2 MATERIALS AND METHODS
2.1. CHEMICAL AND LABORATORY SUPPLIES 1 Radioactive and stable isotopes
NS-isopent-2-enyl[2-3H]adenine (sp. act. 1.25 TBq mmor1
) and trans-[2-3H]zeatin (sp. act. 1.3 TBq mmor1) were purchased from The Institute of Experimental Botany, Academy of Sciences of the Czech Republic Isotope Laboratory IEB, Prague, Czech Republic. cL-cis, trans-[G- 3H]ABA (sp. act. 1.11 TBq mmor1) and 3-[5(n)-3H]IAA (sp. act. 1.11 TBq mmor1
) were both obtained from Amersham International, Buckinghamshire, UK.
2.1.2 Fine chemicals and cofactors
Butylated hydroxytoluene (BHT; 2,6-Di-t-butyl-p-cresol, C1sH240), diethyldithio carbamic acid (DDC; sodium salt, CSH10NS2Na), reduced glutathione (C1oH17N30SS), phenazine methosulphate (PMS; N-methyldibenzopyrazine methyl sulphate salt, C13H11N2'CH3S04), dichloroindophenol (DCIP; C12HsCbN02Na), indole-3-aldehyde (I-aid; C9H7NO), hypoxanthine (6-hydroxypurine, CSH4N40), indole-3-acetaldehyde (lA-aid; sodium bisulfite addition, C10HgNO'NaHS03), linoleic acid (cis-9,cis-12-octadecadienoic acid, C1eH3202), p-aminophenol (4-hydroxyanilin, H2NCsH40H), Coomassie Brilliant Blue G (C47H4eN307S2Na), 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTI; C1eH1SNsSBr), bovine serum albumin (BSA), adenine (6-aminopurine, free base, CsHsNs), allopurinol (allo; 4- hydroxypyrazolo[3,4-d]pyrimidine, CSH4N40), polyethylenimine (Polymin P; 50 % (w/v) aqueous solution, a branched chain polymer), L-cysteine hydrochloride (C3H7N02S'HCI), N- nitroso-N-methylurea (C2HsN30 2), lipoxidase (from Soybean, EC 1.13.11.12) and N,N- dimethyloctylamine (CH3(CH2hN(CH3h) were all purchased from Sigma, St Louis, MO, USA.
Nicotinamide adenine dinucleotide (NAD; free acid, C21H27N7014P2), flavin adenine dinucleotide (FAD; disodium salt, C27H31Ng01SP2Na2) and tris (2-amino-2-(hydroxymethyl)-1,3- propandiol, C4H11N03) were purchased from Boehringer Mannheim GmbH, Mannheim, Germany. Potassium molybdate (K2Mo04) was purchased from Aldrich Chemical Company Inc., Milwaukee, WI, USA. Sodium molybdate (Na2Mo04.2H20), trichloroacetic acid (CCI3COOH) and di-potassium hydrogen orthophosphate (K2HP04) were purchased from Associated Chemical Enterprises, Glenvista, RSA. Magnesium sulphate (hepthydrate, MgS04.7H20) was purchased from .Holpro Analytics (Pty) Ltd, Johannesburg, RSA.
Ammonium sulphate «NH4hS04) and sodium formate (HCOONa) were obtained from Merck Laboratory Supplies (Pty) Ltd, Midrand, RSA. Ethylene diamine tetra-acetic acid (EDTA;
disodium salt, C10H140eN2Na2.2H20) and ammonium hydroxide (25 % (w/v) solution, NH40H)
was purchased from Saarchem, Krugersdorp, RSA. Potassium dihydrogen phosphate (KH2P04) was purchased from Riedel-de Haen GmbH, Seelze, Germany. Ammonium dihydrogen phosphate «NH4)H2P04) was purchased from Merck KGaA, Darmstadt, Germany.
1,4-Dithiothreitol (DTI; C4H1002S2) and Complete~ protease inhibitor tablets were obtained from Roche Diagnostics GmbH, Mannhein, Germany. Insoluble polyvinylpolypyrrolidone (PVP;
Polyclar AT) and calcium chloride (CaCI2) were obtained from BDH Laboratory Supplies, Poole, England. Heptaldehyde (C7H140), citral (C1oH1aO) and 3-methyl-2-butenal (3,3- dimethylacrolein, CsHaO) were purchased from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. Benzaldehyde (C7HaO) and acetaldehyde (C2H40) were purchased from Fluka Chemie Ltd, Buchs, Switzerland. Pico-Fluor™ 40 (Universal LSC cocktail) was purchased from Packard Bioscience Company, Groningen, The Netherlands.
2.1.3 Electrophoresis reagents
Acrylamide (C3HsNO), bromophenol blue (3', 3", 5', 5"-tetrabromophenol-sulfonephthalein, C1gHgBr40SSNa) and glycine (C2HsN02) were purchased from BDH Laboratory Supplies, Poole, England. Bis-acrylamide (N,N'-methylene-bis-acrylamide, C7H10N202) and sodium thiosulphate (pentahydrate, Na2S203.5H20) were obtained from ICN Biomedicals Inc., Aurora, Ohio. Ammonium persulphate «NH4hS20a) was obtained from Biosolve Ltd, Valkenswaard, The Netherlands. N,N,N',N'-Tetramethylethylenediamine (TEMED; CeH1aN2), urease molecular weight markers for native-PAGE (Jack Bean, approx. mol. wt 272 000 (trimer) and 545 000 (hexamer» and formaldehyde (37 % (w/v) solution, CH20) were purchased from . Sigma, St Louis, MO, USA. Silver nitrate (AgN03) was purchased from Merck Laboratory Supplies (pty) Ltd, Midrand, RSA. Sodium carbonate (anhydrous, Na2C03) was purchased from Saarchem, Krugersdorp, RSA.
2.1.4 Tissue culture reagents
Agar Bacteriological (Agar No. 1) was purchased from Oxoid Ltd, Basingstoke, Hampshire, England. Myo-inositol (CeH120e) and nicotinic acid (niacin, pyridine-3-carboxylic acid, CeHsN02) were purchased from Sigma, St Louis, MO, USA. Pyrodixine HCI (vitamin Ba, CeH11 N03·HCI) and thiamin HCI (vitamin B1, C12H17N40SCI·HCI) were purchased from BDH Laboratory Supplies, Poole, England. All other chemicals for the basal medium were obtained from Merck laboratory Supplies (Pty) Ltd, Midrand, RSA and BDH laboratory Supplies, Poole, England.
2.1.5 Growth regulators
Isopentenyladenine (iP; 6-(y,y-dimethylallylamino)-purine, CloH13Ns), isopentenyladenosine (iPA; 6-(y,y-dimethylallylamino)-purine riboside, C1sH21 HS0 4), DL-zeatin (Z; 6-[4-hydroxy-3-
methylbut-2-enylamine]purine, C10H13NsO), trans-zeatin riboside (ZR; 9-[~o-ribofuranosyl]
trans-zeatin, C1sH21 NsOs), DL-dihydrozeatin (DHZ; C10H1SNsO), kinetin (K; 6-furfurylamino purine, C10HgNsO), indole-3-acetic acid (IAA; C10HgN02), indole butyric acid (IBA; C12H13N02), a-naphthalene acetic acid (NAA; C12H1002), cis-trans abscisic acid (ABA; C1sH2004) and authentic methylated abscisic acid (ABAMe) and indole-3-acetic acid (IAAMe) standards were obtained from Sigma, St Loius, MO, USA. 6-chloropurine, 2,6-dichloropurine, 2-methylthio-6- chloropurine and 2-methylthio-6-chloro-9-methylpurine were obtained from OIChemlm Ltd, Olomouc, Czech Republic. Authentic phaseic acid (PA) and dihydrophaseic acid -(DPA) standards were kindly supplied by Professor A.K. Cowan, Research Centre for Plant Growth and Development, University of Natal, Pietermaritzburg.
2.1.6 Solvents
High performance liquid chromatography (HPLC) grade solvents were obtained from Burdick and Jackson, AlliedSignal Inc., Muskegon, MI, USA. Analytical grade solvents and polyoxyethylene (20) sorbitan monooleate (Tween 80®) were supplied by Merck Laboratory Supplies (Pty) Ltd, Midrand, RSA and Associated Chemical Enterprises (Pty) Ltd, Southdale, RSA. Dimethyl sulphoxide (DMSO) was purchased from Sigma, St Loius, MO, USA.
Polyoxyethylene (20) sorbitan monolaurate (Tween 20~ was purchased from Saarchem (Pty) Ltd, Krugersdorp.
2.1.7 Purification of indole-3-acetaldehyde
Commercially purchased lA-aid comes as a bisulfite addition compound. The bisulfite was removed prior to activity staining, as it is a strong reducing agent and will thus reduce MTT to formazan. Free lA-aid was prepared according to a modified method of Bower etal. (1978). A known amount of lA-aid bisulfite was dissolved in 1 mL water and the pH adjusted to 10 with 0.5 N NaOH, which resulted in the precipitation of free lA-aid. The free lA-aid was removed by partitioning three times against an equal volume of hexane/ethyl acetate (9:1, v/v). The hexane/ethyl acetate fraction was dried in vacuo at 35 cC, using a BOchi Rotavapor\1!) R110 (BOchi Laboratoriums-Technik AG, Flawil, Switzerland), resuspended in 0.5 mL acetone and diluted to 20 mL with 0.1 M Tris-HCI buffer (pH 8). The final concentration of free lA-aid was determined by measuring the absorbance at 280 nm in an Anthelie Advanced Spectrophotometer (Secomam CE, Domont Cedex, France) and applying Beer's law with
E/
80=
5400 L mor1cm-1(Brown and Purves 1976).2.1.8 Preparation of xanthoxal
Xanthoxal was produced enzymatically according to the method of Firn and Friend (1972), by coupling violaxanthin oxidation to the enzymatic oxidation of linoleic acid.
Violaxanthin was extracted from orange peel and purified chromatographically using a modified method of Taylor and Burden (1970) and Davies (1976). The outer layer of the flavedo of orange fruit was grated from the fruit and a total of 750 g macerated with 1.5 L methanol in an Osterizer~ blender. This extract was filtered under vacuum and the residue re- extracted twice with diethyl ether (total volume 4 L). The methanol and diethyl ether extracts were combined and partitioned against water containing NaCl, to prevent an emulsion from forming (Davies 1976). The organic fraction was dried in vacuo at 30°C, using a BOchi Rotavapor® R110. Sufficient methanol was then added to dissolve the residue and 1 M KOH added in a ratio of 2 mL KOH to 10 mL solvent. The extract was saponified for 12 h at room temperature, in the dark. The methanol fraction was reduced to the aqueous phase in vacuo at 35°C and a small volume of water was added. This fraction was subsequently partitioned against diethyl ether, until the ether fraction was colourless. The diethyl ether fractions were pooled, dried in vacuo at 30°C and resuspended in a small volume of hexane. This fraction was then subjected to vacuum liquid chromatography (VLC) on a 6 cm x 13 cm silica gel (150 g; Type 60, particle size 0.063-0.2 mm) column. The sample was dissolved in 5-6 g silica gel, placed on top of the column and covered with glass wool. The column was eluted (400 mL solvent/fraction) with increasing concentrations of ethyl acetate in hexane. The fractions eluted with 50:50 (v/v) hexane/ethyl acetate; 40:60 (v/v) hexane/ethyl acetate and 30:70 (v/v) hexane/ethyl acetate were pooled and dried in vacuo at 35°C. The final concentration of violaxanthin was determined by measuring the absorbance at 450 nm in an Anthelie Advanced Spectrophotometer and applying Beer's law withEA450
=
2 500 L mor1cm-1•A solution of 10 mg violaxanthin and 10 mg Tween 80~ in 10 mL 0.2 M Tris-HCI buffer (pH 7.6) was mixed with a solution of 20 mg linoleic acid and 20 mg Tween 80~ in 10 mL 0.2 M Tris-HCI buffer (pH 7.6). The mixed solution was divided into four equal aJiquots and 2 mL soybean lipoxidase (60 IJg mL-1) in 0.2 M Tris-HCI buffer (pH 7.6) was added to each aliquot.
These aliquots were then incubated at 30°C for 30 min, after which the reaction was terminated by the addition of 1 g ammonium sulphate. This solution was then partitioned three times against ethyl acetate. The ethyl acetate fractions were pooled and dried in vacuo at 35 cC. These fractions were loaded onto silica gel plates (GF254) and developed to 15 cm in hexane/ethyl acetate (1:1, v/v). The zone corresponding to XAN (Rf
=
0.21) was removed and eluted with water-saturated ethyl acetate. This was subsequently filtered through glass wool, dried in vacuo at 35°C and re-purified on silica gel (GF254) plates to a height of 15 cm in petroleum ether/acetone (3: 1, vlv). The XAN zone was removed, eluted in water-saturated ethyl acetate and dried in vacuo at 35°C. This fraction was resuspended in 20%
methanol, filtered through a 0.2 IJm syringe filter (Lida ManufactUring Corp., Kenosha, WI, USA) andfurther purified by HPLC (see section 2.9.7 for HPLC method (System