title: Expression studies of gibberellin oxidases in developing pumpkin seeds.
abstract: Two cDNA clones, 3-ox and 2-ox, have been isolated from developing pumpkin (Cucurbita maxima) embryos that show significant amino acid homology to gibberellin (GA) 3-oxidases and 2-oxidases, respectively. Recombinant fusion protein of clone 3-ox converted GA(12)-aldehyde, GA(12), GA(15), GA(24), GA(25), and GA(9) to GA(14)-aldehyde, GA(14), GA(37), GA(36), GA(13), and GA(4), respectively. Recombinant 2-ox protein oxidized GA(9), GA(4), and GA(1) to GA(51), GA(34), and GA(8), respectively. Previously cloned GA 7-oxidase revealed additional 3beta-hydroxylation activity of GA(12). Transcripts of this gene were identified in endosperm and embryo of the developing seed by quantitative reverse transcriptase-polymerase chain reaction and localized in protoderm, root apical meristem, and quiescent center by in situ hybridization. mRNA of the previously cloned GA 20-oxidase from pumpkin seeds was localized in endosperm and in tissues of protoderm, ground meristem, and cotyledons of the embryo. However, transcripts of the recently cloned GA 20-oxidase from pumpkin seedlings were found all over the embryo, and in tissues of the inner seed coat at the micropylar end. Previously cloned GA 2beta,3beta-hydroxylase mRNA molecules were specifically identified in endosperm tissue. Finally, mRNA molecules of the 3-ox and 2-ox genes were found in the embryo only. 3-ox transcripts were localized in tissues of cotyledons, protoderm, and inner cell layers of the root apical meristem, and 2-ox transcripts were found in all tissues of the embryo except the root tips. These results indicate tissue-specific GA-biosynthetic pathways operating within the developing seed.
authors.affiliation: Institut für Pflanzenbiologie der Technischen Universität Braunschweig, Mendelssohnstrasse 4, D-38106 Braunschweig, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Frisse
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Pimenta
authors.forename: Theo
authors.initials: T
authors.lastname: Lange
mesh_terms: D003001:Cloning, Molecular; D028464:Cucurbita / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005875:Gibberellins / Q000378:metabolism; D017403:In Situ Hybridization; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D012639:Seeds / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D005875:Gibberellins; D006899:Mixed Function Oxygenases; C106552:gibberellin 7-oxidase; C121463:gibberellin 2-dioxygenase; C108186:gibberellin 3beta-hydroxylase