Novel inflorescence architecture in gamma radiation-induced faba bean mutant populations.

Purpose: Inflorescence architecture is an important trait in the seed production of grain legumes. As several genes are responsible for this trait, any mutation, on these genes, may cause change in the inflorescence architecture. This study was conducted to evaluate inflorescence architecture in faba bean exposed to gamma radiation and to characterize the inflorescence architecture mutants phenotypically.Materials and methods: Faba bean M2 seeds (4898) generated from M1 generation of cultivars Hassawi 2 and ILB4347 were used in this study. M1 seeds were produced by irradiation treatments at two doses of gamma radiations (25 and 50 Gy). Faba bean M2 seeds were planted under field conditions. A total of 4032 mutant plants out of 4898 M2 seeds were evaluated for their inflorescence architecture.Results: A total of 20 determinate mutants were found and classified into four different types. Determinate type 1 was characterized by the formation of single terminal inflorescence on shoot apical meristem (SAM), type 2 by the formation of multiple inflorescences on SEM and generated upper branches that act as indeterminate type. Type 3 was characterized by the formation of a panicle-like inflorescence. While type 4 was characterized by the formation of primary and secondary panicle-like inflorescence. All of the determinate mutant types had shorter plant height and earlier maturity than control indeterminate type but had lower biological yield and seed yield. Among the determinate mutant types, determinate type 1 was only mutant that had a higher harvest index than the control indeterminate type. This promising mutant can be used to further breeding program to increase biological yield and seed yield.Conclusions: This study indicated potential of gamma radiation in inducing novel inflorescence architecture in faba bean. The mutants developed are valuable resources to study genes related to inflorescence architecture through forward genetics approach.

Responses of flavonoid profile and associated gene expression to solar blue and UV radiation in two accessions of Vicia faba L. from contrasting UV environments.

Blue light and UV radiation shape a plant's morphology and development, but accession-dependent responses under natural conditions are unclear. Here we tested the hypothesis that two faba bean (Vicia faba L.) accessions adapted to different latitudes and altitudes vary in their responses to solar blue and UV light. We measured growth, physiological traits, phenolic profiles and expression of associated genes in a factorial experiment combining two accessions (Aurora, a Swedish cultivar adapted to high latitude and low altitude; ILB938, from the Andean region of Colombia and Ecuador, adapted to low latitude and high altitude) and four filter treatments created with plastic sheets: 1. transparent as control; 2. attenuated short UV (290-350 nm); 3. attenuated UV (290-400 nm); 4. attenuated blue and UV light. In both accessions, the exclusion of blue and UV light increased plant height and leaf area, and decreased transcript abundance of ELONGATED HYPOCOTYL 5 (HY5) and TYROSINE AMINOTRANSFERASE 3 (TAT3). Blue light and short UV induced the accumulation of epidermal and whole-leaf flavonoids, mainly quercetins, and the responses in the two accessions were through different glycosides. Filter treatments did not affect kaempferol concentration, but there were more tri-glycosides in Aurora and di-glycosides in ILB938. Furthermore, fewer quercetin glycosides were identified in ILB938. The transcript abundance was consistently higher in Aurora than in ILB938 for all seven investigated genes: HY5, TAT3, CHALCONE SYNTHASE (CHS), CHALCONE ISOMERASE (CHI), DON-GLUCOSYLTRANSFERASE 1 (DOGT1), ABA INSENSITIVE 2 (ABI2), AUXIN-INDUCIBLE 2-27 (IAA5). The two largest differences in transcript abundance between the two accessions across treatments were 132-fold in CHS and 30-fold in DOGT1 which may explain the accession-dependent glycosylation patterns. Our findings suggest that agronomic selection for adaptation to high altitude may favour phenotypes with particular adaptations to the light environment, including solar UV and blue light.

Orobanche foetida resistance in two new faba bean genotypes produced by radiation mutagenesis.

PURPOSE: Broomrape produces serious damage to many legume crops and, particularly, becomes a limiting factor for faba bean (Vicia faba L.) production in the Mediterranean basin. Currently, several traditional methods of control have been developed, but none has proved to be effective for this parasite. However, breeding for resistance to this pest remains as one of the most feasible and environment-friendly methods for managing broomrape, but the mechanisms governing the interaction between the parasite and the host are not yet well understood. Therefore, we studied the behavior and molecular and enzymatic changes associated with the resistance to Orobanche foetida in faba bean mutants, which were obtained through radiation mutagenesis. MATERIALS AND METHODS: Three faba bean genotypes were used in this study, the variety 'Badï', characterized by high productivity in Orobanche-free soils and susceptibility to O. foetida, and two mutant lines P2M3 and P7M3 (derived from radio mutagenesis program), selected for their higher resistance to O. foetida in a field evaluation. The infection progress and the relative changes in the co-culture response, the enzymatic activities changes and the efficiency of the root extract stimulants from the host plant were followed and evaluated in all the genotypes. RESULTS: Experiments showed that low induction of seed germination is a major component of resistance in these lines against O. foetida. This is confirmed by the in vitro experiments with root exudates. The parallel reduction in infection was accompanied by the continuous enhancement of the peroxidase activity, the polyphenol oxidase activity and the phenylalanine ammonia lyase activity in faba bean roots. CONCLUSION: These data suggest the contribution of these enzymes in faba bean resistance to O. foetida broomrape induced by the use of gamma rays. Management of Orobanche by way of crop selection, based on these enzyme systems is a possible option.

Exposure to 915 MHz radiation induces micronuclei in Vicia faba root tips.

The increasing use of mobile phones and wireless networks raised a great debate about the real carcinogenic potential of radiofrequency-electromagnetic field (RF-EMF) exposure associated with these devices. Conflicting results are reported by the great majority of in vivo and in vitro studies on the capability of RF-EMF exposure to induce DNA damage and mutations in mammalian systems. Aimed at understanding whether less ambiguous responses to RF-EMF exposure might be evidenced in plant systems with respect to mammalian ones, in the present work the mutagenic effect of RF-EMF has been studied through the micronucleus (MN) test in secondary roots of Vicia faba seedlings exposed to mobile phone transmission in controlled conditions, inside a transverse electro magnetic (TEM) cell. Exposure of roots was carried out for 72h using a continuous wave (CW) of 915 MHz radiation at three values of equivalent plane wave power densities (23, 35 and 46W/m(2)). The specific absorption rate (SAR) was measured with a calorimetric method and the corresponding values were found to fall in the range of 0.4-1.5W/kg. Results of three independent experiments show the induction of a significant increase of MN frequency after exposure, ranging from a 2.3-fold increase above the sham value, at the lowest SAR level, up to a 7-fold increase at the highest SAR. These findings are in agreement with the limited number of data on cytogenetic effects detected in other plant systems exposed to mobile phone RF-EMF frequencies and clearly show the capability of radiofrequency exposure to induce DNA damage in this eukaryotic cell system.

Rapid modulation of ultraviolet shielding in plants is influenced by solar ultraviolet radiation and linked to alterations in flavonoids.

The accumulation of ultraviolet (UV)-absorbing compounds (flavonoids and related phenylpropanoids) and the resultant decrease in epidermal UV transmittance (TUV ) are primary protective mechanisms employed by plants against potentially damaging solar UV radiation and are critical components of the overall acclimation response of plants to changing solar UV environments. Whether plants can adjust this UV sunscreen protection in response to rapid changes in UV, as occurs on a diurnal basis, is largely unexplored. Here, we use a combination of approaches to demonstrate that plants can modulate their UV-screening properties within minutes to hours, and these changes are driven, in part, by UV radiation. For the cultivated species Abelmoschus esculentus, large (30-50%) and reversible changes in TUV occurred on a diurnal basis, and these adjustments were associated with changes in the concentrations of whole-leaf UV-absorbing compounds and several quercetin glycosides. Similar results were found for two other species (Vicia faba and Solanum lycopersicum), but no such changes were detected in Zea mays. These findings reveal a much more dynamic UV-protection mechanism than previously recognized, raise important questions concerning the costs and benefits of UV-protection strategies in plants and have practical implications for employing UV to enhance crop vigor and quality in controlled environments.

Oxidative damage and mutagenic potency of fast neutron and UV-B radiation in pollen mother cells and seed yield of Vicia faba L.

In recent years, there has been a great deal of attention toward free radicals, reactive oxygen species (ROS) generated by exposure of crop plant cells to physical radiations. Henceforth, the current study was planned to compare oxidative stress and mutagenic potential of different irradiation doses of fast neutron (FN) and UV-B on meiotic-pollen mother cells (PMCs), pollen grains (PGs) and seeds yielded from irradiated faba beans seedlings. On the cytogenetic level, each irradiation type had special interference with DNA of PMC and exhibited wide range of mutagenic action on the frequency and type of chromosomal anomalies, fertility of PGs and seed yield productivity based on the irradiation exposure dose and radiation sensitivity of faba bean plants compared with un-irradiated ones. On the molecular level, SDS-PAGE and RPAD-PCR analyses of seeds yielded from irradiated seedlings exhibited distinctive polymorphisms based on size, intensity, appearance, and disappearance of polypeptides bands compared with un-irradiated ones. The total values of protein and DNA polymorphisms reached 88% and 90.80% respectively. The neutron fluency (2.3 × 10(6) n/cm(2)) and UV-B dose for 1 hr were recorded as bio-positive effects. The present study proved that genetic variations revealed by cytogenetic test could be supported by gene expression (alterations in RAPD and protein profiles).

[Genetic effects of root extracts of Glycyrrhiza glabra L. on different test-systems].

The antimutagenic and geroprotective activities of root extracts of Glycyrrhiza glabra have been demonstrated both on plant test systems--Allium fistulosum L., Allium cepa L., Vicia faba L. and on animals--Vistar rats. The possibilities of the mobilization of Glycyrrhiza glabra root extracts as antimutagenic agents are discussed.

Ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent hydrogen peroxide synthesis in Vicia faba L.

Ultraviolet B (UV-B) radiation is an important environmental signal for plant growth and development, but its signal transduction mechanism is unclear. UV-B is known to induce stomatal closure via hydrogen peroxide (H(2)O(2)), and to affect ethylene biosynthesis. As ethylene is also known to induce stomatal closure via H(2)O(2) generation, the possibility of UV-B-induced stomatal closure via ethylene-mediated H(2)O(2) generation was investigated in Vicia faba by epidermal strip bioassay, laser-scanning confocal microscopy, and assays of ethylene production. It was found that H(2)O(2) production in guard cells and subsequent stomatal closure induced by UV-B radiation were inhibited by interfering with ethylene biosynthesis as well as ethylene signalling, suggesting that ethylene is epistatic to UV-B radiation in stomatal movement. Ethylene production preceded H(2)O(2) production upon UV-B radiation, while exogenous ethylene induced H(2)O(2) production in guard cells and subsequent stomatal closure, further supporting the conclusion. Inhibitors for peroxidase but not for NADPH oxidase abolished H(2)O(2) production upon UV-B radiation in guard cells, suggesting that peroxidase is the source of UV-B-induced H(2)O(2) production. Taken together, our results strongly support the idea that ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent H(2)O(2) generation.

An enhancing effect of visible light and UV radiation on phenolic compounds and various antioxidants in broad bean seedlings.

Exposure of dark- or ambient visible light-grown broad bean seedlings to low (LL) and high (HL) visible light intensities, UV-A or UV-C, either alone or in combination, induced significant increases in total phenolic compounds as well as in anthocyanins content, throughout the germination period, as compared with the respective levels in control seedlings. In general, as compared with control levels, exposure of both dark- or light-grown broad bean seedlings to LL, HL, UV-A or UV-C, induced significant increases in the contents of non-enzymatic antioxidants (total ascorbate; ASA-DASA and total glutathione; GSSG-GSH) and enzymatic antioxidant activities (superoxide dismutase; SOD, catalase; CAT, ascorbate peroxidase; APO and glutathione reductase; GR). The obtained results are discussed in relation to induced mechanisms of protection and repair from the inevitable exposure to damaging visible light and UV-radiation.

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts.

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.

Gas exchange and photosynthetic water use efficiency in response to light, CO2 concentration and temperature in Vicia faba.

Light and temperature-response curves and their resulting coefficients, obtained within ecophysiological characterization of gas exchanges at the leaf level, may represent useful criteria for breeding and cultivar selection and required tools for simulation models aimed at the prediction of potential plant behaviour in response to environmental conditions. Leaf-scale gas exchanges, by means of an IRGA open-flow system, were measured in response to light intensity (8 levels from 0 up to 2000 micromol m(-2) s(-1)), CO(2) concentrations (ambient-350 micromol mol(-1) and short-term enriched-700 micromol mol(-1)) and air temperature (from 7 up to 35 degrees C) on three Vicia faba L. genotypes, each representing one of the three cultivated groups: major, equina and minor. The net assimilation rate response to light intensity was well described by an exponential rise to max function. The short-term CO(2) enrichment markedly increased the values of light response curve parameters such as maximum photosynthetic rate (+80%), light saturation point (+40%) and quantum yield (+30%), while less homogenous behaviour was reported for dark respiration and light compensation point. For each light intensity level, the major and minor genotypes studied showed assimilation rates at least a 30% higher than equina. The positive effects of short-term CO(2) enrichment on photosynthetic water use efficiency (WUE) indicate a relevant advantage in doubling CO(2) concentration. In the major and minor genotypes studied, similar assimilation rates, but different WUE were observed. The optimum leaf temperature for assimilation process, calculated through a polynomial function, was 26-27 degrees C and no relevant limitations were observed in the range between 21 and 32 degrees C. Analysis at the single leaf level provided both rapid information on the variations in gas exchange in response to environmental factors and selection criteria for the screening of genotypes.

Guard cells in albino leaf patches do not respond to photosynthetically active radiation, but are sensitive to blue light, CO2 and abscisic acid.

Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 microM ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response.

Protein phosphatase 1 positively regulates stomatal opening in response to blue light in Vicia faba.

Phototropins, plant blue light receptors, mediate stomatal opening through the activation of the plasma membrane H(+)-ATPase by unknown mechanisms. Here we report that type 1 protein phosphatase (PP1) positively regulates the blue light signaling between phototropins and the H(+)-ATPase in guard cells of Vicia faba. We cloned the four catalytic subunits of PP1 (PP1c) from guard cells and determined the expression of the isoforms in various tissues. Transformation of Vicia guard cells with PP1c isoforms that had lost enzymatic activity by one amino acid mutation, or with human inhibitor-2, a specific inhibitor protein of PP1c, suppressed blue light-induced stomatal opening. Addition of fusicoccin, an activator of the plasma membrane H(+)-ATPase, to these transformed guard cells induced normal stomatal opening, suggesting that the transformations did not affect the basic mechanisms for stomatal opening. Tautomycin, an inhibitor of PP1, inhibited blue light-induced H(+) pumping, phosphorylation of the plasma membrane H(+)-ATPase in guard cell protoplasts, and stomatal opening. However, tautomycin did not inhibit the blue light-dependent phosphorylation of phototropins. We conclude that PP1 functions downstream of phototropins and upstream of the H(+)-ATPase in the blue light signaling pathway of guard cells.

[Antimutagenic activity of plant extracts from Armoracia rusticana, Ficus carica and Zea mays and peroxidase in eukaryotic cells]. / Antimutagennaia aktivnost' rastitel'nikh ékstraktov iz Armoracia rusticana, Ficus carica, Zea mays i peroksidazy v kletkakh éukariot.

Antimutagene activity and high efficiency of antimutagene action of plant extracts from horseradish roots (Armoracia rusticana), fig brunches (Ficus carica) and mays seedlings (Zea mays) and their ability to decrease the frequency of spontaneous and induced by gamma-rays chromosome aberrations in meristematic cells of Vicia faba and marrow cells of mice have been shown. Comparative assessment of genoprotective properties of peroxidase and the studied extracts has revealed higher efficiency of antimutagene action of peroxidase.

Blue-light- and phosphorylation-dependent binding of a 14-3-3 protein to phototropins in stomatal guard cells of broad bean.

Phototropins are blue-light (BL) receptor serine (Ser)/threonine kinases, and contain two light, oxygen, and voltage (LOV) domains, and are members of the PAS domain superfamily. They mediate phototropism, chloroplast movement, leaf expansion, and stomatal opening of higher plants in response to BL. In stomatal guard cells, genetic analysis has revealed that phototropins mediate activation of the plasma membrane H+-ATPase by phosphorylation and drive stomatal opening. However, biochemical evidence for the involvement of phototropins in the BL response of stomata is lacking. Using guard cell protoplasts, we showed that broad bean (Vicia faba) phototropins (Vfphots) were phosphorylated by BL, and that this phosphorylation of Vfphots reached to the maximum level earlier than that of the H+-ATPase. Phosphorylation of both Vfphots and H+-ATPase showed similar sensitivity to BL and were similarly suppressed by protein kinase and flavoprotein inhibitors. We found that a 14-3-3 protein was bound to Vfphots upon phosphorylation, and this binding occurred earlier than the H+-ATPase phosphorylation. Vfphots (Vfphot1a and Vfphot1b) were expressed in Escherichia coli, and phosphorylation sites were determined to be Ser-358 for Vfphot1a and Ser-344 for Vfphot1b, which are localized between LOV1 and LOV2. We conclude that Vfphots act as BL receptors in guard cells and that phosphorylation of a Ser residue between LOV1 and LOV2 and subsequent 14-3-3 protein binding are likely to be key steps of BL response in stomata. The binding of a 14-3-3 protein to Vfphot was found in etiolated seedlings and leaves in response to BL, suggesting that this event was common to phototropin-mediated responses.

Energy status and its control on embryogenesis of legumes. Embryo photosynthesis contributes to oxygen supply and is coupled to biosynthetic fluxes.

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O(2) levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O(2) concentration (1.1 micro M). Embryo respiration was more strongly inhibited by low O(2) during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O(2) limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O(2) and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic O(2) production elevated the internal level up to approximately 50% of atmospheric O(2) concentration (135 micro M). Upon light conditions, embryos partitioned approximately 3-fold more [(14)C]sucrose into starch. The light-dependent increase of starch synthesis was developmentally regulated. However, steady-state levels of nucleotides, free amino acids, sugars, and glycolytic intermediates did not change upon light or dark conditions. Maturing embryos responded to low O(2) supply by adjusting metabolic fluxes rather than the steady-state levels of metabolites. We conclude that embryogenic photosynthesis increases biosynthetic fluxes probably by providing O(2) and energy that is readily used for biosynthesis and respiration.

Parallel recordings of photosynthetic electron transport and K+-channel activity in single guard cells.

Stomata open in response to red and blue light. Red light-induced stomatal movement is mediated by guard cell chloroplasts and related to K+-uptake into these motor cells. We have combined a new type of microchlorophyll fluorometer with the patch-clamp technique for parallel studies of the photosynthetic electron transport and activity of plasma membrane K+ channels in single guard cell protoplast. In the whole-cell configuration and presence of ATP in the patch-pipette, the activity of the K+-uptake channels remained constant throughout the course of an experiment (up to 30 min) while photosynthetic activity declined to about 50%. In the absence of ATP inward K+ currents declined in a time-dependent manner. Under these ATP-free conditions, photosynthetic electron transport was completely blocked within 8 min. ADP together with orthophosphate was able to prevent inhibition of photosynthetic electron transport and run-down of K+-channel activity. The results demonstrate that the combination of these two techniques is suited to directly study cytosolic factors as common regulators of photosynthesis and plasma membrane transport within a single-cell.

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.

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.

Peculiarities of biological action of hadrons of space radiation.

Biological investigations in space enable one to make a significant contribution on high-energy hadrons to biological effects under the influence of factors of space flights. Physical and molecular principles of the action of high-energy hadrons are analysed. Genetic and somatic hadron effects produced by the secondary radiation from 70 GeV protons have been studied experimentally. The high biological effectiveness of hadrons, great variability in biological effects, and specifically of their action, are associated with strong interactions of high-energy hadrons. These are the probability of nuclear interaction with any atom nucleus, generation of a great number of secondary particles (among them, probably, highly effective multicharged and heavy nuclei, antiprotons, pi(-)-mesons), and the spatial distribution of secondary particles as a narrow cone with extremely high density of particles in its first part. The secondary radiation generated by high- and superhigh-energy hadrons upon their interaction with the spaceship is likely to be the greatest hazard of radiation to the crew during space flights.