Tissues Of Higher Plants Research Articles

Subcellular localization of hexokinase in pea leaves. Evidence for the predominance of a mitochondrially bound form.

Hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) activity was determined in subcellular fractions prepared from pea (Pisum sativum) leaf homogenates. About 60% of the total detectable activity of hexokinase was found associated with a particulate fraction consisting essentially of mitochondria and chloroplasts and free of cytosol contamination. The hexokinase specific activity of the particulate fraction was 2-fold higher than that of the homogenate and about 4-fold higher than that of the cytosol. Using a specially designed isokinetic-isopycnic sucrose density gradient centrifugation method, the distribution of hexokinase activity correlated with that of the mitochondrial marker (cytochrome oxidase) and not with that of the chloroplast membrane marker ( chlorophyll ) or that of the cytosol marker (phosphoenolpyruvate carboxylase). Thus, the hexokinase/mitochondria ratio was close to 1.0 along the entire gradient, while the hexokinase/chloroplast ratio varied over a 10-fold range. The results strongly suggest that hexokinase is predominantly bound to mitochondria of pea leaves, and that pea leaf chloroplasts are essentially devoid of any specifically associated hexokinase activity. This work provides the first demonstration of a specific association of hexokinase with mitochondria from photosynthetic tissues of higher plants.

Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [3H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of3H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.

In Vitro Fatty Acid Synthesis and Complex Lipid Metabolism in the Cyanobacterium Anabaena variabilis

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH(4))(2)SO(4) fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.

Sugar Uptake by Cotton Tissues

The tissue accumulation of sucrose, glucose, and fructose has been studied in cultured cotton (Gossypium hirsutum L.) roots and leaf discs. Sucrose uptake by both tissues from high apoplastic concentrations was independent of pH but has a slightly acidic pH optimum from low concentrations. Like other higher plant tissues, cotton root cells accumulate sucrose via a ;saturable,' inhibitor-sensitive mechanism and a linear, inhibitor-resistant mechanism. The linear mechanism of sucrose uptake is not as pronounced in leaf disc data as it is in root data. Further, sucrose uptake by cotton leaf discs is more resistant than uptake by root cells to pH alterations, inhibitors, and monosaccharides in the uptake medium. The saturable phase of sucrose influx into cotton root is eliminated by glucose, fructose, and high pH. Sucrose influx into both tissues is not altered by osmotica up to 200 milliOsmolar. Sucrose accumulated by both tissues is rapidly converted to other chemical forms, especially in root tissue where only approximately 50% remains as neutral sugars 1 hour following the start of radiolable exposure. Although the entry of radiolabeled sucrose is faster in abraded leaf discs, they give the same response patterns to pH, inhibitors, and monosaccharide as do unabraded discs.The sucrose accumulation kinetics of cotton roots and leaf discs differ. These differences may be related to the physiological roles (source versus sink) of the two tissues in the intact plant.

Polyphosphate Metabolism in Micro-Organisms

Publisher Summary This chapter discusses the physiological role of inorganic polyphosphates. Inorganic polyphosphates are linear polymers in which orthophosphate residues are linked by energy-rich phospho-anhydride bonds. Inorganic polyphosphates have been found in almost all tested representatives of living cells. They have been detected in eubacteria, fungi, algae, mosses, protozoa, insects, and in various tissues of higher plants and animals. In highly organized organisms, polyphosphates perform several specialized functions, being donors of activated phosphate only for quite specific biochemical or physiological processes. The chapter presents new aspects of inorganic pyrophosphate's metabolic and physiological role. Pyrophosphate is only a byproduct of numerous reactions of pyrophosphorolysis involved in biosynthesis of proteins, nucleic acids, lipids, polysaccharides, and nucleoside coenzymes. The energy-dependent synthesis of pyrophosphate during photosynthetic and oxidative phosphorylation is also discussed. The first evidence for an energy-dependent biosynthesis of pyrophosphate is obtained with animal tissue homogenates. Finally, several modern concepts about the role of high molecular-weight polyphosphates and pyrophosphate in evolution of phosphorus metabolism are presented in the chapter.

Measurements of the volumetric and transverse elastic extensibilities of Chara corallina internodes by combining the external force and pressure probe techniques

The transverse and volumetric elastic extensibilities of Chara corallina internodal cell wall tubes were studied by simultaneous application of the external force and pressure probe measuring techniques. It was found that there is a deformation of the cell wall resulting from the concentrated force exerted by the external force, which dominates the transverse extensibility at low turgor pressures, and which can be important at high turgor pressure. The implications of the effect for the use of the external force technique on higher plant tissue are discussed.

Formulae for Determination of Chlorophyllous Pigments Extracted with N,N-Dimethylformamide

The extraction of chlorophylls in higher plant tissue using N,N-dimethylformamide expedites the process and enables the determination of small samples with low pigment level.Absorption spectra of Chl a, Chl b, and Pchl and of their acidified derivatives, the phaeophytins, were recorded. Conversion of Chl b to its corresponding acidified product occurs much more slowly than that of Chl a and Pchl. When acidified, Pchl differs from Chl a and Chl b by the disappearance of the red band in the absorption spectrum. Specific extinction coefficients were determined and formulae for quantitative determination of pigments concentrations were developed. When concentrations of pigments are low, as in etiolated plant material, the absorption spectra of the chlorophylls can be distorted due to the presence of other substances simultaneously extracted; formulae for pigment determination under such circumstances were also derived.

Water relations of immobilized giant algal cells

Cells of Halicystis parvula, Acetabularia mediterranea, and Valonia utricularis were immobilized in a cross-linked alginate matrix (4-6% w/w) in order to simulate water-relation experiments in individual cells of higher plant tissues. The immobilization of these cells did not lead to an increase in the mechanical stability of the cell walls. This was demonstrated by measuring the volumetric elastic modulus of the cell wall and its dependence on turgor pressure with the aid of the non-miniaturized pressure probe. In immobilized cells, no changes in the absolute value of the elastic modulus of the cell wall could be detected for any given pressure. At the maximum turgor pressure at which non-immobilized cells normally burst (about 3-7 bar for V. utricularis; depending on cell size, 3 bar for A. mediterranea and 0.9 bar for H. parvula) reversible decreases in the pressure are observed which are succeeded by corresponding pressure increases. This obvervation indicates that coating the cells with the cross-linked matrix protects them from rapid water and turgor pressure loss. Turgor pressure relaxation processes in immobilized cells, which could be induced hydrostatically by means of the pressure probe, yielded accurate values for the half-times of water exchange and for the hydraulic conductivity of the cell membrane. The results demonstrate that the water transport equations derived for single cells in a large surrouding medium are valid for immobilized cells, so that any influence exerted by the unstirred layer which is caused by the presence of the cross-linked matrix can be ignored in the calculations. On the other hand, the evaluation of the half-times of water exchange and the hydraulic conductivity from turgor pressure relaxation processes, which have been induced osmotically, only yields correct values under certain circumstances. The model experiments presented here show, therefore, that the correct Lp-value for an individual cell in a higher plant tissue can probably only be obtained presently by using the pressure probe technique rather than the osmotic method. The results are also discussed in relation to the possible applications of immobilized cells and particularly of immobilized micro-organisms in catalytic reaction runs on an industrial scale.

Comparative Effects of Substituted Pyrimidines on Growth and Gibberellin Biosynthesis in Gibberella fujikuroi

The fungicide alpha-(2,4-dichlorophenyl)-alpha-phenyl-5-pyrimidine methyl alcohol (triarimol) and four other structural analogs of this substance, in which one or more of the substituents were varied, were tested for their comparative effects on growth and gibberellin biosynthesis in the fungus Gibberella fujikuroi. Each of the five analogs tested was capable of inhibiting growth as measured by dry weight in 5-day-old cultures. Three of them [alpha-(2-chlorophenyl)-alpha-(4-chlorophenyl)-5-pyrimidine methyl alcohol, fenarimol; alpha-(2-chlorophenyl)-alpha-(4-fluorophenyl)-5-pyrimidine methyl alcohol, nuarimol; and triarimol] were effective at appreciably lower concentrations than the other two [alpha-(4-chlorophenyl)-alpha-(1-methylethyl)-5-pyrimidine methyl alcohol, experimental compound EL 509; and alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidine methyl alcohol, ancymidol].All five substances also inhibited gibberellin production as measured by gibberellin content of fungus filtrates. The relative effectiveness of the compounds as inhibitors of growth and gibberellin production were similar. These analogs were also shown to inhibit ent-kaurene oxidation by microsomal preparations from fungal mycelia. Thus, the site of inhibition of gibberellin biosynthesis may be the same for the fungus as the one affected by this group of substances in higher plant tissues.The structure-activity relationships between the analogs are opposite to those observed in higher plant tissues. The fungicides fenarimol, nuarimol, and triarimol, which were most effective in inhibiting growth and gibberellin biosynthesis in the fungus, were much less effective than EL 509 and ancymidol in inhibiting growth and gibberellin biosynthesis in higher plants. These results indicate that the ent-kaurene oxidase systems from the two sources have somewhat different molecular characteristics, and thus, interact differently with this group of substances.

Identification of guanosine 3′:5′-monophosphate in the fruit of Zizyphus jujuba

Evidence is presented here confirming the identification of guanosine 3′: 5′-monophosphate (c GMP) in the tissue of higher plants. The c GMP activity detected in fruits of Zizyphus jujuba was separated from the c AMP activity also present. The separated sample was extensively purified by Bio-Rad AG 1 × 4 and aluminium oxide CC, and by TLC. The purified sample showed the same physicochemical properties as authentic c GMP by TLC using different solvents and by UV spectroscopy, and was decomposable by cyclic nucleotide-specific phosphodiesterase. The identification was further supported by HPLC. The amount of c GMP present increases 90-fold during fruit ripening.

Desaturation of Fatty Acids Associated with Monogalactosyl Diacylglycerol: The Effects of San 6706 and San 9785

The effects of two substituted pyridazinone herbicides, San 6706 and San 9785, on photosynthesis, dark respiration, and fatty acid metabolism were studied in mature leaf tissue of Vicia faba.Both San 6706 and San 9785 inhibited photosynthesis within 2 hours after initial exposure of leaf tissue to the chemicals although San 9785 was more effective in inhibiting photosynthesis than San 6706. Neither San 6706 nor San 9785 had any marked effect on dark respiration.Kinetic studies using (14)CO(2) indicated that synthesis of 18:3 esterified to monogalactosyl diacylglycerol (MGDG) was not completely inhibited by San 9785 up to 48 hours after feeding. Radioactivity was observed to accumulate in MGDG (digalactosyl diacylglycerol [DGDG] and sulpholipid [SL]) 18:2 at the expense of 18:3 but the specific radioactivity of MGDG 18:3 continued to increase throughout the experiment indicating only partial inhibition of MGDG 18:3 synthesis. No significant differences were observed in the metabolism of other fatty acids. The metabolism of fatty acids from leaf tissue was not affected by treatment with San 6706.The data indicate that there are at least two sites for 18:2 desaturation to form 18:3, one associated with MGDG in the chloroplast, which is inhibited by San 9785, and one or more sites not inhibited by San 9785. The fatty acid specific radioactivity data support the hypothesis that there is vectorial transfer of fatty acids from phosphatidyl choline to MGDG to produce the large quantities of MGDG 18:3 found in higher plant tissue.

Quantitative analysis of methyl- and inorganic-mercury from mammalian, fish and plant tissue

A successful method was developed for the quantitative separation of biologically incorporated inorganic- and methyl-mercury from animal, fish or higher plant tissues. The differences in animal and plant tissue composition determine the initial treatment of tissues for the highest efficiency of extraction and the subsequent quantitative separation of the mercury species by thin layer chromatography. Thus, animal or fish tissue was treated initially with ethanolic potassium hydroxide solution whereas plant tissue was treated initially with a strong nitric:perchloric acid mixture. Recoveries of both inorganic- and methyl-mercury exceeded 96%.

The determination of 4,5-dioxovaleric acid in plant preparations and a procedure for the assay of l-alanine:4,5-dioxovaleric acid aminotransferase (EC 2.6.1.43) activity

Usually, 4,5-dioxovaleric acid (DOVA) is determined in biological materials by measuring the absorption at 269 nm of its benzoquinoxaline derivative which is formed by condensation with 2,3-diaminonaphthalene (DAN). Not only must this benzoquinoxaline be separated from unreacted DAN and flavins which have interfering uv absorption but, when working with higher-plant tissues, additional interfering compounds with uv absorption, ionic and solubility properties similar to polyphenols must also be removed. The separation of the DOVA-derived benzoquinoxaline from all these interfering compounds by a series of solvent extractions utilizing the difference in ionic behaviour of the benzoquinoxaline and the interfering contaminants is described. It was found that a small but significant amount of a benzoquinoxaline is formed when 5-aminolaevulinic acid (ALA) was incubated with DAN at pH 8 at 60°C and was due to the prior non-enzymic deamination of a small portion of ALA to DOVA: this benzoquinoxaline was spectrophotometrically, spectrofluorimetrically, and chromatographically indistinguishable from that formed by the condensation of DOVA and DAN. Since formation of this benzoquinoxaline interferes with the assay of l-alanine:4,5-dioxovaleric acid aminotransferase (EC 2.6.1.43), a procedure to measure DOVA formed by this enzyme from ALA and pyruvate is described in which the DOVA is first separated from the ALA by ion-exchange chromatography prior to condensation with DAN: this method permits the separate determination of both DOVA and ALA concentrations in the aminotransferase reaction mixture.

Polyamines Inhibit Biosynthesis of Ethylene in Higher Plant Tissue and Fruit Protoplasts

Ethylene production in apple fruit and protoplasts and in leaf tissue was inhibited by spermidine or spermine. These polyamines, as well as putrescine, inhibited auxin-induced ethylene production and the conversion of methionine and 1-aminocyclopropane-1-carboxylic acid to ethylene. Polyamines were more effective as inhibitors of ethylene synthesis at the early, rather than at the late, stages of fruit ripening. Ca(2+) in the incubation medium reduced the inhibitory effect caused by the amines. A possible mode of action by which polyamines inhibit ethylene production is discussed.

Induction of cytochrome P-450 and monooxygenase activity by 2,4-dichlorophenoxyacetic acid in higher plant tissue

The time-course of changes in cytochrome contents and enzyme activities related to the microsomal cytochrome P-450-dependent monooxygenases has been determined in Jerusalem artichoke ( Helianthus tuberosus L.) tuber slices exposed to 2,4-dichlorophenoxyacetic acid 2,4-D). The activities of the in chain hydroxylating lauric acid hydroxylase (IC-LAH), of NADPH cytochrome c ( P-450) reductase and the titer of cytochrome b 5 were not substantially altered, whereas tr-cinnamic acid 4-hydroxylase (CA4H), NADH cytochrome c ( b 5) reductase and cytochrome P-450 content were significantly enhanced. The effect was highest in slices exposed to herbicidal (5 × 10 −5 M to 5 × 10 −4 M) concentrations of 2,4 D.

Characteristics of Coal Mine Overburden Important to Plant Growth

AbstractThis investigation was conducted to determine physical and chemical properties of mineland overburden, to evaluate plant growth in these materials, and to identify properties of overburden materials before mining that may serve as predictors of potential plant growth.Eighteen overburden materials from five surface coal mines in the Illinois coal basin of southwestern Indiana were sampled and analyzed for 20 physical and chemical properties. Twelve were unconsolidated materials, including A and B horizons, lacustrine sediments, and glacial tills, and six were rock strata that break and weather easily. Growth potential for overburden materials, with and without sewage sludge and fertilizer amendments, was evaluated in greenhouse pot culture using alfalfa, wheat, and white pine. Oats yield, and survival and growth of Virginia pine and yellow‐poplar were evaluated for 10 materials in outdoor containers.Regression analysis of plant growth against chemical and physical properties of the overburden materials did not reveal properties that could be consistently used in a formula approach to predicting plant growth potential of the materials. Electrical conductivity of the material extract and water storage capacity were most frequently significantly related to growth. High plant tissue levels of B, Fe, Mn, and Al suggested toxicity problems on some materials. The general ranking of overburden materials evaluated for plant growth potential was lacustrine sedment ≥ A horizons > B horizons = glacial tills ≥ brown shale > sandstone > gray shale > black fissile shales; however, physical properties unfavorable to extraction and replacement of lacustrine material may limit its use under field conditions. Addition of sewage sludge resulted in vastly improved growth of wheat, and to a lesser extent, alfalfa, on most materials in the greenhouse. Fertilization with N‐P‐K was less effective than sludge but usually produced increased plant growth.

Characterization of Rhodanese from Cassava Leaves and Tubers

Rhodanese activity has been established in the leaves, in the peel, and in the flesh of the tuberous part of Manihot escalenta Crantz. The pattern of distribution of enzyme activity is shown to follow that of the concentration of the cyanogenic glucoside estimated on the basis of HCN released. For the first time, the presence of rhodanese is reported in higher plant tissues other than the leaves. Identity has been established between rhodanese from peel, leaves, and flesh of the cassava plant. The enzyme is inhibited by cyanide in the absence of thiosulphate or cysteine. Rhodanese is suggested to play a role in the detoxification of cyanide in cassava.

The estimation of phenylalanine ammonia-lyase (PAL) activity in intact cells of higher plant tissue

A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33-40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments (3)H is stereospecifically released from the pro-3S-position of L-[2,3-(3)H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, (3)H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.

Uptake and Transport of Fusicoccin in Higher Plant Tissues

The uptake and release of 3H-labelled fusicoccin (FC) fed to pea internode segments and the transport of [3H]FC in whole plants and in excised parts of plants of pea, maize, and pumpkin have been investigated. FC uptake showed three phases apparently corresponding to the pene tration of FC: (1) into the free space; (2) into the cytoplasm; (3) into the vacuole. The first phase is a little thermosensitive and apparently includes adsorption binding to some free space component (cell surface?). The second and third phases are highly temperature sensitive and in part energy-dependent. However, no accumulation of FC against a concentration gradient is observed at high external FC concentration. Accumulation at low concentration seems due to the formation of a poorly exchangeable complex with some cell structure. The efflux of the FC penetrated within the cells is limited by some highly temperature sensitive process. A small fraction of the accumulated FC tends to be retained in the tissue, either because sequestered in some compartment or because bound to some cellular component. FC transport in whole plants as well as in isolated plant parts seems to depend mainly on simple diffusion and on transport by mass flow in the xylem. Long distance transport of the toxin in pumpkin plants mainly occurs in the xylem, while the slow movement in non-vascular tissues seems to depend on diffusion combined with mass flow in the free space, and does not seem heavily influenced by metabolic factors.

Mass spectrometric identification of adenosine 3′:5′-cyclic monophosphate isolated from a higher plant tissue

Mass spectrometric evidence is presented confirming the identification of the adenosine nucleotide previously isolated from tissues of Phaseolus vulgaris as adenosine 3′: 5′-cyclic monophosphate.