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Structural differences between trypsin-inhibitors isolated from Cucurbitaceae family seeds: immunological, NMR and CD studies.

By manifold immunizations of rabbits with virgin or modified trypsin inhibitor III from squash seeds and trypsin inhibitor II b from cucumber seeds, specific antibodies were produced. In double immunodiffusion the anti-squash inhibitor antibody also gave weak precipitate arcs with inhibitor I from squash, inhibitor II from summer squash and with inhibitor I from zucchini, but not with inhibitor II b from cucumber seeds. The genus of Cucurbita trypsin inhibitors, preincubated with the antibody, lost their antitrypsin activity. The antibody showed a significantly weaker effect on the activity of the inhibitor from cucumber sees. 1H-NMR and CD spectra also confirm structural differences between trypsin inhibitors from the genus of Cucurbita and the cucumber (genus of Cucumis) inhibitor.

Antigen-Antibody Complex↗

Toxicity of spinosad in protein bait to three economically important tephritid fruit fly species (Diptera: Tephritidae) and their parasitoids (Hymenoptera: Braconidae).

The feeding toxicity of the natural insecticide spinosad in Provesta protein bait was evaluated for three economically important fruit fly species, the Mediterranean fruit fly, Ceratitis capitata (Wiedemann); the melon fly, Bactrocera cucurbitae Coquillett; and the oriental fruit fly, Bactrocera dorsalis Hendel. Both females and males were evaluated. Spinosad was remarkably similar in toxicity to all three fruit fly species. Male C. capitata (24 h LC50 values and 95% fiducial limits = 2.8 [2.60-3.0] mg/liter spinosad) were significantly, although only slightly more susceptible to spinosadthan females (4.2 [3.8-4.6] mg/liter). Male (5.5 [4.7-6.6] mg/liter) andfemale (4.3 [3.7-4.9] mg/liter) B. cucurbitae were equally susceptible to spinosad. Female (3.3 [3.1-3.6] mg/liter) and male (3.1 [2.9-3.3] mg/liter) B. dorsalis also were equally susceptible to spinosad. Provesta bait containing spinosad also was evaluated against two parasitoids of tephritid fruit flies, Fopius arisanus (Sonan) and Pysttalia fletcheri (Silvestri). These parasitoids did not feed on the bait, so a contact toxicity test was conducted. Significant amounts of mortality were found only after exposure of parasitoids to spinosad-coated glass vials with concentrations > or =500 mg/liter spinosad. Parasitoids were less susceptible than fruit flies to such a degree that use of spinosad in bait spray should be compatible with these parasitoid species. Because the fruit flies tested in this study were so susceptible to spinosad, this product seems to be promising as a bait spray additive and a replacement for malathion for control of these species.

Animals↗

Releases of Psyttalia fletcheri (Hymenoptera: Braconidae) and sterile flies to suppress melon fly (Diptera: Tephritidae) in Hawaii.

Ivy gourd, Coccinia grandis (L.) Voigt, patches throughout Kailua-Kona, Hawaii Island, HI, were identified as persistent sources of melon fly, Bactrocera cucurbitae (Coquillett). These patches had a low incidence of Psyttalia fletcheri (Silvestri), its major braconid parasitoid natural enemy in Hawaii, and were used to evaluate augmentative releases of P. fletcheri against melon fly. In field cage studies of releases, numbers of melon flies emerging from ivy gourd fruit placed inside treatment cages were reduced up to 21-fold, and numbers of parasitoids were increased 11-fold. In open field releases of P. fletcheri into ivy gourd patches, parasitization rates were increased 4.7 times in release plots compared with those in control plots. However, there was no significant reduction in emergence of melon flies from fruit. In subsequent cage tests with sterile melon flies and P. fletcheri, combinations of sterile flies and P. fletcheri produced the greatest reduction (9-fold) in melon fly emergence from zucchini, Cucurbita pepo L. Reductions obtained with sterile flies alone or in combination with parasitoids were significantly greater than those in the control, whereas those for parasitoids alone were not. Although these results suggest that the effects of sterile flies were greater than those for parasitoids, from a multitactic melon fly management strategy, sterile flies would complement the effects of P. fletcheri. Cost and sustainability of these nonchemical approaches will be examined further in an ongoing areawide pest management program for melon fly in Hawaii.

Animals↗

Carotenoid composition and vitamin A value of a squash and a pumpkin from northeastern Brazil.

The carotenoid composition of a squash and a pumpkin from Northeastern Brazil was determined. Nineteen carotenoids were detected in Cucurbita moschata variety "Baianinha"; beta-carotene was the principal carotenoid, contributing about 74% of an average total carotenoid content of 317.8 micrograms/g. In C. maxima variety "Jerimum Caboclo", 11 carotenoids were found with lutein, and beta-carotene as the major pigments accounting for about 60% and 27%, respectively, of an average total carotenoid content of 78.4 micrograms/g. The abundance of beta-carotene in the C. moschata variety "Baianinha" makes this squash one of the richest sources of provitamin A. The average vitamin A value was 43,175 IU (International Units) per 100 g or 4,317 RE (retinol equivalents) per 100 g. Its vitamin A values is more than 11 times that of C. maxima variety "Jerimum Caboclo" and five times that of C. moschata cultivar "Menina Verde", the squash found previously to be highest in provitamin A among the Cucurbita vegetables most commercialized in São Paulo (Southeastern Brazil).

Carotenoids↗

Protein inhibitors of trypsin from the seeds of Cucurbitaceae plants.

Seven trypsin inhibitors were isolated from the seeds of Cucurbitaceae plants: two from cucumber (Cucumis sativus) and red bryony (Bryonia diotica) and one from figleaf gourd (Cucurbita ficifolia), spaghetti squash (Cucurbita pepo var. (vegetable spaghetti) and water melon (Citrullus vulgaris). The inhibitors were purified by fractionation with ammonium sulphate, followed by ion-exchange chromatography and affinity chromatography using immobilized trypsin or anhydro-trypsin. The homogeneous inhibitors from cucumber and water melon are made up of 32 and 30 amino acid residues, respectively, whereas the remaining ones of 29 residues. All inhibitors contain three disulphide bridges and are free of threonine, phenylalanine and tryptophan. Inhibitors from spaghetti squash and CSTI IIb from cucumber are inactivated by acetylation of free amino groups whereas the remaining ones are inactivated by modification of arginine with 1,2-cyclohexanedione. Thus the P1 residues of the reactive sites of the inhibitors are lysine and arginine, respectively.

Amino Acids↗

Analysis of highly polar compounds of plant origin: combination of hydrophilic interaction chromatography and electrospray ion trap mass spectrometry.

The primary goal of metabolomic analysis is the unbiased relative quantification of every metabolite in a biological system. A number of different metabolite-profiling techniques must be combined to make this possible. Here we report the separation and analysis of highly polar compounds in a proof of concept study. Compounds were separated and analyzed using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization (ESI) mass spectrometry. Two types of HILIC microbore columns (Polyhydroxyethyl A and TSK Gel Amide 80) were compared to normal phase silica HPLC columns. The best separations of standards mixtures and plant samples were achieved using the Amide 80 stationary phase. ESI enabled the detection of both positively and negatively charged metabolites, when coupled to a quadrupole ion trap mass spectrometer using continuous polarity switching. By stepwise mass spectrometric fragmentation of the most intense ions, unknown compounds could be identified and then included into a custom mass spectrometric library. This method was used to detect oligosaccharides, glycosides, amino sugars, amino acids, and sugar nucleotides in phloem exudates from petioles of fully expanded Cucurbita maxima leaves. Quantitative analysis was performed using external standards. The detection limit for stachyose was 0.5 ng per injection (Amide 80). The concentration of stachyose in investigated phloem samples was in the range of 1-7 mM depending on the plant.

Amino Acids↗

Ultrasonic treatment stimulates multiple shoot regeneration and explant enlargement in recalcitrant squash cotyledon explants in vitro.

Ultrasonic treatment (0.5-2 min) stimulated multiple shoot regeneration to high levels in vitro from recalcitrant cotyledon explants of commercial squash (Cucurbita pepo L.) cultivars Ma'yan and Bareqet, on Murashige and Skoog [Physiol Plant 15:473-497, 1962] (regeneration) medium augmented with 4.4 microM benzyladenine. At this stage, unsonicated control explants regenerated only a few very small shoots or bud-like structures. Ultrasound also stimulated massive explant growth. Ultrasound treatment resulted in further multiple shoot production (five times greater than control) after explant transfer to elongation medium (Murashige and Skoog [Physiol Plant 15:473-497, 1962] medium with 0.44 microM benzyladenine and 2.9 microM gibberellic acid). Longer ultrasonic treatments (5 or 10 min) promoted multiple shoot regeneration and explant growth accompanied by hyperhydration. Scanning electron microscope observations showed that 2 min ultrasound changed the joint area between epidermal cells and removed some of the surface from the cotyledon epidermal cells, without gross surface injury to the explants. Longer periods of ultrasound (5-10 min) caused further surface erosion. Rubbing the explant contact surface with chloroform or sandpaper emulated the effect of sonication on shoot regeneration and explant growth, demonstrating that ultrasound exerts its morphogenic influence by surface removal. Sonication of explants from other batches of squash seeds (of cultivars Ma'yan and True French), that regenerated without such treatment, reduced regeneration and caused hyperhydration. This is the first report of stimulation of in vitro regeneration by ultrasound treatment.

Cotyledon↗

Xylem sap protein composition is conserved among different plant species.

Xylem sap from broccoli (Brassica oleracea L. cv. Calabrais), rape (Brassica napus L. cv. Drakkar), pumpkin (Cucurbita maxima Duch. cv. gelber Zentner) and cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) was collected by root pressure exudation from the surface of cut stems of healthy, adult plants. Total protein concentrations were in the range of 100 microg ml(-1). One-dimensional gel electrophoresis (SDS-PAGE) resulted in 10-20 visible protein bands in a molecular mass range from 10 to 100 kDa. The main bands were cut out, digested with trypsin, and analysed using tandem mass spectrometry. Fifty bands resulted in amino acid sequence information that was used to perform database similarity searches. Sequences from 30 bands showed high homology to proteins present in databases. Among them, we found mostly peroxidases, but could also identify the lectin-like xylem protein XSP30, a glycine-rich protein, serine proteases, an aspartyl protease family protein, chitinases, and a lipid transfer protein-like polypeptide. Sequence analysis predicted apoplastic secretion signals for all database entries similar to the partial xylem protein sequences. This and the lack of cross-reactivity with phloem protein-specific antibodies suggest that the proteins really originate from the xylem and do not result from phloem contamination. Most of the highly similar proteins probably function in repair and defence reactions. Some of the most abundant proteins (peroxidases, chitinases, serine proteases) were present in xylem exudate of all species analysed, often in more than one band. This indicates an important basic role of these proteins in maintaining xylem function.

Amino Acid Sequence↗

Isotopic enrichment in a phloem-feeding insect: influences of nutrient and water availability.

The isotopic enrichment between an animal and its diet can vary among and within living systems, but the sources of variation are not yet fully understood. Some studies have found that diet quality or an animal's nutritional status can influence the degree of trophic enrichment, while others have dismissed nutrition as a contributing factor. We evaluated the effects of nutrient and water availability on carbon and nitrogen isotopic enrichment in a specialized plant-herbivore system. Aphids are largely sedentary and rely exclusively on nitrogen-poor phloem sap of their host for nutrition. We grew potato aphids [Macrosiphum euphorbiae (Aphididae)] on an accepted host, pumpkin [Cucurbita pepo L. (Cucurbitaceae)], in a glasshouse environment. Twelve pumpkin plants growing under high- and low-watering regimes were inoculated at 4 weeks of age with aphids. During the course of the experiment we collected leaves, phloem sap, aphids and honeydew (i.e., aphid exudates). We found no trophic enrichment between aphids and their phloem sap diet, but significant carbon enrichment of honeydew relative to aphids (2.5 per thousand) and phloem sap (2.1 per thousand). Honeydew was also enriched in nitrogen compared to the phloem sap (1.2 per thousand). Watering treatment had a substantial impact on trophic enrichment. Correlations among tissues, an indication of uniform trophic enrichment among samples, were significant only for the carbon isotopic composition, and then only for plants and aphids grown in the low-water treatment. Diet quality also influenced the degree of isotopic enrichment; trophic enrichment for both carbon and nitrogen isotopic composition increased as diet quality (C/N) declined. We conclude that the degree of trophic enrichment is variable due, in part, to diet quality, but that the scale of variation is small.

Analysis of Variance↗

Plant growth, phosphorus nutrition, and root morphological responses to arbuscular mycorrhizas, phosphorus fertilization, and intraspecific density.

We examined the effects of arbuscular mycorrhizas (AM), phosphorus fertilization, intraspecific density, and their interaction, on the growth, phosphorus uptake, and root morphology of three facultative mycotrophic crops (Capsicum annuum, Zea mays, and Cucurbita pepo). Plants were grown in pots with or without AM at three densities and four phosphorus availabilities for 10 weeks. AM colonization, plant weight, and shoot phosphorus concentration were measured at harvest. Root morphology was assessed for C. annuum and Z. mays. Phosphorus fertilization reduced but did not eliminate AM colonization of all species. AM, phosphorus, and density interacted significantly to modify growth of C. annuum and C. pepo such that increased density and phosphorus diminished beneficial effects of AM. Increased density reduced positive effects of AM on C. annuum and C. pepo shoot phosphorus concentrations. AM altered both Z. mays and C. annuum root morphology in ways that complemented potential phosphorus uptake by mycorrhizas, but increased density and phosphorus diminished these effects. We infer that increased density predominantly influenced plant responses by affecting whether or not carbon (photosynthate) or phosphorus limited plant growth. By exacerbating carbon limitation, high density reduced the benefit/cost ratio of mycorrhizas and minimized their effects.

Capsicum↗

Cytological modifications in zucchini yellow mosaic virus (ZYMV)-infected Styrian pumpkin plants.

The present research demonstrates severe ultrastructural changes induced by zucchini yellow mosaic virus (ZYMV) within the cells of older and younger leaves of Styrian pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca GREB.). Cylindrical inclusions (pinwheels), proliferated endoplasmatic reticulum and filamentous viral particles were found throughout the cytoplasm of ZYMV-infected cells and within sieve elements. ZYMV-infection also induced severe modifications in the number and ultrastructure of chloroplasts, whereas mitochondria, nuclei and peroxisomes remained unaffected. A significantly lower number of chloroplasts was observed in all tissues of both ZYMV-infected leaf types when compared to control plants. Statistical quantification revealed that in chloroplasts of ZYMV-infected older and younger leaves the amount of plastoglobuli and starch increased significantly, whereas the amount of thylakoids significantly decreased. The present research gives a more precise insight in ZYMV-induced modifications within single cells and organelles, and provides statistical data of the most affected chloroplasts.

Chloroplasts↗

A major integral protein of the plant plasma membrane binds flavin.

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Amino Acid Sequence↗

Characterization and activity enhancement of the phloem-specific pumpkin PP2 gene promoter.

The promoter of the pumpkin (Cucurbita moschata) PP2 gene (designated NP) was isolated from the restriction enzyme-digested genomic DNA pool by genome walking and its activity and phloem specificity were examined in transgenic tobacco plants by using GUS as a reporter. Deletion analysis of the promoter revealed that the 473-bp fragment (-465 to + 8 relative to the transcription start site; designated as NPII) exhibited similar activity as the full-length NP promoter and retained its phloem specificity. Furthermore, the sequence from -465 to -171 was shown to contain positive regulatory cis-elements for the promoter activity. An enhanced NP promoter was constructed by duplicating the sequence -465 to -85, and its activity in phloem tissue was shown to be higher than that of the Commelina Yellow Mottle Virus (CoYMV) promoter or a chimeric promoter consisting of the double enhancer sequence from the Cauliflower Mosaic Virus (CaMV) 35S promoter fused upstream to the NPII fragment.

Agrobacterium tumefaciens↗

Reactions, characterization and uptake of ammoxidized kraft lignin labeled with 15N.

Ammoxidation of kraft lignin was carried out in a Parr reactor using (15)NH(3) as the main nitrogen source. Reaction parameters were set up until a total nitrogen content of approximately 13 wt.% in lignin was achieved, in accordance with conditions of previous studies. Analytical tools such as FTIR, Py-GC/MS, and solid state NMR were used in this research. The nature of nitrogen bondings is discussed. The incorporation of the (15)N from ammoxidized lignin was followed in pumpkins (Zucchini cucurbita pepo L.) by means of (15)N emission spectroscopy.

Ammonia↗

Fatty acids by high-performance liquid chromatography and evaporative light-scattering detector.

A high-performance liquid chromatographic (HPLC) separation method with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of fatty acid methyl esters (FAME) in three different oils. Reverse-phased C18 HPLC separation of 13 FAME is achieved using a methanol/water eluent mixture. The retention times (RT) reflect the elution behavior of these compounds on C18 reversed-phase HPLC. The proposed method is tested on: soybean oil (Glycine max L.) as reference sample, rice bran oil (Oryza sativa L.), pumpkin seed oil (Cucurbita pepo L.) and algal oil (Arthrospira platensis Nordst.).

Calibration↗

A zucchini-peroxidase biosensor applied to dopamine determination.

A biosensor modified with peroxidase from crude extract of zucchini (Cucurbita pepo) was developed for the determination of dopamine in pharmaceutical samples. This enzyme catalyses the oxidation of dopamine to dopaminequinone, in presence of hydrogen peroxide, which the electrochemical reduction can be followed at a peak potential of -0.02 V. The recovery of dopamine from the samples ranged from 94.8% to 106% and a rectilinear analytical curve for dopamine concentration from 5.0 x 10(-4) to 3.0 x 10(-3) mol L-1 (r=0.9982) was obtained. The detection limit was 2.6x10(-5) mol L-1 and the relative standard deviation was less than 1.2% for 7.9 x 10(-4) mol L-1 dopamine in 0.1 mol L-1 phosphate buffer solution at pH 6.0 (n=10).

Biosensing Techniques↗

Ascorbic acid oxidase is dynamically regulated by light and oxygen. A tool for oxygen management in plants?

Ascorbic acid oxidase (AAO) is a plant blue-copper protein catalyzing dioxygen reduction to water using ascorbic acid as the electron donor. In spite of extensive molecular characterization the physiological role of AAO is still uncertain. Abundant mRNA, protein and activity of AAO were observed in illuminated leaves of Cucurbita pepo. AAO activity was found to be proportional to light intensity. The light effect was rapidly reversed in dark and activity remained low throughout the dark period. Activity was elicited in dark by increased oxygen concentration. AAO activity increased in the facultative CAM Kalanchoë blossfeldiana upon induction of the CAM cycle and decreased during germination of C. pepo and Zea mays under hypoxic conditions. These results strongly suggest that AAO activity could be part of a dynamic system for oxygen management in plants.

Ascorbic Acid↗

Proteomics of curcurbit phloem exudate reveals a network of defence proteins.

Many different proteins can be separated from the sap of mature sieve tubes of different plant species. To date, only a limited number of those have been identified and functionally characterised. Due to sieve tubes inability of transcription and translation, the proteins are most probably synthesised in the intimately connected companion cells and transported into the sieve elements through plasmodesmata. The specific protein composition of phloem sap suggests an important role of these proteins not only for sieve tube maintenance, but also for whole plant physiology and development. Here we describe a comprehensive analysis of the phloem protein composition employing one- and high-resolution two-dimensional gel electrophoresis and partial sequencing by mass spectrometry. In this study more than 300 partial sequences generated by hybrid mass spectrometry were used to identify a total of 45 different proteins from the phloem exudates of cucumber (Cucumis sativus L. cv. Hoffmanns Giganta) and pumpkin (Cucurbita maxima Duch. cv. Gelber Zentner) plants. In addition to previously described phloem proteins, it was possible to localise proteins with high similarity to an acyl-CoA binding protein, a glyoxalase, a malate dehydrogenase, a rhodanese-like protein, a drought-induced protein, and a beta-glucosidase. The results indicate that the majority of the so far identified proteins are involved in stress and defence reactions.

Cucumis sativus↗