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PubMed2000

Purification and characterization of a membrane-associated 48-kilodalton phospholipase A(2) in leaves of broad bean.

Several lines of evidence indicate that phospholipase A(2) (PLA(2)) plays a crucial role in plant cellular responses through production of linolenic acid, the precursor of jasmonic acid, from membrane phospholipids. Here we report the purification and characterization of a 48-kD PLA(2) from the membrane fractions of leaves of broad bean (Vicia faba). The plant PLA(2) was purified to near homogeneity by sequential column chromatographies from the membrane extracts. The purified 48-kD protein migrated as a single band on a SDS-PAGE gel and its density correlated with the PLA(2) activity. It was further confirmed that this 48-kD protein is the PLA(2) enzyme based on immunoprecipitating the activity with a monoclonal antibody against it and purifying the enzyme to homogeneity with the antibody affinity column. The purified plant PLA(2) preferred 2-linolenoyl-sn-glycerol-3-phosphocholine (GPC) to 2-linoleoyl-GPC, 2-palmitoyl-GPC and 2-arachidonyl-GPC as substrates with a pH optimum at pH 7.0 to 8.0. The plant PLA(2) was activated by calmodulin and inhibited by pretreatment of 5,8,11, 14-eicosatetraynoic acid known as an inhibitor of mammalian PLA(2)s. The enzyme was characterized as a Ca(2+)-independent PLA(2) different from mammalian PLA(2)s. This membrane-associated and Ca(2+)-independent PLA(2) is suggested to play an important role in the release of linolenic acid, the precursor of jasmonic acid, through a signal transduction pathway.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed2000

Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes.

Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

Abscisic Acid↗
PubMed2001

Apparent absence of a redox requirement for blue light activation of pump current in broad bean guard cells.

In guard cells, membrane hyperpolarization in response to a blue light (BL) stimulus is achieved by the activation of a plasma membrane H(+)-ATPase. Using the patch clamp technique on broad bean (Vicia faba) guard cells we demonstrate that both steady-state- and BL-induced pump currents require ATP and are blocked by vanadate perfused into the guard cell during patch clamp recording. Background-pump current and BL-activated currents are voltage independent over a wide range of membrane potentials. During BL-activated responses significant hyperpolarization is achieved that is sufficient to promote K(+) uptake. BL activation of pump current becomes desensitized by three or four pulses of 30 s x 100 micromol m(-2) s(-1) BL. This desensitization is not a result of pump inhibition as maximal responses to fusicoccin are observed after full BL desensitization. BL treatments prior to whole cell recording show that BL desensitization is not due to washout of a secondary messenger by whole cell perfusion, but appears to be an important feature of the BL-stimulated pump response. We found no evidence for an electrogenic BL-stimulated redox chain in the plasma membrane of guard cells as no steady-state- or BL-activated currents are detected with NADH or NADPH added to the cytosol in the absence of ATP. Steady-state- nor BL-activated currents are affected by the inclusion along with ATP of 1 mM NADH in the pipette under saturating red light or by including NADPH in the pipette under darkness or saturating red light. These data suggest that reduced products of photosynthesis do not significantly modulate plasma membrane pump currents and are unlikely to be critical regulators in BL-stimulation of the plasma membrane H(+)-ATPase in guard cells.

Cell Membrane↗
PubMed2001

Specific binding of vf14-3-3a isoform to the plasma membrane H+-ATPase in response to blue light and fusicoccin in guard cells of broad bean.

The plasma membrane H(+)-ATPase is activated by blue light with concomitant binding of the 14-3-3 protein to the C terminus in guard cells. Because several isoforms of the 14-3-3 protein are expressed in plants, we determined which isoform(s) bound to the H(+)-ATPase in vivo. Four cDNA clones (vf14-3-3a, vf14-3-3b, vf14-3-3c, and vf14-3-3d) encoding 14-3-3 proteins were isolated from broad bean (Vicia faba) guard cells. Northern analysis revealed that mRNAs encoding vf14-3-3a and vf14-3-3b proteins were expressed predominantly in guard cells. The 14-3-3 protein that bound to the H(+)-ATPase in guard cells had the same molecular mass as the recombinant vf14-3-3a protein. The H(+)-ATPase immunoprecipitated from mesophyll cell protoplasts, which had been stimulated by fusicoccin, coprecipitated with the 32.5-kD 14-3-3 protein, although three 14-3-3 isoproteins were found in mesophyll cell protoplasts. Digestions of the bound 14-3-3 protein and recombinant vf14-3-3a with cyanogen bromide gave the identical migration profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but that of vf14-3-3b gave a different profile. Mass profiling of trypsin-digested 14-3-3 protein bound to the H(+)-ATPase gave the predicted peptide masses of vf14-3-3a. Far western analysis revealed that the H(+)-ATPase had a higher affinity for vf14-3-3a than for vf14-3-3b. These results suggest that the 14-3-3 protein that bound to the plasma membrane H(+)-ATPase in vivo is vf14-3-3a and that it may play a key role in the activation of H(+)-ATPase in guard cells.

14-3-3 Proteins↗
PubMed2001

Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe.

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.

Fabaceae↗
PubMed2001

Nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress.

Nitric oxide (NO) is a very active molecule involved in many and diverse biological pathways where it has proved to be protective against damages provoked by oxidative stress conditions. In this work, we studied the effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine SNP-treated on the response of wheat (Triticum aestivum) to water stress conditions. After 2 and 3 h of drought, detached wheat leaves pretreated with 150 microM SNP retained up to 15% more water than those pretreated with water or NO(2)(-)/NO(3)(-). The effect of SNP treatment on water retention was also found in wheat seedlings after 7 d of drought. These results were consistent with a 20% decrease in the transpiration rate of SNP-treated detached wheat leaves for the same analyzed time. In parallel experiments, NO was also able to induce a 35%, 30%, and 65% of stomatal closure in three different species, Tradescantia sp. (monocotyledonous) and two dicotyledonous, Salpichroa organifolia and fava bean (Vicia faba), respectively. In SNP-treated leaves of Tradescantia sp., the stomatal closure was correlated with a 10% increase on RWC. Ion leakage, a cell injury index, was 25% lower in SNP-treated wheat leaves compared with control ones after the recovery period. Carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), a specific NO scavenger, reverted SNP action by restoring the transpiration rate, stomatal aperture, and the ion leakage to the level found in untreated leaves. Northern-blot analysis showed that SNP-treated wheat leaves display a 2-fold accumulation of a group three late embryogenesis abundant transcript with respect to control leaves both after 2 and 4 h of drought periods. All together, these results suggest that the exogenous application of NO donors might confer an increased tolerance to severe drought stress conditions in plants.

Adaptation, Physiological↗
PubMed2001

Transpiration rate. An important factor controlling the sucrose content of the guard cell apoplast of broad bean.

Evaporation of water from the guard cell wall concentrates apoplastic solutes. We hypothesize that this phenomenon provides two mechanisms for responding to high transpiration rates. First, apoplastic abscisic acid is concentrated in the guard cell wall. Second, by accumulating in the guard cell wall, apoplastic sucrose (Suc) provides a direct osmotic feedback to guard cells. As a means of testing this second hypothesized mechanism, the guard cell Suc contents at a higher transpiration rate (60% relative humidity [RH]) were compared with those at a lower transpiration rate (90% RH) in broad bean (Vicia faba), an apoplastic phloem loader. In control plants (constant 60% RH), the guard cell apoplast Suc content increased from 97 +/- 81 femtomol (fmol) guard cell pair(-1) to 701 +/- 142 fmol guard cell pair(-1) between daybreak and midday. This increase is equivalent to approximately 150 mM external, which is sufficient to decrease stomatal aperture size. In plants that were shifted to 90% RH before daybreak, the guard cell apoplast Suc content did not increase during the day. In accordance, in plants that were shifted to 90% RH at midday, the guard cell apoplast Suc content declined to the daybreak value. Under all conditions, the guard cell symplast Suc content increased during the photoperiod, but the guard cell symplast Suc content was higher (836 +/- 33 fmol guard cell pair(-1)) in plants that were shifted to 90% RH. These results indicate that a high transpiration rate may result in a high guard cell apoplast Suc concentration, which diminishes stomatal aperture size.

Adaptation, Physiological↗
PubMed1981

A model for predicting ionic equilibrium concentrations in cell walls.

Purified cell walls were prepared from roots of Horse bean (Vicia faba L., var. minor) and Yellow Lupine (Lupinus luteus L.). Two methods were used: (a) grinding and (b) lysis of the endocellular contents by Triton X-100. The Ca(2+), Mg(2+), and K(+) contents were determined after incubation in various solutions in such a manner that the measurements gave access to the undisturbed equilibrium contents. The results were used to test a model which describes the ionic atmosphere in the cell walls as a result of simultaneous electrostatic interactions between free ions (Donnan effect) and specific association equilibria, including acido-basic ones. This model correctly accounted for the whole set of experimental results and predicted the values of the unmeasurable local concentrations and pH.

Journal Article↗
PubMed1984

Phloem Unloading of Amino Acids at the Site of Attachment of Cuscuta europaea.

By washing out (14)C-solutes or (3)H-solutes in 0.5 mm CaSO(4) during a period of 5 to 6 hours, the release of amino acids by excised stem segments of broad bean (Vicia faba L. cv Witkiem) was studied. Three hours after pulse labeling with l-valine, l-asparagine, or alpha-aminoisobutyric acid (AIB), hollow stem segments were excised from the plant and incubated in a washout solution.In experiments with valine and asparagine, stem segments parasitized by Cuscuta europaea released a higher percentage of labeled solutes into the bathing medium than control segments. This can be ascribed to enhanced phloem unloading at the site of attachment of Cuscuta. At low temperature (0 degrees C) and after addition of p-chloromercuribenzene sulfonate to the bathing medium, parasitized segments did not release an enhanced percentage of labeled solutes, in comparison with control segments. These data suggest a metabolic control of the phenomenon of enhanced phloem unloading of amino acids. In experiments with AIB, an enhanced release of labeled solutes could not clearly be observed, but at the site of attachment of Cuscuta an accumulation of labeled solutes was measured. Accumulation of AIB in parenchyma cells, before the start of a washout experiment, will tend to obscure the phenomenon of enhanced phloem unloading.

Journal Article↗
PubMed1985

Identification of 3-(O-beta-Glucosyl)-2-Indolone-3-Acetylaspartic Acid as a New Indole-3-Acetic Acid Metabolite in Vicia Seedlings.

A new indole-3-acetic acid metabolite was isolated from broad bean (Vicia faba L. cv Chukyo) seedlings. It was a conjugate of dioxindole-3-acetic acid, aspartic acid, and glucose and was identified as 3-(O-beta-glucosyl)-2-indolone-3-acetylaspartic acid (molecular weight 484) from ultraviolet, infrared, nuclear magnetic resonance, and mass spectra. Its natural content in 4-day-old Vicia seedlings was estimated to be 8.6 nanomoles per gram fresh weight. It was suggested that oxidation of indole-3-acetic acid not accompanied by decarboxylation might regulate endogenous level of the hormone.

Journal Article↗
PubMed1989

Preparation and Properties of Antibodies against Indoleacetic Acid (IAA)-C5-BSA, a Novel Ring-Coupled IAA Antigen, as Compared to Two Other Types of IAA-Specific Antibodies.

Two types of indoleacetic acid (IAA) antigens have been described in the literature which show marked differences with respect to antibody specificity. In this communication an alternative antigen design is described. In the so-called IAA-C5-BSA conjugate, both the acetic acid group and the pyrrole moiety are presented free, as the covalent linkage between IAA and the carrier molecule is introduced in the benzene moiety. Antibodies were elicited in rabbits against this novel antigen. Using cross-reactivity data and the strategy of successive approximation for the measurements of auxin levels in the internodes of light-grown broad bean (Vicia faba L. cv Superfine), the three types of antibodies are compared.

Journal Article↗
PubMed1989

Polypeptide composition and amino-terminal sequence of broad bean polyphenoloxidase.

Polyphenoloxidase was purified from broad bean (Vicia faba) leaves. The purified enzyme contained two immunocross-reactive proteins of approximately 60 to 65 and 43 to 45 kilodaltons. Further electrophoretic separation resolved these proteins into doublets with molecular mass of 61.5, 60, 44.5, and 43 kilodaltons, respectively. Each of the four polypeptides was transferred to Immobilon and subjected to microprotein sequencing. All the polypeptides showed the same amino acid sequence up to residue 9 but some variations occurred thereafter. The amino-terminal sequence contained a large number of proline and serine residues. These results suggest that the four polypeptides were derived from a common parent form and that a posttranslational modification(s) must have occurred to account for the difference in their apparent size.

Journal Article↗
PubMed1991

Rapid adjustment of guard-cell abscisic Acid levels to current leaf-water status.

Detached broad bean (Vicia faba L.) leaflets were water stressed; within 15 minutes, guard-cell abscisic acid (ABA) concentration increased ninefold. This result eliminates the apparent discrepancy raised by reports of no correlation between initial water-stress effects on stomata and leaf ABA concentration. Six hours after stress relief, guard-cell ABA concentration was near the prestress value, which would seem to implicate other factors in stress aftereffects on stomata.

Journal Article↗
PubMed1991

Colocalization of Polyphenol Oxidase and Photosystem II Proteins.

Polyphenol oxidase (PPO) appears to be ubiquitous in higher plants but, as yet, no function has been ascribed to it. Herein, we report on the localization of PPO based upon biochemical fractionation of chloroplast membranes in Vicia faba (broad bean) into various complexes and immunocytochemical electron microscopic investigations. Sucrose density gradient fractionations of thylakoid membranes after detergent solubilization reveals that PPO protein (by reactivity with anti-PPO antibody) and activity (based upon ability to oxidize di-dihydroxyphenylalanine) are found only in fractions enriched in photosystem II (PSII). Furthermore, of the PSII particles isolated using three different protocols utilizing several plant species, all had PPO. Immunogold localization of PPO on thin sections reveals exclusive thylakoid labeling with a distribution pattern consistent with other PSII proteins (80% grana, 20% stroma). These data strongly indicate that PPO is at least peripherally associated with the PSII complex.

Journal Article↗
PubMed2006

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain.

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.

Amino Acid Sequence↗
PubMed1995

Seed coat-associated invertases of fava bean control both unloading and storage functions: cloning of cDNAs and cell type-specific expression.

We have studied the molecular physiology of photosynthate unloading and partitioning during seed development of fava bean (Vicia faba). During the prestorage phase, high levels of hexoses in the cotyledons and the apoplastic endospermal space are correlated with activity of cell wall-bound invertase in the seed coat. Three cDNAs were cloned. Sequence comparison revealed genes putatively encoding one soluble and two cell wall-bound isoforms of invertase. Expression was studied in different organs and tissues of developing seeds by RNA gel analysis, in situ hybridization, enzyme assay, and enzyme activity staining. One extracellular invertase gene is expressed during the prestorage phase in the thin-walled parenchyma of the seed coat, a region known to be the site of photoassimilate unloading. We propose a model for an invertase-mediated unloading process during early seed development and the regulation of cotyledonary sucrose metabolism. After unloading from the seed coat, sucrose is hydrolyzed by cell wall-bound invertases. Thus, invertase contributes to establish sink strength in young seeds. The resultant hexoses are loaded into the cotyledons and control carbohydrate partitioning via an influence on the sucrose synthase/sucrose-phosphate synthase pathway. The developmentally regulated degradation of the thin-walled parenchyma expressing the invertase apparently initiates the storage phase. This is characterized by a switch to a low sucrose/hexoses ratio. Feeding hexoses to storage-phase cotyledons in vitro increases the sucrose-phosphate synthase/sucrose synthase ratio and changes carbohydrate partitioning in favor of sucrose. Concomitantly, the transcript level of the major storage product legumin B is downregulated.

Base Sequence↗
PubMed1993

Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes.

A new technique is reported for the physical mapping of low copy DNA sequences on plant chromosomes. Individual chromosomes were microisolated and their DNA used as the target for the polymerase chain reaction in order to identify the chromosome carrying a specific gene sequence. The use of defined translocation chromosomes further refined the resolution of the method to a subchromosomal level. To demonstrate the applicability of the procedure genes have been localized coding for vicilin seed storage proteins on the field bean Vicia faba L. in a region which includes the centromere and the proximal parts of the short and the long arms of chromosome II.

Base Sequence↗
PubMed1997

Fabatins: new antimicrobial plant peptides.

Two new antimicrobial peptides related to the gamma-thionine family have been isolated by acid extraction from the broad bean Vicia faba. The extract was separated by ion exchange chromatography, and a fraction showing antibacterial activity was further purified by reverse-phase HPLC. Material from a single HPLC peak was sequenced and revealed the presence of two peptides differing by one amino acid. The peptides were named fabatins. They are 47 amino acids long, have an overall positive charge and contain 8 cysteines that probably form 4 disulfide bridges characteristic of the gamma-thionins. Fabatins were active against both Gram-negative and Gram-positive bacteria, but were inactive against the yeasts Saccharomyces cerevisiae and Candida albicans.

Anti-Bacterial Agents↗