Mechanics of root growth

Summary A model is developed for the rate of elongation of a root tip in terms of the balance of pressures acting on the root. Differentials of this equation give expressions for the changes in root elongation rate with respect to soil water potential and soil mechanical resistance. The model predicts that root cells osmoregulate against both water stress and soil mechanical resistance with predicts that root cells osmoregulate against both water stress and soil mechanical resistance with similar efficiencies which are less than 100%. Analysis of published data leads to the conclusion that root tips of pea osmoregulate with 70% efficiency. A working equation is developed for the elongation rate of roots in conditions of combined water stress and mechanical resistance.     

Influence of various soil factors on the effects of ferulic acid on leaf expansion of cucumber seedlings

Summary Cucumber seedlings were grown in a Portsmouth soil-sand system to study how varying soil clay and organic matter content might modify cucumber seedling response to ferulic acid, a reported allelopathic agent. Leaf area expansion of cucumber seedlings, soil respiration, and soil solution concentrations of ferulic acid were monitored. Leaf area, mean absolute rates of leaf expansion, and shoot dry weight of cucumber seedlings were significantly reduced by ferulic acid concentrations ranging from 10 to 70 μg/g dry soil. Ferulic acid was applied every other day, since it rapidly disappeared from soil solution as a result of retention by soil particles, utilization by microbes and/or uptake by roots. The amount of ferulic acid retained (i.e., adsorbed, polymerized,etc.) by soil particles appeared to be secondary to microbial utilization and/or uptake by roots. Varying clay (5.3 to 9.8 g/cup) and organic matter (2.0 to 0.04g/cup) contents of the soil appeared to have little impact on the disappearance of ferulic acid from soil solution under “ideal” growth conditions for cucumber seedlings unless larger amounts of ferulic acid were added to the soil; in this case 200 μg/g. The addition of ferulic acid to the soil materials substantially increased the activity of the soil microbes. This latter conclusion is based on recovery of ferulic acid from soil solution and soil respiration measurements. Paper No. 10347 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N C 27695-7601. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the product named, nor criticism of similar ones not mentioned.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by l-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by l-phenylalanine. Prephenate dehydratase was strongly activated by l-tyrosine. NAD⁺-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by l-tyrosine. Two aromatic aminotransferases were resolved, one preferring the l-phenylalanine:2-ketoglutarate substrate combination and the other preferring the l-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which l-tyrosine modulates l-phenylalanine synthesis via activation of prephenate dehydratase. l-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.     

The periplasmic nitrate reductase of Rhodobacter capsulatus; purification,characterisation and distinction from a single reductase for trimethylamine-N-oxide,dimethylsulphoxide and chlorate

The periplasmic dissimilatory nitrate reductase from Rhodobacter capsulatus N22DNAR⁺ has been purified. It comprises a single type of polypeptide chain with subunit molecular weight 90,000 and does not contain heme. Chlorate is not an alternative substrate. A molybdenum cofactor, of the pterin type found in both nitrate reductases and molybdoenzymes from various sources, is present in nitrate reductase from R. capsulatus at an approximate stoichiometry of 1 molecule per polypeptide chain. This is the first report of the occurrence of the cofactor in a periplasmic enzyme. Trimethylamine-N-oxide reductase activity was fractionated by ion exchange chromatography of periplasmic proteins. The fractionated material was active towards dimethylsulphoxide, chlorate and methionine sulphoxide, but not nitrate. A catalytic polypeptide of molecular weight 46,000 was identified by staining for trimethylamine-N-oxide reductase activity after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The same polypeptide also stained for dimethylsulphoxide reductase activity which indicates that trimethylamine-N-oxide and dimethylsulphoxide share a common reductase.Abbreviations DMSO dimethylsulphoxide - LDS lithium dodecyl sulphate - MVH reduced methylviologen - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - TMAO trimethylamine-N-oxide     

Root and microbial biomass dynamics under the canopy of the desert shrub Zygophyllum dumosum

Summary A study was done to evaluate the influence of soil moisture and rainfall on root and microbial biomass production under the canopy of the desert shrub Zygophyllum dumosum. During the study period the root biomass production increased following the early rains but subsequently declined, remaining fairly constant thoroughout the season. In contrast microbial biomass and soil organic matter increased during the rainy season and declined with the onset of the dry summer period. Based on our results we suggest that the moisture event and not the amount and the organic matter content regulate root and microbial biomass production at the 0 to 10 cm soil layer.Contribution of the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. paper no. 2617-E, 1989 series     

Induction of hepatic drug metabolizing enzymes by DL-methionine in rats

A single intraperitoneal injection of DL-methionine (500 mg/kg body wt.) to adult male Wistar rats was shown to significantly induce all the components of the hepatic microsomal mixed function oxidase system such as NADPH cytochrome C reductase activity, cytochromes P-450 and b₅, as well as activities of drug metabolizing enzymes such as aminopyrine demethylase and uridine 5′ -diphosphate-glucuronosyltransferase. Combined administration of nicotinamide (250 mg/kg body wt.) and DL-methionine (500 mg/kg body wt.) was shown to bring about an additional increase (25-30%) in the activities of these enzymes as compared to their induction on independent administration of the two endobiotics. In rats bearing Yoshida sarcoma (ascites) tumour as well as in normal rats injected with serum from tumour bearing animals, the decreased activities of hepatic mixed function oxidases could be restored to their normal levels by administration of DL-methionine (500 mg/kg body wt.) to these rats. Whereas actinomycin D (1 mg/kg body wt.) had no effect on the increased incorporation of [¹⁴C] labelled leucine into microsomal proteins following administration of nicotinamide, the enhanced incorporation of the label following DL-methionine administration was completely inhibited by the same dose of actinomycin D. Administration of cycloheximide (0·5 mg/kg body wt.) to rats could completely inhibit the increased incorporation of [¹⁴C] leucine into hepatic microsomal proteins following independent administration of nicotinamide and DL-methionine. Similar inhibitory pattern with actinomycin D and cycloheximide was also demonstrated in case of induction of NADPH cytochromeC reductase activity by both these endobiotics.     

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.Abbreviations ADH l-alanine dehydrogenase - AOAT l-alanine:2-oxoglutarate aminotransferase - Asnase l-asparaginase - GOAT Glycine: oxaloacetate aminotransferase - GOGAT Glutamate synthase - GOT l-aspartate: 2-oxoglutarate aminotransferase - GS Glutamine synthetase - HPLC High-Pressure Liquid Chromatography - MOPS 2-(N-morpholino)propanesulfonic acid - MSX l-methionine-d,l-sulfoximine