Metabolism of Tritiated Gibberellin A₂₀ in Maize

Abstract

After the application of 2.36 Curies per milUmole 12,3-'Hlgibbereuln A20 (GA20) to 21-day-old maize (Zea mays L., hybrid CM7 x CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of 3H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as I'HIGA1 on the basis of co-chromatography with standard I3HIGA1 on SiO2 partition, high resolution isocratic elution reverse phase C1i high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metaboUtes were identified similarly as 13HIGA8 and C/D ring-rearranged I3HIGA2o, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C1i high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA1 and GAs, or with retention times where conjugates of GA20 would be expected to elute. Conversion to I3HIGAi was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedigs, the C/D ring-rearranged I3HIGA2,-like metabolite was the major acidic product, while conversion to I3HIGA1 was low.Hedden et al. (10) have recently characterized GAui,5 GA44, GA17, GAi0, and GA20 from immature maize tassels by GLC-MS.Further, data from selected ion current monitoring GLC-MS indicate that GA1, GA29, and GA8 are probably also native in maize (10). These GAs are members of the probable early 13-hydroxylation pathway, a pathway which begins with GA12-aldehyde. The 13-hydroxylation pathway also exists in Phaseolus vulgaris (9) where GA20 is apparently a precursor of either GA1 or GA29 (19). In other plant systems, two principal metabolites have been tentatively identified following the application of [3HJGA20: 13,16). Since both GA1 and GA29 appear to be native in maize (10), a branch point in the metabolic pathway in maize may exist after GA20. The biologically active GA1 (1) may be produced through C-3 hydroxylation, or alternatively, C-2 hydroxylation may yield the biologically inactive GA29. Given 21-d-Old Maize Plants. Three kernels of the early maturing maize (Zea mays L.) hybrid CM7 x CM49 (17) were planted in each of 12 plastic pots (13 x 20 cm) filled with a mixture of peat moss and sand. Pots were placed in a growth room at 25/15°C (day/night), 14-h photoperiod (300 ,AE m-2 S-1). After emergence, plants were thinned to one per pot. Twenty-one days after planting (at which time the plants were still in the vegetative growth phase), 0.22 ,uCi [2,3-3HJGA20 (2.36 Ci/mmol) in 0.4 ml 60% aqueous ethanol was pipetted into the leaf whorl. Twenty-four, 48, 96, and 144 h after the addition of [3H]GA2o, three plant shoots were excised at the soil surface and 4 cm above the surface, yielding shoot cylinders which contained the apical meristems. These were rinsed with H20 and then homogenized separately at -40°C in MeOH:H20 (80:20, v/v

Reversible Conjugation of Gibberellins In Situ in Maize

“…Whereas these compounds have been previously observed following [3H]GA20 feeds (9,23), the C/D ring rearrangement proceeds readily under acidic conditions (9), and in the present study, extracts were repeatedly exposed to acetic acid and HCOOH during chromatography and evaporation of fractions.…”

“…Peak IV elutes after [3H]GA20 (Fig. 6) and may consist of one or more conjugates of C/D R [3H]GA2o, since C/DR [3HJGA2o elutes 4.6 min after [3H]GA20 (23). The presence of free and conjugated C/D R [3H]GA2o may be an artifact.…”

“…An on-line HPLC-RC (Berthold) was used to detect radioactive peaks anid aliquots of HPLC fractions were also taken for liquid scintillation spectrometry. More complete descriptions ofHPLC-RC and subsequent GLC-RC characterization on some of these samples, and on other maize samples after [3H]GA2o feeds, have already been published (22,23 GAs and their purported conjugates.…”

“…Although one of the native GAs fed to pea (GA20) is a precursor of GA, in one pea genotype (J. B. Reid, University of Tasmania, personal communication) and in other systems (23,30), in dwarf pea GA20 apparently does not form GA,, but is converted to GA29 through 2(i-hydroxylation (10), a step which leads to a high degree ofbiological inactivation. Thus, it is possible that the apparent reversible conjugation observed by Barendse et al (2) may have been an artifact brought about through the exogenous application of a foreign GA (e.g.…”

“…GAs and are readily taken up and metabolized by maize (22,23, and Rood and Pharis, unpublished). Both occur in the GA conversion sequences known for higher plants at positions which provide a reasonable number of acidic products, as well as GA4 and GA20 per se, which could be conjugated during seed maturation.…”