Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.     

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K_m values for GSSG and NADPH were 23 μm and 4.9 μm, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.     

Allelopathic potential of sweet potato (Ipomoea batatas) germplasm resources of Yunnan Province in southwest China

A laboratory bioassay was conducted to determine the allelopathic potentials of aqueous extracts from either roots or leaves of seventeen sweet potato [Ipomoea batatas L. (Lam)] cultivars (SP0, SP1, SP2, SP3, SP4, SP5, SP6, SP7, SP9, SP10, SP11, SP13, SP14, SP15, SP16, SP18, and SP19). Most inhibitory rates on Lactuca sativa calculated for leaf or root extracts from the seventeen sweet potato cultivars exhibited positive values and significantly increased with increasing concentration. Germination was totally inhibited at a concentration of 0.05 g·mL^?1 for leaf water extracts of SP13, SP15, SP18 and at a concentration of 0.05 g·mL^?1 for both leaf and root water extracts of SP19. Inhibition of root length was clearly greater than inhibition of shoot length for both leaf and root water extracts. Biomass inhibition increased with increasing concentration, but some cultivars showed stimulatory effects at low concentrations, and inhibition was generally more pronounced for root water extracts than for leaf water extracts. Moreover, most synthetical inhibitory rates for both leaf and root water extracts from the seventeen cultivars exhibited positive values and significantly increased with increasing concentration. Comparing the synthetical inhibitory rates for both leaf and root water extracts among the seventeen cultivars, SP19, SP6, SP11, and SP7 had the highest allelopathic inhibition. The inhibitory activity on germination index was the greatest, followed by germination rate, root length, biomass, and shoot length in all bioassays. Inhibition by leaf water extracts was generally greater than inhibition by root water extracts, except in the case of shoot length or biomass. Overall, we conclude that all seventeen sweet potato cultivars have strong inhibitory effects on L. sativa, but that these effects vary with cultivar and plant part, with SP19, SP6, SP11, and SP7 exhibiting the highest rates of allelopathic inhibition.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

Loss of phytochrome photoreversibility in vitro: I. Extraction and partial purification of killer

Crude pea (Pisum sativum L. var. Alaska) phytochrome extracts contain a substance, “Killer,” which interacts with the far red-absorbing form of phytochrome causing a net loss of spectrophotometrically detectable phytochrome in vitro. Killer is absent from crude extracts of Avena phytochrome, is separable from pea phytochrome by gel filtration, and is alcohol-extractable from etiolated pea seedlings. Killer activity in alcohol extracts behaved, during partial purification, in a manner identical to that derived from pea phytochrome preparations. The mass extraction and partial purification of Killer are described.     

Zinc, Iron, and Chlorophyll Metabolism in Zinc-toxic Corn

Zinc toxicity and Zn-Fe interactions were studied in corn (Zea mays L. var. Barbecue hybrid) grown in hydroponic culture. High Zn greatly reduced the root and shoot fresh weights; increasing Fe largely, but not completely, restored normal growth. Correlation analyses of root and leaf Zn and Fe contents suggested that Zn may interfere with the translocation of Fe; however, Zn toxicity was not associated with a diminished leaf Fe content. Fe did appear to retard both the absorption and the translocation of Zn. The chlorosis of Zn-toxic plants is not attributable to diminshed total leaf Fe; however, this chlorosis is relieved by increasing nutrient Fe. Zn and Fe probably do interact at some site.     

Synthesis and degradation of nitrite reductase in pea leaves

We have shown in a previous publication (Gupta, Beevers 1983 J Exp Bot 34: 1455-1462) that the level of extractable nitrite reductase activity in pea (Pisum sativum cv Burpeeana) leaves is subject to environmental perturbations. In the current study, we have used rocket immunoelectrophoresis to quantitate the level of nitrite reductase protein in extracts from pea plants subjected to various environmental treatments. The level of nitrite reductase cross-reacting material closely followed the level of assayable nitrite reductase activity. The environmental conditions which enhanced the level of extractable nitrite reductase activity resulted in an increased level of nitrite reductase cross-reacting material in the extracts. In contrast, environmental conditions which resulted in a decrease in the level of extractable nitrite reductase activity produced a decline in cross-reacting material. These results indicate that the environmentally induced modulation of extractable nitrite reductase activity involves alteration of enzyme level and is not mediated by a reversible activation-inactivation of the existing enzyme.     

In vitro and in vivo antimalarial activity and cytotoxicity of extracts,fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.     

Estimation of pyruvate,phosphate dikinase activity in maize leaf tissue by (phosphoenolpyruvate plus pyrophosphate)-dependent phosphorylation of AMP

Crude extracts of maize leaf tissue catalysed the phosphorylation of AMP by ³²PPi in the presence of phosphoenolpyruvate (PEP). The reaction was enhanced by F^? and NH₄⁺. The optimum concentrations of AMP, PEP and PPi were 0.3, 10 and 1 mM, respectively. Under these conditions, ca75% of the AMP phosphorylated by ³²PPi was present as ATP and ca25 % as ADP. The activity was reversibly cold labile. The specific activity of crude extracts in the presence of F^? was proportional to enzyme concentration only at protein concentrations < 25,μg/ml. Partially purified pyruvate, phosphate dikinase (PPD) from maize leaf quantitatively phosphorylated AMP to ATP in a (PEP plus PPi)-dependent reaction with the concomitant production of 0.9 mol of pyruvate per mol of AMP phosphorylated. It was concluded that (PEP plus PPi)-dependent phosphorylation of AMP provides a reliable method for estimating PPD activity in crude extracts of maize. Crude maize extracts also catalysed ³²Pi-ATP and ³²PPi-ATP exchange but these activities were not specific for PPD.     

Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [¹⁴C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [¹⁴C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.

Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

     

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

Summary Eleven green individuals were isolated when 95000 M₂ plants of barley (Hordeum vulgare L.), mutagenised with azide in the M₁, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F₂ progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.     

Inhibition of Rhizobium etli Polysaccharide Mutants by Phaseolus vulgaris Root Compounds

Crude bean root extracts of Phaseolus vulgaris were tested for inhibition of the growth of several polysaccharide mutants of Rhizobium etli biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher levels in roots and nodules than in stems or leaves. Inoculation with either wild-type or polysaccharide-deficient R. etli did not appear to affect the level of activity. Sequential extractions of the crude root material with petroleum ether, ethyl acetate, methanol, and water partitioned inhibitory activity into each solvent except methanol. The major inhibitors in the petroleum ether and ethyl acetate extracts were purified by C₁₈ high-performance liquid chromatography. These compounds all migrated very similarly in both liquid and thin-layer chromatography but were distinguished by their mass spectra. Absorbance spectra and fluorescence properties suggested that they were coumestans, one of which had the mass spectrum and nuclear magnetic resonances of coumestrol. These results are discussed with regard to the hypothesis that one role of rhizobial polysaccharides is to protect against plant toxins encountered during nodule development.     

Radioimmunoassay for the quantitative determination of scopolamine

A radioimmunoassay for the determination of pmol amounts of the tropane alkaloid scopolamine has been developed. The assay uses tritiated [N-C³H₃]scopolamine of high specific activity (0.67 Ci/mmol) as tracer. The measuring range of the assay extends from 0.5 to 50 ng of scopolamine, and as little as 200 pg may be detected. The antiserum raised against a conjugate of scopolamine-N-β-propionic acid-human serum albumin is highly specific, and neither hyoscyamine, 6-hydroxyhyoscyamine, scopine, tropic acid nor other related alkaloids interfere in the scopolamine determination in crude plant extracts. This assay allows for the first time the rapid, sensitive and precise (CV = 2.5 %) determination of this alkaloid in unpurified extracts of scopolamine-containing plants. The distribution of scopolamine in Datura plants, as well as its diurnal changes in leaf concentrations, has been investigated in detail and a preliminary survey on the variability of scopolamine leaf concentrations in a population of Datura sanguinea plants is given.     

Proteins and endotoxin in house dust mite extracts modulate cytokine secretion and gene expression by dermal fibroblasts

House dust mite extracts used for diagnostic tests and immunotherapy contain bioreactive molecules including proteins and endotoxin. These extracts can influence the cytokine secretion and adhesion molecule expression by cells in the skin and lung airways. The aim of this study was to determine the role of proteins and endotoxin in mite extracts in modulating gene expression and cytokine secretion by human dermal fibroblasts. Cultured normal human dermal fibroblasts were stimulated with whole mite extracts, mite extracts boiled to denature proteins, or mite extracts treated with polymyxin B to inactivate lipopolysaccharide. Gene expression and secretion of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) were determined after 6 h of stimulation. Whole Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei extracts induced dose-dependent IL-6 and IL-8 secretion. In addition, D. farinae and E. maynei induced secretion of MCP-1. Dermatophagoides farinae and E. maynei also induced parallel cytokine gene expression. Cells stimulated with boiled D. farinae extract showed moderate to marked reductions in IL-6 and IL-8 secretion. In contrast, boiled D. pteronyssinus and E. maynei extracts induced equal or greater cytokine secretions than untreated extracts. The stimulating properties were reduced for all three extracts following treatment with polymyxin B. Our data suggest that both endotoxin and proteins in mite extracts modulate the secretion of cytokines by dermal fibroblasts. The biological activities of D. farinae, D. pteronyssinus, and E. maynei extracts are not equivalent. There appears to be a lipopolysaccharide-binding protein in some mite extracts.     

Lack of Types 1 and 2A Protein Serine(P)/Threonine(P) Phosphatase Activities in Chloroplasts

Protein phosphatase activity in crude leaf extracts and in purified intact chloroplasts of wheat (Triticum aestivum) and pea (Pisum sativum) was analyzed using exogenously supplied phosphoproteins or endogenous thylakoid proteins. Leaf extracts contain readily detectable amounts of protein phosphatase activity measured with either phosphohistone or phosphorylase a, substrates of mammalian protein phosphatases. No significant chloroplast protein phosphatase activity was detected using these exogenous phosphoproteins. The dephosphorylation of endogenous thylakoid light-harvesting chlorophyll a/b binding proteins in situ was inhibited by fluoride, but not by microcystin-LR or okadaic acid, diagnostic inhibitors of mammalian types 1 and 2A protein phosphatases. Additionally, no evidence for a pea chloroplast alkaline phosphatase activity was found using β-glycerolphosphate or 4-methylum-belliferyl phosphate as substrates. From these results, we conclude that phosphohistone and phosphorylase a are not useful substrates for chloroplast thylakoid protein phosphatase activity and that the chloroplast enzymes may not fit into one of the canonical classifications currently used for protein phosphatases.     

Effect of growth in highly salinized media on the enzymes of the photosynthetic apparatus in pea seedlings

The rate of chlorophyll formation in initially etiolated pea seedlings (Pisum sativum) that are growing in the light in salinized media is slower than in similar plants not subjected to salinity. However, the final steady state level of chlorophyll is the same under both conditions. Growth under saline conditions did not change the ratio of dry weight to wet weight in the plant leaves nor the specific concentration of soluble protein in leaf extracts. Changes in the specific activity of 11 enzymes in leaf extracts during growth in the light were measured. At least six of these enzymes are known to be part of the photosynthetic apparatus and that their synthesis is subject to photocontrol. The changes in specific activity that were observed were slower in the salt-treated plants, but the final steady state concentration of each was the same as in the control plants. It is concluded that salinity impairs growth of pea plants but that formation of enzymes and other proteins are always in balance with growth.     

Purification of leaf nucleotides and nucleosides on insoluble polyvinylpyrrolidone

Of the 19 nucleotides and nucleosides tested, all were eluted by 1 mM HCl in less than 60 ml from 2 × 6-cm columns of Polyclar AT (an insoluble polyvinylpyrrolidone). Recoveries were good and, with the possible exceptions of ADPG and UDPG, the presence of cotton leaf extract did not decrease recovery of known nucleotides and nucleosides.Passing leaf extracts through Polyclar AT removed most, but not all, of the uv-absorbing impurities that interfere with quantitation of nucleotides and nucleosides. The optimum pH for purification of HClO₄ extracts from leaves of alfalfa, cotton, grape, and orange appeared to be between 2.0 and 3.0. In this pH range Polyclar AT removed from 59 to 91% of the substances in leaf extracts that absorbed at 230 nm and from 93 to 97% of the substances that absorbed at 320 nm.Extraction of leaf extract with isoamyl alcohol was relatively ineffective and extraction with ether was almost completely ineffective in removing uv-absorbing impurities.Because nucleotides and nucleosides quickly pass through a short column of Polyclar AT at pH 3.0 while plant phenols are retained, this procedure provides a simple and rapid method for bulk purification of leaf extracts prior to chromatography and assay of nucleotides and nucleosides.     

The rates of shoot and root growth in intact plants of pea mutants in leaf morphology

Isogenic lines of pea (Pisum sativum L.) with the genetically determined changes in leaf morphology, afila (af) and tendril-less (tl), were used to study the relationship between shoot and root growth rates. The time-course of shoot and root growth was followed during the pre-floral period in the intact plants grown under similar conditions. The af mutation produced afila leaves without leaflets, whereas in the case of the tl mutations, tendrils were substituted with leaflets, and acacia-like leaves were developed. Due to the changes in leaf morphology caused by these mutations, pea genotypes differed in leaf area: starting from day 7, the leaf area was lower in the af plants and larger in the tl plants as compared to the wild-type plants. Such divergence was amplified in the course of plant development and reached its maximum immediately before the transition to flowering. Plants of isogenic lines did not notably differ in stem surface areas. In spite of significant difference in total leaf area, the wild type and tl plants did not differ in leaf dry weight. Starting from leaf 9, the af plants lagged behind two leaflet-bearing genotypes (wild type and tl) in leaf dry weight, whereas stem dry weight was similar in the wild type and tl forms and slightly lower in the af plants. Root dry weights were practically similar in the wild type and tl plants until flowering. The reduction of leaf area in the af plants drastically reduced root dry weight. In other words, the latter index was related to the total weight and total area of leaves and stems. The correlation analysis demonstrated an extremely low relationship between leaf and stem area and dry weight and those of roots early in plant development (when plants develop five to seven leaves). Later, immediately before flowering (nine to eleven leaves), root weight was positively related to leaf weight and area; however, stem area and root weight did not correlate. Thus, in three genotypes (wild type, af, and tl), at the end of their vegetative growth phase, leaf and root biomass accumulated in proportion, independently of leaf area expansion.     

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ

At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.     

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30–40% of the measured rate of CO₂ fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2–5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.Abbreviation GOGAT l-Glutamine:2-oxoglutarate aminotransferase