Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (CA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from Arabidopsis fhaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the CA-deficient Arabidopsis mutant gal-2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized CA,, at C-20 to GA,,, CAZ4, and the C,, compound CA,, a precursor of bioactive CAs; the C, , tricarboxylic acid compound CA,, was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate CA,,, but less effectively than CA,,. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing diques, and YAPl69 in diques only. In the floral shoots of the gal-2 mutant, transcript levels corresponding to each cDNA decreased dramatically after CA, application, suggesting that CA biosynthesis may be controlled, at least in part, through downregulation of the expression of the 20-oxidase genes.

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In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in Agtll was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA2s and of GA53 to GA17, as well as the formation of the C,,-GAs, GA1, GA,, and GA20, from their respective aldehyde precursors, GA23, GA2,4, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted Mr (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.The gibberellins (GAs) form a large group of diterpenoid natural products. Certain GAs function as hormones in plants, controlling many aspects of development, including stem extension, fruit set, and seed germination (1). The later steps of the GA biosynthetic pathway are catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which oxidize the GA skeleton at the 20, 3,8, and 2p3 positions (see Fig.

Abstract. The plant growth retardant paclobutrazol, (PP333) (2RS,3RS)-1 -(4-chlorophenyl) -4,4 -dimethyl -2 -(1,2,4-triazol-l-yl)pentan-3-ol, inhibits specifically the three steps in the oxidation of the gibberellin-precursor ent-kaurene to ent-kaurenoic acid in a cell-free system from Cttcurbita maxima endosperm. The KIs0 for this inhibition is 2 x 10 -8 M.The KIs0 values for the separated 2S,3S, and 2R,3R enantiomers of paclobutrazol in this system are 2 x 10 -8 M and 7 x 10 -7 M, respectively. A cell-free preparation from immature Mahts pumila embryos converts entkaurene to gibberellin A 9, whereas no conversion occurs in a similar preparation from Malus endosperm. The conversion of ent-kaurene by the embryo preparation is inhibited by paclobutrazol with KIs0 values for the 2S,3S and 2R

Cell-free extracts capable of converting 14C1-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [i4C1-labeled GAs, prepared enzymically from I'4Clmevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions:GA,2-+ GA53-+ GA44--GA,9 --GA20. The pH optima for the enzymic conversions of I'4CiGAs3 I`4CIGA44 and I`4CIGA,, were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe2, a-ketoglutarate and 02 for activity, and ascorbate stimulated the conversion of I'4CIGAs3 and I'4C]GA19. Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD.The results indicate that the activities of the enzymes oxidizing GA53 and GA,9 are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA," remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA20 content of spinach plants in LD than in SD.Spinach (Spinacia oleracea L.) is a LD rosette plant in which stem growth in LD is mediated by GAs2 (19,20). Six 13-hydroxylated GAs have been identified in spinach leaves (14). Of these, GA53, GA44, GA,9, GA20, and GA29 are related metabolically as shown in Figure 1. Although all the steps of this pathway have not been unequivocally demonstrated in spinach, the evidence strongly suggests that the pathway does operate (5,16).When spinach plants grown in SD are transferred to LD the level of endogenous GA,9 decreases, while levels of GA20 and GA29 increase ( 15). This suggests that the photoperiod regulates the conversion of GA,9 to GA20, which is essential for stem growth. Further evidence for this hypothesis was put forward by Gianfagna et al. (5) GA metabolic pathways have been extensively studied using