Uptake and metabolism of 3H-Gibberellin A1 by barley aleurone layers: Response to abscisic acid

Self Cite

The major metabolite produced during incubation of ['H] ['H]ampho GA1 in barley aleurones begins after a period of 2.5 hours. As judged by degradation studies as well as Sephadex column chromatography, GA1 appears to be linked to a peptide; positions C-3 and C-7 were ruled out as conjugation sites.Among the most studied effects of gibberellins on plant tissues is the induction of certain hydrolytic enzymes in barley aleurone layers (5,7,10,11,12 2 Ernest Sondheimer's research was characterized by his consistent efforts to provide a chemical basis for an understanding of plant hormone action. He understood that simply measuring a change in concentration of a hormone is not sufficient to explain its function in a process. Not only was he concerned with the occurrence of hormones but also with their identity. The dedication of this volume to mark his lamentably early death should serve to focus the attention of those engaged in growth regulator research on the need to attain a fuller comprehension of the role of hormone biosynthesis and turnover in relation to physiological processes. If this occurs his contribution will remain as much more than a footnote in the annals of hormone research. Generally, the incubation procedures folowed were those described by Chrispeels and Varner (3). The buffer was 2 mM in sodium acetate and 20 mm in CaCl, adjusted to pH 4.8. One hundred fifteen isolated aleurone layers were incubated at 25 C in 6 ml of buffer which was 48 ,uM in L'H]GA1 (10 ug, 2.6 X 10' dpm, per 6.0 ml) and 19 FM in ABA (30 ,g per 6 ml). Most of the layers were incubated 24 hr, but nine lots of three layers each were withdrawn at earlier intervals to determine the onset of formation of I (Fig. 1). After 24 hr of incubation, radioactive materials in the remaining layers were extracted with methyl alcohol. The methyl alcohol extract was concentrated by distillation under high vacuum so that the temperature never exceeded 45 C. The concentrate was refined by TLC on ChromAR, using isopropanol-NH,OH-water (8: 1: 1). In this system, I was the only radioactive material remaining at the origin.The chromatographic systems and the RF values of the compounds studied are shown in Table I. Details on these procedures have been reported previously (8) and are summarized in Table II along with the retention times of the compounds under investigation.High voltage paper electrophoresis was done using sheets of Whatman No. 3 MM paper having basic weight = 185 g/m', 0.33 mm thickness, medium flow rate, and smooth surface. In all runs the voltage was 1500 v, and the time was 1.5 hr. Zones were detected by radiochromatogram scanning. Buffers were: pH 10.6, NH4OH (0.065 M); pH 5.9, pyridine-acetic acid-H,O (100:15:900); pH 2.9, pyridine-acetic acid-H.0 (3:100:1900); pH 2.2, 2.5% formic acid-8% acetic acid, with water. RESULTS AND DISCUSSION

supporting

confidence: 86%

mentioning

confidence: 99%

An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

Nadeau

Rappaport²

1974

Self Cite

“…The first was received from H. Kende (9) and had a specific radioactivity of 420 mCi/mmole. The second was received from L. Rappaport (14) with a specific radioactivity of 48 Ci/mmole. Experiments were conducted with 3H-GA1 concentrations of 1 iLM to 10 nM.…”

Section: Methodsmentioning

confidence: 99%

Transport and Metabolism of ³H-Gibberellin A₁ in Dioecious Cucumber Seedlings

Rudich

Sell²,

Baker³

1976

The transport of 3H-GA1 through hypocotyl segments of cucumber (Cucumis sadivus L.) was found to be nonpolar. The transport of 3H-GA1 was increased by pretreatment with relatively high concentrations of either IAA or Ethephon (2-chloroethylphosphonic acid). Hypocotyl segments from plants of a gynoecious genotype transported more 3H-GA1 than those of an androecious. The metabolism of 3H-GA1 in hypocotyl segments was neither related to the sex genotype of the cucumber plant nor influenced by pretreatment with Ethephon. The primary metabolite of GA, was suggested to be GA8. Two other suspected metabolites were not identified. Differences in the endogenous GA of gynoecious and androecious plants could not be accounted for by transport differences.The sex expression of cucumber is influenced by the hormonal balance of auxin, ethylene, GA and ABA (1,5,6,18,20,21). Male flower production in cucumber is associated with endogenous GA levels (7,21,26). Exogenous application of GA3, GA4, GA7, and GA9 also increase the production of male flowers on both monoecious and gynoecious phenotypes (2, 26). Gynoecious phenotypes contain far less GA than monoecious (7,21), whereas the qualitative GA composition of both seeds and green plants is similar for all sex phenotypes. Most endogenous GA is present as GA, and GA3 with traces of GA4 and GA7 (7). Conjugated derivatives of GA have also been identified from cucumber seed (8), but appear to play no regulatory role in sex expression. The primary hormones associated with female flower production are ethylene (1,19,20) and auxin (5, 6). Treatment of cucumber plants with Ethephon actually decreases the endogenous GA activity (21).Accordingly, one possibility for the control of sex expression in cucumber is via ethylene biosynthesis and its effect on GA. Other possibilities (12) for hormonal control of sex expression might be a conversion from biologically active to inactive forms via conjugation or degradation, differences in biosynthesis, or differences in transport. We investigated the possibility that GA1 transport and/or metabolic differences as affected by ethylene might regulate the difference between male and female expression in cucumber. MATERIALS Hypobaric Ventilation. These experiments were conducted with explants under a reduced pressure of 0.2 atm to decrease internal ethylene concentration (1). Experiments were conducted over a 24-hr period with a gas mixture of 99.85% 02 and 0.15% C02. For ethylene treatments, the gas mixture was 99.85% 02, 3 ,u1/l C2H4, and 0.15% CO2. All experiments were conducted at a temperature of 25 C with continuous light (400 ft-c). All treatments were replicated three times in a completely randomized design.Radioactive 3H-GA1. Two sources of 3H-GA1 were used. The first was received from H. Kende (9) and had a specific radioactivity of 420 mCi/mmole. The second was received from L. Rappaport (14) with a specific radioactivity of 48 Ci/mmole. Experiments were conducted with 3H-GA1 concentrations of 1 iLM to 10 nM.Agar Block Preparation. ...

“…The results herein and recent results by Sponsel and MacMillan (19), also using an in vivo method, contrast to their earlier results (18) which were consistent with the conclusions from their in vitro method. In this culture system [14,15,17-3HJGA29 was apparently not metabolized, except perhaps very slowly. This stability of the substrate may have been due to lack of resolution of the labeled products (19) or to the in vitro system used.…”

Section: Discussionmentioning

confidence: 82%

“…It has been assumed that a major pathway of GA deactivation is by C-2 hydroxylation and conjugation to molecules such as glucose (4). The formation of GA8 and GA8 glucoside from GA1 has been demonstrated in barley aleurone layers (14) and immature (20) and germinating (12) bean seed. Although GA20 was converted to GA29 (as well as GA1) in immature bean seeds, the conversion to GA29 glucoside was not reported (20).…”

mentioning

confidence: 99%

Metabolism of Tritiated Gibberellin A₂₀ in Immature Seeds of Dwarf Pea, cv. Meteor

Durley

Sassa²,

Pharis³

1979

Tritium-labeled gibberellin A20 (13HIGA2o) was applied via the pedicel to immature pods and seeds of dwarf peas and three harvests were made at days 5, 10, and 23 (mature) after application. Of the five metabolites of 13HIGA20, the three in highest yield were GA29, an a,fl-unsaturated ketone, and a compound (B), whose structure was only tentatively assigned.The metabolic sequence GA20 --GA29 -* compound B -* the ketone was indicated. The amount of 13HIGA29 in both seeds and pods was highest at day 5 and declined to its lowest level at maturity. The amount of the I3Hlketone in the seed increased with time to its highest level at maturity.It is suggested that compound B and the ketone represent the major pathway of catabolism of GA2,, a 2#-hydroxylated GA of low biological activity, and that the ketone is not metabolized, or only slowly metabolized, during seed maturation.The roles of gibberellins (GAs) in the growth of seeds and fruit have yet to be adequately resolved. Many GAs are present in substantially higher levels in immature seeds (4, 7) than in other parts of the plant. These high levels of GAs may be causally correlated with seed and fruit growth (1, 10) or may ensure that adequate levels are stored in the seed for use in growth processes during germination (1). Evidence for the latter role has been cast in doubt by recent work with 3H-GAs (cited in ref. 1).The major biologically active GA in immature pod and seed of pea is GA20 (7,8). In seeds of dwarf pea high levels of the less biologically active GA29 are present together with much smaller amounts of GA9, GA17, GAu, GA44, and GA51 (18).