Ascorbate oxidase from Cucurbita maxima

Ascorbate oxidase is present in homogenates of the flesh of Cucurbita maxima fruits. Its activity is independent of ascorbate concentration over th     

Metabolic networks of Cucurbita maxima phloem

Metabolomic analysis aims at a comprehensive characterization of biological samples. Yet, biologically meaningful interpretations are often limited by the poor spatial and temporal resolution of the acquired data sets. One way to remedy this is to limit the complexity of the cell types being studied. Cucurbita maxima Duch. vascular exudates provide an excellent material for metabolomics in this regard. Using automated mass spectral deconvolution, over 400 components have been detected in these exudates, but only 90 of them were tentatively identified. Many amino compounds were found in vascular exudates from leaf petioles at concentrations several orders of magnitude higher than in tissue disks from the same leaves, whereas hexoses and sucrose were found in far lower amounts. In order to find the expected impact of assimilation rates on sugar levels, total phloem composition of eight leaves from four plants was followed over 4.5 days. Surprisingly, no diurnal rhythm was found for any of the phloem metabolites that was statistically valid for all eight leaves. Instead, each leaf had its own distinct vascular exudate profile similar to leaves from the same plant, but clearly different from leaves harvested from plants at the same developmental stage. Thirty to forty per cent of all metabolite levels of individual leaves were different from the average of all metabolite profiles. Using metabolic co-regulation analysis, similarities and differences between the exudate profiles were more accurately characterized through network computation, specifically with respect to nitrogen metabolism.     

Different AT-rich satellite DNAs in Cucurbita pepo and Cucurbita maxima

Summary The AT-rich highly repeated satellite DNA of Cucurbita pepo (zucchini) and Cucurbita maxima (pumpkin) were cloned and their DNA structure was investigated. DNA sequencing revealed that the repeat length of satellite DNA in Cucurbita pepo is 349–352 base pairs. The percentage of AT-base pairs is about 61%. This satellite is highly conserved in restriction enzyme pattern and DNA sequence; sequence heterogeneity is about 10%. In contrast, the satellite DNA of Cucurbita maxima has a repeat length of 168–169 base pairs. This satellite is also rich in AT-base pairs (64%), existing in at least three different variants as revealed by restriction enzyme analysis and DNA sequencing. The sequence heterogeneity between these variants is about 15%. The two satellite DNAs showed no cross-hybridization to each other and sequence homology is only limited. Nevertheless, we found in the C. pepo genome a high amount of sequences resembling the satellite of C. maxima. In contrast, the satellite repeat of C. pepo is found in the C. maxima DNA only in a few copies. These observations were discussed with respect to satellite DNA evolution and compared to the data received from monocotyledonous species.     

Cucurbitacin 19-hydroxylase in Cucurbita maxima

The enzymatic hydroxylation of the C-19-methyl group of cucurbitacin B and D was observed in partly purified preparations obtained from the unripe fruit of Cucurbita maxima. Assay methods were developed and the pH optimum, cofactor requirements, and substrate specificity determined.     

笋瓜光合特性研究

对 无杈早 笋瓜品种光合特性的研究表明:笋瓜单个真叶旺盛光合时期约4周,在展叶后7～35d;壮龄叶片在陆地自然条件下具有明显的光合 午休 现象; 无杈早 笋瓜壮龄叶片的光合适宜温度在24℃左右,CO2补偿点与CO2饱和点分别为16.2和1395μL·L-1,光补偿点与饱和点分别为40.3和1196μmolCO2·m-2·s-1;幼龄叶片的可利用光强范围与羧化效率明显小于壮龄叶片.着果能够明显增强笋瓜植株座果节位及邻近叶片的光合能力.     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Synthesis of P-protein in mature phloem of Cucurbita maxima

Summary Cotyledons of Cucurbita maxima Duch. seedlings were provided with ¹⁴C-labeled amino acids for 12 h. Besides the bulk of labeled amino acids the sieve-tube exudate also carried labeled proteins. 80% of the incorporated radioactivity was found in the P-protein, 20% in a neutral protein, and traces were found in acidic proteins after fractionation on diethyl-aminoethyl cellulose columns. The radioactive elutes were characterized by autoradiographs of both disc- and sodium dodecyl sulfate-gelelectropherograms, and by isoelectric focusing. The P-protein fraction appeared with the void volume from the diethylaminoethyl-cellulose column. Obviously, this is the protein that gels when oxidized and that is reversibly precipitable giving rise to filaments when processed for electron microscopy. Its main component has a molecular weight of 115,000 Dalton. By isoelectric focusing this fraction separated into 3 proteins with isoelectric points of 9.8, 9.4, and 9.2. The isoelectric point 9.2-protein probably is identical with an oligomer of a 30,000 Dalton protein with neutral isoelectric point, which keeps 20% of the incorporated label. Microautoradiographs suggest that the labeled proteins were synthesized in companion cells. The results indicate that P-protein of Cucurbita maxima is synthesized continuously in mature phloem. It can be assumed that P-protein has a relatively high turn-over rate. Therefore it seems unlikely that P-protein is a structural protein.Abbreviations DEAE diethylaminoethyl - SDS sodium dodecyl sulfate - pI isoelectric point Supported by Deutsche Forschungsgemeinschaft.     

P-protein distribution in mature sieve elements of Cucurbita maxima

Summary Portions of the hypocotyls of 16-day-old Cucurbita maxima plants, from which the cotyledons and first foliage leaves had been removed 2 days earlier, were fixed in glutaraldehyde and postfixed in osmium tetroxide for electron microscopy. In well over 90% of the mature sieve elements examined the P-protein was entirely parietal in distribution in both the lumina and sieve-plate pores. In addition to the parietal P-protein, the unoccluded sieve-plate pores were lined by narrow callose cylinders and the plasmalemma. Segments of endoplasmic reticulum also occurred along the margins of the pores.     

Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima

Phloem proteins (P-proteins) were isolated from exudates of both the fruit skin and the stem of the pumpkin, Cucurbita maxima, and the cucumber, Cucumis sativus, and were purified by ammonium sulphate precipitation and chromatography over Sephadex, DEAE-cellulose, and CM-cellulose. It was found that the fruit skin phloem is an excellent source for large-scale preparations of P-proteins which are biochemically and structurally identical with those from stem exudates. Besides fractionation on columns the P-protein preparation was characterized by electrophoresis in polyacrylamide gels (in SDS or acidic urea solutions) and on cellulose acetate, by precipitation studies using salts, acids and thiol reagents, by centrifugation techniques, by determination of amino acid composition, by cyanogen bromide cleavage, by immunodiffusion and immunoelectrophoresis using anti-P-protein-sera obtained from mice, and by electron microscopy of negatively stained and ultrathin sectioned preparations of native and reaggregated P-proteins. P-proteins were identified as a class of at least eight different but related basic (IEPs from pH 9.6 to 10.4) proteins and polypeptides of molecular weights 14000, 17000, 44000, 59000, 116000 and 158000 D which are rich in lysine, leucine, glycine, glutamic acid (plus glutamine) and aspartic acid (plus asparagine). Structurally they were pleomorphic and formed, at various proportions, floccules, fibrils, doughnuts and 100 Å lamellae with a membrane-like ultrastructure. The P-proteins showed in several properties (amino acids, IEPS, retention on CM-cellulose, antigenic sites, molecular weights of the smaller components) a strong similarity to proteins extracted from a ribosomal fraction prepared from cucurbit seedlings, in particular to a weakly basic subtraction of ribosomal proteins. It is hypothesized that P-proteins are formed during sieve element maturation by aggregation and oxidative disulphide cross-linking of preexisting proteins which, at least in the Cucurbitaceae, are basic and may include ribosomal or ribosome-associated proteins. Possible functions of these aggregates are discussed.     

Microbial population of the laimosphere of squash (Cucurbita maxima)

Summary The laimosphere, a term analogous to rhizosphere, describes the zone of influence of below-ground portions of shoots on soil microbial populations. Squash hypocotyls influenced microbial populations in soils 0–3 mm from hypocotyl surfaces. In this region, the laimosphere/soil ratio was 7, 36, and 7, respectively, for general bacteria, fluorescent pseudomonads, and actinomycetes.     

A comparative analysis of phloem exudate proteins from Cucumis melo,Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing

Summary Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.Abbreviations DTT dithiothreitol - Bis N,N-methylenebisacrylamide - SDS Sodium dodecyl sulphate - TCA trichloroacetic acid     

Filament formation in vitro of a sieve tube protein from Cucurbita maxima and Cucurbita pepo

Summary Phloem proteins of the sieve tube exudate from Cucurbita maxima Duch. and Cucurbita pepo L. were investigated as to their filament forming ability in vitro. From the two main proteins (116000 dalton, 30000 dalton) only the 116000 dalton protein was found to form reversibly distinct filaments of 6–7 nm diameter upon removal of SH-protecting agents from the buffer, whereas the 30000 dalton protein was precipitated as amorphous material under these conditions. The protein filaments were similar to the filaments ocurring within the sieve tube cells in vivo.Abbreviations SDS sodium dodecyl sulfate - TCA trichloroacetic acid     

Multiple forms of phytase in germinating cotyledons of Cucurbita maxima

Multiple forms of phytase (myoinositol hexaphosphate phosphohydrolase, EC 3.1.3.8) have been isolated in highly purified forms from germinating Cucurbita maxima cotyledons using acetone and ammonium sulphate fractionation, Sephadex gel filtration and ion exchange chromatography on DEAE- and CM-cellulose. Gel filtration produced two peaks of phytase activity; phytase I (high MW) and phytase II (low MW). Phytase I was further resolved into 4 distinct species on CM-cellulose and these were designated phytase IA, IB, IC and ID, according to their elution order. On the other hand, phytase II remained as a single species with a purification of 35-fold. The MWs of each phytase I species were identical (MW 66 500 ± 4000) and they were twice the MW of phytase II (MW 32 400 ± 4000) indicating that I and II may be structurally related. The properties of various molecular forms were compared. The difference in properties between phytase II and phytase I isoenzymes (IA, IB, IC and ID) was more pronounced than that observed among the isoenzymes of phytase I alone.     

Protein composition of Cucurbita maxima and C. moschata seeds

Seeds of Cucurbita maxima, C. moschata and their interspecific hybrids were used to evaluate the intrapopulational and interpopulational variation of their protein composition. Three immunoprecipitating systems common to all the studied samples were detected by the Ouchterlony technique. Fourteen protein bands were identified by polyacrylamide gel electrophoresis (PAGE) whereas 23 bands were identified by sodiumdodecylsulfate (SDS)-PAGE. Using Western blotting (WB) also 23 bands were detected. The Jaccard's index of similarity calculated from SDS-PAGE and WB varied between 91 and 100 % for all the compared pairs of samples. These results demonstrate a high uniformity in the protein composition of all the samples and do not allow for their clear characterization.     

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

A new product obtained by incubation of [2-¹⁴C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent-kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent-kaurene, ent1-kaurenol, ent-kaurenal and ent-kaurenoic acid, but not ent-7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent-kaurenoic acid and proceeds via the dienoic acid.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.