Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development.

PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgaris). One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv 15-11, Pv73-1 and Pv85-26) from bean. Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting [14C]GA12 to [14C]GA9. The intermediates in this three-step reaction, GA15 and GA24, were also identified as products. The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA53 to GA20, as effectively as they did GA12. On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6). Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries. Treatment of unpollinated ovaries with GA3 to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2,4-D, although inducing fruit-set, did not reduce the levels of these transcripts. Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod. The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv 15-1 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons, while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant. Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves. Southern blot analysis indicated that the bean GA 20-oxidases are each encoded by single-copy genes, whereas one more gene, homologous to Ps27-12, could also exist in pea.

Gibberellin biosynthesis from gibberellin A12-aldehyde in endosperm and embryos of Marah macrocarpus.

Soluble enzyme preparations from embryos and endosperm of Marah macrocarpus (previously Echinocystis macrocarpa) were incubated with [14C4]gibberellin(GA)12-aldehyde, [14C4]GA12, [14C1] GA9, 2,3-didehydro[14C1]GA9, [14C1]GA20, and [17-13C, 3H]GA5. Embryo preparations converted GA12-aldehyde, GA12, and GA9 to GA4 and GA7; 2,3-didehydroGA9 to GA7; GA5 to GA3; and GA20 (incompletely) to GA1 and GA60, but not to GA3. Endosperm preparations converted GA12-aldehyde and GA12 to GA15, GA24, and GA9, but, unlike embryo preparations, not to GA4 or GA7. However, GA4 and GA7 were formed from GA9 and GA7 was formed from 2,3-didehydroGA9. Metabolism of GA5 to GA3 and GA20 to GA1 was low. 2,3-DidehydroGA9 accumulated when GA9 was incubated with a desalted endosperm preparation. A cDNA clone (M3-8), selected from an embryo-derived cDNA library using a DNA fragment generated by reverse transcriptase polymerase chain reaction, was expressed in Escherichia coli. The fusion protein converted GA12 to GA9 (major) and GA25 (minor); GA53 was metabolized less effectively and only to GA44. Thus, the M3-8 protein is functionally similar to GA 20-oxidases from Arabidopsis thaliana, Spinacia oleracea, and Pisum sativum, but different from that from Cucurbita maxima seeds, to which its amino acid sequence is most closely related. mRNA hybridizing to M3-8 accumulated in embryos and endosperm of M. macrocarpus, but was absent in vegetative tissues.

Effect of probenecid and benzbromarone on gluconeogenesis in isolated rat liver cells.

The effect of benzbromarone on gluconeogenesis from several gluconeogenic substrates in isolated rat liver cells is reported. Benzbromarone inhibited glucose synthesis from all substrates employed when the drug was used at concentrations half to ten times greater than its therapeutic plasma levels. This inhibition was more pronounced from lactate and pyruvate than from fructose and glycerol. The results are compared with those obtained for probenecid, a classical uricosuric drug. We found that probenecid inhibited the pathway in the same way as benzbromarone.

Inhibition of purine catabolism by benzbromarone in isolated rat liver cells. Comparison with allopurinol and probenecid.

Benzbromarone, a potent uricosuric agent, inhibited allantoin production in isolated hepatocytes at concentrations half to ten times greater than therapeutic plasma levels of the drug. In addition, the drug at these concentrations also markedly inhibited xanthine oxidase (EC 1.2.1.37), an enzyme involved in the regulation of this pathway. We found that allopurinol is several times superior to benzbromarone in the lowering of allantoin production (if they are compared in terms of their relative therapeutic levels), and that probenecid had no effect on it.