Metabolism of [3H]gibberellin A5 by immature seeds of apricot (Prunus armeniaca L.)

Abstract

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A5 (GA5) as 1- and 1,2-[3H]GA5 (5.3 Curies per millimole to -16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [3H]GA-like metabolites were separated from the highly H2O-soluble [3H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted → isocratic eluted reversed-phase Cl8 high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 x expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [3H] GA5 were identified as GA1, GA3, and GA6 by GC-SIM. The major highly water soluble metabolite of [3H]GA5 at all levels of substrate GA5 had chromatographic characteristics similar to authentic GA1-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [3H]GA5 feeds (2 x expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA1, GA3, GA5 methyl ester, GA6, GA22, GA2, (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA1, GA3, GA5, and GA8 (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA6 and GA29 would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GAs. Increasing substrate GA5 levels increased the proportion of metabolites with HPLC Rts similar to GA1, GA6, and especially GA1 glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA3, GA3-glucosyl ester, and a postulated conjugate of GA6. There was evidence that high doses of substrate GA5 induced new metabolites which often, but not always, differed from GA1 GA3, and GA6 in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M+ and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities. Immature seeds or fruits are rich in Gas,4 and the initial characterizations of GAs in higher plants has been from these tissues (5, 10, 11, 17, 25, 26, and see references in Bearder [2]). Even though related species contain structurally similar GAs (2), the interconversion pathways and rapidity of metabolism may differ with the plant organ and its developmental stage (10-12). Gibberellin A5 has been identified by GC-SIM as being native to immature seeds of apricot (Prunus armeniaca) (4). It is a potential precursor of GA1. (14), GA3 (9, 14), and GA29 (14), of which GA1, and GA29 are known to be native to immature seeds of apricot (4). Gibberellin A5is also a possible, though unlikely, precursor of GA32. In the present work we examine the metabolism of [3H]GA5 of varying substrate amounts in this tissue.