Site of action of chlorsulfuron: inhibition of valine and isoleucine biosynthesis in plants.

The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I(50) values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.

Herbicide-resistant mutants from tobacco cell cultures.

Several mutants resistant to the herbicides chlorsulfuron and sulfometuron methyl were isolated form cultured cells of Nicotiana tabacum. Resistance was inherited as a single dominant or semidominant mutation in all cases. Linkage analysis of six mutants identified two unlinked genetic loci. Studies of plants homozygous for one mutation showed the mutant plants to be completely resistant to treatment with a concentration of chlorsulfuron 100 times higher than that which produces symptoms of phytotoxicity on normal plants.

The Azolla-Anabaena azollae Relationship : XI. PHYCOBILIPROTEINS IN THE ACTION SPECTRUM FOR NITROGENASE-CATALYZED ACETYLENE REDUCTION.

Visible absorption spectra are presented for the Azolla caroliniana Willd.-Anabaena azollae Strass. association and the individual partners. Although absorption by the phycobiliproteins of the endophytic cyanobacterium clearly complements the absorption by the fern pigments, their contribution to the absorption spectrum of the association is effectively concealed by the preponderance of the Azolla pigments. Action spectra for nitrogenase-catalyzed C(2)H(2) reduction in both the Azolla-Anabaena association and the endophytic Anabaena demonstrate that quanta absorbed by the phycobiliproteins is as effective as that absorbed by chlorophyll a in driving this photosystem I-linked process. Under anaerobic conditions, the inhibition of photosystem II activity by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron did not selectively decrease the relative quantum yields in the region of phycobiliprotein absorption. At the well-below saturating light intensities used for the action spectra studies, the absolute rates of C(2)H(2) reduction were increased uniformly via respiratory-linked processes under aerobic conditions. The occurrence of phycobiliproteins in heterocysts of the endophytic Anabaena was demonstrated using fluorescence microscopy of intact filaments. Fluorescence micrographs of Anabaena cylindrica filaments are presented for comparison.

Effects of norepinephrine and 5-hydroxytryptamine reuptake inhibitors on electrically-induced spinal cord seizures in rats.

Nisoxetine and desipramine (inhibitors of norepinephrine reuptake) each exerted a suppressant effect on spinal cord seizures by decreasing the duration of tonic extension. In addition, desipramine increased the duration of tonic flexion and nisoxetine decreased the total duration of seizure. In contrast, citalopram (an inhibitor of 5-hydroxytryptamine reuptake) did not affect any of these seizure components. These observations support the concept that spinal cord noradrenergic, but not 5-hydroxytryptaminergic neurons act as attenuators of convulsive activity.

Effects of fluoxetine on electrically-induced spinal cord seizures in rats.

The effects of fluoxetine [an inhibitor of 5-hydroxytryptamine (5-HT) reuptake] on electrically-induced spinal cord seizures were determined by monitoring alterations in the total duration of seizure, as well as the durations of tonic flexion and extension. The capacity of fluoxetine to inhibit 5-HT reuptake in the spinal cord was confirmed by measurements of 5-hydroxyindoleacetic acid (5-HIAA) content. When given in a dose of 10 mg/kg, fluoxetine failed to produce an effect on spinal cord seizures or on 5-HIAA content. However, a dose of 20 mg/kg did significantly reduce 5-HIAA content although it did not alter seizure activity. These results further support our earlier suggestion that 5-HT containing neurons in the spinal cord do not modulate electrically-induced seizures.

The effects of reserpine, iproniazid and L-dopa on electrically-induced spinal cord seizures.

The effects of selected drug treatments on spinal cord norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) were compared to the effects of these same treatments on electrically-induced spinal cord seizure. Depletion of monoamine stores by reserpine facilitated spinal cord seizures. In contrast, L-DOPA, given to animals pretreated with iproniazid, exerted an anticonvulsant effect and elevated spinal cord NE and DA levels. L-DPOA administered alone produced a substantial elevation in DA levels but had no effect on spinal cord seizures. Iproniazid had no effect on monoamines, whereas it facilitated seizure activity. These observations support the concept that spinal cord noradrenergic, but not 5-hydroxytryptaminergic or dopaminergic neurons act as attenuators of convulsive activity. The effect of iproniazid on spinal cord seizures, in the absence of an observed alteration in monoamine levels, provides evidence that this effect is mediated through non-monoaminergic mechanisms.

Azolla-Anabaena Relationship: VIII. Photosynthetic Characterization of the Association and Individual Partners.

Photosynthesis in the Azolla-Anabaena association was characterized with respect to photorespiration, early products of photosynthesis, and action spectra. Photorespiration as evidenced by an O(2) inhibition of photosynthesis and an O(2)-dependent CO(2) compensation concentration was found to occur in the association, and endophyte-free fronds, but not in the endophytic Anabaena. Analysis of the early products of photosynthesis indicated that both the fern and cyanobacterium fix CO(2) via the Calvin cycle. The isolated endophytic Anabaena did not release significant amounts of amino acids synthesized from recently fixed carbon. The action spectra for photosynthesis in the Azolla-Anabaena association indicated that the maximum quantum yield is between 650 and 670 nanometers, while in the endophyte the maximum is between 580 and 640 nanometers. Although the endophytic cyanobacterium is photosynthetically competent, any contribution it makes to photosynthesis in the intact association was not apparent in the action spectrum.

Azolla-Anabaena Relationships: VII. Distribution of Ammonia-assimilating Enzymes, Protein, and Chlorophyll between Host and Symbiont.

The N(2)-fixing Azolla-Anabaena symbiotic association is characterized in regard to individual host and symbiont contributions to its total chlorophyll, protein, and levels of ammonia-assimilating enzymes. The phycocyanin content of the association and the isolated blue-green algal symbiont was used as a standard for this characterization. Phycocyanin was measured by absorption and fluorescence emission spectroscopy. The phycocyanin content and total phycobilin complement of the symbiotic algae were distinct from those of Anabaena cylindrica and a free-living isolate of the Azolla endophyte. The algal symbiont accounted for less than 20% of the association's chlorophyll and protein. Acetylene reduction rates in the association (based solely on the amount of algal chlorophyll) were 30 to 50% higher than those attained when the symbiont was isolated directly from the fern. More than 75% of the association's glutamate dehydrogenase and glutamine synthetase activities are contributed by the host plant. The specific activity of glutamate dehydrogenase is greater than that of glutamine synthetase in the association and individual partners. Both the host and symbiont have glutamate synthase activity. The net distribution of these enzymes is discussed in regard to the probable roles of the host and symbiont in the assimilation of ammonia resulting from N(2) fixation by the symbiont.

Effects of Ro 4-1284 on electrically-induced spinal cord seizures and on spinal cord norepinephrine and 5-hydroxytryptamine levels.

The time course effects of the benzoquinolizine Ro 4-1284 on spinal cord norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels were compared to the effects of this same drug on electrically-induced spinal cord seizures. The data show that a significant decrease in spinal cord NE levels and a facilitating effect on spinal cord seizures are apparent 15 minutes after Ro 4-1284 (10 mg/kg s.c.) and that both of these effects persist for at least 24 hours. Forty-eight hours after injection, the effects of Ro 4-1284 on seizure and on NE levels are completely dissipated. A significant decrease in 5-HT levels is not apparent until 1 hour after Ro 4-1284. These data suggest that noradrenergic neurons of the spinal cord act as attenuators of seizure activity. The possibility that spinal cord 5-HT also subserves a seizure attenuating function is not precluded.

Oxaloacetate as the Source of Carbon Dioxide for Photosynthesis in Bundle Sheath Cells of the C(4) Species Panicum maximum.

3-Mercaptopicolinic acid (3-MPA), an inhibitor of phosphoenolpyruvate carboxykinase, was employed to study the role of organic acid decarboxylation during C(4) photosynthesis. Treatment of detached Panicum maximum leaves with 5 mm 3-MPA inhibited photosynthesis 70 to 75%. Oxygen was found to have no effect on the degree of inhibition. The postillumination (14)CO(2) burst associated with P. maximum photosynthesis was almost abolished by 5 mm 3-MPA. The turnover rates of malate and aspartate during C(4) photosynthesis were severely reduced as well as the rates of formation of C(3) cycle intermediates in P. maximum leaves treated with 3-MPA. These results are interpreted as direct evidence for the fixation of CO(2), arising from the decarboxylation of oxaloacetate, by the C(3) cycle in bundle sheath cells of P. maximum leaves.

Characterization of Phosphoenolpyruvate Carboxykinase from Pineapple Leaves Ananas comosus (L.) Merr.

Phosphoenolpyruvate carboxykinase has been partially purified from pineapple (Ananas comosus [L.]) leaves. Specific activities obtained show it to be a major activity in this tissue. Above 15 C, the respective activation energies for decarboxylation and carboxylation are 13 and 12 kcal/mol. Below 15 C, there are discontinuities in Arrhenius plots with an associated large increase in activation energy. The adenine nucleotides are preferred to other nucleotides as substrates. The apparent Km values in the carboxylation direction are: ADP 0.13 mm, HCO(3) (-) 3.4 mm, and phosphoenolpyruvate 5 mm. In the decarboxylation direction, the apparent Km values are: ATP 0.02 mm, ADP 0.05 mm, and oxaloacetate 0.4 mm. The decarboxylation activity had an almost equal velocity with either ADP or ATP. The pH optima are between 6.8 and 7. Inhibition of the carboxylation reaction by ATP, pyruvate, and carbonic anhydrase was demonstrated. Decarboxylase specific activities are over twice carboxylation activities. The data support a model in which phosphoenolpyruvate carboxykinase is of physiological significance only during the light period and then only as a decarboxylase.

Characterization of Phosphoenolpyruvate Carboxykinase from Panicum maximum.

Phosphoenolpyruvate carboxykinase, EC 4.1.1.32 (PEPCK), was purified 43-fold from the grass Panicum maximum. Michaelis constants (Km) were determined for the exchange reaction, the carboxylation reaction, and the decarboxylation reaction. The Km values for oxaloacetate and ATP in the decarboxylation reaction were found to be lower than the Km values for the substrates used in the exchange reaction and in the carboxylation reaction. Phosphoenolpyruvate carboxylase was not detectable in the purified PEPCK preparation.Studies on the nucleotide specificity of the oxaloacetate decarboxylation reaction indicate that ATP serves as the best nucleotide for this reaction and that ADP is about 60% as effective as ATP. The pH optimum for decarboxylase activity is near 6.8. The decarboxylation reaction has a divalent cation requirement with both Mn(2+) and Mg(2+) needed for full activity.Temperature curves of the three PEPCK reactions indicate optimum activities between 38 and 45 C. There is a pronounced drop in the decarboxylation and carboxylation activities as the temperature is decreased from these optima. Below 30 C the energy of activation was 8.2 kcal/mol for the decarboxylation reaction.These studies are consistent with the proposal that under physiological conditions PEPCK catalyzes the decarboxylation of oxaloacetate in the bundle sheath cells of Panicum maximum leaves during C(4) dicarboxylic acid photosynthesis.

Inhibition of oxalacetate decarboxylation during C4 photosynthesis by 3-mercaptopicolin acid.

3-Mercaptopicolinic acid specifically inhibits phosphoenolpyruvate carboxykinase in leaves of the C4 plant Panicum maximum. Both the ATP- and ADP-dependent decarboxylation of oxalacetate and the carboxylation activity of phosphoenolpyruvate carboxykinase are inhibited by 3-mercaptopicolinic acid while phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase are not inhibited. 3-Mercaptopicolinic acid inhibits the fixation of 14CO2 by illuminated P. maximum bundle sheath strands which is dependent upon oxalacetate and ATP but does not affect C3 photosynthesis in bundle sheath strands nor C4 photosynthesis in mesophyll cells. 3-Mercaptopicolinic acid treatment reduced P. maximum leaf photosynthesis 25% while raising the photosynthetic CO2 compensation point from near zero to 18 to 45 mul of CO2/liter of air.