The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.

Abstract

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14. point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GAS locus encodes GA 20-oxidase. Expression of the GAS gene in Arabidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.The gibberellins (GAs) are tetracylic diterpenoid compounds that play an important role in many aspects of plant growth and development, such as promotion of cell division and extension, seed germination, stem growth, and fruit set (1). The biosynthesis of GAs has been studied extensively in developing seeds (2, 3), but their function in immature seeds, if any, is unknown. In contrast, relatively little is known about GA biosynthesis in vegetative tissues, because GA levels in these tissues are low compared with levels in immature seeds, and active enzyme preparations for GA conversions are difficult to obtain. It is, therefore, desirable to extend studies on regulation of GA biosynthesis to green plants in which GAs have definite roles. One of these roles is to mediate photoperiodic control of stem elongation in rosette plants (4-6). Therefore, to understandThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6640the mechanism by which long days (LD) cause stem elongation, it is necessary to elucidate how daylength regulates GA biosynthesis at the biochemical and molecular levels.In Arabidopsis thaliana, a long-day plant (LDP) suitable for molecular genetic studies (7), a number of GA-responsive dwarf mutants have been isolated (8). It has been demonstrated that one of these mutants carrying thegaS mutation has reduced levels of Cl-GAs, indicating that the product of the GAS gene catalyzes elimination of C-20 at the aldehyde level in the GA biosynthetic pathway. The gaS dwarf mutant also has increased levels of certain C20-GAs, indicating existence of an additional control, possibly hydroxylation of C-20 (9). This suggests that the GAS locus encodes a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-]. In pumpkin endosperm, the enzyme activities for the steps between GA53 and GA20 ( Fig. 1) reside on a single polypeptide (10), indicating that GA 20-oxidase is a multifunctional enzyme that catalyzes the oxidation and elimination of C-20 and thus plays a pivotal role in the conversion of C20-to C19-GAs. Recently, cDNA clones encoding GA 20-oxidases that differ by one amino acid have been isolated from developing cotyledons (11) and liquid endosperm (12) of devel...

Loss-of-function of a Rice Gibberellin Biosynthetic Gene, GA20 oxidase (GA20ox-2), Led to the Rice 'Green Revolution'.

“…Degenerate primers were designed from the consensus sequences of the GA20 oxidase gene (GA20ox) (Toyomasu et al 1997) from rice and Arabidopsis (GA5) (Xu et al 1995) (sense: 5′YTNCCNTGGAAYGARACNYT3′ and antisense: 5′GTNGGRTCRCARTGNGG3′). The DNA fragment was amplified by PCR with the genomic DNA from a japonica rice variety, TC65.…”

“…Since the Arabidopsis genome carries three GA 20-oxidase genes and the products function in a redundant manner (Xu et al 1995), we assumed that SD1 might encode another GA20ox. In an attempt to isolate a novel GA20ox gene, we designed degenerate primers based on the conserved domain between the rice and Arabidopsis GA20ox genes (Toyomasu et al 1997, Xu et al 1995. Two amplified DNA fragments were obtained; one corresponded to the previously identified GA20ox-1, and the other was a novel GA20ox gene (GA20ox-2).…”

Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli

“…A GA 20-oxidase cDNA previously has been cloned from pumpkin cotyledons (7), and the cloning of another 20-oxidase from pumpkin endosperm was reported, without details of the nucleotide sequence, but stating that it differed from the first by one amino acid (8). The coding regions of the cDNA sequences of the 20-oxidases from the endosperm, described here, and from cotyledons (7), are highly conserved.…”

“…26). There are many reports on the expression of recombinant enzymes retaining catalytic activity as fusion proteins (7)(8)(9)(10)(11)(12)(13)(14)(15)(26)(27)(28)(29). Therefore, the method described here should be applicable for cloning a wide range of enzymes, provided that these are not involved in the metabolism of or are toxic to E. coli itself.…”

“…The cloning of another 20-oxidase gene from pumpkin endosperm was reported, but details of this work were not documented (8). Subsequently, many GA 20-oxidase genes have been cloned from various plant sources (8-15) using PCR-based cloning procedures.…”