Changes in gravitational forces induce modifications of gene expression in A. thaliana seedlings.

By comparing the expression patterns of selected genes from Arabidopsis thaliana (L.) Heynh. grown either at 1 g or on a clinostat (horizontally or vertically inverted, 1 rpm), and either used directly or after hypergravity stimulation, we have shown that the pattern of expression did not proceed in a stereotypical manner. Rather, the selected genes fell into different classes. These classes include (i) those insensitive to the gravitational conditions, (ii) those that are regulated in an opposite manner by hypergravity and clinostat conditions, (iii) those that are desensitised to hypergravity by long-term culture on a clinostat, and (iv) those enhanced by such a treatment. Our data suggest that rapid reorientation of gene expression is likely to occur in response to changes in the gravitational conditions.

A 45-kDa protein kinase related to mitogen-activated protein kinase is activated in tobacco cells treated with a phorbol ester.

Phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinases in animals, elicits the transient activation of a 45-kDa protein kinase in tobacco cell-suspension cultures. The 45-kDa protein kinase preferentially phosphorylates myelin basic protein (MBP), a general substrate for MAPK. Studies using cycloheximide indicated that protein synthesis is not required for the activation of the kinase. Treatment of tobacco cell extracts containing the activated kinase with either serine/threonine-specific or tyrosine-specific protein phosphatase abolished the kinase activity, which consequently appears to be regulated by phosphorylation. By using an immune complex kinase assay with antibodies specific for stress-responsive MAPKs, we show that the PMA-activated kinase is immunologically related to the wound-induced protein kinase (WIPK), and not to the salicylic acid-induced protein kinase (SIPK), two representative members of the tobacco MAPK family, known to be activated by extracellular stimuli. Furthermore, the activated kinase was recognized by phospho-specific MAPK antibodies. Collectively, these results indicate that phorbol ester promotes the activation of a 45-kDa protein kinase related to WIPK in tobacco cells. Activation of WIPK in response to PMA is associated with protein phosphorylation but not with an increase in protein level.

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.

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.

[Basis for the calcium specificity in the plant response to extracellular stimulation]. / Les supports de la spécificité calcique dans la réponse des plantes aux stimuli extracellulaires.

The Ca2+ cation is fully recognized as an important intracellular second messenger coupling a wide range of extracellular stimuli to characteristic responses in plant cells. Such a pleiotropic effect raises questions regarding the mechanisms by which the signalling pathways, all of then involving an increase in intracellular calcium concentration, can be specific to a given stimulus. Here, we present recent results which shed light into different concepts which may explain the response specificity in signalling processes, such as "the cross-talk between signalling pathways", "the Ca2+ signatures" and "the compartmentation of Ca(2+)-signalling".

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.

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.

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.

Ligand specificity of a high-affinity binding site for lipo-chitooligosaccharidic Nod factors in Medicago cell suspension cultures.

Rhizobial lipo-chitooligosaccharides (LCOs) are signaling molecules involved in host-range recognition for the establishment of the symbiosis with leguminous plants. The major LCO of Rhizobium meliloti, the symbiont of Medicago plants contains four or five N-acetylglucosamines, O-acetylated and N-acylated with a C16:2 fatty acid on the terminal nonreducing sugar and O-sulfated on the reducing sugar. In this paper, the ligand specificity of a high-affinity binding site (Nod factor binding site 2 or NFBS2), enriched in a plasma membrane-enriched fraction of Medicago cell suspension cultures, is reported. By using chemically synthesized LCOs, the role of structural elements, important for symbiotic activities, as recognition motifs for NFBS2 was determined. The results show that the substitutions on the nonreducing sugar of the LCOs (the O-acetate group, the fatty acid, and the hydroxyl group on the C4 of the sugar) are determinants for high-affinity binding to NFBS2. In contrast, the sulfate group, which is necessary for all biological activities on Medicago, is not discriminated by NFBS2. However, the reducing sugar of the LCO seems to interact with NFBS2, because ligand binding is affected by the reduction of the free anomeric carbon and depends on the number of N-acetyl glucosamine residues. These results suggest that the recognition of the LCOs by NFBS2 is mediated by structural elements in both the lipid and oligosaccharidic moities, but not by the sulfate group.

Plasma membrane depolarization-activated calcium channels, stimulated by microtubule-depolymerizing drugs in wild-type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half-life in ton 2 mutant cells affected in the organization of cortical microtubules.

Depolarization-activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch-clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole-cell configuration, due most probably to protein-protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch-clamp studies showed that depolarization-activated calcium channel activities in ton 2 protoplasts were 10-fold higher and their relative half-life three-times longer than in wild-type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild-type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild-type protoplasts. It is suggested that the down-regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.

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.

Functional expression of a tobacco gene related to the serine hydrolase family -- esterase activity towards short-chain dinitrophenyl acylesters.

We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.

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.

Characterization of a binding site for chemically synthesized lipo-oligosaccharidic NodRm factors in particulate fractions prepared from roots.

This paper describes the characteristics of a binding site for the major, lipo-oligosaccharide Nod factor of Rhizobium meliloti in roots of the symbiotic host plant, Medicago truncatula. Chemically synthesized NodRm-IV(Ac, S, C16:2) was labelled by tritiation to a specific activity of 56 Ci mmol-1 and this ligand was shown to be biologically active in the root hair deformation assay at 10(-11) M. Binding of the ligand to a particulate fraction from roots of M. truncatula was found to be saturable and reversible with an affinity (Kd) of 86 nM and the binding characteristics were consistent with a single class of binding sites. Competition with modified Nod factors showed that the binding was independent of both the O-acetyl and the sulphyl group and did not depend on the unsaturation of the fatty acid. However, both moieties of the lipo-oligosaccharide are required for high-affinity binding since tetra-N-acetyl-chitotetraose and palmitate were found to be poor competitors of ligand binding. A binding site with analogous characteristics was also found in a similarly prepared particulate fraction of tomato roots. This binding site for Nod factors, termed NFBS1, which is present in both a leguminous and a non-leguminous plant, may have a more general role than symbiosis.

Enzymatic radiolabelling to a high specific activity of legume lipo-oligosaccharidic nodulation factors from Rhizobium meliloti.

In this paper we describe the two-step coupled 35S-radiolabelling of the lipo-oligosaccharidic nodulation (Nod) factors of the bacterium Rhizobium meliloti to a specific radioactivity of 800 Ci/mmol. These radiolabelled Nod factors bind to a particulate fraction from roots of the bacterium's symbiotic host, Medicago truncatula, with an equilibrium dissociation constant (KD) of 117 nM, similar to that observed with a synthetic tritiated ligand. The first step of the 35S-labelling involves the synthesis of 3'-phosphoadenosine 5'-phospho[35S]sulphate ([35S]PAPS) from ATP and [35S]sulphate using yeast enzymes. The second step exploits the sulphotransferase activity of the R. meliloti NodH protein, which has been expressed in Escherichia coli, to transfer the labelled sulphate group from PAPS to non-sulphated Nod factors. This enzyme was found to be active in E. coli cultured at 18 degrees C but not 37 degrees C. NodH could also transfer the sulphate group from PAPS to a model substrate, tetra-N-acetyl chitotetraose, with apparent Km values of 56 and 70 microM respectively, and exhibited an apparent Km value for non-sulphated Nod factors of 28 microM. Coupling the two steps of the radiolabelling resulted in an efficiency of 35S incorporation from inorganic sulphate to the Nod factors of approximately 10%. These labelled factors will be a valuable tool in the search for high-affinity receptors for the lipo-oligosaccharidic nodulation factors.

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.

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.

Molecular characterization of a 70 kDa heat-shock protein of bean mitochondria.

A bean cDNA clone that specifies a 70 kDa heat-shock protein (hsp70) has been isolated and sequenced. The nucleotide sequence analysis shows that the cDNA could encode a 72 kDa protein that is highly related to prokaryotic and mitochondrial members of the hsp70 family. The predicted protein was found to contain an amino-terminal extension typical of transit sequences. The in vitro transcription/translation product of the cDNA behaved as a 72 kDa polypeptide as predicted from the longest open reading frame. This polypeptide could be imported into isolated mitochondria and recovered as a 68 kDa product. The imported protein is identical in size to a mitochondrial protein that cross-reacts with hsp70-specific antibodies. The import data and Western blot analysis suggest that the cDNA clone encodes a mitochondrial member of the hsp70 family. Electrophoretic and immunoblot analysis reveal that the protein is loosely associated to the mitochondrial envelope and also exists as discrete soluble protein aggregates of about 270 and 420 kDa. Hsp70 of bean mitochondria can be in vitro phosphorylated on threonine residues in a calcium-dependent manner, and the modified protein was detected as an oligomer of about 160 kDa only. The data are discussed with respect to the chaperone function of hsp70 in mitochondria.