Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

Grass species and soil type effects on microbial biomass and activity

We evaluated plant versus soil type controls on microbial biomass and activity by comparing microbial biomass C, soil respiration, denitrification potential, potential net N mineralization and nitrification in different soils supporting four grass species, and by growing a group of 10 different grass species on the same soil, in two experiments respectively. In the first experiment, none of the microbial variables showed significant variation with grass species while all variables showed significant variation with soil type, likely due to variation in soil texture. In the second experiment, there were few significant differences in microbial biomass C among the 10 grasses but there were significant relationships between variation in microbial biomass C and potential net N mineralization (negative), soil respiration (positive) and denitrification (positive). There was no relationship between microbial biomass C and either plant yield or plant N concentration. The results suggest that 1) soil type is a more important controller of microbial biomass and activity than grass species, 2) that different grass species can create significant, but small and infrequent, differences in microbial biomass and activity in soil, and 3) that plant-induced variation in microbial biomass and activity is caused by variation in labile C input to soil.     

Anemophilous plants select pollen from their own species from the air

In wind-tunnel experiments, Niklas (1985) has demonstrated the ability of anemophilous plants to select pollen from their own species from the airstream. However, there have been no field experiments to establish whether this operates in nature. We surveyed the pollents on the stigmas of four different, coextensive, dioecious anemophilous species surrounded by a Pinus radiata plantation. The alien pine pollen was overrepresented relative to background levels on only one of the four species. For all three indigenous species with both male and female plants in the area, the highest proportion — in all cases more than 40% — of the pollen found on their stigmas came from their own species. One indigenous species lacked male plants in the area; consequently, the results from this species are difficult to interpret. However, for all four species, there was at least 15% pine pollen, and some pollen of other indigenous species. These results suggest that there is some pollen selection, but that the mechanisms are may be not as effective as Niklas has suggested.     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

Factors affecting production of COS and CS2 in Leucaena and Mimosa species

Carbon disulfide (CS₂) and carbonyl sulfide (COS) are colorless, foul-smelling, volatile sulfur compounds with biocidal properties. Some plants produce CS₂ or COS or both. When used as an intercrop or forecrop, these plants may have agronomic potential in protecting other plants. Most of the factors which affect production of these plant-generated organic sulfides are unknown. We determined the effects of sulfate concentration, plant age, nitrogen fixation, drought stress, root injury (through cutting), and undisturbed growth on COS production in Leucaena retusa or Leucaena leucocephala and the effect of some of these factors on CS₂ production in Mimosa pudica. In addition, we determined if organic sulfides were produced in all Leucaena species. When L. retusa and M. pudica seedlings were grown in a plant nutrient medium with different sulfate concentrations (50 to 450 mg SL^-1), COS or CS₂ from crushed roots generally increased with increasing sulfate concentration. COS production was highest (74 ng mg^-1 dry root) for young L. retusa seedlings and declined to low amounts (<5 ng mg^-1 dry root) for older seedlings. Nitrogen fixation reduced the amounts of COS or CS₂ produced in L. leucocephala and M. pudica. Under conditions of undisturbed growth, root cutting, or drought stress, no COS production was detected in 4-to 8-weeks-old L. retusa plants. COS or CS₂ or both was obtained from crushed roots or shoots of all 13 known Leucaena species.     

The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms

The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.We are very grateful to Dr. Roger Ellis (SCRI, Dundee, Scotland) for the gift of barley seeds, and to Drs Roger Ellis, Alan Schulman and Cathie Martin for helpful advice and comments during the course of this work.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5-phosphosulfate sulfotransferase and -glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than -glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of³⁵S from [³⁵S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of³⁵S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.Abbreviations APSSTase adenosine 5-phosphosulfate sulfotransferase - EC -glutamylcysteine - GR glutathione reductase - OSH glutathione - NR nitrate reductase We thank M. Suter for preparing [³⁵S]adenosine 5-phosphosulfate, Dr. A. Fleming (both our Institute) for correcting the English and M. Soldati (Eschlikon, Switzerland) for his help with the plant material. This work was supported by COST 814 Crop development for the wet and cool regions of Europe.     

Resetting biological oscillators—A stochastic approach

We present a stochastic approach to phase-resetting of an ensemble of oscillators. In order to describe stimulation-induced dynamical phenomena we develop a stochastic model which consists of an ensemble of phase oscillators interacting via random forces. Every single oscillator is submitted to a phase stimulus. The ensemble's dynamics is determined by a Fokker-Planck equation. The stationary states are calculated explicitly, whereas the transients are analysed numerically. If the stimulus of a given (non-vanishing) intensity is administered at a critical initial cluster phase for a critical duration T _crit the ensemble's synchronized oscillation is annihilated. A transition from type 1 resetting to type 0 resetting occurs when the stimulation duration exceeds T _crit. Stimulation causes a shift of the mean frequency of every single oscillator. This frequency shift is explicitly calculated by deriving the mean first passage time. The model shows that there is a subcritical intensity which is connected with an enhanced vulnerability to stimulation. The desynchronized states, the so-called black holes, are typically associated with a double peak in the ensemble's phase distribution. This is important for analysing experimental data because simple peak-detection algorithms are not able to extract the underlying dynamics.Our results are discussed from the experimentator's point of view so that the insights derived from our model can improve data analysis and design of stimulation experiments.