and more specifically sodium) that drives the influx of calcium.

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s).

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.

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. (32)PO4 was incorporated into the purified enzyme when the cell cultures were supplemented with labeled phosphate in vivo. Moreover, (32)P from [γ-(32)P]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.

Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin.

Quinate:NAD(+) 3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD(+) oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca(2+). Cyclic AMP at concentrations up to 5 muM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD(+) oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca(2+). However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD(+) oxidoreductase is activated by a Ca(2+) - and calmodulin-dependent plant protein kinase.