Showing papers in "Plant Physiology in 1968"

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

Abstract: The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

Relationship of Water Potential to Growth of Leaves

Abstract: A thermocouple psychrometer that measures water potentials of intact leaves was used to study the water potentials at which leaves grow Water potentials and water uptake during recovery from water deficits were measured simultaneously with leaves of sunflower (Helianthus annuus L), tomato (Lycopersicon esculentum Mill), papaya (Carica papaya L), and Abutilon striatum Dickson Recovery occurred in 2 phases The first was associated with elimination of water deficits; the second with cell enlargement The second phase was characterized by a steady rate of water uptake and a relatively constant leaf water potential Enlargement was 70% irreversible and could be inhibited by puromycin and actinomycin D During this time, leaves growing with their petioles in contact with pure water remained at a water potential of -15 to -25 bars regardless of the length of the experiment It was not possible to obtain growing leaf tissue with a water potential of zero It was concluded that leaves are not in equilibrium with the potential of the water which is absorbed during growth The nonequilibrium is brought about by a resistance to water flow which requires a potential difference of 15 to 25 bars in order to supply water at the rate necessary for maximum growthLeaf growth occurred in sunflower only when leaf water potentials were above -35 bars Sunflower leaves therefore require a minimum turgor for enlargement, in this instance equivalent to a turgor of about 65 bars The high water potentials required for growth favored rapid leaf growth at night and reduced growth during the day

Photoreduction and photophosphorylation with tris-washed chloroplasts.

Abstract: The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e2 ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.

Sequential Induction of Phenylalanine Ammonia-lyase and a Lyase-inactivating System in Potato Tuber Disks

Abstract: The light induced synthesis of phenylalanine ammonia-lyase in disks cut from potato tubers is very sensitive to cycloheximide. Synthesis is inhibited 50% in disks cultured on 5 mum cycloheximide instead of water and almost completely in disks aged in the presence of 10 mum inhibitor. Inhibition is irreversible. Fresh disks exposed only 1 hour to 10 mum cycloheximide do not synthesize enzyme during the subsequent 24 hours.Normally a maximal enzyme activity develops in disks about 24 hours after being cut from the tuber. Thereafter enzyme activity declines. The disappearance of enzyme is not affected by concentrations of cycloheximide sufficient to inhibit the synthesis of enzyme initially. No disappearance of enzyme is noted during the initial phase of induction if enzyme synthesis is inhibited by cycloheximide. However, enzyme does disappear from the tissue if more than half the maximal enzyme content is allowed to form before synthesis is inhibited. If cycloheximide at a concentration 10-fold that needed to inhibit synthesis completely is added to disks after they have attained a maximal enzyme level, then subsequent loss of enzyme activity from the tissue is prevented. The initial stability of the enzyme in the absence of further synthesis and the inhibition of enzyme disappearance by high concentrations of cycloheximide suggest A) that early phases of induction involve synthesis of enzyme protein in the absence of turnover, B) that a system capable of degrading or inactivating the lyase subsequently forms in the tissue, and C) that the formation of the degrading or inactivating system requires protein synthesis.The effect of cycloheximide on uptake and incorporation of l-isoleucine-U-(14)C into soluble and insoluble proteins of tuber disks was also examined. During induction the rate of uptake increased 3 to 4-fold, and the rate of incorporation into protein, corrected for change in uptake, increased 25-fold. Cycloheximide inhibited incorporation of isoleucine-(14)C into proteins of fresh disks more than 80%. It did not prevent activation of general protein synthesis during induction and inhibited incorporation in induced disks only 20%. At all times incorporation of amino acid into the soluble, lyase-rich, protein fraction was more sensitive to cycloheximide than the insoluble fraction.

Inhibition of Chloroplasts by UV-Irradiation and Heat-Treatment

Abstract: The site of inhibition in UV-irradiated and heat-treated chloroplasts was examined by using artificial electron donor compounds such as p-phenylenediamine and hydroquinone which donated electrons specifically to photosystem II. In both cases the electron donors restored the photoreduction of nicotinamide adenine dinucleotide phosphate and the restored activity was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. The fluorescence of variable yield was eliminated by both inhibitory treatments and was partially restored by the electron donors in the heat-treated but not the UV-irradiated chloroplasts. The results suggest that the sites of inhibition of UV-radiation and heat treatment are in the photosynthetic electron transport chain between water and photosystem II.

Characterization of glyoxysomes from castor bean endosperm.

Abstract: Electron micrographs are presented which establish the identity of the components of the 3 major bands observed after sucrose density centrifugation of the crude particulate fraction from the endosperm of germinating castor bean seedlings. These are: mitochondria (density 1.19 g/cc), proplastids (density 1.23 g/cc) and glyoxysomes (density 1.25 g/cc). Further evidence is provided on the enzymatic composition of the glyoxysomes. Essentially all of the particulate malate synthetase, isocitrate lyase, catalase, and glycolic oxidase is present in these organelles. The distribution of glyoxysomal enzymes on sucrose density gradients is contrasted with that of the strictly mitochondrial enzymes fumarase, NADH oxidase, and succinoxidase. Malate dehydrogenase and citrate synthetase are present in both organelles. The functional role of glyoxysomes and their relationship to cytosomes from other tissues is discussed.

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. II. Responses to KCl Concentration and the Role of Potassium Absorption

Abstract: The stimulation by KCl of stomatal opening in isolated epidermal strips of Vicia faba was examined. In dark + normal air the opening response was maximal at 100 mm KCl while in light + CO(2)-free air it was maximal at about 10 mm KCl. CO(2)-free air was more influential than light in reducing the KCl concentration required for maximal opening. K(+) was essential while Cl(-) seemed to be of secondary importance in these processes.The use of (86)Rb(+) as a tracer for K(+) showed that the increase in stomatal aperture under various conditions was well correlated with K(+) uptake. The estimated amount of K(+) taken up by guard cells, along with a counter ion, was sufficient to account for the changes in solute potential associated with opening. It is suggested that the absorption of extracellular solutes, such as K(+), may be the primary mechanism of stomatal opening. Both opening and K(+) absorption are stimulated by light + CO(2)-free air.The increase in stomatal aperture was also well correlated with the decrease in stainable starch in guard cells under all conditions. It is suggested that this is a secondary change, although perhaps closely linked to K(+) absorption.

Ethylene, plant senescence and abscission.

Abstract: Evidence supporting the hypothesis that ethylene is involved in the control of senescence and abscission is reviewed. The data indicate that ethylene causes abscission in vivo by inhibiting auxin synthesis and transport or enhancing auxin destruction, thus lowering the diffusible auxin level. Studies with isolated leaves and explants suggest that the gas also may influence abscission by accelerating senescence and through an action on plant cell walls. Freshly prepared explants produce ethylene at a rate which must be high enough to maximally affect the tissue and this may explain why these explants (stage I) cannot respond to applied ethylene.

Protein synthesis in relation to ripening of pome fruits.

Abstract: Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. (14)C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases during the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment with cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis.

Ethylene formation in pea seedlings; its relation to the inhibition of bud growth caused by indole-3-acetic Acid.

Abstract: Indole-3-acetic acid stimulates ethylene production in the nodal region of pea stems, and the gas inhibits bud growth. At all concentrations of IAA there is a close correlation between the intensity and duration of ethylene production and the bud inhibition which results. Kinetin reverses the inhibitory actions of ethylene and IAA on bud growth. It is concluded that auxins suppress bud development by stimulating ethylene formation. The possibility that auxin induced ethylene formation controls apical dominance is considered.

The isolation of spinach chloroplasts in pyrophosphate media.

Abstract: A simplified procedure (involving disruption in sorbitol-pyrophosphate mixtures) permits the separation of spinach chloroplasts which retain the ability to catalyze the photosynthetic assimilation of carbon dioxide and its associated oxygen evolution

Effects of Kinetin, IAA, and Gibberellin on Ethylene Production, and Their Interactions in Growth of Seedlings.

Abstract: Kinetin in concentrations of 10 −8 to 10 −4 m, stimulated ethylene production in 3 and 4-day old etiolated seedlings of Alaska pea ( Pisum sativum L. var. Alaska). Seedlings of other species responded similarly. The response to kinetin depended on the age of the seedlings. Kinetin alone did not influence ethylene production in 6-day old stem sections, but it greatly increased the enhancing effect of IAA. Gibberellic acid had no effect on ethylene production by pea seedlings during the first 6 days of growth. Ethylene and gibberellic acid are antagonistic in their effects on growth of the seedlings; ethylene interfered severely with the action of gibberellic acid but did not completely suppress it. The inhibitors cycloheximide, cupferron, and N -ethylmaleimide, caused considerable inhibition of kinetin-induced ethylene production but were much less effective in the endogenous ethylene-forming system.

Seasonal Course of Photosynthesis, Respiration, and Distribution of 14C in Young Pinus resinosa Trees as Related to Wood Formation

Abstract: Rates of net photosynthesis and dark respiration, and distribution of 14 C were determined for new (current season9s) and old (previous season9s) needles at 10 times during the seasonal development of young Pinus resinosa Ait. trees. The seasonal changes in these factors associated with the development of the new shoot were related to known seasonal patterns of wood formation. Net photosynthesis per gram of needle dry weight (photosynthetic efficiency) was maximum in the old needles at the time of first new needle elongation; at the same time translocation of 14 C from old to new needles was greatest. Photosynthetic efficiency of new needles was maximum at the end of the period of rapid new needle elongation, when the new needles also began exporting much greater quantities of 14 C to other plant parts. In particular, the amount translocated from the new needles to the stem was greatly increased. At this time thick-walled xylem cells were first observed in the stem. These results, together with those of previous studies, indicate that the production of thick-walled xylem tracheids normally associated with latewood is physiologically correlated with maturation of the current season9s needles. Because there is a lesser demand for photosynthate in the new shoot and a high rate of photosynthesis in the whole plant at the time of new needle maturity, a sharply increased amount of photosynthate becomes available for wall synthesis by cambial derivatives.

Some Characteristics of Nitrate Reductase From Higher Plants

Abstract: With respect to cofactor requirements, NADH, and FMNH(2) were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH(2) required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH(2) was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH(2)).All attempts to separate the NADH(2)- and FMNH(2)-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH(2) activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH(2)-dependent activities were not additive.A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35 degrees in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.In those instances where NADH activity but not FMNH(2) activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH(2) as cofactor. However the high concentration of FMNH(2) required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).

Diurnal Pattern of Water Potential in Woody Plants

Abstract: The dynamic relationship between the rates of water loss and uptake controls plant water status. Marked diurnal variations in water potential of both leaves and fruit occurred in all plants studied. Variations in water status during the day were most clearly related to changes in evaporative demand of the air and were different for the east and west sides of a tree. At night, the plant water potential reflected the soil moisture status.Changes in the water potential of pear fruit were correlated with changes in fruit diameter. Since water loss from fruit occurred mostly through the pedicel into the xylem of the tree, the fruit could be used as a crude gauge of xylem water potential, which also showed dramatic changes during the day.

Regulation of Starch Biosynthesis in Plant Leaves: Activation and Inhibition of ADPglucose Pyrophosphorylase

Abstract: The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 65° for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate. Fructose-6-phosphate and fructose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents. The A 0.5 (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 × 10 −6 m while for the sorghum enzyme it was 3.7 × 10 −4 m. Inorganic phosphate proved to be an effective inhibitor of ADPglucose synthesis. The I 0.5 (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 × 10 −5 m (barley) to 1.9 × 10 −4 m (sorghum). This inhibition was reversed or antagonized by the activator 3-phosphoglycerate. These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis.

Growth Physics in Nitella: a Method for Continuous in Vivo Analysis of Extensibility Based on a Micro-manometer Technique for Turgor Pressure

Abstract: The view that the plant cell grows by the yielding of the cell wall to turgor pressure can be expressed in the equation: rate = cell extensibility × turgor. All growth rate responses can in principle be resolved into changes in the 2 latter variables. Extensibility will relate primarily to the yielding properties of the cell wall, turgor primarily to solute uptake or production. Use of this simple relationship in vivo requires that at least 2 of the 3 variables be measured in a growing cell. Extensibility is not amenable to direct measurement. Data on rate and turgor for single Nitella cells can, however, be continuously gathered to permit calculation of extensibility (rate/turgor). Rate is accurately obtained from measurements on time-lapse film. Turgor is estimated in the same cell, to within 0.1 atm or less, by measurement of the ability of the cell to compress gas trapped in the closed end of a capillary the open end of which is in the cell vacuole. The method is independent of osmotic equilibrium. It operates continuously for several days, over a several fold increase in cell length, and has response time of less than one minute. Rapid changes in turgor brought on by changes in tonicity of the medium, show that extensibility, as defined above, is not constant but has a value of zero unless the cell has about 80% of normal turgor. Because elastic changes are small, extensibility relates to growth. Over long periods of treatment in a variety of osmotica the threshold value for extensibility and growth is seen to fall to lower values to permit resumption of growth at reduced turgor. A brief period of rapid growth (5× normal) follows the return to normal turgor. All variables then become normal and the cycle can be repeated. The cell remains essentially at osmotic equilibrium, even while growing at 5× the normal rate. The method has potential for detailed in vivo analyses of “wall softening.”

Cell wall dissolution and enzyme secretion during leaf abscission.

Abstract: Cell wall changes leading to the formation of the separation layer during abscission of unifoliate (Phaseolus vulgaris) leaves are reviewed. Based on evidence from explants and intact plants, dissolution of pectic substances between cells of adjacent tissue regions (petiole and pulvinus) is necessary and may be sufficient to form the separation layer. Initially, the abscission zone is not structurally weak. The decline in break strength accompanying formation of the separation layer correlates with the appearance of pectinase activity. Pectinase activity is not detectable in freshly harvested explants but increases to about 0.09 mug per abscission zone at the time of 50% separation. At the same time, water extractable pectin fractions increase with a corresponding decline in the pectin fraction extractable with dilute acid. Separation is aided by internal shear forces generated by differential growth and hydrostatic pressure or both.

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds I. An Analytical Study

Abstract: Time-sequence analyses of carbohydrate breakdown in germinating rice seeds shows that a rapid breakdown of starch reserve in endosperm starts after about 4 days of germination. Although the major soluble carbohydrate in the dry seed is sucrose, a marked increase in the production of glucose and maltooligosaccharides accompanies the breakdown of starch. Maltotriose was found to constitute the greatest portion of the oligosaccharides throughout the germination stage. α-Amylase activities were found to parallel the pattern of starch breakdown. Assays for phosphorylase activity showed that this enzyme may account for much smaller amounts of starch breakdown per grain, as compared to the amounts hydrolyzed by α-amylase. There was a transient decline in the content of sucrose in the initial 4 days of seed germination, followed by the gradual increase in later germination stages. During the entire germination stage, sucrose synthetase activity was not detected in the endosperm, although appreciable enzyme activity was present in the growing shoot tissues as well as in the frozen rice seeds harvested at the mid-milky stage. We propose the predominant formation of glucose from starch reserves in the endosperm by the action of α-amylase and accompanying hydrolytic enzyme(s) and that this sugar is eventually mobilized to the growing tissues, shoots or roots.

Studies on the Mode of Action of Tomatine as a Fungitoxic Agent

Abstract: Removal of 1 or more sugar residues from the α-tomatine molecule markedly decreased its fungitoxicity. While partial hydrolysis of α-tomatine did not greatly affect its surfactant properties, it did destroy the ability of this alkaloid to form a complex with cholesterol. Only unprotonated α-tomatine was capable of binding cholesterol; the protonated form did not. Since α-tomatine was far more toxic at a high pH than at a low pH, this suggests that the unprotonated alkaloid is the active form and that it acts by complexing with fungal sterols.

Studies on Cytokinin-Controlled Bud Formation in Moss Protonemata

Abstract: Application of cytokinins to moss protonemata of the proper physiological age causes bud formation on specific cells (caulonema). During the early stages of their development, buds revert to protonemal filaments if the cytokinin has been removed by washing the protonemata. This indicates that the hormone is not acting as a "trigger" but has to be present during a critical period of time until differentiation is stabilized. Autoradiographs of protonemata treated with a labeled cytokinin, benzyladenine-benzyl-7-(14)C, show a striking accumulation of the radioactivity in caulonema cells which are in the stage of bud formation, and in the buds themselves. Cells which did not react to the hormone contained very little radioactivity. The accumulation of benzyladenine in the "target cells" may be due to the presence of binding sites which, in turn, may distinguish responding cells from non-responding ones.

Effect of Temperature, CO2 Concentration, and Light Intensity on Oxygen Inhibition of Photosynthesis in Wheat Leaves

Abstract: The effect of 21% O(2) and 3% O(2) on the CO(2) exchange of detached wheat leaves was measured in a closed system with an infrared carbon dioxide analyzer. Temperature was varied between 2 degrees and 43 degrees , CO(2) concentration between 0.000% and 0.050% and light intensity between 40 ft-c and 1000 ft-c. In most conditions, the apparent rate of photosynthesis was inhibited in 21% O(2) compared to 3% O(2). The degree of inhibition increased with increasing temperature and decreasing CO(2) concentration. Light intensity did not alter the effect of O(2) except at light intensities or CO(2) concentrations near the compensation point. At high CO(2) concentrations and low temperature, O(2) inhibition of apparent photosynthesis was absent. At 3% O(2), wheat resembled tropical grasses in possessing a high rate of photosynthesis, a temperature optimum for photosynthesis above 30 degrees , and a CO(2) compensation point of less than 0.0005% CO(2). The effect of O(2) on apparent photosynthesis could be ascribed to a combination of stimulation of CO(2) production during photosynthesis, and inhibition of photosynthesis itself.

Rapid Starch Synthesis Associated With Increased Respiration in Germinating Lily Pollen

Abstract: In vitro studies of germinating Lilium longiflorum pollen showed that starch increased more rapidly during the first 30 minutes of incubation than during the next several hours. The period of rapid starch formation coincided with the first period of high respiration. An estimate was made of the extra ATP utilized to form extra starch during the first 30 minutes, and this estimate indicates that starch synthesis accounts for a significant portion of the initial high rate of respiration. This pattern of respiration and starch synthesis was not altered when pollen germinated in a pentaerythritol medium that could not be metabolized instead of the standard sucrose medium.Sucrose was the predominant sugar in mature lily pollen. This sugar decreased 50% during several hours incubation in pentaerythritol culture medium. Reducing sugars remained low during incubation which may indicate that sucrose breakdown is regulated by the rate of utilization of hexose units.

Phosphorylases I and II of Maize Endosperm

Abstract: Two phosphorylases have been found in the endosperm of Zea mays. Phosphorylase I is found through all stages of endosperm development and seed germination investigated. The other enzyme, phosphorylase II appears only at the stage of rapid starch biosynthesis and is not found during germination. At 22 days after pollination, the activity of phosphorylase II is 10 times that of phosphorylase I. These 2 phosphorylases are separable by column chromatography and behave differently in several respects.Phosphorylase I cannot utilize maltose as a primer while phosphorylase II does so readily. Furthermore, phosphorylase II can synthesize an amylose-like polymer from a "primer free" system after a lag phase.Phosphorylase II is inhibited severely at pH 5.8 by ATP, GTP, ADP, and GDP, and less drastically by UTP, CTP, UDP and CDP. Phosphorylase I is somewhat inhibited by purine nucleotides but not by pyrimidine nucleotides. In all cases, the inhibition is pH-dependent. Phosphorylase I is inhibited competitively by ATP while phosphorylase II is inhibited non-competitively.Phosphorylase II is markedly stimulated by 10 mm Mg(2+) and by 2 mm ethylenediamine tetraacetic acid while phosphorylase I is relatively little affected.

The Respiratory Chain Components of Higher Plant Mitochondria

Abstract: TIGHTLY COUPLED MITOCHONDRIA HAVE BEEN PREPARED FROM A VARIETY OF PLANT SOURCES: white potato (Solanum tuberosum), Jerusalem artichoke (Heliantus tuberosus), cauliflower buds (Brassica oleracea), and mung bean hypocotyls (Phaseolus aureus). Mitochondria with no appreciable coupling were also prepared from skunk cabbage spadices (Symplocarpus foetidus).Room temperature difference spectra show that these mitochondria are very similar in the qualitative and quantitative composition of their electron carriers. The different cytochromes are present in the amounts of 0.1 to 0.3 mmumole per mg of mitochondrial protein. The molar ratios of the different electron carriers are, on the average: 0.7:0.7:1.0:3 to 4:10 to 15 respectively for cytochrome aa(3), cytochromes b, cytochromes c, flavoproteins, and pyridine nucleotides.From low temperature difference spectra carried out under particular experimental conditions, it can be deduced that these mitochondria contain 3 b cytochromes whose alpha bands are located at 552, 557, and 561 mmu, and 2 c cytochromes, one of which, a c(1)-like cytochrome, is firmly bound to the mitochondrial membrane. Cytochrome oxidase can be optically resolved into its 2 components a and a(3).For all kinds of mitochondria, the rates of oxidation of succinate are similar as well as the turnover of cytochrome oxidase (50-70 sec(-1)), regardless of the metabolic activities of the tissues. The number of mitochondria per cell appears to be the controlling factor of the intensity of tissue respiration.

Effect of Sterols on the Permeability of Alcohol-Treated Red Beet Tissue

Abstract: Alcohols and hydrogen peroxide altered the permeability of membranes of Beta vulgaris root cells. Generally alcohols increased the permeability of membranes without going through an induction period except methanol which required a 10- to 15-hour induction period. The membrane effect of methanol could be inhibited with CaCl(2), cholesterol, beta-sitosterol, and stigmasterol. Cholesterol was the most effective inhibitor, followed by beta-sitosterol and stigmasterol; and at the same concentration, the sterols were more effective than CaCl(2), the classic membrane stabilizer.Ergosterol increased the methanol-initiated betacyanin leakage. Since none of the tested sterols reversed the betacyanin efflux induced by hydrogen peroxide, the sterols do not apparently act as antioxidants. The results are explained in terms of sterol-phospholipid interaction, based on stereochemistry and charge distribution.

Water relations of pine seedlings in relation to root and shoot growth

Abstract: The effects of water stress on growth and water relations of loblolly and white pine seedlings were studied during series of drying cycles. As mean soil water potential decreased, growth of roots, needles, and buds decreased. Growth of roots during successive severe drying cycles was not uniform, however. A study of needle and root extension showed that of the total growth of roots for 3 7-day drying cycles, only 6% occurred during the third cycle, while needle extension was uniform for the 3 cycles. The difference in response of needles and roots to drying cycles may be attributed primarily to the effect of water stress on the growing region. When subjected to a severe stress, roots matured toward the tip and became dormant, resulting in less growth during subsequent drying cycles. The intercalary growing region of needles, however, was not altered seriously enough by the stress to cause a difference in amount of growth during each drying cycle.Transpiration of loblolly pine was lower in the second drying cycle than in the first. Needle water potential after rewatering was as high as that of control plants watered daily; root resistance was apparently not important in restricting transpiration during a second drying cycle. Needle diffusion resistance of loblolly pine, measured with a low-resistance diffusion porometer, was slightly higher during the second drying cycle than during the first. In addition, many primary needles were killed during the first period of stress. These factors contributed to the reduction of transpiration during the second drying cycle. Diffusion resistance of Coleus increased and transpiration ceased during the first drying cycle while water potential remained relatively high. After rewatering, both leaf resistance and transpiration returned to the control level, presumably because the stress during the first period of drying was not severe. The diffusion resistances observed for well-watered plants were 30 to 50 sec.cm(-1) for loblolly pine, 3 to 5 sec.cm(-1) for Coleus, and 4 to 6 sec.cm(-1) for tomato. These values agree closely with those reported by other workers.

Investigation on Photorespiration With a Sensitive 14C-Assay

Abstract: A leaf disk assay for photorespiration has been developed based on the rate of release of recently fixed (14)CO(2) in light in a rapid stream of CO(2)-free air at 30 degrees to 35 degrees . In tobacco leaves (Havana Seed) photorespiration with this assay is 3 to 5 times greater than the (14)CO(2) output in the dark. In maize, photorespiration is only 2% of that in tobacco.The importance of open leaf stomata, rapid flow rates of CO(2)-free air, elevated temperatures, and oxygen in the atmosphere in order to obtain release into the air of a larger portion of the (14)CO(2) evolved within the tissue in the light was established in tobacco. Photorespiration, but not dark respiration, was inhibited by alpha-hydroxy-2-pyridinemethanesulfonic acid, an inhibitor of glycolate oxidase, and by 3-(4-chlorophenyl)-1,1-dimethylurea (CMU), an inhibitor of photosynthetic electron transport, under conditions which did not affect the stomata. These experiments show that the substrates of photorespiration and dark respiration differ and also provide additional support for the role of glycolate as a major substrate of photorespiration. It was also shown that at 35 degrees the quantity of (14)CO(2) released in the assay may represent only 33% of the gross (14)CO(2) evolved in the light, the remainder being recycled within the tissue.It was concluded that maize does not evolve appreciable quantities of CO(2) in the light and that this largely accounts for the greater efficiency of net photosynthesis exhibited by maize. Hence low rates of photorespiration may be expected to be correlated with a high rate of CO(2) uptake at the normal concentrations of CO(2) found in air and at higher light intensities.

Abscisic Acid Levels and Seed Dormancy

Abstract: Dormant seeds from Fraxinus species require cold-temperature after-ripening prior to germination. Earlier, we found that abscisic acid (ABA) will inhibit germination of excised nondormant embryos and that this can be reversed with a combination of gibberellic acid and kinetin. Using Milborrow's quantitative "racemate dilution" method the ABA concentration in 3 types of Fraxinus seed and pericarp were determined. While ABA was present in all tissues, the highest concentration was found in the seed and pericarp of dormant F. americana. During the chilling treatment of F. americana the ABA levels decreased 37% in the pericarp and 68% in the seed. The ABA concentration of the seed of the nondormant species, F. ornus, is as low as that found in F. americana seeds after cold treatment. Experiments with exogenously added ABA solutions indicate that it is unlikely that the ABA in the pericarp functions in the regulation of seed dormancy. However, the ABA in the seed does seem to have a regulatory role in germination.

Control of abscission in agricultural crops and its physiological basis.

Abstract: Some naphthalene and phenoxy compounds prevent preharvest drop of apples, pears, and citrus fruits. These studies have been complicated by an unrecognized high level of ethylene produced by leaves and fruit on trees sprayed with these growth regulators. An apparent contradiction is the effectiveness of both 2,4-dichlorophenoxyacetic acid and n-dimethylaminosuccinamic acid (a growth retardant which retards biosynthesis of auxin) in preventing abscission of apples. Thus, in the presence of low auxin concentrations in the tissue, this growth retardant prevents fruit abscission even more effectively than 2,4-dichlorophenoxyacetic acid at high auxin concentrations in the tissue. This anomaly is clarified on the basis that n-dimethylaminosuccinamic acid, in the presence of a known low ethylene biosynthesis, delays maturity of the fruit and thus prevents fruit abscission. On the other hand, 2,4-dichlorophenoxyacetic acid prevents abscission by direct growth hormone action, in spite of the side effects of ethylene production which speeds ripening of the fruit. With the promotion of abscission of leaves and fruit of agricultural crops, attention is given to the use of chemicals which induce ethylene production when applied to the plant, but which have no growth promotion effect to retard abscission. We can distinguish 5 kinds of such chemicals. One group includes gibberellic and abscisic acids that induce treated leaves to produce ethylene and abscise (under certain circumstances). However, they do not induce ethylene production by fruit and do not promote fruit abscission. A second group includes ascorbic acid, which, when used at relatively high levels, induces fruit to produce enough ethylene to promote abscission. Ascorbic acid-treated leaves also produce ethylene but not enough to cause much defoliation. A third group includes protein-synthesis inhibitors, such as cycloheximide. When low concentrations (about 30 mumoles/l) are sprayed on the fruit, the rapid effect of the freely moving ethylene (produced by the treated fruit) appears to mask temporarily any potential effect of the slowly moving inhibitor. A fourth group includes 2-chloroethylphosphonic and cupric ethylenediaminetetracetic acids, which induce ethylene production of fruit and leaves; production by leaves is substantially greater than by fruit and substantial defoliation results. A fifth group includes the cotton defoliation chemicals which clearly produce ethylene primarily as a result of chemical injury to the leaf blade. Another group of compounds, represented by beta-hydroxyethylhydrazine, produces ethylene by a chemical reaction with formaldehyde and water, and the presence of leaves or fruit is not required. At this time we are unaware of how chemicals in groups one to four act to promote ethylene evolution in leaves and fruit, but possible biological and chemical paths of ethylene production are discussed.