Identification of Pea Gibberellins by Studying [ 14 C]GA 12 -Aldehyde Metabolism

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To interpret the metabolism of radiolabeled gibberellins A12-aldehyde and A12 in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [14C]GA12 and [14C]GA12-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC).[14C]GA12 was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [14C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GAN, GA", GA19 and GA20 was demonstrated and their HPLC peak identity ascertained. The 14C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GAN to 4% in GA20. Calculated levels of GA2o, GA19, GA.", and GAN, were 42, 8, 10, and 0.5 nanograms/ gram, respectively.The identification of endogenous gibberellins provides the necessary starting point from which metabolism and physiological studies can begin. The endogenous GAs of pea seeds and shoots have been extensively studied but evidence for some metabolic steps in vivo is still lacking (7,14). In the G2 genetic line of.neas (Pisum sativum L.) growth is indeterminate in short photoperiods while in long photoperiods the senescence of the plants takes place (12). Indeterminate growth is correlated with a higher level of biologically active GAs (13) which were subsequently associated with GAs identified by GC-MS as GA20 and GA,9 (5). In addition, the presence of GA29 and GA29-catabolite was shown in shoot extracts (5). When ['4C]GA,2-aldehyde is metabolized by G2 pea shoots many products are formed (4). The levels of the 14C metabolites tentatively identified as GA53, GA44, GA19, and GA20 were higher in shoots grown in SD than in those grown in LD. Since the products of ['4C]GA,2-aldehyde were tentatively identified from the HPLC retention time of en-

Section: Gc-ms Analysissupporting

confidence: 73%

“…By following the radioactivity in the peaks the endogenous materials are copurified (9). GC-MS provided full scan mass spectra of each peak.…”

Section: Gc-ms Analysismentioning

confidence: 99%

Further Identification of Endogenous Gibberellins in the Shoots of Pea, Line G2

Halinska¹,

Davies²,

Lee³

et al. 1989

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“…In contrast to the results with peas (19), there were no detectable UV-absorbing (205 nm) peaks with the same Rt as the radioactive markers. Even so, each fraction was analyzed for nonlabeled GAs by GC-MS, and by GC-SIM for characteristic ions of appropriate 'early 3-OH' or 'early 3,13-OH' GAs (5).…”

contrasting

confidence: 51%

“…Cyathea australis may have both pathways except for the dehydrogenation of GA4 to GA7; this fern probably further oxidized GA4 and GA9 to 11-OH and 12-OH GAs (29). Soybeans may use the pathway to GA7 just described since: (a) In (19) allowed us to isolate GA7 and the GA, component with sufficient purity to obtain clean mass spectra by GC-MS. Purification using bioassay alone probably would have been insufficient to yield fractions pure enough to identify by GC-MS. In fact, the bioactivity associated with GA7 ( Fig.…”

mentioning

confidence: 99%

Metabolism of Gibberellin A₁₂-7-Aldehyde by Soybean Cotyledons and Its Use in Identifying Gibberellin A₇ as an Endogenous Gibberellin

Birnberg¹,

Brenner²,

Mardaus³

et al. 1986

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The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill-about 10 nanograms GA3 equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. elucidating the pathways for their synthesis is a first step in understanding the roles that GAs play in the physiology of this major crop. Since seeds of many legumes are relatively rich sources of GAs (e.g. up to 125 gg/g fresh weight in Phaseolus coccineus [8]), this suggests soybean seeds should be the first tissue examined for this purpose.The GAs in extracts of higher plants usually have been prepared for identification by solvent partitioning, followed by either silica gel partition chromatography or reverse phase HPLC (5). After one or two such LC steps, fractions demonstrating biological activity, or pools of fractions with LC Rts similar to known GAs can usually be analyzed by GC-MS (e.g. 1). However, this approach has not yielded detectable levels of any GAs in immature seeds or seedlings of soybean (V Sponsel, personal communication to P Birnberg and M Brenner).The failure of these traditional methods to identify any GAs in soybeans suggests either that soybean GA levels are lower than in most seeds, or that their presence is masked by compounds which are not removed by the minimal purification procedures used. Therefore, we decided to study soybean cotyledon metab-

“…Several GAs are now known in both seeds and shoots of peas (2 and references therein; 7,13). These GAs are all derived from GA,2 aldehyde, the first GA compound on the GA synthetic pathway (20).…”

mentioning

confidence: 99%

Photoperiod Modification of [¹⁴C]Gibberellin A₁₂ Aldehyde Metabolism in Shoots of Pea, Line G2

Davies¹,

Birnberg²,

Maki³

et al. 1986

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In G2 peas (Pisum sativum L.) apical senescence occurs only in long days (LD), and indeterminate growth is associated with elevated gibberellin (GA) levels in the shoot in short days (SD). Metabolism of GA12 aldehyde was investigated by feeding shoots grown in SD or LD with I"CiGA,2 aldehyde through the cut end of the stem for 0.5 to 6 hours in the light and analyzing the tissue extract by high performance liquid chromatography. More radioactive products were detected than can be accounted for by the two GA metabolic pathways previously known to be present in peas. Three of the major products appear to be GA conjugates, but an additional pathway(s) of GA metabolism may be present. The levels of putative C20 GAs, '4CiGAs3, 4qC]GA44, ['4CJGA,,, and/or I'4C1 GA17, were all elevated in SD as compared to LD. Putative 114CIGA, was slightly higher in LD than in SD. Putative I'4C]GAs3 was a major metabolite after 30 minutes of treatment in SD but had declined after longer treatment times to be replaced by elevated levels of putative I'4C] GA44 and 114CIGA9/,,1. Metabolism of GA2o was slow in both photoperiods. Although GA2o and GA,9 are the major endogenous GAs as determined by gas chromatography-mass spectrometry, putative j'4CJGA20 and I'4CIGA,, were never major products of I'4C]GA,2 aldehyde metabolism. Thus, photoperiod acts in G2 peas to change the rate of GA53 production from GA12 aldehyde, with the levels of the subsequent GAs on the 13-OH pathway being determined by the amount of GA53 being produced.In the G2 genetic line of peas (Pisum sativum L.) senescence of the apical bud, which is a prelude to the senescence of the whole plant, takes place only in long photoperiods (17). In short photoperiods growth is indeterminate. The prevention of senescence by SD is associated with the presence of an elevated level