Rapid [gamma]-Aminobutyric Acid Synthesis and the Inhibition of the Growth and Development of Oblique-Banded Leaf-Roller Larvae.

The hypothesis that rapid [gamma]-aminobutyrate (GABA) accumulation is a plant defense against phytophagous insects was investigated. Increasing GABA levels in a synthetic diet from 1.6 to 2.6 [mu]mol g-1 fresh weight reduced the growth rates, developmental rates, and survival rates of cultured Choristoneura rosaceana cv Harris larvae. Simulation of the mechanical damage resulting from phytophagous activity increased soybean (Glycine max L.) leaf GABA 10- to 25-fold within 1 to 4 min. Pulverizing leaf tissue resulted in a value of 2.15 ([plus or minus]0.11 SE) [mu]mol GABA g-1 fresh weight.

The Synthesis of [gamma]-Aminobutyric Acid in Response to Treatments Reducing Cytosolic pH.

[gamma]-Aminobutyric acid (GABA) synthesis (L-glutamic acid + H+ -> GABA + CO2) is rapidly stimulated by a variety of stress conditions including hypoxia. Recent literature suggests that GABA production and concomitant H+ consumption ameliorates the cytosolic acidification associated with hypoxia or other stresses. This proposal was investigated using isolated asparagus (Asparagus sprengeri Regel) mesophyll cells. Cell acidification was promoted using hypoxia, H+/L-glutamic acid symport, and addition of butyrate or other permeant weak acids. Sixty minutes of all three treatments stimulated the levels of both intracellular and extracellular GABA by values ranging from 100 to 1800%. At an external pH of 5.0, addition of 5 mM butyrate stimulated an increase in overall GABA level from 3.86 (0.56 [plus or minus] SE) to 20.4 (2.16 [plus or minus] SE) nmol of GABA/106 cell. Butyrate stimulated GABA levels by 200 to 300% within 15 s, and extracellular GABA was observed after 10 min. The acid load due to butyrate addition was assayed by measuring [14C]butyrate uptake. After 45 s of butyrate treatment, H+-consuming GABA production accounted for 45% of the imposed acid load. The cytosolic location of a fluorescent pH probe was confirmed using fluorescent microscopy. Spectrofluorimetry indicated that butyrate addition reduced cytosolic pH by 0.60 units with a half-time of approximately 2 s. The proposal that GABA synthesis ameliorates cytosolic acidification is supported by the data. The possible roles of H+ and Ca2+ in stimulating GABA synthesis are discussed.

The production and efflux of 4-aminobutyrate in isolated mesophyll cells.

The pathway of 4-aminobutyric acid (GABA) production and efflux was investigated in suspensions of mesophyll cells isolated from asparagus (Asparagus sprengeri Regel) cladophylls. Analysis of free amino acids demonstrated that, on a molar basis, GABA represented 11.4, 19, and 6.5% of the xylem sap, intact cladophyll tissue, and isolated mesophyll cells, respectively. l-Glu, a GABA precursor, was abundant in intact cladophylls and isolated cells but not in xylem sap. When cells were incubated with l-[U-(14)C]Glu, intracellular GABA contained less than 10% of the radioactivity found in intracellular Glu. However, GABA in the medium contained 78% of the radioactivity found in extracellular l-Glu metabolites. Incubation with l-[1-(14)C]Glu resulted in the appearance of unlabeled GABA, demonstrating its production through decarboxylation at carbon 1. GABA released to the medium from cells incubated with l-[U-(14)C]Glu had a specific activity of 18 nanocuries per nanomole, whereas GABA remaining in the cell had a specific activity of 2.25 x 10(-1) nanocuries per nanomole. In the presence of exogenous l-Glu, amino acid analysis and cell volume measurements indicated intracellular Ala and GABA concentrations of 4.2 and 1.4 millimolar, respectively. In the medium, however, the corresponding concentrations were 2 and 57 micromolar. The data indicate that l-Glu entering the cell is decarboxylated to GABA, and that specific and passive efflux is from this pool of recently synthesized GABA and not from a previously synthesized unlabeled pool of GABA.

Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells.

Addition of l-[U-(14)C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-(14)C]glutamate, and (c) efflux of [(14)C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H(+)/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H(+)/l-Glu symport may involve both H(+) consumption during 4-aminobutyrate production and ATP-driven H(+) efflux.

l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism?

Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H(+) efflux from the cells by 111% and the uptake of l-[U-(14)C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO(2) evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and (14)CO(2) evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas (14)CO(2) evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-(14)C]glutamate were higher in the light while rates of (14)CO(2) evolution were higher in the dark. The major labeled product of glutamate decarboxylation, gamma-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO(2) and gamma-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H(+)/l-glutamate uptake process.

A plasmamembrane redox system and proton transport in isolated mesophyll cells.

Potassium ferricyanide (K(3)Fe[CN](6)) was added to aerated and stirred nonbuffered suspensions of mechanically isolated photosynthetically competent Asparagus sprengeri Regel mesophyll cells. Rates of Fe(CN)(6) (3-) reduction and H(+) efflux were measured with or without illumination. On the addition of 1 millimolar Fe(CN)(6) (3-) to nonilluminated cell suspensions acidification of the medium indicated an H(+) efflux of 1.54 nanomoles H(+)/10(6) cells per minute. Simultaneous Fe(CN)(6) (3-) reduction occurred at a rate of 1.55 nanomoles Fe(CN)(6) (3-)/10(6) cells per minute. Illumination stimulated these rates 14 to 17 times and corresponding values were 26.1 nanomoles H(+)/10(6) cells per minute and 22.9 nanomoles Fe(CN)(6) (3-)/10(6) cells per minute. These two processes appeared to be tightly coupled and were rapidly inhibited when illuminated suspensions were transferred to darkness or treated with 1 micromolar 3-(3,4-dichlorophenyl)-1,1 dimethylurea. Addition of 0.1 millimolar diethylstilbestrol eliminated ATP dependent H(+) efflux in illuminated or nonilluminated cells but had no influence on Fe(CN)(6) (3-) dependent H(+) efflux. Recent reports indicate that a transmembrane redox system spans the plasma membrane of root cells and is coupled to the efflux of H(+). The present report extends these observations to photosynthetically competent mesophyll cells. The results indicate a transport process independent of ATP driven H(+) efflux which operates with a H(+)/e(-) stoichiometry of one.

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells.

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO(4). Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H(+)/10(6) cells.minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-(14)C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K(+) efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.

Inhibition of yeast respiration and fermentation by benomyl, carbendazim, isocyanates, and other fungicidal chemicals.

The inhibition of yeast (Saccharomyces cerevesiae) metabolism by fungicidal chemicals was investigated. Glucose- or ethanol-dependent yeast respiration was measured with an oxygen electrode, and manometric determination of carbon dioxide release was used to measure fermentation. Both respiration and fermentation were inhibited more by benomyl than by identical molar concentrations of its breakdown product, carbendazim. Butyl isocyanate, another benomyl breakdown product, inhibited respiration more but inhibited fermentation less than the parent compound. Of the isocyanates tested, hexyl isocyanate was the most inhibitory towards both activities. Captan was more active and iprodione less active than benomyl. Because benomyl rapidly broke down to carbendazim when it was prepared in 80% ethanol, only 59% of the dissolved benomyl was intact when it was added to yeast to determine its effect on respiration or fermentation.

An investigation into the role of photosynthesis in regulating ATP levels and rates of h efflux in isolated meosphyll cells.

Aerated and stirred 10-ml suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used for simultaneous measurements of net H(+) efflux and steady-state ATP levels.Initial rates of medium acidification indicated values for H(+) efflux in the light and dark of 0.66 and 0.77 nanomoles H(+)/10(6) cells per minute, respectively. When the medium pH was maintained at 6.5, with a pH-stat apparatus, rates of H(+) efflux remained constant. Darkness or DCMU, however, stimulated H(+) efflux by 100% or more. Darkness increased ATP levels by 33% and a switch from dark to light reduced ATP levels by 31%. In the absence of aeration, illumination prevented the accumulation of respiratory CO(2) and the buffering capacity of the medium was about 50% less than that found in the nonilluminated nonaerated medium. As a result, rates of pH decline were similar even though the dark rate of H(+) efflux was approximately 50% greater.Proposals that photosynthesis stimulates H(+) efflux are based on changes in the rate of pH decline. The present data indicate that photosynthesis inhibits H(+) efflux and that changes in rates of pH decline should not be equated with changes in the rate of H(+) efflux.

Disulfiram metabolism in isolated mesophyll cells and inhibition of photosynthesis and cyanide-resistant respiration.

Tetraethylthiuram disulfide (disulfiram) stimulated medium acidification when added at a concentration of 0.4 millimolar to illuminated or nonilluminated suspensions of Asparagus sprengeri Regel mesophyll cells. Similar concentrations inhibited photosynthesis and cyanide-resistant respiration. The reduction product of disulfiram, diethyldithiocarbamic acid, accumulated in concentrations sufficient to account for the observed acidification.

Stimulation of h efflux and inhibition of photosynthesis by esters of carboxylic acids.

Suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the influence of various carboxyester compounds on rates of net H(+) efflux in the dark or light and photosynthetic O(2) production. Addition of 0.15 to 1.5 millimolar malathion, alpha-naphthyl acetate, phenyl acetate, or p-nitrophenyl acetate stimulated H(+) efflux and inhibited photosynthesis within 1 minute. In contrast, the more polar esters methyl acetoacetate or ethyl p-aminobenzoate had little or no effect on either of these two processes. A 0.15 millimolar concentration of alpha-naphthylacetate stimulated the normal rate of H(+) efflux, 0.77 nanomoles H(+) per 10(6) cells per minute by 750% and inhibited photosynthesis by 100%. The four active carboxyester compounds also stimulated H(+) efflux after the normal rate of H(+) efflux was eliminated with 0.01 milligrams per milliliter oligomycin or 100% N(2). Oligomycin reduced the ATP level by 70%. Incubation of cells with malathion, alpha-naphthyl acetate, or p-nitrophenyl acetate resulted in the generation of the respective hydrolysis products ethanol, alpha-naphthol, and p-nitrophenol. It is proposed that inhibition of photosynthesis and stimulation of H(+) efflux result when nonpolar carboxyester compounds enter the cell and generate acidic carboxyl groups when hydrolyzed by esterase enzymes.

An investigation into the roles of photosynthesis and respiration in h efflux from aerated suspensions of asparagus mesophyll cells.

Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H(+) efflux. Rates varied between 0.12 and 1.99 nanomoles H(+) per 10(6) cells per minute or 3 and 40 nanomoles H(+) per milligram chlorophyll per minute. The mean rate of H(+) efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H(+) efflux from illuminated cells. Bubbling with N(2) or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H(+) efflux in light or dark. In the absence of aeration, H(+) efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO(2)-free air, rates of H(+) efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO(2) fixation but not photosynthetic CO(2) fixation. It is suggested that H(+) efflux is dependent on respiration and dark CO(2) fixation, but independent of photosynthesis.

The effect of light on the gibberellin metabolism and growth of Phaseolus coccineus seedlings.

Light inhibits the rate of stem elongation of Phaseolus coccineus L. seedlings. Gibberellin A4 (GA4), an endogenous component of Phaseolus seedlings (Bowen et al., Phytochem. 12, 2935-2941, 1973) promotes stem growth in the light but not in darkness. Dark-grown seedlings contain larger GA pools than light-grown plants. Apically applied [(3)H]GA4 in etiolated bolised more extensively in the light. The slower rate of metabolism of [(3)H]GA4 in etiolated seedlings is not a consequence of isotopic dilution by the endogenous GA4 pool or a lack of penetration of the labelled material. While it can be concluded that the capacity of seedlings to metabolise [(3)H]GA4 is greater in the light than in darkness, it does not necessarily follow that there is a more rapid rate of turnover of endogenous GA4 in light-grown tissues. The results are discussed in relation to the involvement of GAs in the inhibitory effects of ligh on stem elongation.

A Synergistic Stimulation of Avena sativa Coleoptile Elongation by Indoleacetic Acid and Carbon Dioxide.

The ability of 0.03% CO(2) to stimulate growth has been investigated using etiolated Avena coleoptile sections maintained in buffered solution. This concentration of CO(2)-stimulated growth after a lag period of 12 to 15 minutes, and a synergistic relationship between indoleacetic acid and CO(2) in stimulating growth has been demonstrated. The response to CO(2) is inhibited by cycloheximide and is lost approximately 10 minutes after exposure to CO(2)-free air. Malate can replace CO(2) in stimulating growth. In the light of these data and recent literature on a growth response of coleoptile sections to CO(2)-saturated solutions, the existence of two mechanisms of CO(2)-simulated growth is proposed. In addition, it is suggested that growth promotion by 0.03% CO(2) is mediated by a process involving dark CO(2) fixation.

The Influence of 0.03% Carbon Dioxide on Protein Metabolism of Etiolated Avena sativa Coleoptiles.

The influence of indoleacetic acid, 0.03% CO(2), and malate on protein metabolism of etiolated Avena sativa coleoptile sections has been investigated. All three were found to elevate both the rate of incorporation of labeled leucine into protein, and the level of soluble protein. The combination of indoleacetic acid and CO(2) stimulated these values in an additive or weakly synergistic manner, in contrast to the nonadditive influence of malate and CO(2). Evidence is presented that cyclo-heximide inhibited the stimulation of protein synthesis by CO(2), and that indoleacetic acid increased the incorporation of (14)C-bicarbonate into protein. These data are discussed in the context of CO(2)-stimulated growth of etiolated tissue, and proposals that CO(2)-stimulated growth involves dark CO(2) fixation.