Plant Hormones Abscisic Acid Auxins Cytokinins Ethylene Gibberellins

Gibberellin extraction, purification and analysis

Qualitative analysis

Samples were homogenized in cold (4°C) CH,OH:water (4:1 v/v)(l L). Small amounts (833 Bq each) of the following tritiated GA standards, GA19 , GA20, GA1 and GA8 were added to the homogenate and the extract stirred overnight in the cold (4°C). After filtration, the residue was reextracted with CH30H (1 L) for 4 h and refiltered.Methanol was removed from the combined filtrates under reduced pressure at 35°C, the aqueous residue adjusted to pH 2.5 (2 M HCI), and partitioned against ethyl acetate (3 x equal volumes). The combined organic phases were partitioned against 5% (w/v) sodium bicarbonate (3 x l/5 volume). The aqueous phases were acidified to pH 3.0 (2 M HCI) and partitioned against ethyl acetate (3 x equal volume), which was then reduced to dryness in varuo at 35°C.

The extract was dissolved in 5 ml of water, adjusted to pH 8.0 (1 M KOH) and loaded onto a PVP column (3 ml bed volume) preequilibrated with 0.1 M KH2P04 (pH 3). After loading, the column was washed with water (2 x 5 mt) at pH 8. The eluate was combined and loaded onto a QAE Sephadex A-25 anion exchange column (5 mL bed volume) preequilibrated with sodium formate (0.5 M) then washed with formic acid (0.2 M) and water (pH 8). After loading, the column was washed with water (pH 8) (15 ml) and GAs were eluted with 0.2 M formic acid (20 ml) and applied directly to a preequilibrated C18 cartridge . After washing with water (pH 3) (5 ml), GAs were eluted with CH3OH:water (4:1 v/v) (5 mL) which was then evaporated to dryness in vacuo.

GAs were resolved by reverse phase HPLC using a 4.9 mm i.d x 250 mm column containing Hypersil 5 um octadecyl- siloxane (ODS) and a linear gradient of increasing CH3OH in 2 mM acetic acid (28% CH3OH to 100% CH3OH over 40 min) at a flow rate of 1 mL min-1. Samples were dissolved in 80uL CH3OH, made up to 400 uL with 2 mM acetic acid, and injected onto the column using a Rheodyne 7125 valve fitted with a 500 uL loop. Forty 1-ml fractions were collected and aliquots(l/20) removed for scintillation counting to locate GA8, GA1, GA20, and GA19. Fractions, based on the location of these GAs, were combined and taken to dryness in vacuo. The dried fractions were methylated with excess ethereal diazomethane, transferred to glass ampoules and trimethylsilylated with MSTFA (5 uL) at 90°C for 30 min. Derivatized samples were analyzed using a Kratos MS80 RFA GC-MS system. Samples (1 uL) were coinjected with Parafilm in hexane (to determine KRI values) into a fused silica WCOT SE-52/54 capillary column (25 m x 0.22 mm x 0.25 um film thickness) at an oven temperature of 50°C with the injector splitter closed. After 0.5 min the splitter (50:1) was opened and 1 min later the oven temperature was increased at 15"C min-1 to 240'C and then at 4°C min-1 to 300°C. The He inlet pressure was 0.04 MPa and the injector and interface temperatures were 250°C. After 12 min, positive ion electron impact mass spectra were acquired, scanning from 700 to 50 atomic mass units at 1 s per mass decade. The electron energy was 70 eV and the source temperature 200"C.

Quantitative analysis

Samples were extracted as described above and in addition to the tritiated standards, [17-2H2-GA8, [17-2H2]- GA1, [17-2H2]-GA3, [17-2H2]-GA20, and [17-2H2]-GA1 were added as internal standards. The amounts of standards added depended on the weight of tissue and were based on results from preliminary experiments. After purification, as above, but omitting the PVP step, samples were analyzed using a Hewlett-Packard 5890 gas chromatograph coupled to an HP5970 mass selective detector . Samples(l uL) were injected into a fused silica WCOT capillary column (25 m x 0.22 mm x 0.25 um film thickness) coated with either BP-1 or SE52/54 (Thames Chromatography) at an oven temperature of 60°C. After 0.5 min, the splitter (50:1) was opened and 1 min later the temperature was increased at 20°C min-1 to 240°C and then at 4°C min-1 to 300°C. The He inlet pressure was 0.09 MPa and the injector, interface and MS source temperatures were 220, 270, and 200'C, respectively. Characteristic ions were monitored with dwell times of 50 ms (GA8, m/z 594,448; [17-2H2]-GA8, m/z 596,449; GA1m/z 506,448;[1 7-2H2]GA1 m/z 508,450; GA3, m/z 504; [17-2H2]-GA3, m/z 506; GA20, m/z 418, 375; [17-2H2]-GA20, m/z 420, 377; GA19, m/z 434, 402; [17-2H2]- GA19, m/z 436, 404. The concentration of GAs in the original extracts was determined from previously established calibration curves of the peak area ratios for unlabeled and deuterated GAs plotted against varying molar ratios of the two compounds . The same stock solutions of labeled GAs were used for production of the calibration curves.