Gibberellin biosynthesis in developing pumpkin seedlings. (41/160)

A gibberellin (GA) biosynthetic pathway was discovered operating in root tips of 7-d-old pumpkin (Cucurbita maxima) seedlings. Stepwise analysis of GA metabolism in cell-free systems revealed the conversion of GA(12)-aldehyde to bioactive GA(4) and inactive GA(34). Highest levels of endogenous GA(4) and GA(34) were found in hypocotyls and root tips of 3-d-old seedlings. cDNA molecules encoding two GA oxidases, CmGA20ox3 and CmGA3ox3, were isolated from root tips of 7-d-old LAB150978-treated seedlings. Recombinant CmGA20ox3 fusion protein converted GA(12) to GA(9), GA(24) to GA(9), GA(14) to GA(4), and, less efficiently, GA(53) to GA(20), and recombinant CmGA3ox3 protein oxidized GA(9) to GA(4). Transcript profiles were determined for four GA oxidase genes from pumpkin revealing relatively high transcript levels for CmGA7ox in shoot tips and cotyledons, for CmGA20ox3 in shoot tips and hypocotyls, and for CmGA3ox3 in hypocotyls and roots of 3-d-old seedlings. Transcripts of CmGA2ox1 were mainly found in roots of 7-d-old seedlings. In roots of 7-d-old seedlings, transcripts of CmGA7ox, CmGA20ox3, and CmGA3ox3 were localized in the cap and the rhizodermis by in situ hybridization. We conclude that hypocotyls and root tips are important sites of GA biosynthesis in the developing pumpkin seedling.  (+info)

Cucurbit phloem serpins are graft-transmissible and appear to be resistant to turnover in the sieve element-companion cell complex. (42/160)

Serpins are unique inhibitors of serine proteases that are located in various plant tissues and organs. An orthologue of the pumpkin (Cucurbita maxima) phloem serpin CmPS-1 was amplified from cucumber (Cucumis sativus) RNA by RT-PCR, cloned, and designated as CsPS-1 (GenBank accession no. AJ866989). Alternative amino acid sequences in the reactive centre loop suggest distinct inhibitory specificity between CmPS-1 and CsPS-1. A difference in the electrophoretic mobility of these serpins was used in heterografts to establish that serpins are phloem-mobile. Immuno light microscopy revealed that the phloem serpins are localized exclusively to sieve elements (SE), while the phloem filament protein CmPP1, used as a reference, is localized to both SEs and companion cells (CCs). Similar to CmPS-1, CsPS-1 accumulates over time in phloem exudates, indicating that serpins differ from other phloem-mobile proteins whose concentrations appear to be stable in phloem exudates. These differences could reflect alternative mechanisms regulating protein turnover and/or inaccessibility of protein degradation. The functionality of the pore/plasmodesma units connecting SEs and CCs was tested with graft-transmitted CmPP1 as a transport marker. The occurrence of CmPP1 in the CCs of the Cucumis graft partner shows that translocated 88 kDa phloem filament protein monomers can symplasmically exit the SE and accumulate in the CC. By contrast, serial sections probed with the serpin antibody demonstrate that the 43 kDa serpin does not enter CCs. Collectively, these data indicate that CCs play a decisive role in homeostasis of exudate proteins; proteins not accessing the CCs accumulate in SEs and display a time-dependent increase in concentration.  (+info)

Selenium species determination in selenium-enriched pumpkin (Cucurbita pepo L.) seeds by HPLC-UV-HG-AFS. (43/160)

Pumpkins were treated by spraying the leaves in the flowering period with a water solution containing 1.5 mg Se per liter in the form of Na2SeO4. The average total selenium content of seeds was found to be 0.19 microg g(-1) in nontreated pumpkins and 1.1 microg g(-1) in exposed ones. For speciation analysis, enzymatic hydrolysis with different amounts of Protease XIV was carried out. Under optimal conditions of enzymatic hydrolysis, 90% of the total selenium was found in soluble forms. Separation of species was performed using HPLC on anion and cation exchange columns and for detection UVHG-AFS was applied. In enzymatic hydrolysis extracts, the main fraction of selenium was bound as selenomethionine (SeMet), representing on average of 81 +/- 8% of the total Se content in the sample.  (+info)

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants. (44/160)

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.  (+info)

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Reconstructing the origins and dispersal of the Polynesian bottle gourd (Lagenaria siceraria). (45/160)

The origin of the Polynesian bottle gourd (Lagenaria siceraria), an important crop species in prehistoric Polynesia, has remained elusive. Most recently, a South American origin has been favored as the bottle gourd could have been introduced from this continent with the sweet potato by Polynesian voyagers around A.D. 1,000. To test the hypothesis of an American origin for the Polynesian bottle gourd, we developed seven markers specific to bottle gourd (two chloroplast and five nuclear). The nuclear markers were developed using a new technique where polymorphic inter simple sequence repeat (ISSR) markers are converted into single-locus polymerase chain reaction and sequencing markers--an approach that will be useful for developing markers in other taxa. All seven markers were sequenced in 36 cultivars of bottle gourd from Asia, the Americas, and Polynesia. The results support a dual origin for the Polynesian bottle gourd: the chloroplast markers are exclusively of Asian origin, but the nuclear markers show alleles originating in both the Americas and Asia. Because hybridization of Polynesian bottle gourds with post-European introductions cannot be excluded, ancient DNA from archaeological material will be useful for further elucidating the prehistoric movements of this species in Polynesia. This work has implications not only for the dispersal of the Polynesian bottle gourd but also for the domestication and dispersal of the species as a whole.  (+info)

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9. (46/160)

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.  (+info)

Serine proteinase inhibitor from wax gourd (Benincasa hispida [Thunb] Cogn.) seeds. (47/160)

Two squash family protease inhibitors were obtained from wax gourd (Benicasa hispida [Thunb] Cogn.). Even though they were distinctly separated by reversed-phase chromatography, the amino acid sequences of two inhibitors were identical. Both inhibitors were converted into each other, perhaps due to cis-trans isomerization of characteristic Pro in the C-terminal region.  (+info)

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata. (48/160)

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml(-1), all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.  (+info)