Changes in beta-1,3-Glucan Synthase Activity in Developing Lima Bean Plants.

A plasma membrane-enriched fraction was isolated from various tissues of developing lima bean seedlings, Phaseolus lunatus var Cangreen, to study beta-1,3-glucan synthase activity changes. All tissues contained an active beta-glucan synthase, including the cotyledons that will be senescent in mature lima bean plants. Young primary leaves exhibited a very active beta-glucan synthase; but this activity dropped markedly, about fivefold, as the leaves gained weight and became photosynthetic. Some tissues, such as the hypocotyl and young stem, exhibited an increase in beta-glucan synthase activity as the tissues were growing and a decrease as the growth rate slowed. Roots exhibited a high activity early in development that only decreased slightly, about 30%, as root growth increased. Surprisingly the senescent cotyledons contained an activity equivalent to some other tissues that was maintained over our measurement time of 21 days. Perhaps this callose synthesis activity is related to translocation processes as the cotyledons transfer their reserves to the growing seedling. We concluded that beta-glucan synthase was not a good indicator of sink strength in these lima bean tissues. The plasma membrane fractions also were tested for other enzymes that might be present because an electron microscope study revealed a low contamination by other types of membranes. The membrane fractions had low but detectable activities of sucrose synthase, UDPglucose pyrophosphorylase, UDPase, alkaline invertase, and a general phosphatase; but these enzymes exhibited no consistent pattern(s) of activity change with plant development.

Fructose 2,6-bisphosphate inhibition of phosphoglucomutase.

Fructose 2,6-bisphosphate inhibits phosphoglucomutase noncompetitively with respect to the cofactor glucose 1,6-bisphosphate. Previous studies from our laboratory had shown that phosphoglucomutase was activated by fructose 2,6-bisphosphate in the absence of added glucose 1,6-bisphosphate. The fructose 2,6-bisphosphate activation previously reported was due to the presence of glucose 1,6-bisphosphate in the commercial preparation of fructose 2,6-bisphosphate.

Changes in the C-Labeled Cell Wall Components with Chase Time after Incorporation of UDP[C]Glucose by Intact Cotton Fibers.

Intact, in vitro-grown cotton fibers will incorporate [(14)C]glucose from externally supplied UDP[(14)C]glucose into a variety of cell wall components including cellulose; this labeled fraction will continue to increase up to 4 hours chase time. In the fraction soluble in hot water there was no significant change in total label; however, the largest fraction after the 30 minute pulse with UDP[(14)C]glucose was chloroform-methanol soluble (70%) and showed a significant decrease with chase. The lipids that make up about 85% of this fraction were identified by TLC as steryl glucosides, acylated steryl glucosides, and glucosyl-phosphoryl-polyprenol. Following the pulse, the loss of label from acylated steryl glucosides and glucosylphophoryl-polyprenol was almost complete within 2 hours of chase; steryl glucosides made up about 85% of the fraction at that chase time. The total loss in the lipid fraction (about 100 picomoles per milligram dry weight of fiber) with chase times of 4 hours approximates the total gain in the total glucans.

Levels of Free and Conjugated Abscisic Acid in Developing Floral Organs of the Navel Orange (Citrus sinensi [L.] Osbeck cv Washington).

The levels of abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate (putatively identified as the glucosyl ester, abscisyl-beta-d-glucopyranoside) were determined by enzyme immunoassay in the organs of developing navel orange (Citrus sinensis [L.] Osbeck cv Washington) flowers. Although both compounds were detected in every tissue, developmentally related differences between organs in the total and relative contents were observed. The highest ABA levels were observed in the stigma/style shortly after anthesis (11.5 +/- 0.6 nanomoles ABA per gram fresh weight and 4.8 +/- 0.6 nanomoles ABA-conjugate per gram fresh weight); whereas, the highest ABA-conjugate levels were observed at the same time in the floral disc (hypogynous disc plus calyx; 3.5 +/- 0.1 nmol nanomols ABA per gram fresh weight and 11.8 +/- 0.9 nanomoles ABA-conjugate per gram fresh weight). These results suggest that differences in ABA content reflect tissue-specific variation in the facility for ABA conjugation. Increased ABA levels were observed in the stigma/style near anthesis; however, a relationship with pollination is discounted, since ;Washington' navel orange flowers are male sterile and devoid of pollen.

The Occurrence of Abscisic Acid and Abscisyl-beta-d-Glucopyranoside in Developing and Mature Citrus Fruit as Determined by Enzyme Immunoassay.

The contents of (+)-cis-abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate(s) were analyzed by means of enzyme immunoassay in partially purified extracts of developing and mature sweet orange fruit (Citrus sinensis [L.] Osbeck cv Washington navel). A relatively small increase in ABA was observed in the fruit exocarp during the natural color transition from green to orange. At the same time, the ABA-conjugate level increased approximately 12-fold in this tissue. The contents of ABA and ABA-conjugate equaled 15.0 +/- 0.7 and 107.8 +/- 2.1 nanomoles per gram fresh weight, respectively, in the exocarp at harvest. Other tissues also contained considerable quantities of these compounds. Whereas the highest ABA content was observed in the exocarp, the highest ABA-conjugate content was observed in the central vascular axis of the fruit and equaled 187.0 +/- 10.3 nanomoles per gram fresh weight. The only immunoreactive conjugate found in significant quantity in mature fruit was identified as abscisyl-beta-d-glucopyranoside (ABA-GE) based on (a) immunological cross-reactivity, (b) thin layer chromatography co-chromatography with authentic standards in two solvent systems, (c) susceptibility to both chemical and enzymic degradation, and (d) mass spectroscopy.

Incorporation of UDPGlucose into Cell Wall Glucans and Lipids by Intact Cotton Fibers.

The [(14)C] moiety from [(3)H]UDP[(14)C]glucose was incorporated by intact cotton fibers into hot water soluble, acetic-nitric reagent soluble and insoluble components, and chloroform-methanol soluble lipids; the [(3)H] UDP moiety was not incorporated. The (3)H-label can be exchanged rapidly with unlabeled substrate in a chase experiment. The cell wall apparent free space of cotton fibers was in the order of 30 picomoles per milligram of dry fibers; 25 picomoles per milligram easily exchanged and about 5 picomoles per milligram more tightly adsorbed. At 50 micromolar UDPglucose, 70% of the [(14)C]glucose was found in the lipid fraction after both a short labeling period and chase. The percent of [(14)C]glucose incorporated into total glucan increased slightly with chase, but the fraction of total glucans incorporated into insoluble acetic-nitric reagent (cellulose) did increase within a 30-minute chase period. The data supports the concept that glucan synthesis, including cellulose, as well as the synthesis of steryl glucosides, acetylated steryl glucosides, and glucosyl-phosphoryl-polyprenol from externally supplied UDPglucose occurs at the plasma membrane-cell wall interface. The synthase enzymes for such synthesis must be part of this interfacial membrane system.

In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate.

The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K(a)) values were obtained for each bisphosphate were: for glucose 1-6-P(2), 0.5 to 1.8; fructose 2,6-P(2), 6 to 11.7; and fructose 1,6-P(2), 7 micromolar, respectively. Fructose 2,6-P(2) is known to occur in both plant and animal tissues at changing levels encompassing the K(a) values found in this study; hence, these results implicate fructose 2,6-P(2) as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P(2) has not been found in plants, and the method for measuring glucose 1,6-P(2) by monitoring the activation of phosphoglucomutase is not specific.

Effects of Boron Deficiency on Rubidium Uptake and Photosynthesis in the Diatom Cylindrotheca fusiformis.

Culturing the diatom Cylindrotheca fusiformis under boron-deficient conditions leads to changes in (86)Rb uptake and photosynthesis prior to any effect on the rate of cell division. The influx rate of (86)Rb into boron-deficient cells was 79% of the control rate after 5 to 5.5 hours culture. Despite lowered (86)Rb influx, however, boron-deficient diatoms accumulated more (86)Rb than did control cells; this was due to the deficient cells' lower efflux rate. After 24 hours culture, boron-deficient cells had accumulated 30% more (86)Rb than had control cells, while releasing (86)Rb at only one-half the control rate. Increased photosynthetic rates were another effect of boron deficiency during this early stage of culture. Prior to 20 hours boron-deficient culture, diatoms had photosynthetic rates 37% greater than those of control cells. Corresponding to the increase in photosynthesis, boron-deficient diatoms had 12% more carbohydrate than control cells after 16 hours culture.

Pyrimidine Pathway in Boron-deficient Cotton Fiber.

Cotton ovules cultured in an insufficiency of boron (10 micromolar), showed inhibition of fiber growth by the ninth day in culture. Averaging data from eight to eleven days of culture under these conditions, total incorporation of [6-(14)C]orotic acid into fiber was inhibited by 59%. Inhibition was evident in all radioactively labeled pools, indicating that the effect may be at the membrane transport level or at an early stage of orotic acid metabolism. On a per cent basis, incorporation into RNA under boron deficiency was higher than under sufficiency. The effect is greater on the eighth day of culture, with a decreasing difference from controls up to the eleventh day. Conversely, the per cent incorporation into UDP-glucose was lower under boron deficiency than in controls, having a more or less constant value from 8 to 11 days of culture. Thus, a primary event of boron deficiency in cotton fiber culture is an alteration in the flow of metabolites through the pyrimidine synthesis pathway.

Effect of Boron on the Incorporation of Glucose from UDP-Glucose into Cotton Fibers Grown in Vitro.

Boron is required for fiber growth and development in cotton ovules cultured in vitro. Incorporation of [(14)C]glucose by such fiber from supplied UDP-[(14)C]glucose into the hot alkali-insoluble fraction is rapid and linear for about 30 minutes. Incorporation of [(14)C]glucose from such substrate by fibers grown in boron-deficient ovule cultures is much less than in the case with fibers from ovules cultured with boron in the medium. Total products (alkali-soluble plus alkali-insoluble fractions) were also greater in fibers from ovules cultured with boron. The fraction insoluble in acetic-nitric reagent was a small part of the total glucans; however, in the boron-sufficient fibers, there was significantly more of this fraction than in fibers from boron-deficient ovule cultures. The hot water-soluble glucose polymers from the labeled fibers had a significant fraction of the total [(14)C]glucose incorporated from UDP-[(14)C]glucose. Both beta-1,4- and beta-1,3- water-soluble polymers were formed in the boron-sufficient fibers, whereas the same water-soluble fraction from the boron-deficient fibers was predominantly beta-1,3-polymers. The incorporation of [(14)C]glucose from GDP-[(14)C]glucose by the fibers attached to the ovules was insignificant.

Interaction of boron with components of nucleic Acid metabolism in cotton ovules cultured in vitro.

Cotton (Gossypium hirsutum L.) ovules grown in a defined nutrient medium undergo normal morphogenesis, including fiber production. In identical medium lacking boron, ovules callus and accumulate brown substances. Boron deficiency-like symptoms were induced by 6-azauracil and 6-azauridine in ovules growing in boron-sufficient media. Other nucleoside base analogs either reduced or had no effect on over-all growth, but did not cause typical boron-deficient callus growth of cotton ovules. Orotic acid and uracil countered the effects of 6-azauracil. Actinomycin D, fluorodeoxyuridine, and ethidium bromide reduced not only fiber production on ovules growing in boron-sufficient media but also callusing of ovules in boron-deficient media.Similarities between symptoms of boron deficiency and 6-azauracil injury, and the ability of uracil to suppress both, suggest that boron deficiency symptoms are related to reduced activity in the pyrimidine biosynthetic pathway. Growth inhibition by most nucleoside base analogs tested, actinomycin D, fluorodeoxyuridine, and ethidium bromide, as compared to callusing brought on by boron deficiency and 6-azauracil, indicates that boron deficiency symptoms are not related to a reduction in nucleic acid biosynthesis. Based on this information, a discussion of the possibility that boron deficiency causes reduced synthesis of UDP-glucose is presented.

Boron Deficiency in Unfertilized Cotton (Gossypium hirsutum) Ovules Grown in Vitro.

Boron deficiency and phytohormone interactions have been studied in unfertilized cotton (Gossypium hirsutum) ovules grown in vitro. Such ovules required exogenous indoleacetic acid and/or gibberellic acid for fiber elongation. Boron also was required for maintenance of fiber elongation and normal morphogenesis throughout 14 days of culture. The amount of exogenous boron necessary for maximum fiber elongation varied among experiments, presumably in relation to endogenous boron levels at anthesis. Some ovular epidermal cells distant from the liquid medium could be induced to elongate into fiber even after 6 days in boron-deficient medium in response to the later addition of boron.Boron deficiency, in the presence of exogenous indoleacetic acid, was characterized by lack of fiber development on the inundated ovular surface and reduced fiber growth on the ovular surface exposed to air. In the presence of gibberellic acid, boron deficiency was characterized by complete absence of fiber and callusing of the entire ovular surface. When both indoleacetic acid and gibberellic acid were added, the lack of boron resulted in proliferation of callus laterally and upward from the inundated epidermis, accumulation of brown pigments (presumably phenolic compounds) in the callus, and restriction of fiber to a small area of the upper ovular surface.

Observations on the mechanism of copper damage in chlorella.

Addition of excess copper to nongrowing cells of a normal, green Chlorella caused a reduction in total pigments and a blue shift of chlorophyll absorption, concurrent with the inhibition of photosynthesis. Chlorophylless yellow and white mutant strains of the same alga showed a rise in nonspecific absorption (i.e., change in light scatter) within 5 to 10 minutes after the addition of CuSO(4); concomitantly a lowering of packed cell volume and a rise in respiration occurred. Glutathione prevented all copper-induced changes, whereas MnCl(2) protected only partially. Selective inhibition of some responses to copper was observed when O(2) was absent or an antioxidant present.

Inhibitory effect of peroxyacetyl nitrate on cyclic photophosphorylation by chloroplasts from black valentine bean leaves.

The inhibitory effect of peroxyacetyl nitrate on the cyclic photophosphorylation of chloroplasts isolated from Black Valentine variety bean leaves (Phaseolis vulgaris L.) has been studied. Peroxyacetyl nitrate caused inhibition to photophosphorylation, in either the dark or the light, by affecting the chloroplast. Evidence is presented which suggests that peroxyacetyl nitrate could oxidize sulfhydryl groups on enzymes necessary for photophosphorylation. The inhibition to photophosphorylation caused by peroxyacetyl nitrate cannot be reversed by glutathione, even when added in large amounts, whereas the inhibition to photophosphorylation caused by para-chloromercuriphenylsulfonic acid is easily reversed by small quantities of glutathione. This suggests that if peroxyacetyl nitrate is oxidizing sulfhydryl groups necessary for photophosphorylation, this oxidation is proceeding beyond the disulfide state.

CO(2) Metabolism in Corn Roots. I. Kinetics of Carboxylation and Decarboxylation.

The kinetics of (14)CO(2) carboxylation and decarboxylation in corn root tips were determined to ascertain the sequence of product formation and subsequent utilization, and to obtain further evidence to predict the enzymes mediating the carboxylation and decarboxylations. The carboxylation data indicated that the first product was oxaloacetate followed by malate and aspartate. Malate was the first stable product which could be detected. Decarboxylation data indicated that a large fraction of the (14)CO(2) release and turnover of (14)C was accountable for by a decrease in malate: however, essentially all labeled amino acids turned over rapidly and at a greater rate than organic acids. The data generally support the hypothesis that CO(2) fixation in corn root tips is via P-enolpyruvate carboxylase and malic dehydrogenase and that subsequent malate metabolism is for the most part by direct decarboxylation, possibly by the malic enzyme.