Isolation and characterization of two peroxidases from Cucumis sativus

Two heme peroxidases of 35.2 and 36.5 kDa have been isolated from cucumber (Cucumis sativus) peelings and characterized through electronic and 1H NMR spectra in the pH range 3.5-10.5. Their spectroscopic and catalytic properties, which are closely similar, are characteristic of highly homologous isoenzymes. Both proteins, as isolated, exist as a mixture of two ferric forms containing a high-spin and a low-spin heme in an approximately 2:1 molar ratio. The latter form likely contains a hydroxide ion axially coordinated to the heme iron and is proposed to be the result of partial irreversible protein inactivation due to the purification procedure. Both proteins in the reduced form are fully high-spin. The high-spin ferric form is sensitive to two acid-base equilibria with apparent pKa values of approximately 5 and 8.5, which have been assigned to the distal histidine and the arginine adjacent to it, respectively. These equilibria also affect the catalytic activity and the interaction with inorganic anions such as azide and fluoride. The reactivity of both proteins is closely similar to that of other plant peroxidases, primarily horseradish peroxidase; however, they also show spectroscopic properties similar to those of cytosolic ascorbate peroxidase. Therefore, overall, these two species show molecular, spectroscopic and catalytic features which are rather peculiar among plant peroxidases.     

Immunochromatographic purification of gibberellins from vegetative tissues of Cucumis sativus L: Separation and identification of 13-hydroxy and 13-deoxy gibberellins

Immunological methods are described for the separation and purification of 13-hydroxy and 13-deoxy-gibberellins of Cucumis sativus. Qualitative and quantitative data show that 13-deoxygibberellins predominate over 13-hydroxygibberellins in stems and leaves of this species.Abbreviations FCS foetal calf serum - FW fresh weight - GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - IAC immunoaffinity chromatography David Marshall is thanked for help with the preparation of the extracts. The authors also thank ICI Plant Protection, Jealotts Hill, Bracknell, Berks., UK, the Science and Engineering Research Council, the Agricultural and Food Research Council, and also the National Science Foundation (grant DCB 8718540 to V.M. Sponsel) for financial support.     

Isolation and Characterization of a Chromoplast-Specific Carotenoid-Associated Protein from Cucumis sativus Corollas

The differentiation of chloroplasts to chromoplasts in corollas of cucumber (Cucumis sativus) is subject to developmental control. To study factors involved in the chloroplast-chromoplast conversion, a chromoplast-specific protein of 35 kD was isolated, and polyclonal antibodies were prepared against it. This protein was found to be a principal component of the carotenoid-protein complex resolved from chromoplast membranes by nondenaturing gel electrophoresis. Immunological studies revealed that expression of this protein is regulated in a temporal and tissue-specific manner. Its steady-state level increased in parallel with flower development and carotenoid accumulation, peaking in mature flowers and then rapidly decreasing to very low levels. The protein was not detectable in cucumber leaves or fruits. To ascertain whether an organ-specific system regulates the chloroplast-chromoplast conversion and to enable future molecular studies of factors involved in this regulation, an in vitro bud culture system was established. Patterns of expression of the 35-kD protein and carotenoids in corollas of detached buds were similar to those in intact buds.     

Acylated flavone C-glycosides from Cucumis sativus

Leaves of Cucumis sativus plants treated with silicon and infected with Sphaerotheca fuliginea yielded five new acylated flavone C-glycosides identified as isovitexin 2"-O-(6"'-(E)-p-coumaroyl)glucoside (6), isovitexin 2"-O-(6"'-(E)-p-coumaroyl)glucoside-4'-O-glucoside (7), isovitexin 2"-O-(6"'-(E)-feruloyl)glucoside-4'-O-glucoside (11), isoscoparin 2"-O-(6"'-(E)-p-coumaroyl)glucoside (9), and isoscoparin 2"-O-(6"'-(E)-feruloyl)glucoside-4'-O-glucoside (12). The known flavone-glycosides isovitexin (1), saponarin (2), saponarin 4'-O-glucoside (3), vicenin-2 (4), apigenin 7-O-(6"-O-p-coumaroylglucoside) (5), isovitexin 2"-O-(6"'-(E)-feruloyl)glucoside (8) and isoscoparin 2"-O-(6"'-(E)-feruloyl)glucoside (10), were also identified in this plant material.     

A C15 aldehyde from Cucumis sativus

cis-8-Pentadecenal was isolated from a concentrate of cucumber volatiles and characterized by spectral analyses and ozonolysis. The biochemical origin of this compound and other long chain aldehydes isolated from cucumber is discussed.     

Biological activity of some conjugated gibberellins

Summary The biological activity of several gibberellin (GA) conjugates was studied and compared with that of the corresponding free GAs. The following conjugates were included: O(3)--d-glucopyranosides of GA₁, GA₃ and GA₄; O(13)--d-glucopyranosides of GA₁, GA₃ and GA₅; O(13)--d-glucopyranosyl-GA₅--d-glucopyranosyl ester; GA₃--d-glucopyranosyl ester and GA₃--d-glucopyranosyl ester; N-GA₃-oyl-glycine, its methyl ester, N-GA₃-oyl-glycylglycine, and N-GA₃-oyl-proline. All compounds were synthesized chemically but some of them are known to occur as endogenous plant products, or to be formed in plants upon application of a free GA. Activity was determined in the dwarf pea, dwarf corn, dwarf rice, and lettuce hypocotyl bioassays. The GA conjugates were found to posses different relative activities depending on the chemical structure, the bioassay system, and the site of application (shoot or roots). It is concluded that the activity of GA conjugates as measured in different bioassays is based upon the ability of plant enzymes and possibly of certain microorganisms to hydrolyze glucosidic, glucosyl ester, and amide-like linkages.Gibberellins-XLVIII. Part XLVII=Adam et al. (1976b)     

黄瓜藤的化学成分研究

从丽江产黄瓜藤甲醇提取物的氯仿部位分离得到9个化合物,经理化和波谱分析鉴定为α-菠甾醇(1)、α-菠甾醇-3-O-β-D-葡萄糖苷(2)、β-谷甾醇(β-sitosterol,3)、豆甾-7-烯-3-O-β-D-葡萄糖苷(4)、22-亚甲基-9,19-环羊毛甾烷-3β-醇(5)、(2S,3S,4R,10E)-2-(2′,3′-二羟基二十四烷酰氨基)-10-十八烯-1,3,4-三醇(6)、(2S,3S,4R,10E)-2-[(2′R)-2-羟基二十四烷酰氨基]-10-十八烯-1,3,4-三醇(7)、(2S,3S,4R,10E)-1-(β-D-葡萄糖苷)-2-[(2′R)-2-羟基二十四烷酰氨基]-10-十八烯-1,3,4-三醇(8)、大豆脑苷(9),除化合物3外,其它化合物均为首次从该植物中分离得到.     

High pressure liquid chromatography of conjugated gibberellins

The application of high pressure liquid chromatography to the purification and identification of conjugated gibberellins was examined. Two kinds of reversed phase columns, octadesylsilanized and dimethylsilanized silica gel, were useful for the isolation and identification of gibberellin A₃ glucoside and gibberellenic acid glucoside from immature seeds of Quamoclit pennata.     

Isolation of a novel stable peptide from cultivated Raphanus sativus with peroxidase activity

Isolation of a novel stable peptide from cultivated Raphanus sativus with peroxidase activity has been achieved using zinc ion precipitation followed by aqueous two phase extraction with a polyethylene glycol (PEG)/phosphate system. The best result was achieved with PEG 600/phosphate (28.8/13% w/v) which extracted 96.15% of total enzyme activity. The peptide has a molecular weight of 5.85 kD as determined by HPLC size exclusion method with an optimized pH of 6.     

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings

N⁶-(Δ²-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N⁶-(Δ²-isopentenyl) adenosine antibodies retained tRNAs containing N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N⁶-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N⁶-(Δ²-isopentenyl) adenosine, and N⁶-(Δ²-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNA^Phe and tRNA^Tyr contained cytokinins whereas tRNA^Ala did not.     

Inheritance of seed weight in Cucumis sativus (L.) var. sativus and var. hardwickii (Royle) Kitamura

Summary A series of experiments was conducted to determine the inheritance of seed weight in cucumber. Matings between a Cucumis sativus var. sativus (Cs) L. inbred line (USDA WI 1606; P₁) and a C. sativus var. hardwickii (Royle) Kitamura (Ch) collection (PI 215589; P₂) were made to produce seed of reciprocal F₁, F₂, and BC₁ families. Families were grown under field and greenhouse conditions, and seeds were extracted from fruit 55 to 60 days post-pollination. Seed of F₁ and F₂ families was obtained using the Cs inbred WI2808 (P₁₂) and the Ch collection LJ 90430 (P₁₀), and seed of F₁ families were produced using a North Carolina Design II mating scheme in which three Cs (P₃= GY-14; P₄=WI 1379; P₅=WI 1909) inbreds were used as maternal parents and seven Ch collections (P₂; P₆= PI462369; P₇=486336; P₈=LJ91176; P₉=273469; P₁₀= 2590430; P₁₁=PI187367) were used as paternal parents. Mean seed weights of F₁ progeny reflected the dominance of genes of the C. sativus var. sativus parent. Transformation to number of seeds per unit weight resulted in increased variance homogeneity within generations and a broad-sense heritability ranging between 26% to 56%. Additive and dominance effects were important in the expression of seed weight in P₁×P₂ progeny produced in the greenhouse and additive effects were important in field grown progeny resulting from P₁×P₂ and P₁₀×P₁₂ matings. The estimated number of factors or loci involved ranged between 10 to 13, depending on the method of calculation.     

Reproduction and cytogenetic characterization of interspecific hybrids derived from Cucumis hystrix Chakr. x Cucumis sativus L

Interspecific hybrids between Cucumis hystrix Chakr. (2n = 2 x = 24) and Cucumis sativus L. (2n = 2 x = 14) were produced by means of F(1) (2n = 19) embryo rescue and subsequent chromosome doubling. The hybridity was confirmed by genomic in situ hybridization (GISH) and chromosome analysis. The amphidiploid (2n = 38) was self-pollinated and backcrossed to cucumber resulting in lines with improved crossability to C. sativus. Examination of shape, stainability, and germination rate of pollen grains and yield as a function of mature fruit set per ten pollinated flowers indicated a tendency for increased fertility in BC(1)S(1) progeny when compared to F(1) and amphidiploid offspring. Cytogenetic characterization of F(1) and amphidiploid progeny was performed. Generally normal meioses produced viable pollen grains, and fertilization resulted in partial fertility restoration in amphidiploid progeny. Chromosome anomalies such as "frying-pan trivalent", chromosome lagging and spindle mis-orientation were also observed. In most of the PMCs of the F(1) diploid hybrid progeny, 19 univalents were observed at diakinesis and MI. In the amphidiploid, more than 90% of the configurations at MI consisted of the predicted 19 bivalents and less than 5% contained multivalents [trivalents (2.3%) + quadrivalents (0.3%)], suggesting the presence of preferential pairing, and a distinctive parental genome as well. The chiasmata observed between homoeologous chromosomes further demonstrated the introgression of the C. hystrix genome into that of C. sativus.     

Effect of ovular receptivity on seed set and fruit development in cucumber (Cucumis sativus L.)

Summary Seed set and fruit development in cucumber (Cucumis sativus L.) were studied in relation to female flower receptivity from day — 2 before anthesis to day + 2 after anthesis. The female cucumber flower is protogynous. The pistil was receptive 2 days before anthesis. The iso-electric focusing (IEF) patterns of the stigma/style proteins, were identical from day -5 to day +2. In pollinated flowers in vivo germination and pollen-tube growth in the ovary were affected by pistil age from day -2 to day +2. In addition, differences in sectorial filling in full seeds were observed within the fruits. A negative correlation was observed between the frequency of fertilized ovules in the pedoncular part of the fruit and ovary length at the time of pollination. In the whole fruit, significant differences in the number of full seeds and fruit size at maturity were found, and these were observed to be correlated with the various stages of female flower maturation at pollination. The day -2 and day +2 stages yielded the smallest fruits with few full seeds compared to the day -1, day 0 and day +1 stages, which had the biggest fruits and a large number of full seeds. A strong positive correlation was found between total seed number (including full and empty seeds), fruit length and weight at maturity. All these results suggest that both seed set in the different parts of the fruit and fruit development are controlled by ovular receptivity rather than by stigma/style receptivity.     

Isolation of water-soluble gibberellins from immature seeds of Pharbitis nil

Summary The presence of three water-soluble gibberellins was confirmed in immature seeds of morning-glory (Pharbitis nil). The structure of the main component has been elucidated as 2-O--glucosyl-gibberellin A₃. It shows marked growth-promoting activity on rice seedlings but is far less active on dwarf maize mutants d ₁, d ₂ and d ₅.     

Isolation of two new gibberellins from immature seeds of Canavalia

Summary Two new gibberellins, Canavalia gibberellin-I (CG-I) and Canavalia gebberellin-II (CG-II), were isolated from immature seeds of sword bean, Canavalia gladiata DC. The molecular formulas C₁₉H₂₂O₇ and C₁₉H₂₂O₆, were assigned to CG-I and CG-II, respectively, on the basis of high resolution mass, infrared and NMR spectra. The growth-promoting effects of CG-I and CG-II on dwarf maize mutants d₁ and d₅, rice seedlings and dwarf peas, Progress No. 5, are reported.     

黄瓜韧皮部的类血影蛋白

以黄瓜 (CucumissativusL .)叶柄为实验材料 ,应用胶体金免疫电镜技术证明类血影蛋白存在于韧皮部的筛管_伴胞复合体中 ,广泛分布于筛分子中的韧皮蛋白纤丝以及筛分子网络结构上 ,并且分布在伴胞的细胞质和线粒体膜以及筛分子与伴胞之间的分支状胞间连丝上 ,表明该蛋白可能由伴胞合成并经由二者之间的胞间连丝运输到筛分子中。用免疫印迹技术证明 ,黄瓜韧皮部汁液蛋白中存在类血影蛋白 ,其分子量约为 2 6 0kD ,与动物细胞中血影蛋白的分子量接近     

In vitro culture of Cucumis sativus L.

Summary The ability to regenerate plants from leaf explants has been tested for three highly inbred cucumber lines (B, G, S), their reciprocal hybrids, F₂ and BC₁ generations. The lines differed from each other in their regenerating ability, which was expressed by the percentage of explants regenerating embryoidal callus and mean number of plantlets per plant. Thus, the lines could be classified as frequently (B), intermediately (G) or occasionally regenerating ones (S). There were no reciprocal cross differences in the regeneration. It was found that the intermediately and intensively regenerating lines contain two pairs of dominant genes responsible for plant regeneration, characterized by complementary and probably additive interaction. The frequently regenerating line differed from the intermediately regenerating in the effect of one gene. It is supposed that the above-mentioned genes belong to three different loci. The ability to regenerate plants from leaf expiants had high heritability.