Changes in Levels of Naturally Occurring Gibberellin-like Substances during Germination of Seed of Lycopersicon esculentum Mill.

Naturally occurring gibberellin-like substances possessing acidic,basic, and neutral properties were detected, by paper partitionchromatography, in ethanolic extracts of tomato seed and ofetiolated seedlings after 72 and 116 hours' growth. Dwarf maizemutants of the d-1 and d-5 types, ‘Meteor’ pea seedlingsand young ‘Potentate’ tomato plants were used asbioassay material. Hydrolysis of seed and seedling proteinsby ficin in phosphate buffer, pH 6.2, after removal of ethanol-solublesubstances, liberated more and different ‘bound’gibberellin-like substances. It is suggested that protein hydrolysisduring germination is an important means of liberating thesesubstances at different stages of seedling development. Acidic substances were present in all the extracts prepared,but in general two with Rfs 0.25 and 0.55 in iso-propanol: ammonia:water : : 10:1:1 v/v were differentiated on d-2 and d-5 maizerespectively. Neutral substances in dry seed extracts chromatographedin the same solvent, had Rfs of 0.05, 0.35, and 0.95 and thesewere found only in the ethanolic (‘free’) extracts.They were active on d-1 and d-2 maize and ‘Meteor’pea. Basic gibberellin-like substances with Rfs of 0.05 and0.35 were found in ‘free’ extracts of both dry seedand etiolated seedlings after 116 hours' growth which were activeon d-2 maize only. Two others with Rfs 0.45 and 0.95 were extractedfrom seedlings after 72 hours' growth and these were activeon young ‘Potentate’ tomato plants. It is suggested that certain gibberellin-like substances, capableof reversing dwarfism in test plants, may be responsible formorphogenetic or other responses not involving stem extensionin the parent species. Changes were found in the levels of gibberellin-likesubstances but there was no evidence of changes in levels ofseed inhibitors relative to seed growth substances.     

Antibacterial properties of extracts from selected planktonic freshwater cyanobacteria – a comparative study of bacterial bioassays

Aqueous and methanol extracts from five selected cyanobacteria were examined for antibacterial properties in six different bacterial bioassays. All five cyanobacteria revealed antibacterial properties. Methanol extracts made from Tychonema bourrellyi, Aphanizomenon flos-aquae and Cylindrospermopsis raciborskii showed the most pronounced inhibitory effects. Aqueous extracts made from Microcystis aeruginosa and T. bourrellyi possessed evident antibacterial properties. The bacterial bioassays were based on agar diffusion tests and included pour-plate methods commonly used to detect residues of antibacterial substances in food. In addition, a pour-plate bioassay with Aeromonas hydrophila was developed and described. Antibacterial effects were observed in five of the six bacterial bioassays. No antibacterial effect was observed in the Micrococcus luteus bioassay. Bioassays based on Aer. hydrophila, Bacillus cereus and B. subtilis grown in Antibiotic Medium 8, pH 5·85, seemed to be sensitive and suitable. The MIC value of diluted MeOH extracts made from C. raciborskii and T. bourrellyi against Aer. hydrophila corresponded to 38 mg freeze-dried cyanobacteria. Bacillus subtilis was more sensitive when grown in a culture medium with pH 5·85 than 7·9. The antibacterial properties of extracts from the cyanobacteria examined differed from defined cyanotoxins and antibacterial substances. The pattern of inhibition in the bacterial bioassays indicated that various antibacterial substances are involved.     

Female mating receptivity inhibited by injection of male-derived extracts in Callosobruchus chinensis

The effects of male-derived extracts on female receptivity to remating were investigated in Callosobruchus chinensis (Coleoptera: Bruchidae). Injection of aqueous extracts of male reproductive tracts into the abdomen of females reduced receptivity. When aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: <3, 3-14, and >14 kDa, the filtrate containing MW substances <3 kDa reduced female receptivity 3h and 1 day after injection, whereas the fraction containing MW substances >14 kDa inhibited receptivity 2 and 4 days after injection. Finally, male reproductive tract organs were divided into accessory gland, seminal vesicle, and testis. Aqueous extracts of testis reduced receptivity of females on the second day and at 3h, and aqueous extracts of accessory gland reduced receptivity of females on the second day after injection. On the other hand, aqueous extracts of seminal vesicle did not reduce female receptivity. The results indicate that more than one mechanism may be involved in producing the effects of male-derived substances on female receptivity; low MW male-derived substances, which possibly exist in testis, cause short-term inhibition, while high MW substances, which possibly exist in the accessory gland, inhibit female mating later than low MW substances in C. chinensis.     

STUDIES OF VANDADIUM-BROMOPEROXIDASE USING SURFACE AND CORTICAL PROTOPLASTS OF MACROCYSTIS PYRIEFERA (PHAEOPHYTA)1

Aqueous Tri-SO₄ buffer (pH 8.3) extracts of cortical and surface protoplasts of Macrocystis pyrifera (L.) C. Ag. Catalyzed the bromination of monochlorodimedone (2-chloro-5, 5-dimethyl-1, 3-dimedone, MCD) in the presence of hydrogen peroxide and bromide. The apparent bromo-peroxidase activity as measured by the bromination of MCD was inhibited by the presence of endogenous compounds which are probably polyphenolics compounds (i.e. polymers of phloroglucinol) or other inhibitors. The bromoperoxidase activity of the protoplast extracts increased substantially when the extracts were washed extensively with Tris-SO₄ buffer (pH 8.3) by ultrafiltration. The bromoperoxidase activity of both surface and cortical protoplast extracts was dependent on the presence of vanadium, indicating that the bromoperoxidase present in cortical and surface cells of M. pyrifera is vanadium-bromoperoxidase. Halogenated compounds constitute one of the most significant classes of marine natural products. Since bromoperoxidases are assumed to be involved in the biosynthesis of these compounds, elucidation of the location of BrPO with in the algal tissue is important.     

Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts

Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and fractions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heat-treated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at room temperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate content relative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations and cyanogenic glucosides. which were present, could not account for the anthelmintic activity detected. Thus, our results are best explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds are extracted.     

Biosynthesis of ethylene in higher plants: the metabolic site of inhibition by phosphate*

Abstract. Phosphate inhibited endogenous as well as 1-aminocyclopropane-1-carboxylic acid (ACC)-stimulated ethylene synthesis in slices of tomato fruit, segments of carrot root and pea hypocotyls. ACC concentrations of up to 10 mol m^?3 did not overcome this inhibition. Phosphate inhibited the conversion of ¹⁴C ACC to ethylene in tomato fruit and vegetative tissue. Enzymatic conversion of ACC to ethylene by pea seedling homogenate was also inhibited by phosphate with a linear concentration dependency. The formation of ACC from S-adenosylmethionine (SAM) by extracts of pink tomatd fruit was slightly, but not significantly, affected by phosphate. However, the SAM to ACC conversion was greater when extracts from tomato fruit were made in phosphate rather than in HEPES-KOH buffer. Non-enzymatic ethylene synthesis from ACC in a model system was stimulated by phosphate. We suggest that phosphate is an inhibitor of ethylene biosynthesis in higher plants and that one site of its control is the conversion of ACC to ethylene.     

Induction of oviposition by injection of male-derived extracts in two Callosobruchus species

In some insect species, certain substances in the seminal fluid of males induce egg production and laying in females. We determined the effects of male-derived substances on female oviposition behaviour in two Callosobruchus species, C. chinensis and C. maculatus. Aqueous extracts of the accessory gland; testis; and seminal vesicle, including the ejaculatory duct, were prepared. The injection of these extracts into abdomen of females induced oviposition in both species. Oviposition was induced by the testis and seminal vesicle extracts in C. chinensis and by the accessory gland extracts in C. maculatus. The extracts were separated into three fractions by ultrafiltration: fractions I, molecular weight (MW) <3 kDa; fraction II, 3-14 kDa; and fraction III, >14 kDa. Fraction III induced oviposition in both species. These results suggest that in these two species, the substances that induce oviposition have similar MW but are present in different organs. Oviposition was induced by high-MW (>14 kDa) substances in the testis and seminal vesicle in C. chinensis, and by high-MW substances in accessory gland in C. maculatus. Here, we have discussed the relationship between oviposition and the abovementioned male-derived substances.     

Antimicrobial effects of aqueous plant extracts on the fungi Microsporum canis and Trichophyton rubrum and on three bacterial species

Aqueous extracts of 10 plants were tested for their ability to inhibit Trichophyton rubrum and Microsporum canis, the aetiological agents of dermal fungal infections in humans. These extracts were also evaluated for their activity against some bacteria. Aqueous extracts from the leaves of Inula viscosa produced detectable antifungal activity against these dermatophytes.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Mycosphaerella pinodes in pea seeds

A polyclonal antiserum was raised against soluble mycelial extracts of Mycosphaerella pinodes aiming at pathogen detection in infected pea seeds by ELISA. When tested against the homologous antigen, it allowed the detection of 5 ng fungal soluble protein ml^-1 buffer, by double-antibody sandwich ELISA (DAS-ELISA). Positive reactions were obtained with isolates of M. pinodes of wide geographical origins but also with all tested isolates of Ascochyta pisi and Phoma medicaginis var. pinodella, two closely related pathogens forming with the target organism the Ascochyta complex. Out of the 11 other genera of pea seed-borne fungi tested, only two (Alternaria sp. and Stemphylium sp.) cross-reacted strongly by both antigen-coated plate (ACP-ELISA) and DAS-ELISA. Cross-absorption of the crude antiserum could not lead to a species-specific antiserum; however, a combination of P. medicaginis var. pinodella and Stemphylium sp. antigens resulted in an antiserum preferentially recognising A. pisi and M. pinodes. The cross-absorbed antiserum detected 50 and 500 ng of fungal protein ml^-1 buffer and healthy seed extracts respectively. DAS-ELISA proved suitable for the detection and quantification of M. pinodes in infected pea seeds tested singly.     

The Presence of Gibberellins in Excised Tomato Roots

Substances having similar physiological properties to the gibberellins(located by the ‘Meteor’ dwarf pea bio-assay) havebeen detected in extracts from excised tomato root. The chromatographicbehaviour of the most active zone is similar to that of gibberellinA₁. Experiments using the d-1 and d-5 mutants of mazie did not indicatethe presence of substances with differential effects on thesetwo mutants.     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Arginine Decarboxylase and Putrescine Oxidase in Ovaries of Pisum sativum L. (Changes during Ovary Senescence and Early Stages of Fruit Development)

Enzymatic activities involved in putrescine metabolism in ovaries of Pisum sativum L. during ovary senescence and fruit set were investigated. Accumulation of putrescine was observed during incubation of extracts from gibberellic acid-treated unpollinated ovaries (young developing fruits) but not in extracts from untreated ovaries (senescent ovaries). Extracts from pea ovaries showed arginine decarboxylase (ADC) activity, but ornithine decarboxylase and arginase activity were not detected. ADC activity decreased in presenescent ovaries and increased markedly after induction of fruit set with gibberellic acid. Increases in ADC activity were also observed with application of other plant growth substances (benzy-ladenine and 2,4-dichlorophenoxyacetic acid), after pollination, and in the slender (la crys) pea mutant. By contrast, putrescine oxidase activity increased in presenescent ovaries but did not increase during early fruit development. All of these results suggest that ADC and putrescine oxidase are involved in the control of putrescine metabolism. Ovary senescence is characterized by the absence of putrescine biosynthesis enzymes and increased levels of putrescine oxidase and fruit development by an increase in ADC and a constant level of putrescine oxidase.     

Effect of moderately acidic pH on heat resistance of Clostridium sporogenes spores in phosphate buffer and in buffered pea puree.

The effect of pH in the range 5.0 to 7.0 on the thermal destruction of spores of Clostridium sporogenes putrefactive anaerobe 3679 was examined by three methods: a capillary tube method in which spores were suspended in phosphate buffers, a thermoresistometer method in which spores were suspended in buffered pea puree adjusted to the same set of pH values, and a thermal death time can method in which spores were again suspended in buffered pea puree. The results indicated that increasing acidity is, in general, accompanied by decreasing heat resistance, although the pH effect was more pronounced at the higher than at the lower processing temperatures. Certain pH values appear to be critical, as they produced, in all three sets of experiments, effects which would not be predicted by the overall relationship between acidity and spore heat resistance. Differences between heat resistance in phosphate buffer as compared with that in pea puree adjusted to the same pH were also noted. D-values in buffer were found to be lower than those in pea puree, except at the highest temperatures coupled with the lowest pH values. The differences between buffer D-value and pea puree D-value were found to increase with increasing pH and with decreasing temperature. On the other hand, at all pH values examined, z-values determined in buffer were somewhat higher than those determined in pea puree adjusted to the same pH.     

Presence of a Root Malformation Factor in Culture Filtrates of Fusarium moniliforme var. subglutinans1

The presence of a root malformation factor, different from plant growth substances viz. NAA, GA₃, Kinetin and reduced Glutathion was demonstrated in culture filtrates of Fusarium moniliforme var. subglutinans, a fungus commonly associated with malformed mango tissues. Culture filtrate extracts of the fungus caused curling and stunting of maize and pea roots and formation of flap like growth at the tip of wheat roots, but failed to induce malformation in bean plants as reported, for malformin.     

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. Objectives: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.     

Forward mutation of S. typhimurium by smokeless tobacco extracts

Mutagenicity of 4 popular brands of smokeless tobaccos was studied using a S. typhimurium forward mutation assay. Aqueous extracts of 4 brands and dichloromethane and methanol extracts of 1 of the 4 brands of smokeless tobacco's did not induce significant mutagenicity either in the presence or absence of metabolic activation. Aqueous and organic extracts were however mutagenic when treated with physiological levels of sodium nitrite (0.25 mM) at acidic pH and without metabolic activation. The results indicate that smokeless tobacco contain polar and non-polar chemicals which become mutagenic to S. typhimurium under nitrosation conditions.     

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.     

Insecticidal effects of various concentrations of selected extractions of Cestrum parqui on adult and immature Ceratitis capitata

Aqueous extracts of Cestrum parqui L'Héritier (Solanaceae) were evaluated at different concentrations in several stages of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). For adults, the study was extended to extracts obtained with several solvents of an increasing degree of polarity. Aqueous extracts from C. parqui showed a high toxicity to neonate larvae when ingested through diet, inhibiting pupation at a concentration above 0.6%. Lower concentrations delayed the larval development and reduced the percentages of pupae formed and adult emergence. An LC50 = 0.9% after 3 d of continuous ingestion of C. parqui aqueous extracts could be calculated, whereas extracts obtained with organic compounds were nearly innocuous except with the use of the solvent methanol/water (80:20), the more polar of those tested, that killed 12.5% of adults. Aqueous extracts were also harmful to adults by diminishing the reproductive potential, which implies a significant effect on the offspring. Egg contact with insecticide or dipping third instars did not cause any adverse effect, supporting the hypothesis that only by means of ingestion can the toxic compounds of C. parqui reach the target. Our results showed that C. parqui components causing C. capitata mortality are mostly dissolved in water and not in organic solvents, which point out their polar character.     

Lack of fructose-1,6-bisphosphatase in a range of higher plants that store starch.

The aim of this work was to discover whether fructose-1,6-bisphosphatase (FBPase) is present in higher-plant cells that synthesize storage starch. The following were examined: suspension cultures of soybean (Glycine max), tubers of potato (Solanum tuberosum), florets of cauliflower (Brassica oleracea), developing endosperm of maize and of sweet corn (Zea mays), roots of pea (Pisum sativum), and the developing embryos of round and wrinkled varieties of pea. Unfractionated extracts of each tissue readily converted fructose 1,6-bisphosphate to fructose 6-phosphate in assays for both plastidic and cytosolic FBPase. These conversions were not inhibited by 20 microM-fructose 2,6-bisphosphate. Except in extracts of pea embryos and sweet-corn endosperm, treatment with affinity-purified antibodies to pyrophosphate: fructose-6-phosphate 1-phosphotransferase reduced the above fructose 6-phosphate production to the rate found with boiled extracts. The antibody-resistant activity from sweet corn was slight. In immunoblot analyses, antibody to plastidic FBPase did not react positively with any protein in extracts of soybean cells, potato tuber, cauliflower florets, maize endosperm and pea roots. Positive reactions were found for extracts of embryos of both round and wrinkled varieties of peas and endosperm of sweet corn. For pea embryos, but not for sweet-corn endosperm, the Mr of the recognized protein corresponded to that of plastidic FBPase. It is argued that soybean cells, potato tuber, cauliflower florets, maize (var. White Horse Tooth) endosperm and pea roots lack significant activity of plastidic FBPase, but that this enzyme is present in developing embryos of pea. The data for sweet corn (var. Golden Bantam) are not decisive. It is also argued that, where FBPase is absent, carbon for starch synthesis does not enter the amyloplast as triose phosphate.     

Solubilization of ribulose-1,5-bisphosphate carboxylase from the membrane fraction of pea leaves

The solubilization of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from the membrane fraction was studied in whole leaf extracts and chloroplasts from pea. The amount of membrane-bound Rubisco was dependent on the pH of the chloroplastic lysate buffer. Maximum binding was found at pH 8.0, with about 8% of total leaf Rubisco being bound. The binding of Rubisco to the membranes was strong, and it was not released by repeated washing with hypotonic buffer or by changing ionic strength. Detergents such as Triton X-100, Tween 20, deoxycholate and dodecylsulfate were effective in solubilizing the membrane-bound Rubisco. Triton X-100 was most effective in the range of 0.04% to 0.2% and it solubilized Rubisco from the membrane without any decrease in enzyme activity.Abbreviations BSA bovine serum albumin - CABP carboxyarabinitol-1,5-bisphosphate - DTT dithiothreitol - LDS lithium dodecylsulfate - LHC light-harvesting chlorophyll protein complex - RuBP ribulose-1,5-bisphosphate - Rubisco RuBP carboxylase/oxygenase - SDS sodium dodecylsulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis