Phosphorylated Compounds in Plants: II. THE ESTIMATION OF HEXOSE PHOSPHATES AND ADENOSINE PYROPHOSPHATES IN PLANT TISSUE 3

Modifications of the method of Slater for the estimation ofhexose phosphates and adenosine pyrophosphates in plant tissueare described. Triphosphatea of nucleosides other than adenosineinterfere with the estimation of adenosine pyrophosphates inplant tissue, but other interference is not serious if the concentrationof hexose phosphates or adenosine pyrophosphates is not lowerthan about 0.15 µmoles per ml. of tissue extract.     

GROWTH PATTERNS OF PEA SEEDLINGS IN DARKNESS AND IN RED AND WHITE LIGHT

Thomson , Betty F., and Pauline Monz Miller . (Connecticut Coll., New London.) Growth patterns of pea seedlings in darkness and in red and white light. Amer. Jour. Bot. 48(3): 256–261. Illus. 1961.—Seedlings of peas were grown in vermiculite at 22°C. and exposed 16 hr. daily to red or white light or kept in darkness. Others were grown in soil in the greenhouse. Samples harvested daily to 16 days were dissected, the length of each internode and leaf measured and the total number of leaves and leaf primordia counted. The form of the stem apex and youngest primordia and interrodes is the same in light as in darkness. Leaf production is accelerated very slightly and the growth of leaves and internodes is decidedly accelerated by light. Leaf-leaf, leaf-internode and internode-internode correlations indicate that the morphogenetic effect of light is limited to later stages of organ growth. Dry weight is consumed more rapidly in light than in darkness, probably because of more rapid growth and slightly greater amounts of respiring tissue in light-grown plants.     

A KINETIC STUDY OF GROWTH MOVEMENTS AND PHOTOMORPHOGENESIS IN ETIOLATED PEA SEEDLINGS

The kinetics of the effects of inductive photomorphogenically active light on etiolated peas have been studied by means of time-lapse photography. The effects noted include: (1) A light-induced decrease in the rate of stem elongation, beginning about 6 hr after the light treatment, and ending about 18 hr later. (2) A light-induced opening of the apical hook, beginning ca. 2–5 hr after light treatment, and reaching its peak rate ca. 6 hr later. (3) A light-stimulated circumnutation, starting usually about 15–22 hr after the light, and resulting in a decreased period of oscillation (from ca. 86 to ca. 76 min) and an increased amplitude (from 15° total angular displacement to about 40°). (4) A promotion of terminal bud growth, known from previous work to start at about 4 hr after irradiation and to reach a peak about 12 hr later. (5) A “bobbing” movement of the apex, apparently involving reversible hook and stem oscillations, which appears to be of endogenous origin and insensitive to light. These data furnish a kinetic background against which proposed biochemical mechanisms of de-etiolation can be assessed.     

MAXIMUM ACTIVITIES, PROPERTIES AND DISTRIBUTION OF 5''NUCLEOTIDASE, ADENOSINE KINASE AND ADENOSINE DEAMINASE IN RAT AND HUMAN BRAIN

Abstract— The maximum activities of 5'nucleotidase, adenosine kinase and adenosine deaminase have been measured in several areas of rat and human brain. There is no major difference between the activities of nucleotidase and kinase between rat and human brain, but the activity of deaminase is considerably higher in human brain. The activities of all these enzymes are similar in three areas of rat brain and nine areas of human brain, except for hind brain of the human, which has a low activity of adenosine deaminase. This variation may indicate the existence of different steady-state concentrations of adenosine in certain areas of the brain.
Subcellular fractionation of different areas of rat brain showed that, whereas adenosine kinase and deaminase activities were located mainly in the soluble fractions, 5'nucleotidase was present in all subcellular fractions (i.e. membrane, synaptosomal, mitochondrial and soluble). In particular, there was no major localisation within the synaptosomal fraction. Thus it is unlikely that the regulation of the activities of these enzymes is dependent upon changes within a specific compartment (e.g. synaptosomes) in the brain.     

THE DISTRIBUTION OF FORMYLTETRAHYDROFOLATE SYNTHETASE IN PLANTS, AND THE PURIFICATION AND PROPERTIES OF THE ENZYME FROM PEA SEEDLINGS

1. Formyltetrahydrofolate synthetase (E. C. 6. 3. 4. 3) was foundto be widely distributed in higher plants and the high enzymeactivity was observed in green leaves of Brassica and Alliumspecies, spinach, and in pea seedlings. In pea seedlings, theenzyme activity changed during the course of germination, andmost of the enzyme activity was located in a soluble fractionof the cytoplasm.
2. The enzyme was labile and lost the activityrapidly, even whenstored at 5 in the presence of 0.1 M mercaptoethanol.It was,however, found that ammonium sulfate was very effectivein stabilizingthe enzyme activity.
3. The enzyme has been purifiedapproximately 500-fold from extractsof pea seedlings by treatmentswith ammonium sulfate, protaminesulfate, hydroxylapatite, calciumphosphate gel, and DEAE-cellulosecolumn chromatography.
4. Thepurified enzyme was specific for formate, ATP and FAH₄,andthe Michaelis constants for these reactants were 2.1 10^–2M, 5.1 10^–4 M, and 5.6 10^–3 M, respectively.
5. The optimum pH was found to be 8.0, and the optimal temperaturewas observed at 37. Both NH₄^$ and a divalent cation (Mg^SS orMn^SS) were required for the optimal activity.

¹ Studies on the Enzymatic Synthesis and Metabolism of FolateCoenzymes in Plants. II. (For the previous paper see reference(8)) A part of this paper was presented at the Meeting of theKansai Division of the Agricultural Chemical Society of Japan,Kyoto, January 29, 1966.     

ON THE CORRELATION OF PRIMARY ROOT LENGTH,MERISTEM SIZE AND PROTOXYLEM TRACHEARY ELEMENT POSITION IN PEA SEEDLINGS

Pea seedlings (Pisum sativum L. cv. Alaska) were darkgrown in vermiculite. Roots of various lengths were cleared, stained and measured to determine the relative meristem height (MH), width and volume and the distance to the most proximal protoxylem tracheary element (PTE). A correlation was found between root length, MH and PTE position as follows: in roots from 4–40 mm as the root elongated the MH lengthened and PTE position increased its distance from the body/cap juncture; in roots 40–80 mm MH and PTE position remained approximately constant; in longer roots (80–120 mm) MH became shorter, and PTE position closer to the tip as the root elongated. The relationship, using our measurement procedure was that for every 0.19 mm change in MH, the PTE position changed by 1 mm. This response was partially growth rate dependent since short roots (4–80 mm) grew at a constant rate and longer roots (80–140 mm) grew slower. Root manipulations and trifluralin treatment, to inhibit cell division, caused tip swelling and modulated the position of the PTE toward the root tip. The control of the spatial relationship between meristem size and maturation position is discussed.     

ACRYLAMIDE GEL ELECTROPHORESIS OF SOLUBLE PLANT PROTEINS: A STUDY ON PEA SEEDLINGS IN RELATION TO DEVELOPMENT

The application of the technique of acrylamide gel electrophoresis to the separation of components of the soluble proteins of plants is described. The reproducibility of the results is demonstrated with reference to Neurospora proteins, and suitable procedures to document the results are described. These techniques have been applied to the soluble protein complement of segments along the axis of the root of Pisum sativum and of other parts of the seedling. The observations are interpreted in the light of current views on protein synthesis and cellular development.     

The Translocation of Sulphonamides in Higher Plants: I. UPTAKE AND TRANSLOCATION IN BROAD BEANS

The uptake and translocation of sulphanilamide, sulphacetamide,sulphaguani-dine, sulphapyridine, sulphadiazine, sulphathiazole,and 4:4-diaminodiphenyl-sulphone by broad bean plants growingin water culture has been studied. After varying times of exposureto the compounds at 100/µg./ml., total sulphonamide presentin the roots, stems, and leaves was determined in acid-hydrolysedmacerates by diazotization and coupling of the primary aminogroup. These compounds were identified in the leaves of treatedplants by paper chromatography. Accumulation of sulphonamide in the roots appears to be relatedsimply to time, and the concentration of sulphonamide may, eventually,be far higher than that in the treating solution. Movement fromthe roots to the stems and leaves depends on transpiration.Sulphanilamide and the sulphone passed rapidly into the leaves;sulphacetamide, sulphapyridine, sulphadiazine, and sulphathiazolemoved less rapidly. There was, however, a marked accumulationof sulphacetamide, sulphapyridine, sulphadiazine, sulphathiazole,and the sulphone in the roots. Sulphaguanidine was poorly absorbedfrom the treating solution.     

Effects of Glyphosate on Metabolism of Phenolic Compounds: V. l-alpha-AMINOOXY-beta-PHENYLPROPIONIC ACID AND GLYPHOSATE EFFECTS ON PHENYLALANINE AMMONIA-LYASE IN SOYBEAN SEEDLINGS

The phenylalanine ammonia-lyase (PAL) inhibitor l-alpha-aminooxy-beta-phenylpropionic acid (AOPP) was root-fed to light-exposed soybean seedlings alone or with glyphosate [N-(phosphonomethyl)glycine] to test further the hypothesis that PAL activity is involved in the mode of action of glyphosate. Extractable PAL activity was increased by 0.01 and 0.1 millimolar AOPP. AOPP reduced total soluble hydroxyphenolic compound levels and increased phenylalanine and tyrosine levels, indicating that in vivo PAL activity was inhibited by AOPP. The increase in extractable PAL caused by AOPP may be a result of decreased feedback inhibition of PAL synthesis by cinnamic acid and/or its derivatives. AOPP alone had no effect on growth (fresh weight and elongation) at either concentration, but at 0.1 millimolar it slightly alleviated growth (fresh weight) inhibition caused by 0.5 millimolar glyphosate after 4 days. Reduction of the free pool of phenylalanine by glyphosate was reversed by AOPP. These results indicate that glyphosate exerts some of its effects through reduction of aromatic amino acid pools through increases in PAL activity and that not all growth effects of glyphosate are due to reductions of aromatic amino acids.     

CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA-PIG CEREBRAL CORTICAL SLICES: STUDIES ON THE ROLE OF ADENOSINE

Abstract— In guinea-pig cerebral cortical slices levels of cyclic AMP increase in response to adenosine to about 200pmol/mg protein within 10 min and stay at that level up to 30 min. In the absence of calcium ions and the presence of 1mm -EGTA in the Krebs-Ringer-bicarbonate medium the effect of adenosine is enhanced, cyclic AMP levels rise to about 600 pmol/mg protein within 30 min. In normal and calcium deficient media restimulation of cyclic AMP formation with adenosine is possible after a prior stimulation with adenosine. When slices are preincubated for various periods of time with histamine or adenosine before addition of the complementary agent i.e. adenosine or histamine cyclic AMP levels obtained are unaltered compared to levels seen when adenosine and histamine are added together. Slices which are rendered unresponsive to stimulation with histamine + noradrenaline by a prior incubation with these agents do not regain any response during a 100 min period of incubation in medium. The PDE inhibitors diazepam, SQ 66007 and isobutylmethylxanthine are capable of restoring the sensitivity of the slices to histamine + noradrenaline. This suggests an involvement of PDE in the unresponsive phase of the slices. Addition of adenosine to slices not affected by histamine + noradrenaline does reestablish the response of these slices to the neurohormones. A dose-response curve of adenosine for the interaction with histamine + noradrenaline yields an ED₅₀ of 16 μM using sensitive or desensitized slices. An adenosine concentration of only 7 μM is necessary to restore the original increase of cyclic AMP in response to histamine + noradrenaline to slices insensitive to the biogenic amines. The data are discussed in terms of a possible activation of PDE within cerebral cortical slices from guinea-pig. Adenosine may reverse this activation. The possibility of inactivation of adenylate cyclase during stimulation of cyclic AMP formation and the role of adenosine and PDE inhibitors in this process is being considered.     

小麦幼苗及不同育性花药中P5C还原酶的研究

从小麦幼苗和花药中提取的二氢吡咯-5-羧酸(P5C)还原酶,在可育花药中活性很高,约为幼苗中活性的7～13倍,表明花药有很高的脯氨酸合成能力。从减数分裂期到单核靠边期酶活性逐渐升高,到双核初期明显降低。在不育花药中,减数分裂期酶活性高于可育花药,到单核初期酶活明显下降,仅为可育花药活性的一半。 初步纯化的小麦幼苗P5C还原酶的最适pH为7.2左右。对P5C和NADH的K_m值分别为400μmol/L和370/μmol/L。以NADPH为供氢体,其酶活性为NADH的35％。NADP~+、NAD~+、ATP、ADP对酶活性均有强烈的抑制作用。脯氨酸浓度在10m mol/L以上对酶活有轻微抑制作用。     

GROWTH OF THE BARLEY COLEOPTILE. I. ITS RELATIONSHIP TO CELL NUMBER AND LENGTH IN NORMAL AND γ-IRRADIATED SEEDLINGS

The number of cells per vertical column in barley coleoptiles differs in various growth classes; it is highest in tall coleoptiles, intermediate in medium ones, and lowest in short ones. In those that elongate early and grow rapidly, cells per column increased from 88–218 between 12 and 44 hr after the seeds were placed on water; in short coleoptiles they increased to only 85 per column after 115 hr because elongation and division are restricted in these. Cell number does not increase in coleoptiles from seeds irradiated with 250 krad. Variation in the growth pattern of irradiated coleoptiles was similar to that of normal ones, although the range in lengths was reduced. Although the number of cells is much higher in tall controls than in irradiated coleoptiles, the latter can become tall; therefore an increase in cell number during germination does not seem to be a prerequisite for tallness. Coleoptiles 32 mm long, from irradiated seeds, have the same number of cells per column as the shortest ones (6 mm) after 119 hr.     

The Utilization of P--N Compounds by Plants: I. Root-mediated Hydrolysis of Phosphodiamidate

The uptake and utilization of sodium phosphodiamidate, a compoundcontaining covalent P—N bonds, was investigated. Growthanalysis showed that this compound could serve as the sole sourceof phosphorus for tomato plants grown in culture solutions,although the growth rate of plants supplied with phosphodiamidatewas slightly less than that of plants utilizing diammonium phosphate.Chromatographic analysis of xylem sap showed that phosphodiamidatewas not transported in the unhydrolysed form in the sap of tomatoplants supplied with this compound. Tomato plants supplied withphosphodiamidate as the sole source of both phosphorus and nitrogenassimilated some nitrogen in a form other than unhydrolysedphosphodiamidate. Comparison with plants supplied with diammoniumphosphate showed that the plants receiving phosphodiamidatehad lower nitrogen contents, suggesting that the rate of hydrolysisof the compound may have been limiting nitrogen assimilationby the plants. Measurements of the hydrolysis of phosphodiamidatein culture solutions in the absence of plants showed that theplants assimilated more nitrogen than that released by normalchemical hydrolysis of the compound occurring in the absenceof plants. The excess nitrogen assimilated, over and above thatproduced by normal chemical hydrolysis, could not be accountedfor solely by the uptake of unhydrolysed phosphodiamidate asthis would require the concomitant uptake of more phosphorusthan was actually present in the plant. Thus it was inferredthat the presence of the plant roots in the culture solutionsomehow caused extra hydrolysis of the phosphodiamidate.     

CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: POSSIBLE REGULATION OF PHOSPHODIESTERASE ACTIVITY BY CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE AND CALCIUM IONS

Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) accumulates in guinea pig cerebral cortical slices during incubation with histamine, histamine + noradrenaline and adenosine. Noradrenaline does not enhance cyclic AMP formation. In the absence of Ca²⁺ ions and presence of 1 mM-EGTA in the Krebs-Ringer bicarbonate medium the effects of histamine, histamine + noradrenaline and adenosine are significantly enhanced and noradrenaline elicits an increase in cyclic AMP over control levels. When histamine is used as stimulant, cyclic AMP levels start to decline after only 5 min. However, in the absence of calcium and in the presence of EGTA in the medium this decline is not observed and cyclic AMP levels continue to rise for a considerable period of time. In normal medium, responses to restimulation by histamine or histamine + noradrenaline are greatly reduced in magnitude after a prior stimulation by these putative neurotransmitters. In contrast, when calcium is omitted from the incubation medium and 1 mM-EGTA is included, cyclic AMP levels increase to normal values at a second stimulation with histamine or histamine + noradrenaline. When slices are preincubated for various periods of time with histamine before addition of noradrenaline, the accumulation of cyclic AMP is significantly reduced as compared to levels obtained when histamine + noradrenaline were added simultanously. This decline in the overall response to histamine + noradrenaline is not observed when preincubation with histamine and subsequent incubations with histamine + noradrenaline are performed in Ca²⁺-free, 1 mM-EGTA containing buffer. Also preincubation with noradrenaline in normal, calcium-containing medium does not affect the total amount of cyclic AMP accumulating in the brain slices. The results are discussed in terms of an activation of phosphodiesterase within the cerebral cortical slices by increased levels of intracellular, freely available calcium which is mediated by the elevation of cyclic AMP concentration following hormonal stimulation.     

ACCUMULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE IN BRAIN SLICES: INTERACTION OF LOCAL ANAESTHETICS AND DEPOLARIZING AGENTS

Abstract— Membrane depolarizing agents such as veratridine, ouabain and high concentrations of potassium ions elicit a remarkable accumulation of cyclic AMP in brain slices incubated in vitro , and this accumulation, but not that elicited by biogenic amines, is prevented by a membrane stabilizer, cocaine. The effect of various local anaesthetics (compounds which are known to stabilize the membrane of peripheral sensory nerves) on the accumulation of cyclic AMP elicited by depolarizing agents in incubated slices of guinea pig brain has now been examined. At optimal concentrations the anaesthetics inhibited by more than 95 per cent the accumulation of cyclic AMP elicited with veratridine, ouabain, and high concentrations of potassium ions. The order of the inhibitory potency vs. veratridine was: dibucaine (ED₅₀= 9.5 ± 10⁻⁶ M) > tetracaine > cocaine (ED₅₀= 1·3 ± 10⁻⁴ M) > lidocaine > procaine (ED₅₀= 1.7 ± 10⁻³M). This order is consistent with the order of their local anaesthetic potency, but is not consonant with the order of the relative toxicity of these agents when used as spinal anaesthetics.     

ADENOSINE 3'',5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: EFFECTS OF α- AND β-ADRENERGIC AGENTS, HISTAMINE, SEROTONIN AND ADENOSINE

—Norepinephrine and epinephrine, in combination with either adenosine or histamine, enhanced the accumulation of cyclic AMP in guinea pig cerebral cortical slices. Isoproterenol had only marginal effects under the same conditions. Studies with d- and l-norepinephrine and with the α- and β-adrenergic blocking agents, phenoxybenzamine, phentolamine, dihydroergokryptamine, propranolol and sotalol, indicated that the effect of catecholamines on cyclic AMP levels in this tissue was stereo-specific and was mediated primarily via interaction with a classical α-adrenergic receptor. Studies with the antihistaminics, diphenhydramine and pheniramine, and the antiserotonin agent, methysergide, indicated that guinea pig cerebral cortical slices contain receptors for histamine and serotonin, whose activation also stimulates an enhanced accumulation of cyclic AMP in the presence of adenosine.     

ACCUMULATION OF CYCLIC ADENOSINE 3', 5'-MONOPHOSPHATE IN CEREBRAL CORTICAL SLICES FROM RAT AND MOUSE: STIMULATORY EFFECT OF α- AND β-ADRENERGIC AGENTS AND ADENOSINE

Abstract— Norepinephrine, epinephrine, isoproterenol, and adenosine elicit enhanced accumulations of cyclic AMP in incubated slices of rat cerebral cortex. Combinations of norepinephrine, epinephrine, isoproterenol, or histamine with adenosine have a greater than additive effect on cyclic AMP levels. The effects of isoproterenol appear to be mediated via a classical β-adrenergic receptor whereas the effects of norepinephrine appear due to interactions with both α- and β-adrenergic receptors. The presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, potentiates the effects of the catecholamines and reveals a histamine-mediated increase in cyclic AMP levels. After an initial stimulation of cyclic AMP formation with norepinephrine, followed by washing of the slices, the cyclic AMP-generating system is unresponsive to norepinephrine but does respond to an adenosine-norepinephrine combination. In mouse cerebral cortical slices, catecholamines appear to elicit an accumulation of cyclic AMP primarily via interaction with a β-adrenergic receptor.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: I. IDENTIFICATION IN tRNA FROM ETIOLATED PHASEOLUS VULGARIS L. SEEDLINGS

The cytokinin-active ribonucleosides present in tRNA from etiolated Phaseolus vulgaris L. seedlings have been isolated and identified as cis-ribosylzeatin, 2-methylthio-ribosylzeatin, and N⁶-(Δ²-isopentenyl)-adenosine. The structures of the compounds were established on the basis of their chromatographic properties and the mass spectra of their permethylated and perdeuteromethylated derivatives. Cis-ribosylzeatin was the major cytokinin-active constituent of tRNA from this source.