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.

Figure 1

Principal GA biosynthetic pathways in developing pumpkin seeds. In pumpkin embryos, reactions are catalyzed by GA 7-oxidase (A), seed-specific GA 20-oxidase (B), recently cloned GA 20-oxidase from pumpkin seedlings (C), GA 3-oxidase (D), and GA 2-oxidase (E). Structures and metabolic relations are discussed in the text. Bold arrows indicate major pathways.

Figure 2

Phylogenetic analysis of deduced amino acid structures of selected GA oxidases from diverse species. The tree was generated using ClustalW (version 1.8) and visualized using TREEVIEW. Shown are GA 7-oxidase from pumpkin (Cm 7-ox, accession no. U61386); GA 20-oxidases from Citrullus lanatus (Cl 20-ox, AF074710), Marah macrocarpus (Mm 20-ox, Y09112), pumpkin (Cm 20-ox, X73314; and Cm 20-ox-RT, AJ308480), and Arabidopsis (At 20-ox, X83379); GA 3-oxidases from C. lanatus (Cl 3-ox, AF074710), pumpkin (Cm 2, 3-ox, U63650; Cm 3-ox, AJ006453; and Cm 3-ox-RT, AJ302040), and Arabidopsis (At 3-ox, L37126); GA 2-oxidases from pumpkin (Cm 2-ox, AJ302041) and Arabidopsis (At 2-ox, AJ132437); and a dioxygenase of unknown function from M. macrocarpus (Mm dioxygenase, Y09113).

Figure 3

Localization of GA 7-oxidase (A, F, and K), seed-specific GA 20-oxidase (B, G, and L), recently cloned GA 20-oxidase from seedlings (C, H, and M), GA 3-oxidase (D, I, and N), and GA 2-oxidase (E, J, and O) mRNA in pumpkin seeds by in situ hybridization. All samples were sectioned longitudinally in a 90° angle with the plane of seeds at 50% (A, B, D, F, G, I, K, L, and N) or 36% (C, E, H, J, M, and O) mature index according to Graebe (1972). The sections were hybridized to either an antisense (A–J) or a sense (K–O) RNA probe of the entire respective cDNA (A, B, D, F, G, I, K, L, and N) or the predicted ORF (C, E, H, J, M, and O) that was labeled with digoxigenin. c, Cotyledon; gm, ground meristem; mp, micropylar end; p, protoderm; qc, quiescent center; ram, root apical meristem; sc, inner layer of the seed coat. Bar = 0.1 mm.