Plant Physiol, March 2003, Vol. 131, pp. 1220-1227

Expression Studies of Gibberellin Oxidases in Developing Pumpkin Seeds¹

Institut für Pflanzenbiologie der Technischen Universität Braunschweig, Mendelssohnstrasse 4, D-38106 Braunschweig, Germany

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 GA₁₂-aldehyde, GA₁₂, GA₁₅, GA₂₄, GA₂₅, and GA₉ to GA₁₄-aldehyde, GA₁₄, GA₃₇, GA₃₆, GA₁₃, and GA₄, respectively. Recombinant 2-ox protein oxidized GA₉, GA₄, and GA₁ to GA₅₁, GA₃₄, and GA₈, respectively. Previously cloned GA 7-oxidase revealed additional 3-hydroxylation activity of GA₁₂. 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 2,3-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.