1 5 References
2.1 Sample Preparation for Hormone Analysis
2.1.1 Extraction and preliminary purification
Fruit tissue was harvested, immediately dissected into core, inner pericarp and outer pericarp tissue and snap frozen in liquid nitrogen. Fruit tissue was lyophilised, ground to a fine powder in a bench grinder (1 mm screen) and stored at -70°C until required. Sap was stored at -70°C in polypropylene tubes.
Up to 400 mg fruit tissue (dry weight) was weighed out on a balance (Mettler PM200) accurate to 0. 1 mg. Tritiated abscisic acid CH-ABA, Amersham) and tritiated isopentenyl-adenosine CH-IPA dialcohoI, see section 2.2. 1 ) were added to each sample as internal standards. B ieleski solution (Bieleski, 1 964) is often advocated for cytokinin extraction to avoid the conversion of nucleotide cytokinins to nucleosides (Morris, 1 986). Phosphatase activity in 80% methanol extracts was measured by conversion of p nitrophenoI phosphate to p-nitrophenol, but was not found to be significantly differentftom
e
Biel§ki solution extracts. Therefore 80% methanol was used for routine extraction. Tissue was extracted in 1 00 times (v/w) of 80% methanol containing 10 mgll butylated hydroxy toluene (anti-oxidant) for 24 hours at 1 °C with continuous shaking. Extracts were rapidly filtered using a vacuum filtration apparatus fitted with Whatman no. 1 qualitative filter paper and then washed with a further 2 x 5ml methanol. Extracts were taken to near dryness, re-dissolved in 5ml 0.02M ammonium acetate and adjusted to pH 7 with 1 0% NH40H.
Before use, polyvinyI-polypyrolidine (PVPP) powder (Sigma P6755) was soaked in milIipure water one day prior to use and fines were decanted five times to improve the flow rate. DEAE Sephadex A-25 (Pharmacia Biotech) was initially soaked in O. l M ammonium acetate for two days prior to use. After use, sephadex was rejuvenated by
2-2 2: General Materials and Methods. Sample Preparation
passmg 2M NaCl in 0. 1 M NaOH through columns until the eluent was colourless , repeating with methanol and finally washing with 0. 1 M ammonium acetate. Rejuvenated Sephadex A-2S was stored in excess 0. 1 M ammonium acetate containing 200 mg.r1
NaN}.
A S ml bed volume PVPP column was connected in series to a 3ml bed volume Sephadex A-2S column and they were pre-conditioned with l OmI 0. 1 M ammonium acetate followed by 20ml 0.0 1 M ammonium acetate. A C li, Sep-pak pre-conditioned for cytokinin retention with Sml methanol and 2 x 4ml O.O I M ammonium acetate was connected to the base of the PVPP and sephadex columns. Fruit extract or sap, adjusted to pH 7, was loaded onto the PVPP column and washed through with 20ml O.O I M ammonium acetate. The Sep-pak retaining cytokinins was removed, washed with 5rnI H20 and cytokinins were eluted with 5ml 70% methanol. A second Sep-pak was connected to the bottom of the sephadex column, the PVPP column discarded and ABA eluted from the sephadex with 1 0ml 1 M acetic acid. The second Sep-pak was removed, washed with Sml H20 and eluted with 5ml 80% methanol. Both extracts were taken to dryness using a Savant AS290 automatic speedvac (speedvac) .
Cytokinin extract was re-dissolved in 400 III o f the High performance liquid chromatography (HPLC) starting solvent ( 1 0% acetonitrile in TEA, see section 2 . 1.2) , filtered though a 0.451lm filter (Gelman GHP acrodisc 1 3mm syringe filter with low hold-up volume). A further 1 00 III of solvent was passed through the filter to minimise loss.
ABA extract was re-dissolved in 1 ml TBS buffer (see section 2.3.2) and duplicate aliquots removed for radioactivity counting to determine recovery immediately prior to assay. Recovery of ABA usually exceeded 80%.
2.1.2 High performance liquid chromatography separation of cytokinins
High performance liquid chromatography (HPLC) was used to separate the different cytokinins for analysis. Separation was made on a 220 x 4.6 octadecyl silica (ODS)
2: General Materials and Methods. Sample Preparation 2-3
column in line with a 3 x 4.6 ODS guard column (Applied Biosystems inc. Brownlee spheri-5).
Solvent delivery to the HPLC system was via a Waters 50 1 HPLC pump and a Waters 5 1 0 HPLC pump controlled by a Waters automated gradient controller. All solvents were HPLC grade and made up fresh prior to each run. TEA solvent was a 40mM acetic acid solution adjusted to pH 3 .38 with re-distilled triethylamine. Both TEA and acetonitrile solvents were vacuum filtered through a 0.211m nylon filter and dissolved gases were removed by sonication for 30 minutes. The retention times of cytokinin standards was established with a programmable multiwavelength detector (Waters 490E) at 268nm. Retention time of IP A dialcohol was established using a flow through radioactivity monitor (�Ram, InfUs systems N .J . , U.S.A.) with Inflow-3 liquid scintillant for homogenous counting cells at a ratio of 3ml scintillant to 1 ml eluent, or by counting radioactivity of collected fractions on a liquid scintillation counter (Wallac 1 409 series).
Prior to each HPLC run, retention times of cytokinins and dialcohols were checked. Retention times were found to be extremely consistent between runs.
Purified samples (500111) were loaded via a Rheodyne injection port and separated with a solvent gradient (Table 2. 1 ). After each inj ection, the column was returned to the starting conditions and allowed to re-equilibrate for 1 0 minutes. The column was cleaned by passing 1 00% acetonitrile through after every five samples.
Table 2. 1 Solvent gradient used for separation of cytokinins on an octadecyl silica (ODS) column used in High performance liquid chromatography (HPLC.
Time (minutes) %Acetonitrile %TEA Curve
o 1 0 90 *
30 40 60 6
33 40 60 7
One minute fractions were collected directly into 1 .5ml RIA assay tubes in an ISCO 568 automated fraction collector. Fractions containing 3H-dialcohol internal standard
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(fractions 22-24) were taken to dryness in the speedvac, re-dissolved in 200 III methanol and counted for radioactivity. Other fractions were taken to dryness and assayed by radioimmunoassay (see section 2.2.4) using the appropriate antiserum. Typical results for retention times and assays are shown in Figure 2. 1 .