An extract from 8-day-old cotton ovules (Gossypiumii hirsutuint L.) was partitioned into three fractions and each fraction was derivatized and analyzed separately. Gas-liquid chromatography and computer-controlled gas-liquid chromatography-mass spectrometry were used to separate, measure, and identify the naturally occurring plant hormones. A single extract contained abscisic acid, indoleacetic acid, and gibberellins A1, A3, A4. A7, A9, and A13 in the first fraction; ethyl indole-3-acetate and indole-3-aldehyde in the second fraction; and the cytokinins 6-(3-methyl-4-hydroxybutylamino)purine (dihydrozeatin), 6-(4-hydroxy-3-methyl-2-trans-butenylamino) purine (zeatin), 6-(3-methyl-2-butenylamino)purine (2iP), 6-(3-methyl-2-butenylamino) .9 -A -D -ribofuranosylpurine (2iPA), and 6-(4-hydroxy-3-methyl-2-trans-butenylamino) -9-/3-D-ribofuranosylpurine (zeatin riboside) in the third fraction.Many research workers have attempted to identify and measure hormone levels in cotton plants by using various bioassays (1,6,7,9,24,25,28). Because plant hormones are present in microgram quantities, the usefulness of bioassays has been limited because of interference of excessive quantities of impurities in extracts containing the hormones (2,14).Recently, development of GLC has provided a physicalchemical assay of ABA (11,19), auxins (13,29), cytokinins (30), and gibberellins (8), demonstrating the feasibility of qualitative as well as quantitative measurements of naturally occurring plant hormones. Gas-liquid chromatography-mass spectrometry has been used for further identification of some of these hormones (15,21,22,31).Computer-controlled GLC-MS4 has been used for identifica- 6-(4-hydroxy-3-methyl-2-trans-butenylamino)purine; dihydrozeatin: 6-(3-methyl-4-hydroxybutylamino)purine; zeatin riboside: 6-(4-hydroxy-3-methyl-2-trans-butenylamino)-9-PB-D-ribofuranosylpurine; 2iP: 6-(3-methyl-2-butenylamino)purine; 2iPA: 6-(3-methyl-2-butenylamino)-9-fl-D-ribofuranosylpurine: 3iPA: 6-(3-methyl-3-butenylamino)-9-I3-D-ribofuranosylpurine.tion of cytokinins (31), gibberellins, and ABA (21). Abscisic acid (11) and IAA (32) have been identified from cotton tissues by GLC but we have found no report in which ABA, gibberellins, cytokinins, and auxins were measured in a single extract by physical-chemical assays.The objective of this investigation was to utilize GLC and computer-controlled GLC-MS to separate, identify, and measure individual plant hormones of four major groups from a single extract of ovules from 8-day-old cotton fruit. MATERIALS AND METHODSCotton plants (Gossypiu,n hirsutum L., cv. Acala SJ-1) were grown in a greenhouse. Cotton flowers were tagged at anthesis and bolls were harvested 8 days thereafter. The developing ovules were separated from the fruits, frozen immediately on Dry Ice, and stored at -20 C until the time of extraction. Extraction Procedure. In different experiments, 2 to 5 g of frozen ovules were ground in cold 80% (v/v) aqueous methanol with a mortar and pestle. The macerate was transferred to a fla...

A bstract. The effect of exogenous growth regulators on movement of assimilates into flowers and young fruits of 'Black Corinth' grapes was studied. Clusters were treated with growth regulator and after 0.5 hr to 5 davs the leaves above the clusters were exposed to 14CO. Control shoots received 14CO9 but no growth regulator. At harvest, counting and radioautographic techniques were used to ascertain amount and distribution of activity in clusters. Clusters were dipped in 4-CPA (4-chlorophenoxyacetic acid), GA3 (gibberellic acid), or BA (benzyladenine). All berries were heavier than controls within 3 days. Total counts in the fruits were increased by 4-CPA, and the distribution of radioactivity among the sugar, organic acid, and amino acid fractions was usually altered by all treatments. In a time series experiment, within 6 hr after treatment of fruits with GA3 there was almost an 8-fold increase in total counts relative to the control. After 12 hr there was about a 9-fold and 6-fold increase in counts in tartaric and malic acids, respectively, and in y-aminobutyric acid, pipecolic acid, and valine increases of 56, 150, and 330 ters at the late bloom or early fruit-set stage were used. One cluster per shoot was retained and all clusters were trimmed to about the same size. Clusters or portions of clusters were dipped momentarilv in soluitions of 6 X 10-5 M gibberellic acid (GA3), 1.3 X 10-4 M of the auxin 4-chlorophenoxyacetic acid (4-CPA), or 8.9 X 10-3 M of the cytokinin benzvladenine (BA), using B-1956 at 0.05 % as a wetting agent. These concentrations of growth regulators were utilized because they had been previously shown to cause a large increase in size of 'Black Corinth' flowers and fruits (16,18,19). Two shoots were utilized per vine, and in all instances the same treatment was made to these 2 shoots on a given vine. Previous research revealed there is little or no movement of growth regulator out of a cluster, as they are importing photosynthate and olher compounds (17,18). Treatments were made between 8:00 and 9:00 AM. After a time interval ranging from 0.5 hr to 5 days the leaf immediately ibove and on the same side of the stem as the cluster was treated with 14CO2 for 30 min. A plastic bag was placed around the leaf to be treated.

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High specific radioactivity (26.3 mc/mmole) racemic 2-14C-abscisic acid was synthesized. An aliquot of abscisic acid, 1.2 X 10'-M in aqueous methanolic solution, was applied to the surface of either a cotyledon or the first true leaf of 8-to 32-dayold cotton seedlings (Gossypium hirsutum L.). After various intervals (6-192 hours), the seedlings were processed for autoradiography, counting, and identification of the radioactivity. After 6 hours, radioactivity was observed moving basipetally out of the treated leaf toward the roots. Four days later, radioactivity could be detected throughout the whole seedling. After 8 days, 10% of the recovered radioactivity was found in the roots, and 80% remained in the treated leaf blade. Neither leaf type nor age had any effect on the abscisic acid movement or pattern of distribution. Isolated radioactivity from the roots was identified as abscisic acid, based on comparison with an authentic standard by thin layer chromatography, gas-liquid chromatography, or gas-liquid chromatography-mass spectrometry.Abscisic acid has been isolated from cotton plants and identified (6, 24, 25). It has also been found in a large number of plant species (3, 11,18,22,23 MATERIALS AND METHODS Synthesis of High Specific Radioactivity 2-14C-Abscisic Acid. Although a synthesis of 1-"4C-methyl (triphenylphosphoranylidene) acetate has been published (5), the description given is incomplete, and the yield of the 1-4C-compound reported is lower than the yields in the macrosynthesis (17). A similar Wittig reagent was prepared with good yield as follows. Ethyl-2-"C-bromoacetate (21.0 mg) 28.7 mc/mmole, (ICN-Tracerlab) in 0.25 ml of benzene was transferred to a 15-ml graduated centrifuge tube, and the shipping vial was rinsed five times with 0.45 ml of benzene. Triphenylphosphine (132 mg, 0.503 mmole) was added, dissolved in the benzene, and the tube was covered with aluminum foil and allowed to stand at room temperature for 24 hr. The tube was then spun for 15 min at 3000 rpm, and the benzene was decanted. Fresh benzene (2.5 ml) was added, the tube was shaken and then centrifuged again for 15 min at 3000 rpm, and the benzene rinse was decanted. The tube was then dried 2 hr in a vacuum oven at about 250 mm Hg and 40 C to yield 50.22 mg of salt (93.0% of theory). Previous trial runs had given quantitative yields. The salt was dissolved in 2.5 ml of distilled water, and a drop each of 0.1% phenolphthalein alcohol solution and 1% Tween 20 solution was added followed by 4 drops of 10% KOH to a pink end point. The precipitated ethyl (triphenylphosphoranylidene)-2-'4C-acetate was spun down for 15 min at 3000 rpm, the supernatant was decanted, the precipitate was rinsed with 2.5 ml of water and recentrifuged, and the water was decanted. The product was then dried overnight at 250 mm Hg and 60 C to yield 35.67 mg, 87.5% of theory for this step and 81.4% over-all. Trial runs had given 84 + 2% of theory in this step.Next, 4-(2, 6, 6-trimethyl-1 -hydroxy-4-keto-2-cyclohexen-1-yl)-3-buten-2-one, 0.1024 mmole...