Rice momilactone gene cluster: transcriptional response to barnyard grass (Echinochloa crus-galli).

Expression of genes involved in diterpene biosynthesis, especially momilactone and gibberellins (GAs), in rice plants (Oryza sativa L.) in response to barnyard grass (Echinochloa crus-galli) stress was examined. The three analyzed class II diterpene synthases had the highest fold change expression. Transcription patterns of genes for two homologs of momilactone synthases, OsMAS and OsMAS2, suggests their distinct roles in response to the presence of barnyard grass.

Monitoramento do ácido chiquímico no manejo de plantas daninhas com glifosato em pomar comercial de citros / Shikimic acid monitoring in weed control with glyphosate in a commercial citrus orchard

Com o objetivo de verificar o acúmulo de ácido chiquímico em plantas de laranja pêra (Citrus sinensis) num pomar comercial manejado com glifosato, um herbicida sistêmico de amplo espectro, foram coletadas amostras na Fazenda Jequitibá, tradicional no cultivo de citros, situada no Município de Santo Antônio de Posse, SP. O produtor aplicou de forma convencional Roundup® Original a 1.440 g.ha-1 de equivalente ácido (e.a.) do sal de isopropilamino de glifosato em 19/12/ 2006 na entrelinha de 15 plantas, deixando outras cinco como testemunha. A reaplicação de glifosato a 1.260 g.ha-1 de e.a. foi realizada em 2/4/2007. Em ambos os casos, imediatamente antes da aplicação e aos 3, 7, 10, 15, 20 e 35 dias após, foram coletadas 20 folhas de cada planta tanto da área tratada como da não tratada, analisando-se o teor de ácido chiquímico por cromatografia líquida de alta eficiência (CLAE) de forma isocrática após extração por micro-ondas. Os resultados mostraram não ocorrer acúmulo do ácido chiquímico nas plantas de laranja pêra, não havendo diferenças significativas nos teores deste composto entre o material proveniente da área tratada com glifosato e o daquela capinada manualmente.

In order to check the accumulation of shikimic acid in a traditional commercial grove of citrus "Pêra" cultivar (Citrus sinensis) managed for weed control with glyphosate, a systemic herbicide with wide spectrum, samples were collected at Fazenda Jequitibá, in Santo Antonio de Posse County, São Paulo State, Brazil. The producer applied the following treatments of Roundup Original® glyphosate at 1,440 g.ha-1 a.e. of the isopropylamine salt on 19 December 2006 between rows of 15 plants, leaving five others as control. The reapplication of glyphosate at 1,260 g ha-1 was done on 2 April 2007. In both cases, immediately before application and at 3, 7, 10, 15, 20 and 35 days thereafter, 20 leaves from each treated and untreated plants were collected for analysis of the content of shikimic acid by isocratic high performance liquid chromatography (HPLC) assisted with microwave. The results showed no significant differences in levels of shikimic acid between the material from the area treated with glyphosate and that weeded manually.

Repellency of callicarpenal and intermedeol against workers of imported fire ants (Hymenoptera: Formicidae).

Callicarpenal and intermedeol are two insect-repellent terpenoids isolated from leaves of American beautyberry (Callicarpa americana L.; Verbenaceae) and Japanese beautyberry (Callicarpa japonica Thunb.). The repellency of these two terpenoids against workers of red imported fire ants, Solenopsis invicta Buren, black imported fire ants, Solenopsis richteri Forel, and a hybrid of these two species was evaluated using digging bioassays. In a multiple choice digging bioassay using two colonies from each species and their hybrid, callicarpenal showed significant repellency at concentration as low as 50 ppm against both red imported fire ant colonies and 6.25 ppm against all black imported fire ant and hybrid colonies. Intermedeol showed significant repellency at concentration as low as 1.50 ppm against both red imported fire ant colonies and 6.25 ppm against all black imported fire ant and hybrid colonies. In total, 15 colonies, five colonies from each species and the hybrid, were tested on callicarpenal and intermedeol at 50 ppm in a two-choice digging bioassay. Both callicarpenal and intermedeol showed repellency against all colonies, and intermedeol showed significantly greater repellency than callicarpenal against both species and their hybrid.

Isolation and identification of mosquito bite deterrent terpenoids from leaves of American (Callicarpa americana) and Japanese (Callicarpa japonica) beautyberry.

Essential oil extracts from Callicarpa americana and Callicarpa japonica were investigated. Bioassay-guided fractionation of C. americana extracts using the yellow fever mosquito, Aedes aegypti, led to the isolation of alpha-humulene, humulene epoxide II, and intermedeol and a newly isolated terpenoid (callicarpenal). Similar work involving C. japonica resulted in the isolation of an additional compound, spathulenol, as well as the four compounds isolated from C. americana. Structure elucidation was performed on all isolated compounds using a combination of gas chromatography-mass spectrometry-electron ionization, high-resolution liquid chromatography-MS-electrospray ionization, and one- and two-dimensional NMR experiments. Heretofore, 13,14,15,16-tetranorclerodane, callicarpenal, has never been identified from natural sources. Complete (1)H and (13)C NMR assignment data are provided for this compound. In bite deterrent studies, spathulenol, intermedeol, and callicarpenal showed significant repellent activity against A. aegypti and Anopheles stephensi.

Synthesis, herbicidal activity, and mode of action of IR 5790.

IR 5790, an arylthiadiazolone herbicide structurally related to oxadiargyl and oxadiazon, was synthesized. The herbicidal activity and mode of action of IR 5790 were investigated. This herbicide has broad-spectrum pre-emergence activity against both dicotyledonous and monocotyledonous weeds. The phenotypic responses of susceptible plants, such as interruption of growth and light-dependent development of necrotic areas on the foliage, are consistent with those observed with protoporphyrinogen oxidase-inhibiting herbicides. Tissues exposed to IR 5790 in darkness accumulated protoporphyrin IX, which led to a photodynamic loss of membrane integrity upon exposure to light. Consistent with these physiological symptoms, IR 5790 strongly inhibited protoporphyrinogen oxidase, with an I(50) value of 3 nM. The presence of a sulfur atom did not significantly alter the molecular properties of the thiadiazolone ring, relative to the oxadiazolone ring of oxadiargyl, which explains why IR 5790 has the same mode of action as this herbicide.

Amino- and urea-substituted thiazoles inhibit photosynthetic electron transfer.

Amino- and urea-substituted thiazoles exhibited in vivo herbicidal activity on duckweed (Lemna paucicostata Hegelm. strain 6746) cultures and appeared to act via inhibition of photosynthetic electron transport system. A small number of the thiazole derivatives tested were active but only at relatively high concentrations. The most active structures were the amino-substituted thiazoles with isopropyl and n-butyl side chains and the urea-substituted thiazole with p-chlorophenyl side chain. Decreasing the length of the side chain had a negative effect on the PSII inhibitory activity. The urea-substituted series was as a group less active than the amino series, and the free acid series had no biological activity. The most active compounds competed for the same binding site as atrazine on PSII. Computer modeling highlighted the structural similarities between some of the thiazoles and the commercial herbicides diuron and atrazine.

Composition and some biological activities of the essential oil of Callicarpa americana (L.).

The essential oil profile of Callicarpa americana was examined. Samples were collected from Lafayette county in north central Mississippi, and GC-MS data and retention indices were used to identify 67 oil components. Humulene epoxide II (13.9%), alpha-humulene (10.0%), 7-epi-alpha-eudesmol (9.4%), beta-pinene (8.8%), and 1-octen-3-ol (8.5%) were the major components of the steam-distilled oil. The oil was selectively toxic toward the cyanobacterium Oscillatoria perornata compared to Oscillatoria agardhii and the green alga Selenastrum capricornutum, with complete growth inhibition at 28.5 microgram/mL. The oil was only mildly phytotoxic and antifungal.

Inhibition of plant asparagine synthetase by monoterpene cineoles.

Asparagine (Asn) synthetase (AS) is the key enzyme in Asn biosynthesis and plays an important role in nitrogen mobilization. Despite its important physiological function, little research has been done documenting inhibitors of plant AS. Plant growth inhibition caused by the natural monoterpene 1,4-cineole and its structurally related herbicide cinmethylin was reversed 65% and 55%, respectively, by providing 100 microM Asn exogenously. Reversion of the phytotoxic effect was dependent on the concentration of Asn. The presence of either 1,4-cineole or cinmethylin stimulated root uptake of [(14)C]Asn by lettuce (Lactuca sativa) seedlings. Although the physiological responses suggested that both compounds affected Asn biosynthesis, biochemical analysis of AS activity showed that the natural monoterpene was a potent inhibitor (I(50) = approximately 0. 5 microM) of the enzyme, whereas the commercial product was not inhibitory up to levels of 10 mM. Analysis of the putative metabolite, 2-hydroxy-1,4-cineole, showed that the cis-enantiomer was much more active than the trans-enantiomer, suggesting that the hydroxyl group was involved in the specific ligand/active site interaction. This is the first report that AS is a suitable herbicide target site, and that cinmethylin is apparently a proherbicide that requires metabolic bioactivation via cleavage of the benzyl-ether side chain.

Glutathione-dependent oxidative modification of protoporphyrin and other dicarboxylic porphyrins by mammalian and plant peroxidases.

Protoporphyrin, an intermediate in heme and chlorophyll biosynthesis, can accumulate in human and plant tissues under certain pathological conditions and is a photosensitizer used in cancer phototherapy. We previously showed that protoporphyrin and the related non-natural dicarboxylic porphyrin deuteroporphyrin are rapidly oxidized by horseradish peroxidase in the presence of some thiols, especially glutathione. This study reports that bovine lactoperoxidase, but not leucocyte myeloperoxidase, can also catalyze this reaction and that Tween and ascorbic acid are inhibitors. Exogenous hydrogen peroxide is not required and cannot replace glutathione. Deuteroporphyrin was oxidized to a unique green chlorin product with two oxygen functions added directly to the characteristic reduced pyrrole ring of the chlorin. Spectroscopic and chromatographic results suggest that protoporphyrin was oxidized not to a green chlorin, but to a much more polar red porphyrin modified by oxidative addition to the two vinyl side chains. Two related nonnatural dicarboxylic porphyrins, with ethyl or hydroxyethyl instead of vinyl side chains, are not substrates or products for this enzymatic conversion.

Thiol-dependent degradation of protoporphyrin IX by plant peroxidases.

Protoporphyrin IX (PP) is the last porphyrin intermediate in common between heme and chlorophyll biosynthesis. This pigment normally does not accumulate in plants because its highly photodynamic nature makes it toxic. While the steps leading to heme and chlorophylls are well characterized, relatively little is known of the metabolic fate of excess PP in plants. We have discovered that plant peroxidases can rapidly degrade this pigment in the presence of thiol-containing substrates such as glutathione and cysteine. This thiol-dependent degradation of PP by horseradish peroxidase consumes oxygen and is inhibited by ascorbic acid.

A new photosystem II electron transfer inhibitor from Sorghum bicolor.

Our study of the mechanism(s) by which sorgoleone (1) acts as a photosystem II (PS II) inhibitor led to the isolation of a new benzoquinone derivative, 2-hydroxy-5-ethoxy-3-[(Z,Z)-8',11', 14'-pentadecatriene]-rho-benzoquinone (2), from the root exudate of sorghum. The structure of 2, which is being given the name 5-ethoxy-sorgoleone, was determined by spectroscopic means. A methoxy derivative (3) of 1 was also prepared. Both 2 and 3 caused a reduction in oxygen evolution by thylakoid membranes and induced variable chlorophyll fluorescence. These compounds, however, were less active inhibitors of PS II than 1.

Horseradish peroxidase-dependent oxidation of deuteroporphyrin IX into chlorins.

Chlorins are cyclic tetrapyrrole derivatives of great interest for use in photodynamic therapy. We have found that horseradish peroxidase (EC 1.11.1.7) (HRP) can convert deuteroporphyrin IX (Deutero) into chlorins. Some characteristics of this enzymatic transformation were investigated. The formation of chlorins was determined spectrophotometrically by monitoring the change in absorbance in the Q-band region (638 nm). The reaction occurred without addition of H2O2 and had a pH optimum of 7.5. The presence of thiol-containing reductants, with a great preference for reduced glutathione, was required and could not be substituted by adding H2O2. Ascorbic acid acted as a potent inhibitor of the reaction, while other organic acids (citric and benzoic) had little to no inhibitory effect. The requirement for O2 was suggested by the inhibitory effect of sodium hydrosulfite and was confirmed by carrying the assay in nitrogen-saturated solutions. Though the reaction occurred without adding H2O2, low amounts of H2O2 (3-30 microM) were stimulatory to the assay. However, concentrations of 300 microM H2O2 or higher were inhibitory. Similarly, light was not required, but was stimulatory at low levels and inhibitory at high levels. Catalase and deferoxamine were inhibitory, but superoxide dismutase and mannitol had no effects. Kinetic analysis and respiratory studies suggest that HRP may initially react with reduced glutathione in a reaction that does not consume much oxygen. The ensuing steps, probably involving an oxygen free radical and porphyrin radical intermediates, consume a large amount of O2 to oxidize Deutero into chlorin.

Oxidation of porphyrinogens by horseradish peroxidase and formation of a green pyrrole pigment.

When humans or plants are exposed to certain chemicals which interfere with heme biosynthetic enzymes, porphyrinogen intermediates accumulate and are oxidized to cytotoxic porphyrins. Here we have investigated the role of peroxidases in porphyrinogen oxidation. Horseradish peroxidase (HRP) rapidly oxidizes uroporphyrinogen to uroporphyrin and this is inhibited by ascorbic acid. HRP also oxidizes deuteroporphyrinogen (a synthetic porphyrin similar to protoporphyrinogen), but the yield of porphyrin is lower than with uroporphyrinogen as substrate. This low yield is in part due to a rapid, HRP-dependent conversion of deuteroporphyrin (but not uroporphyrin) to a green compound with spectral characteristics of a chlorin with a large peak at 638 nm. This reaction requires addition of a sulfhydryl reductant such as glutathione and is inhibited by ascorbic acid. These findings suggest that cellular peroxidases and ascorbic acid levels may play a role in modifying the phototoxic tetrapyrroles which accumulate in plants and humans after certain environmental exposures.

Fumonisin- and AAL-Toxin-Induced Disruption of Sphingolipid Metabolism with Accumulation of Free Sphingoid Bases.

Fumonisins (FB) and AAL-toxin are sphingoid-like compounds produced by several species of fungi associated with plant diseases. In animal cells, both fumonisins produced by Fusarium moniliforme and AAL-toxin produced by Alternaria alternata f. sp. lycopersici inhibit ceramide synthesis, an early biochemical event in the animal diseases associated with consumption of F. moniliforme-contaminated corn. In duckweed (Lemna pausicostata Heglem. 6746), tomato plants (Lycopersicon esculentum Mill), and tobacco callus (Nicotiana tabacum cv Wisconsin), pure FB1 or AAL-toxin caused a marked elevation of phytosphingosine and sphinganine, sphingoid bases normally present in low concentrations. The relative increases were quite different in the three plant systems. Nonetheless, disruption of sphingolipid metabolism was clearly a common feature in plants exposed to FB1 or AAL-toxin. Resistant varieties of tomato (Asc/Asc) were much less sensitive to toxin-induced increases in free sphinganine. Because free sphingoid bases are precursors to plant "ceramides," their accumulation suggests that the primary biochemical lesion is inhibition of de novo ceramide synthesis and reacylation of free sphingoid bases. Thus, in plants the disease symptoms associated with A. alternata and F. moniliforme infection may be due to disruption of sphingolipid metabolism.

Cellular Localization of Protoporphyrinogen-Oxidizing Activities of Etiolated Barley (Hordeum vulgare L.) Leaves (Relationship to Mechanism of Action of Protoporphyrinogen Oxidase-Inhibiting Herbicides).

Seven-day-old, etiolated barley (Hordeum vulgare L. var Post) leaves were fractionated into crude and purified etioplast, microsomal, and plasma membrane (PM) fractions. Protoporphyrinogen oxidase (Protox) specific activities of crude etioplast, purified etioplast, microsome, and PM fractions were approximately 29, 26, 23, and 12 nmol h-1 mg-1 of protein, respectively. The herbicide acifluorfen-methyl (AFM), at 1 [mu]M, inhibited Protox activity from crude etioplasts, purified etioplasts, microsomes, and PM by 58, 59, 23, and 0% in the absence of reductants. Reductants (ascorbate, glutathione [GSH], dithiothreitol [DTT], and NADPH) individually reduced the Protox activity of all fractions, except that microsomal Protox activity was slightly stimulated by NADPH. Ascorbate, GSH, or a combination of the two reductants enhanced Protox inhibition by AFM, and AFM inhibition of Protox was greatest in all fractions with DTT. NADPH enhanced AFM inhibition significantly only in etioplast fractions. Uroporphyrinogen I (Urogen I) and coproporphyrinogen I (Coprogen I) oxidase activities were found in all fractions; however, etioplast fractions had significantly more substrate specificity for protoporphyrinogen IX (Protogen IX) than the other fractions. Urogen I and Coprogen I oxidase activities were unaffected by AFM in all fractions, and 2 mM DTT almost completely inhibited these activities from all fractions. Diethyldithiocarbamate inhibited PM Protox activity by 62% but had less effect on microsome and little or no effect on etioplast Protox. Juglone and duroquinone stimulated microsomal and PM Protox activity, whereas the lesser effect of these quinones on etioplast Protox activity was judged to be due to PM and/or microsomal contaminants. These data indicate that there are microsomal and PM Protogen IX-oxidizing activities that are not the same as those associated with the etioplast and that these activities are not inhibited in vivo by AFM. In summary, these data support the view that the primary source of high protoporphyrin IX concentrations in AFM-treated plant tissues is from Protogen IX exported by plastids and oxidized by AFM-resistant extraorganellar oxidases.

Effect of diphenyl ether herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and plasma membrane enriched fractions of barley.

In barley (Hordeum vulgare L.) root cells, activity for oxidizing protoporphyrinogen to protoporphyrin (protoporphyrinogen oxidase), a step in chlorophyll and heme synthesis, was found both in the crude mitochondrial fraction and in a plasma membrane enriched fraction separated by a sucrose gradient technique utilized for preparing plasma membranes. The specific activity (expressed as nanomoles of protoporphyrin formed per hour per milligram protein) in the mitochondrial fraction was 8 and in the plasma membrane enriched fraction was 4 to 6. The plasma membrane enriched fraction exhibited minimal cytochrome oxidase activity and no carotenoid content, indicating little contamination with mitochondrial or plastid membranes. Etioplasts from etiolated barley leaves exhibited a protoporphyrinogen oxidase specific activity of 7 to 12. Protoporphyrinogen oxidase activity in the barley root mitochondrial fraction and etioplast extracts was more than 90% inhibited by assay in the presence of the diphenyl ether herbicide acifluorfen methyl, but the activity in the plasma membrane enriched fraction exhibited much less inhibition by this herbicide (12 to 38% inhibition) under the same assay conditions. Acifluorfen-methyl inhibition of the organellar (mitochondrial or plastid) enzyme was maximal upon preincubation of the enzyme with 4 mm dithiothreitol, although a lesser degree of inhibition was noted if the organellar enzyme was preincubated in the presence of other reductants such as glutathione or ascorbate. Acifluorfen-methyl caused only 20% inhibition if the enzyme was preincubated in buffer without reductants. Incubation of barley etioplast extracts with the earlier tetrapyrrole precursor coproporphyrinogen and acifluorfen-methyl resulted in the accumulation of protoporphyrinogen, which could be converted to protoporphyrin even in the presence of the herbicide by the addition of the plasma membrane enriched fraction from barley roots. These findings have implications for the toxicity of diphenyl ether herbicides, whose light induced tissue damage is apparently caused by accumulation of the photoreactive porphyrin intermediate, protoporphyrin, when the organellar protoporphyrinogen oxidase enzyme is inhibited by herbicides. Our results suggest that the protoporphyrinogen that accumulates as a result of herbicide inhibition of the organellar enzyme can be oxidized to protoporphyrin by a protoporphyrinogen oxidizing activity that is located at sites such as the plasma membrane, which is much less sensitive to inhibition by diphenylether herbicides.

Physiological basis for differential sensitivities of plant species to protoporphyrinogen oxidase-inhibiting herbicides.

With a leaf disc assay, 11 species were tested for effects of the herbicide acifluorfen on porphyrin accumulation in darkness and subsequent electrolyte leakage and photobleaching of chlorophyll after exposure to light. Protoporphyrin IX (Proto IX) was the only porphyrin that was substantially increased by the herbicide in any of the species. However, there was a wide range in the amount of Proto IX accumulation caused by 0.1 millimolar acifluorfen between species. Within species, there was a reduced effect of the herbicide in older tissues. Therefore, direct quantitative comparisons between species are difficult. Nevertheless, when data from different species and from tissues of different age within a species were plotted, there was a curvilinear relationship between the amount of Proto IX caused to accumulate during 20 hours of darkness and the amount of electrolyte leakage or chlorophyll photobleaching caused after 6 and 24 hours of light, respectively, following the dark period. Herbicidal damage plateaued at about 10 nanomoles of Proto IX per gram of fresh weight. Little difference was found between in vitro acifluorfen inhibition of protoporphyrinogen oxidase (Protox) of plastid preparations of mustard, cucumber, and morning glory, three species with large differences in their susceptibility at the tissue level. Mustard, a highly tolerant species, produced little Proto IX in response to the herbicide, despite having a highly susceptible Protox. Acifluorfen blocked carbon flow from delta-aminolevulinic acid to protochlorophyllide in mustard, indicating that it inhibits Protox in vivo. Increasing delta-aminolevulinic acid concentrations (33-333 micromolar) supplied to mustard with 0.1 millimolar acifluorfen increased Proto IX accumulation and herbicidal activity, demonstrating that mustard sensitivity to Proto IX was similar to other species. Differential susceptibility to acifluorfen of the species examined in this study appears to be due in large part to differences in Proto IX accumulation in response to the herbicide. In some cases, differences in Proto IX accumulation appear to be due to differences in activity of the porphyrin pathway.