The metabolism of steviol to 13-hydroxylated ent-Gibberellanes and ent-kauranes

Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with '4"CGA1r7-aldehyde (['4CGA12ald) would serve as appropriate I'Cimarkers for the recovery of naturally-occurring gibberellins (GAs). The I'CIGA12Ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using 14,5-ClCmevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high perfornumce liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as I'4C1markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co-eluting with each 114Cimarker. These '4"C and UV-absorbing peaks were shown to contain GAI3, GA4, GA2, GA,, and GA17 by GC-MS. The fmding of GAS3 is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using ['4CjGA,2ald metabolites as I'4Cimarkers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species.

mentioning

confidence: 99%

Identification of Pea Gibberellins by Studying [¹⁴C]GA₁₂-Aldehyde Metabolism

Maki

Brenner²,

Birnberg³

et al. 1986

“…The remaining plant material was frozen in liquid N2, lyophilized, and stored at -15 C prior to extraction. Approximately 2,500 plants yielded 2 Fractions which contained biological activity, or eluted at the same step as authentic GAs, were subjected to final purification using analytical reverse phase HPLC with a ,uBondapak C18 column as described earlier (11). Elution of GAs from this column was achieved with either a 30-100% linear gradient in methanol in 1% aqueous acetic acid (grad A), or a 10-70% linear gradient (grad B).…”

mentioning

confidence: 99%

Identification of Six Endogenous Gibberellins in Spinach Shoots

Metzger¹,

Zeevaart²

1980

Analysis of highly purified extracts from spinach shoots by combined gas chromatography-mass spectrometry has demonstrated the presence of six 13-C-hydroxylated gibberellins (GAs): GA53, GA", GA1, GA17, GA20, and GA29. The major GAs were GA17, GA19, and GA2o, whereas the other three GAs occurred in trace amounts. Structural considerations suggest that the six GAs identflSed in spinach are related in the following metabolic sequence: GAI3 -. GA4 -. GA1, -. GA17 -. GA20 -) GA29.LD3 rosette plants respond to a transfer from SD to LD with increased stem elongation and subsequent flower formation. In many instances application of GAs to plants grown under SD will cause stem growth (10). Work in this laboratory with spinach (18,19) demonstrated that photoperiodic control of stem elongation is mediated by GAs. It was shown that although LD did not act to increase the total GA content of spinach plants, GA turnover was enhanced (18). It was also found that the levels of individual GAs were altered. However, before the mechanism of photoperiodic regulation of GA metabolism can be fully investigated, the chemical identity of the endogenous GAs should be determined. In this paper we report the identification of six GAs in spinach and discuss the possible metabolic relationships between those GAs. MATERIALS AND METHODS

“…Such a pathway may include enzymes in the GA biosynthetic pathway that are acting on non-GA substrates, e.g. antheridiogen specific substrates, or enzymes distinct from GA biosynthesis that catalyze similar cyclization and hydroxylation steps (1).…”

Section: Gametophyte Culturementioning

confidence: 99%

Evidence for a Gibberellin Biosynthetic Origin of Ceratopteris Antheridiogen

Warne¹,

Hickok²

1989

The species-specific chemical messenger, antheridiogen Ac*, mediates the differentiation of male gametophytes in the fern Ceratopteris. In order to investigate the biochemical origin of antheridiogen, the effect of the inhibitors, 2'-isopropyl-4'-(trimethylammoniumchloride)-5'-methylphenylpiperidine-1-carboxylate (AMO-1618), 2-chloroethyl trimethylammonium chloride (CCC), and a-cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) on gametophytic sex expression was determined in C. richardli. Both AMO-1618 (24,26). At sexual maturity a dimorphic gametophytic population consists of large cordate and small spatulate individuals. Cordates are either female or hermaphroditic and possess a defined meristematic region, whereas males are ameristic and bear only antheridia. Characteristic frequencies of sexual types depend upon population dependent antheridiogen production and accumulation, the sensitivity of gametophytes, and asynchrony in population development (17,24,29,31,32). A genetic basis for this trait, suggested by extensive phenotypic variability, has recently been identified and described (15,29,33 MATERIALS AND METHODS Plant MaterialSpores of the diploid (n = 39) homosporous fern, Ceratopteris richardii from three strains were used in this study, the 2 Abbreviations: AMO-1618, 2'-isopropyl-4'-(trimethylammoniumchloride)-5'-methylphenylpiperidine-1-carboxylate; CCC, 2-chloroethyl trimethylammonium chloride; ancymidol, a-cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidine methyl alcohol. Plant Physiol. Vol. 89, 1989 wild type (Hn-n) (12), and two derived mutants, HaA48,(13,14), and HaC18 (33). HaA48 is an abscisic acid tolerant strain that exhibits enhanced sensitivity to antheridiogen, whereas HaC 18 was selected for reduced sensitivity to antheridiogen. Based upon the responses of gametophytic populations (maximum percent males) to antheridiogen supplementation in the medium, these strains form a series of decreasing sensitivity to antheridiogen, i.e. HaA48 > Hn-n > HaC18. Gametophyte CultureGametophytic cultures were axenically established according to standard procedures in 24-well tissue culture dishes (14, 34). About 100 spores/well were sown onto 4.2 mL of 1% agar solidified medium containing Parker's macronutrients and Thompson's micronutrients (16) (v/v %) into medium prior to pH titration and autoclaving. InhibitorsInhibitors (ancymidol, AMO-1618, CCC) or solvents alone (controls) were filter sterilized (0.2 ,um) into autoclaved and cooled medium. For AMO-1618 and CCC, 0.5% DMSO was used as a solvent (final concentration 0.05% for 1 mm treatment and controls). For ancymidol, a mixture of0.5 % DMSO and 5% ethanol (95%) were used as solvents (final concentrations; 0.05% DMSO and 0.5% ethanol (95%) for the 1 mM treatments and controls). Severe effects on gametophyte development were evident with 0.5% DMSO and greater (TR Warne, LG Hickok, unpublished data). CCC and AMO-1618 were purchased from Sigma Chemical Co. and Calbiochem Co., respectively. AnalysisAt 14 d following...