Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10515-10518, September 1996 Plant Biology The dwarf-i (dl) mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway (biological activity/GC-MS identification/metabolism) CLIVE R. SPRAY*, MASATOMO KOBAYASHI*, YOSHIHITO SUZUKI*, BERNARD 0. PHINNEY*, PAUL GASKINt, AND JAKE MACMILLANt *Department of Biology, University of California, Los Angeles, CA 90095-1606; and tlnstitute of Arable Crops Research-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, BS18 9AF, United Kingdom Contributed by Bernard 0. Phinney, May 29, 1996 ABSTRACT In plants, gibberellin (GA)-responding mu- linear and there is no evidence for a metabolic grid with the tants have been used as tools to identify the genes that control other pathways, as has been shown for cell-free preparations specific steps in the GA-biosynthetic pathway. They have also obtained from seeds of bean, cucumber, and pea (for review, been used to determine which native GAs are activeper se, i.e., see ref. 3). further metabolism is not necessary for bioactivity. We The biological significance of the metabolic studies in maize present metabolic evidence that the Dl gene of maize (Zea comes from the use of GA mutants that exhibit a dwarf mays L.) controls the three biosynthetic steps: GA20 to GA1, phenotype, yet respond by normal growth to applied GAs. GA20 to GA5, and GA5 to GA3. We also present evidence that Thus, the relative responses of the mutants to specific GAs three gibberellins, GA1, GA5, and GA3, have per se activity in together with information on the role of the genes in control- stimulating shoot elongation in maize. The metabolic evidence ling specific steps in the pathway have led to the conclusion comes from the injection of [17-13C,3H]GA20 and [17- that only a limited number of GAs in the pathway are active 13C,3H] GA5 into seedlings ofdl and controls (normal and d5), per se, i.e., they do not require further metabolism to be followed by isolation and identification of the 13C-labeled bioactive (for reviews, see refs. 10-12). metabolites by full-scan GC-MS and Kovats retention index. The purpose of the present study was to examine the For the controls, GA20 was metabolized to GA1, GA3, and GA5; metabolic steps, GA20 to GA5, GA5 to GA3 and GA20 to GA1 GA5 was metabolized to GA3. For the dl mutant, GA20 was not in relation to the dl mutation (blockage after GA20). To this metabolized to GA1, GA3, or to GA5, and GA5 was not end, [17-13C,3H]GA2o and [17-13C,3H]GAs were fed to dl metabolized to GA3. The bioassay evidence is based on dosage seedlings and to controls (normal and d5 seedlings) and the response curves using dl seedlings for assay. GA1, GA3, and metabolites from the feeds were analyzed by full-scan GC-MS. GA5 had similar bioactivities, and they were 10-times more In addition, the bioactivities of GA20, GA1, GA5, and GA3 were active than GA20. determined using dl seedlings for assay. The gibberellins (GAs) are tetracarbocyclic diterpenes that occur naturally in higher plants (1). There is continued interest MATERIALS AND METHODS in the biosynthetic origin of the GAs since some of them are GAs. The [17-13C,3H]GA2o (0.915 atoms 13C per molecule, known to act as native regulators controlling a range of growth 1.79 GBq/mmol) was synthesized as described by Ingram et al. responses, including seed germination, floral development, (13) and purified as described by Kobayashi et al. (14). and shoot elongation (for reviews, see refs. 2 and 3). [17-13C,3H]GAs (0.915 atoms 13C per molecule, 1.51 GBq/ All GAs are biosynthesized from trans-geranylgeranyl mmol) was synthesized by Fujioka et al. (5) and purified before diphosphate (GGDP) via ent-copalyl diphosphate (CDP) and use by HPLC on a LiChrosorb C-18 column. Both compounds the tetracyclic hydrocarbon, ent-kaurene. ent-Kaurene is se- were radiochemically pure; no chemical impurities were de- quentially oxidized to ent-7a-hydroxykaurenoic acid, which is tected by GC-MS. then rearranged to GA12-aldehyde and oxidized to GA12. At The unlabeled GA1, GA3, GA5, and GA20, used for the least three pathways diverge from GA12-aldehyde and GA12: bioassays, were 99% pure as determined by GC-MS. No GA (i) the early-non-3,13-hydroxylation pathway, (ii) the early-3- contaminants were detected. For bioassays, each GA was hydroxylation pathway, and (iii) the early-13-hydroxylation dissolved in (Me)2CO/H20 (1:1, vol/vol). pathway. The early-non-3,13-hydroxylation pathway and early- Plant Material. The dl and dS maize mutants were seg- 3-hydroxylation pathway were initially shown to be present in regants from CC5/L317 hybrid seed stocks. The Dl gene the fungus, Gibberella fujikuroi, and later in higher plants (for controls a step(s) late in the pathway, whereas the D5 gene review, see ref. 4). The studies with higher plants also showed controls a step early in the pathway (5). For each stock, the presence of the early-13-hydroxylation pathway, which is seedlings segregated in the ratio of 3:1 normal/dwarf. Seeds unique to higher plants (for review, see ref. 3) and is the main were soaked in H20 for 24 hr and grown in vermiculite/soil pathway in vegetative shoots of Zea mays (maize). Our evi- (1:1) in the University of California (Los Angeles) greenhouse. dence for the presence of the early-13-hydroxylation pathway Ten-day-old seedlings (1-leaf stage) were used for bioassay; in maize was based on the GC-MS identification from vege- 3-week-old seedlings (4-leaf stage) were used for metabolic tative shoots of the 10 gibberellins (GA12, GA53, GA44, GA19, studies. GA17, GA20, GA29, GA1, GA5, and GA3) (5, 6) and the Bioassay Studies. The dl seedlings were treated by adding placement of these GAs in a series of metabolic steps by 50 ,ul of the GA solution into the axil of the first unfolding isotope labeling studies (7-9). Except for the terminal steps, leaf. Ten days later, the lengths of the first and second leaf the early-13-hydroxylation pathway in maize appears to be sheaths were measured and averaged to give the response data. The publication costs of this article were defrayed in part by page charge Abbreviations: GA, gibberellin; AE, acidic ethyl acetate soluble; NB, payment. This article must therefore be hereby marked "advertisement" in n-butanol soluble; NBE, ethyl acetate soluble-hydrolyzed neutral accordance with 18 U.S.C. §1734 solely to indicate this fact. butanol; KRIs, Kovats retention indices. 10515 Downloaded by guest on September 23, 2021 10516 Plant Biology: Spray et aL Proc. Natl. Acad. Sci. USA 93 (1996) Five dosage levels were used for each compound. Each assay 60°C and, after a 2 min isothermal hold, was programmed at point is the mean of measurements from 10 plants (standard 100 min-' to 150°C and then at 30 min-' to 300°C (OV-1) or errors are shown as vertical bars when greater than the point 280°C (OV-1701) with a 10-min isothermal hold at the end of size). the program. The pressure of the helium carrier gas was 100 Metabolic Studies. Each labeled substrate was dissolved in kPa. The column effluent was led directly to the ion source of EtOH/H20 (1:1, vol/vol) and 2 ,ul was injected into the basal a VG Analytical (Manchester, U.K.) 70-250 computerized part (about 1 cm below the ligule of the first leaf) of individual GC-MS with a source temperature of 220°C and an interface seedlings. Shoots were harvested 12 hr after treatment. temperature of 280°C. The ionizing potential was 24 eV and For [17-13C,3H]GA2o feeds, 10 each of dl, d5, and normal the mass spectra were recorded at 1 Hz. seedlings were injected with 4983 Bq per seedling. For [17- The metabolites (Tables 1 and 2) were identified by full-scan 13C,3H]GAs feeds, five seedlings of each genotype were in- GC-MS. A mixture of n-alkanes (C16-C36, ca. 1-15 ng of each) jected with 1800 Bq per seedling. was coinjected with each sample to provide a KRI (17) for each After harvest, the shoots were frozen in dry ice and homog- GC peak. enized with a mortar and pestle. The powdered plant material was added to a 10-fold excess (vol/wt) of MeOH/H20 (4:1, vol/vol) and stored at -20°C for 24 hr. The mixture was RESULTS filtered and the solid residue was extracted a second time with After treatment with the substrates [17-13C,3H]GA2o and a excess 10-fold (vol/wt) of MeOH/H20 (4:1, vol/vol). The [17-13C,3H]GAs, fractions were recovered from normal, dS, two aqueous methanolic extracts were combined and concen- and dl seedlings. The information from these fractions is trated in vacuo to give an aqueous residue (60 ml) that was shown in Tables 1 and 2. Each metabolite was tentatively solvent fractionated as described by Fujioka et al. (5) to give identified from its HPLC retention volume and conclusively the acidic ethyl acetate soluble (AE) fraction. The AE fraction established by comparing full-scan GC-MS and KRI data with was purified as described by Kobayashi et al. (15) by successive unlabeled reference spectra (16). Typical GC-MS data are use of Bond-Elut C18 and Bond-Elut DEA columns, followed shown in Table 3.