Long-day rosette plants, such as lettuce and spinach, grow as rosettes during the short days of winter, and produce stems and flowers during late spring and summer when the days are long. Previous work has shown that production of gibberellins (GAs), a class of plant growth hormones, is stimulated by long days, and the resulting increase in GA content of the shoot tips causes stem growth. The objective of the proposed research is to investigate how the daylength regulates gibberellin production in connection with stem growth of rosette plants. The work focuses on expression of the gene for GA 20-oxidase which encodes a multifunctional enzyme that catalyzes three successive steps in the GA biosynthetic pathway. A full-length cDNA for GA 20-oxidase with an open reading frame encoding a putative 43-kD was isolated from spinach. When this cDNA clone was expressed in E. coli, the fusion protein catalyzed the biosynthetic sequences GA53 GA44 GA19 GA20, and GA19 GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of carbon-20. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in long- than in short days. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in short- and long-day conditions. GA 20-oxidase will be expressed in E. coli for the preparation of antibodies. The fusion protein will be analyzed by MALDI mass spectrometry to determine if the protein undergoes post-translational modifications.
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