The Plant Journal (2000) 24(4), 551±558
TECHNICAL ADVANCE
Application of high-performance liquid chromatography with photodiode array detection to the metabolic pro®ling of plant isoprenoids Paul D. Fraser, M. Elisabete S. Pinto, Daniel E. Holloway² and Peter M. Bramley* School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK Received 27 April 2000; revised 8 August 2000; accepted 14 September 2000. *For correspondence (fax +44 1784 434326; e-mail
[email protected]). ² Present address: Department of Biology and Biochemistry, University of Bath, South Building, Claverton Down, Bath BA2 7AY, UK.
Summary The application of high-performance liquid chromatography (HPLC) using a C30 reverse-phase stationary matrix has enabled the simultaneous separation of carotenes, xanthophylls, ubiquinones, tocopherols and plastoquinones in a single chromatogram. Continuous photodiode array (PDA) detection ensured identi®cation and quanti®cation of compounds upon elution. Applications of the method to the characterization of transgenic and mutant tomato varieties with altered isoprenoid content, biochemical screening of Arabidopsis thaliana, and elucidation of the modes of action of bleaching herbicides are described to illustrate the versatility of the procedure. Keywords: high-performance liquid chromatography, isoprenoids, metabolic pro®ling.
Introduction Advances in DNA technologies have facilitated the genetic manipulation of metabolic pathways and physiological processes in plants. It is predicted that within the next two years the ®rst completed genomic sequence from a higher plant (Arabidopsis thaliana) will be publicly available. The challenge to the scienti®c community will then be concerned with assigning function to the 20 000± 25 000 genes and fully characterizing their role in plant metabolism. The extraction, separation and identi®cation of a wide range of compounds in a single measurement (metabolic pro®ling) will be essential for the progression of such studies (Trethewey et al., 1999). Traditionally, analytical biochemists have focused their efforts on optimizing extraction, separation and detection methodologies for speci®c compounds rather than for a range of metabolites in a single sample. These procedures are, in general, timeconsuming, require relatively large amounts of material, and are limited in the number of compounds that can be analysed simultaneously. Therefore, their value in functional genomics or metabolic engineering of quality traits in plants is rather low. ã 2000 Blackwell Science Ltd
The chemical diversity and varying abundance of plant metabolites make it dif®cult, if not impossible, to employ a procedure that is applicable for all, or a majority, of plant metabolites. However, advances in analytical instrumentation and chromedia have improved the feasibility of metabolic pro®ling. In this study, we have focused on the metabolic pro®ling of plant isoprenoids (particularly carotenes, xanthophylls, tocopherols, plastoquinones, chlorophylls and ubiquinone). Over 22 000 members of the isoprenoid family are known, comprising one of the largest classes of natural products in the plant kingdom (Connolly and Hill, 1992). The complex array of isoprenoids is in part due to specialized isoprenoid formation occurring in different plant tissues, for example monoterpenes in trichomes of peppermint (Croteau and Gershenzon, 1994) and carotenoids in chromoplast tissues (Hugueney et al., 1995). Despite this complexity, all isoprenoids are derived from the common precursor isopentenyl pyrophosphate (IPP; Chappell, 1995). The biosynthetic pathway of isoprenoid formation involves many interconnecting branch points (Figure 1). In general, the intermediates of the pathways are transient and it is 551
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Figure 1. Summary of isoprenoid biosynthesis. Different classes of isoprenoid compounds are shown in bold and boxed, the prenyl phosphate precursors are not.
the end-products that are detectable under normal growth conditions. For example, the plant growth hormone abscisic acid, cytokinins, gibberellins and the brassinosteroids are isoprenoids of very low abundance, making their analysis dif®cult in a routine pro®ling procedure. The value in identifying and quantifying isoprenoids may be illustrated with two examples. First, for some decades, isoprenoid biosynthesis has been an important target site for bleaching herbicides, and quantitative structure±activity relationships (QSAR) need to be determined (BoÈger and Sandmann, 1989). Secondly, the healthpromoting properties of certain anti-oxidant isoprenoids such as carotenoids and vitamin E have resulted in intense interest in determining and manipulating their levels in plants (Grusak and DellaPenna, 1999). The amenability of crops to genetic manipulation and the availability of isoprenoid biosynthetic genes (Cunningham and Gantt, 1998; Scolnik and Bartley, 1996) enables the isoprenoid content of food crops such as tomato (RoÈmer et al., 2000) and rice (Ye et al., 2000) to be altered. In order to assess the utility of genes and fully evaluate the effects of these manipulations, rapid and comprehensive isoprenoid analyses are required. The thermal instability of isoprenoids such as carotenoids excludes their separation by GC. In addition, their chemical diversity restricts effective separation of broad isoprenoid classes on traditional HPLC stationary phases, whilst those with no characteristic absorption spectra cannot be detected spectrophotometrically. In the present study, isoprenoids have been separated on a reverse-phase C30 HPLC column and identi®ed by characteristic spectral properties derived from an online photodiode array (PDA) detector (Bramley, 1992). Applications of the method for the analysis of isoprenoids in a variety of plants are described to illustrate the utility of this procedure.
The chemical nature of many isoprenoids means that they are unstable to heat, light, acid and in some cases alkali (Britton, 1985). Therefore, such unfavourable conditions were avoided or minimized in the study. The extraction procedure described in Experimental procedures can be used with both fresh and freeze-dried tissue. Freeze-dried material is, however, more amenable to small-scale analyses. The use of a representative proportion from a larger pool of ground material reduces plant, tissue and sample variability, and alleviates the necessity for large numbers of replicates. Hexane, light petroleum (boiling point 40±60°C), diethyl ether, ethyl acetate and chloroform were evaluated as extraction solvents. Chloroform was the most suitable, ef®ciently extracting a broad range of isoprenoids. Recoveries obtained for tocopherols, quinones, carotenoids and xanthophylls from spiked samples were 96 6 2%, 94 6 4%, 98 6 5% and 97 6 3% (n = 3), respectively. After two extractions, further reextraction, even with an increased volume, removed virtually no more isoprenoids. The suitability of chloroform for extractions was further endorsed by its ability to ef®ciently extract the lycopene present in tomato tissue at very high concentrations (approximately 200 mg g±1 fresh weight). The total amount of carotenoid extracted by chloroform from tomato tissue was 10-fold, 3-fold and 1.5fold greater than using hexane, ethyl acetate and diethyl ether, respectively. Misleading lycopene:b-carotene ratios (e.g. 3:1 with hexane compared to 9:1 with chloroform) were also detected due to the differential extractability of b-carotene and lycopene in solvents other than chloroform. Partitioning of the chloroform extract against 25 mM Tris±HCl pH 7.5 prior to HPLC improved online PDA spectra by eliminating unwanted UV interference. Further puri®cation of the crude extracts by solid-phase or ®ltration procedures was not necessary. Extracts could be stored dry, under an atmosphere of nitrogen, for at least 1 year at ±20°C. The presence of anti-oxidants such as butylated hydroxytoluene (BHT) had no observable effect on the stability of the compounds during extraction or storage over this period. HPLC system for the analysis of isoprenoids Prior to HPLC analysis, samples were dissolved in ethyl acetate. Hexane, methanol or the initial running solvent were not suitable for this purpose due to precipitation at 4°C, whilst chloroform was prone to evaporation. Reverse-phase C18 and normal-phase silica have often been the stationary phases of choice when analysing isoprenoids by HPLC. Numerous systems have been devised to separate carotenes and xanthophylls (Craft, ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 551±558
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Table 1. Isoprenoids separated on a reverse-phase C30 HPLC system and spectral characteristics (in the eluting solvent) used in identi®cation from photodiode array detection
Figure 2. HPLC pro®le of isoprenoid standards recorded at (a) 287 nm and (b) 460 nm. In both (a) and (b), the left and right panels are the chromatogram and derived spectra, respectively. The compounds in (a) are (1) vitamin K3; (2) plastoquinone; (3) g-tocopherol; (4) d-tocopherol; (5) a-tocopherol; (6) a-tocopherol acetate; (7) vitamin K1; (8) ubiquinone-7; (9) ubiquinone-9. In (b), the compounds are (1) astaxanthin; (2) lutein; (3) zeaxanthin; (4) canthaxanthin; (5) b-cryptoxanthin; (6) a-carotene; (7) b-carotene; (8¢) 13cis-lycopene; (8¢¢) 9-cis-lycopene; (8) all-trans lycopene. The solvent system is described in Experimental procedures.
1992; Fraser et al., 1993; Indyk, 1987; Weber, 1987) and tocopherols (Abidi and Mounts, 1997; Indyk, 1990). While evaluating these systems for potential use in isoprenoid pro®ling, it became clear that they had been tailored to a speci®c group of compounds or isoprenoid class. Simultaneous carotenoid and tocopherol separations do exist, but their application is principally related to clinical samples (Barua and Olsen, 1998; Schuep et al., 1995), and the number of compounds that can be resolved makes their use restrictive. An alternative, C30 reverse-phase matrix (Sander et al., 1994), with a methanol/tert-methyl butyl ether-based mobile phase (as described in Experimental procedures) was assessed. Using the latter system, tocopherols, plastoquinones, vitamin K and ubiquinone (Figure 2a) as well as carotenoids (Figure 2b) were separated in one chromatographic run. The online PDA detector facilitates identi®cation from the characterã Blackwell Science Ltd, The Plant Journal, (2000), 24, 551±558
Isoprenoid
Retention time (min)
Spectral characteristics (nm at lmax)
Carotenoids 15-cis-phytoene All-trans-phytoene Phyto¯uene-1 Phyto¯uene-2 Phyto¯uene-3 z-carotene-1 z-carotene-2 z-carotene-3 z-carotene-4 z-carotene-5 Neurosporene cis-lycopene-1 cis-lycopene-2 All-trans-lycopene Poly-cis-lycopene d-carotene a-carotene b-carotene b-cryptoxanthin Zeaxanthin Lutein Violaxanthin Neoxanthin Astaxanthin Canthaxanthin
24.69 25.62 27.05 27.70 28.23 28.96 29.61 32.03 32.50 34.70 33.98 33.88 36.42 40.69 30.90 32.40 26.79 28.22 22.78 16.79 15.00 11.17 10.04 13.50 18.56
275.8, 285.6, 297.3 278.0, 286.0, 301.6 330.8, 346.3, 366.4 330.1, 346.3, 366.4 330.1, 347.5, 366.4 374.8, 397.7, 421.8 371.2, 396.4, 418.2 379.6, 400.1, 425.4 379.6, 400.1, 425.4 379.6, 400.1, 425.4 416.2, 440.2, 469.3 439.9, 465.3, 496.9 441.2, 467.8, 496.9 446.0, 472.6, 504.2 403.0, 426.6 424.0, 448.0, 482.5 423.0, 446.3, 474.1 ±, 453.2, 478.0 430.0, 451.2, 477.8 425.0, 451.2, 477.8 421.0, 443.9, 477.8 419.0, 439.1, 469.3 415.0, 440.3, 466.9 ±, 474.1, ± ±, 477.8, ±
Tocopherols a-Tocopherol a-Tocopherol acetate d-Tocopherol g-Tocopherol
11.81 13.83 9.23 10.68
290.0 285.0 295.0 298.0
Quinones Ubiquinone-7 Ubiquinone-9 Ubiquinone-10 Plastoquinone Vitamin K3 Vitamin K1
17.55 23.75 28.20 5.60 3.77 14.83
274.5 274.5 278.0 278.1 339.8 329.1
Chlorophylls Chlorophyll a Chlorophyll b
21.33 16.69
432.6, 657 467.8, 656
Data provided are for all-trans isomers unless otherwise stated. Isomer con®gurations are assigned from spectral and chromatographic characteristics (Britton, 1985). In cases where no isomer can be assigned (e.g. z-carotene), numbers have been used.
istic spectral properties of these molecules. The range of isoprenoids separated and identi®ed using this protocol is given in Table 1. Since astaxanthin and canthaxanthin are not found in higher plant tissues, they were used as internal standards to determine recoveries, relative reten-
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tion times and for quanti®cation. Calibration curves revealed linearity, with R2 values exceeding 0.9 (peak area versus amount). Ratios of peak area versus amount were within 1% in the range 10 ng to 1 mg. The limit of detection was 0.0004 AU, typically in the 2±10 ng range per compound. Retention times were consistent, the maximum ¯uctuation being within 6 0.15% over eight chromatograms. Analysis of tomato samples revealed variations of 5% for ubiquinone (n = 3), 8% for a-tocopherol (n = 3), 5% for plastoquinone (n = 3), 8% for lutein (n = 3) and 5% for b-carotene (n = 6). Application of isoprenoid pro®ling by HPLC±PDA Arabidopsis thaliana. Wild-type extracts were analysed using the C30 HPLC system. Four xanthophylls, lutein (200 6 40 mg g±1 dry weight, n = 3), violaxanthin (86 6 13 mg g±1 dry weight, n = 3), neoxanthin (4 6 0.6 mg g±1 dry weight, n = 3) and zeaxanthin (48 6 6 mg g±1 dry weight, n = 3) were detected. b-carotene (160 6 8.4 mg g±1 dry weight, n = 3) was the principal carotene present. Other isoprenoids determined were a-tocopherol (170 6 18 mg g±1 dry weight, n = 3), plastoquinone (140 6 13 mg g±1 dry weight, n = 3), ubiquinone (200 6 8.6 mg g±1 dry weight, n = 3) and chlorophylls (677 6 23 mg g±1 dry weight, n = 3). These compounds could be detected in as little as 2.0 mg of freeze-dried tissue, which is equivalent to one lea¯et (about 10 mm in length). Thus, the method is suitable for the analysis of Arabidopsis lines grown on agar for 2±3 weeks in Petri dishes (140 mm in diameter; approximately 200 plants per dish) and frozen at ±20°C. No degradation of the isoprenoids present in the tissue extract stored in this way occurred for at least 1 year. In order to evaluate the system for the detection of atypical isoprenoid pro®les, Arabidopsis was grown on four inhibitors of isoprenoid biosynthesis with different modes of action. Nor¯urazon inhibits carotenoid formation by inhibiting phytoene desaturation (Sandmann, 1993). Sulcotrione is an inhibitor of 4-hydroxyphenylpyruvate dioxygenase, an enzyme involved in plastoquinone formation (Schulz et al., 1993). Inhibition of plastoquinone formation has the secondary effect of inhibiting carotenoid synthesis by preventing the electron transfer necessary for desaturation to proceed (Norris et al., 1995). Both Sulcotrione and Nor¯urazon-treated plants are bleached in appearance. HPLC±PDA analyses using the C30 system show the accumulation of cis- and trans-phytoene in the nor¯urazon and Sulcotrione-treated Arabidopsis extracts (Figure 3b,c). No phytoene was detectable in the wild-type grown under identical conditions (Figure 3a). Therefore, the analysis con®rms that the phytoene desaturation is inhibited in both cases. More signi®cantly, however, the ability to identify simultaneously other isoprenoids (plastoquinone, ubiquinone and tocopherols) shows that
Figure 3. HPLC chromatograms recorded at 287 nm of (a) Arabidopsis wild-type, (b) Arabidopsis following Nor¯urazon treatment, and (c) Arabidopsis following Sulcotrione treatment. The peaks are (1) plastoquinone; (2) a-tocopherol; (3) 15-cis-phytoene; (4) all-trans phytoene; (5) ubiquinone-9. Approximately 2 mg of freeze-dried material was extracted as described in Experimental procedures.
treatment with Sulcotrione, but not Nor¯urazon, results in a reduction of plastoquinone (Figure 3b,c). The ratio of plastoquinone:phytoene was approximately 1 following nor¯uazon treatment but approximately 5 with Sulcotrione. Thus, the inhibition of phytoene desaturation has been identi®ed and the indirect mechanism causing the Sulcotrione-phenotype elucidated. Therefore, this system may be used to differentiate between the different ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 551±558
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Figure 4. HPLC chromatograms recorded at 450 nm of (a) Arabidopsis wild-type, (b) Arabidopsis following treatment with CPTA, and (c) Arabidopsis following treatment with Clomazone. Both (d) and (e) are recorded at 287 nm, and show wild-type and Clomazone-treated Arabidopsis, respectively. Peaks are (1) neoxanthin; (2) violaxanthin; (3) chlorophyll b; (4) lutein; (5) chlorophyll a; (6) bcarotene; (6¢) cis-b-carotene; (7) a-carotene; (8) d-carotene; (9) all-trans lycopene; (9¢) cis-lycopene; (10) zeaxanthin; (11) plastoquinone; (12) atocopherol; (13) 15-cis phytoene; (14) ubiquinone-9. Both inhibitors were used at a concentration of 100 mM. In all cases, 6 mg freeze dried tissue was extracted.
modes of action of bleaching herbicides. Such an approach will also facilitate the identi®cation of mutants with a bleached phenotype but not blocks in carotenoid synthesis (e.g. Norris et al., 1995). In order to inhibit lycopene cyclization and also the non-mevalonate isoprenoid pathway (Rohmer, 1999), Arabidopsis was treated with CPTA (Bramley, 1994) and Clomazone (Scott et al., 1994), respectively. HPLC pro®les of extracts showed accumulation of lycopene and d-carotene in the case of CPTA (Figure 4b), indicating that the b-lycopene cyclase had been inhibited, whilst Clomazone-treated tissue revealed no speci®c accumulation of a carotenoid precursor (Figure 4c,d). However, the reduction in plastid-synthesized isoprenoids (chlorophyll, carotenoids, tocopherols) provides a characteristic pro®le that indicates inhibition of the non-mevalonate pathway. Tomato fruit. Isoprenoids from transgenic ripe tomato fruit expressing constitutively an additional bacterial phytoene desaturase (crtI) show a signi®cant increase in ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 551±558
b-carotene accumulation compared to the wild-type (Figure 5a,b). Increases in lutein and cyclic carotenoids such as a-carotene are also detectable. Several cis isomers of lycopene are separated under these chromatographic conditions, which cannot be resolved by C18 columns (Table 1, Figure 4b). Isomers of lycopene are of interest with respect to dietary absorption (Holloway et al., 1999). Detection of eluates at 290 nm demonstrates that tocopherols and quinones (Figure 5c) can be analysed simultaneously, thus enabling effects on terpenoid quinones to be detected. This is also illustrated by the analysis of extracts of the R mutant of tomato, which contains negligible amounts of carotenoids (Fray and Grierson, 1993). As shown in Figure 6, higher levels of plastoquinone and tocopherols are found in the fruit, presumably as a consequence of an elevated pool of GGPP due to it not being converted into carotenoids. The chromatogram of crtI extracts (Figure 5) illustrates the ability of the system to detect all carotenoid pigments that have resulted from the expression of the transgene.
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Figure 6. Comparison of isoprenoid levels in ripe fruit from Ailsa Craig and R mutant tomato varieties. A logarithmic scale has been used to relate changes in the R mutant compared with Ailsa Craig. Values are 6 standard error (n = 3). Values < 0 represent a decrease in metabolite levels in the R mutant, whilst values > 0 indicate increases in the mutant. When metabolites were not detected in the R mutant, but present in Ailsa Craig, they are shown as solid bars, and values represent the maximum difference found. Metabolites present in the R mutant but absent in Ailsa Craig are shown as hatched bars. PL, plastoquinone; DT, d-tocopherol; YT, g-tocopherol; AT, a-tocopherol; UBQ, ubiquinone-9; LUT, lutein; CHL, chlorophyll; LY, lycopene; BC, b-carotene; PH, phytoene; NE, neurosporene; PF, phyto¯uene; ZC, z-carotene.
Figure 5. Chromatograms of (a) wild-type ripe tomato fruit, (b) transgenic crtI-containing ripe tomato fruit, recorded at 460 nm, and (c) transgenic crtI-containing ripe tomato fruit recorded at 290 nm. Peaks are (1) lutein; (2) a-carotene; (3) b-carotene; (4) all-trans lycopene; (4¢) 13-cis-lycopene; (4¢¢) 9-cis-lycopene; (5) plastoquinone; (6) a-tocopherol; (7) 15-cis-phytoene; (7¢), all-trans phytoene; (8) ubiquinone-9.
In summary, the methodology reported provides an improved simultaneous analysis of isoprenoids with UV or visible absorption spectra compared to existing methods. It allows the identi®cation and quanti®cation of thermally labile compounds, without derivitization, and separates geometric isomers. The procedure can be automated, is rigorous (over 2800 runs have been carried out in two years, with just one change of guard column per year) and reproducible. The procedure could make an important contribution to aspects of functional genomics, elucidation of the modes of action of herbicides, and analysis of metabolic engineering of quality traits derived from the isoprenoid pathway. Experimental procedures Materials
Spectrophotometric analyses cannot detect these changes accurately. Indeed, quanti®cation of coloured carotenoids by the spectrophotometric procedure of Lichtenthaler and Wellburn (1983) or using a general absorption coef®cient for A1%/1 cm of 2500 (Britton, 1985) results in underestimations of 51 6 2% (n = 4) and 56 6 2% (n = 4), respectively, compared to HPLC determinations. Estimating the carotenoid content from the spectrophotometric analysis using the percentage composition obtained from HPLC also under-estimated the amount by 56% 6 4 (n = 4), while quanti®cation by comparison to a known amount of external standard under-estimated the amount by 30 6 4% (n = 4).
Chemicals and solvents were obtained from Merck (Lutterworth, Leicestershire, UK) unless stated otherwise. Analytical grade chemicals were used. Ammonium acetate and all solvents used in HPLC, extraction and sample preparation were of HPLC grade.
Standards and inhibitors The crystalline reference carotenoids lycopene, b-carotene, bcryptoxanthin, zeaxanthin, astaxanthin and canthaxanthin were obtained from Hoffman-La Roche (Basel, Switzerland). Lutein was kindly provided by Kemin Industries (Des Moines, IA, USA). a-carotene was purchased from Sigma Chemical Co. (Poole, Dorset, UK). The acyclic carotenes phytoene, phyto¯uene and z-carotene were puri®ed from the Phycomyces blakesleeanus S442 mutant, as described by Fraser et al. (1991). Neurosporene was obtained from Escherichia coli harbouring the plasmid ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 551±558
Metabolic pro®ling of isoprenoids pACCAR-EBI (Albrecht et al., 1995) containing the phytoene desaturase gene from Rhodobacter capsulatus. d-carotene was puri®ed from ripe fruit of the del tomato. Violaxanthin, neoxanthin and antheraxanthin were isolated from tomato leaf tissue. a-, d- and g-tocopherols and a-tocopherol acetate and quinones were purchased from Sigma Chemical Co. The bleaching herbicides Nor¯urazon (4-chloro-5-(methylamino)-2-(3-(tri¯uoromethyl)phenyl-3(2H)-pyridazinone) and Sulcotrione (2-[2-chloro4-methanesulphonyl benzol] cyclohexane-1,3-dione), CPTA (2-(4chlorophenylthio)-triethylamine) and Clomazone (2-[2-chlorophenyl]-4,4-dimethyl-3-isoxazolidinone) were kindly provided by Dr K. Pallett (Aventis Crop Science, Ongar, Essex, UK) and Dr S. Ridley (Zeneca Agrochemicals, Bracknell, Berkshire, UK).
Plant material, growth conditions and application of inhibitors Wild-type Arabidopsis thaliana (ecotype Columbia) were surfaced-sterilized and germinated on MS medium (Murashige and Skoog, 1962) supplemented with 0.5% w/v sucrose and 0.2% w/v Phytagel (Sigma Chemical Co.). Seedlings were grown in controlled environmental growth chambers with a 16 h photoperiod provided by ¯uorescent lights (250 mmol m±2 sec±1). Day and night temperatures were 23°C and 19°C, respectively. Relative humidity was maintained above 70%. Inhibitors were prepared in methanol (typically as 3 100 stock solutions) and stored at ±20°C. Media containing the inhibitors were prepared by adding an aliquot from the stock until a ®nal concentration of typically 100 mM was obtained. For the controls, an identical amount of methanol was added. Following germination, Arabidopsis plants were grown for a further 14 days. Plants were harvested by removal from the agar, washed gently with dH2O, frozen or freeze-dried and then stored at ±20°C. Alternatively, when analysing mutant collections, complete agar plates were frozen directly. Tomato plants (Lycopersicon esculentum Mill. cv Ailsa Craig and R mutant) were grown in a greenhouse with supplementary lighting. Fruit was typically harvested at 7 days postbreaker (i.e. red ripe). Fruit were cut in half, seeds removed and then frozen at ±20°C.
Extraction of isoprenoids Freeze-dried material of Arabidopsis (1±10 mg) or tomato fruit (10±200 mg) was ground into a powder with a mortar and pestle or hand-held homogenizer. Small-scale extractions were carried out in micro-centrifuge tubes (1.5 ml) or alternatively screwcapped Pyrex test tubes (15 ml). Whenever possible, all subsequent manipulations were carried out on ice and shielded from strong light. For extraction of 1±2 mg of ground freeze-dried material, methanol (100 ml) was added, along with the internal standard (e.g. canthaxanthin, 1 mg). The suspension was mixed by inversion for 5 min at 4°C. Tris±HCl (50 mM, pH 7.5) (containing 1 M NaCl) was then added (100 ml) and a further incubation at 4°C for 10 min carried out. Chloroform (400 ml) was added to the mixture and incubated on ice for 10 min. A clear partition was formed by centrifugation at 3000 g for 5 min at 4°C. The hypophase was removed with a Pasteur pipette and the aqueous phase re-extracted with chloroform (400 ml). The pooled chloroform extracts were dried under a stream of nitrogen or by centrifugal evaporation. Dried residues were stored under an atmosphere of nitrogen at ±20°C prior to HPLC. In order to ef®ciently extract larger quantities (e.g. fresh tissue or 50±500 mg dry powder), the volume of buffer (50 mM Tris±HCl pH 7.5) and ã Blackwell Science Ltd, The Plant Journal, (2000), 24, 551±558
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methanol was increased to 1.5 ml and that of chloroform to 4 ml. If fresh tissue was used, homogenization was carried out with an Ultra-Turrax T25 (Janke and Kunkel, Staufen, Germany). When necessary, saponi®cation was performed by adding 60% w/v KOH and methanol to the suspension of ground tissue until a ®nal concentration of 6% (w/v) was reached. The mixture was heated at 60°C for 30 min in darkness. Buffer (50 mM Tris±HCl pH 7.5) was then added and the extraction performed as described above.
Isoprenoid separation and detection by HPLC±PDA Samples were prepared for HPLC by dissolving the residues in ethyl acetate. Chromatography was carried out on either a Waters system (Watford, Hertfordshire, UK) consisting of a no. 616 pump, no. 996 diode array detector and no. 717 auto-sampler or a Waters Alliance 2600S system with no. 996 diode array. Data were collected and analysed using the Waters Millennium32 software supplied. Throughout chromatography, the eluate was monitored continuously from 200 to 600 nm. Column temperature was maintained at 25°C by a no. 7955 column oven (Jones Chromatography, Hengoed, Mid-Glamorgan, UK). A reversephase C18 Nucleosil 5 mm (250 3 4.6 mm or 150 3 4.6 mm) column (Jones Chromatography) coupled to a 5 mm (10 3 4.6 mm) Phase Sep C18 guard column (Phase Separations, Deeside, Clwyd, UK) was used. Acetonitrile-based mobile phases with either methanol, water, isopropanol or ethyl acetate modi®ers were used as described by Fraser et al. (1992), Sandmann (1993) and Holloway et al. (1999). A reverse-phase C30, 5 mm column (250 3 4.6 mm) coupled to a 20 3 4.6 mm C30 guard (YMC Inc., Wilmington, NC, USA) with mobile phases consisting of methanol (A), water/methanol (20/80 by volume) containing 0.2% ammonium acetate (B) and tert-methyl butyl ether (C) was also used. The gradient elution used with this column was 95%A, 5% B isocratically for 12 min, a step to 80% A, 5% B, 15% C at 12 min, followed by a linear gradient to 30% A, 5% B, 65% C by 30 min. A conditioning phase (30±60 min) was then used to return the column to the initial concentrations of A and B. In addition, a normal phase Inert Sil 5 mm, 250 3 4.6 mm column with identical guard unit (10 3 4.6 mm) purchased from Chrompak (Walton, Surrey, UK) was used. The mobile phases with this column were either 0.5% ethanol in n-hexane run isocratically, or gradient elution from 20% ethyl acetate in hexane to 100% ethyl acetate over 25 min, maintaining ethyl acetate for a further 10 min. In all cases, ¯ow rates of 1 ml min±1 were used.
Quanti®cation Peak areas of the standards were determined at the wavelength providing maximum absorbance using the Waters Millennium32 software supplied.
Acknowledgements This work was partly funded by an EU Programme (PL 962077), a BBSRC/ROPA award (111/9810714), The Royal Society (grant G503) and the UK Ministry of Agriculture, Fisheries and Food (grant AN 0444). We would like to thank Drs K. Pallett and S. Ridley for supplying herbicides, Kemin and Hoffman La-Roche for carotenoid standards, and Zeneca Agrochemicals for ef®cient cultivation of tomato plants.
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