Planta(1996) 200:159 166 P l a n t n (c) Springer-Verlag 1996 Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L. David N. Martin 1, William M. Proebsting ~, T. Dawn Parks 2, William G. Dougherty 2, Theodor Lange 3, Mervyn J. Lewis 4, Paul Gaskin 4, Peter Hedden 4 1 Department of Horticulture and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331-7304, USA 2 Department of Microbiology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis, OR 97331-7304, USA 3 Pflanzenphysiologisches Institut und Botanischer Garten der Universit~it G6ttingen, Untere Karspiile 2, D-37073 G6ttingen, Germany 4 IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Bristol BS18 9AF, UK Received: 21 March 1996/Accepted: 12 May 1996 Abstract. Treatment of tall and dwarf (3[3-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C2o-GA, GA19. In shoots of the slender (la cry s) mutant, the con- tent of C19-GAs was lower and GA]9 content was higher than in those of the tall line. Metabolism of GA]9 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feed- back regulation of the 20-oxidation and 3[3-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase eDNA from pea. The eDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this eDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C~9- GA, GAg, which was the major product. The 13-hy- droxylated substrate GAs3 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20. Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treat- ment with GA3, suggesting that there is feed-back regula- tion of GA 20-0xidase gene expression. Key words: Gibberellin biosynthesis (regulation) - Gib- berellin 20-oxidase (eDNA cloning) Pisum The EMBL accession number for the eDNA clone described in this article is X91658 Abbreviations: GA = gibberellin; GC-SIM = gas chromatogra- phy-single ion monitoring; IPTG = isopropyl-[~-D-thiogalactoside; PCR = polymerase chain reaction Correspondence to: P. Hedden; FAX: 44 (1275)394281; Tel.: 44 (1275) 549 263; E-mail: peter.hedden@bbsrc.ac.uk Introduction Gibberellins (GAs) are major determinants of stem height in plants. Genes altering either GA biosynthesis or sensi- tivity have had a profound impact on agriculture and the development of new plant varieties. Efforts to understand regulation of GA biosynthesis and stem elongation have focused on GA1, the principal bioactive GA in many species (Phinney 1984; Ingram et al. 1986). The major pathway of GA1 biosynthesis in pea shoots is considered to involve early 13-hydroxylation (Davies et al. 1982), as shown in Fig. 1. In this metabolic sequence, GAI2 is 13-hydroxylated to GAs3, which is then converted suc- cessively to GA44, GA19 and GA2o by 20-oxidation (Fig. 1). Oxidation of GA19 at C-20 can yield either the biologically inactive C2o compound, GA17, or GA20 , a C19-GA, by loss of C-20. The GA2o can then be 313- hydroxylated to the bioactive GAb or 2~-hydroxylated to form biologically inactive GA29. Deactivation of GA~ by 213-hydroxylation to GAs also occurs. Gibberellin 20- oxidases have been cloned from several species and shown to catalyze each of the 20-oxidation steps (Lange et al. 1994; Phillips et al. 1995; Xu et al. 1995; Wu et al. 1996). Recently, the GA4 gene of Arabidopsis thaliana, assumed to control 313-hydroxylation, was cloned by T-DNA tag- ging (Chiang et al. 1995). Many genetic mutations result in decreased GA] con- tent and, thereby, reduce stem growth (Ross 1994). In pea, for example, four loci (ha, le, lh, ls) contain alleles that reduce GA biosynthesis and produce varying degrees of dwarfing (Reid and Ross 1993). In these mutants, a normal tall phenotype is restored by application of active GAs or by biosynthetic precursors occurring after the genetic block. Dwarfing may also result from reduced sensitivity to GA (Phinney 1961; Gale and Marshall 1973; Koornneef et al. 1985; Reid and Ross 1993). Although these muta- tions do not affect GA biosynthesis directly, GA metabol- ism may be affected nonetheless. For instance, the Rht alleles (dwarfing) of wheat cause accumulation of GA1 and GA2o, and reduce GA19 content in the leaf sheath compared to rht (wild type), which accumulates GA19