The Plant Journal (2000) 24(4), 551558

TECHNICAL ADVANCE

Application of high-performance liquid chromatography

with photodiode array detection to the metabolic proling
of plant isoprenoids
Paul D. Fraser, M. Elisabete S. Pinto, Daniel E. Holloway 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 p.bramley@rhbnc.ac.uk).

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
identication and quantication 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 proling.

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 scientic
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 identication of a wide
range of compounds in a single measurement (metabolic
proling) 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
specic compounds rather than for a range of metabolites
in a single sample. These procedures are, in general, time-
consuming, require relatively large amounts of material, and
are limited in the number of compounds that can be
analysed simultaneously. Therefore, their value in func-
tional genomics or metabolic engineering of quality traits in
plants is rather low.
The chemical diversity and varying abundance of plant
metabolites make it difcult, if not impossible, to employ a
procedure that is applicable for all, or a majority, of plant
metabolites. However, advances in analytical instrument-
ation and chromedia have improved the feasibility of
metabolic proling. In this study, we have focused on the
metabolic proling 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 iso-prenoids is in part
due to specialized isoprenoid formation occurring in different
plant tissues, for example mono-terpenes 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

2000 Blackwell Science Ltd 551

552 Paul D. Fraser et al.
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 brassino-steroids are
isoprenoids of very low abundance, making their analysis
difcult in a routine proling 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 structureactivity
relationships (QSAR) need to be deter-mined (Boger and
Sandmann, 1989). Secondly, the health-promoting
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 (Romer 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 caroten-oids
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 identied 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.
Blackwell Science Ltd, The Plant Journal, (2000), 24, 551558
Results and Discussion
Optimization of extraction 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
4060C), diethyl ether, ethyl acetate and chloroform were
evaluated as extraction solvents. Chloroform was the most
suitable, efciently 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 re-extraction, even with an increased
volume, removed virtually no more isoprenoids. The
suitability of chloro-form for extractions was further
endorsed by its ability to efciently extract the lycopene
present in tomato tissue at very high concentrations
1
(approximately 200 mg g fresh weight). The total amount
of carotenoid extracted by chloroform from tomato tissue
was 10-fold, 3-fold and 1.5-fold 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 chloro-form. Partitioning of the
chloroform extract against 25 mM TrisHCl pH 7.5 prior to
HPLC improved online PDA spectra by eliminating
unwanted UV interference. Further purication of the crude
extracts by solid-phase or ltra-tion procedures was not
necessary. Extracts could be stored dry, under an
atmosphere of nitrogen, for at least 1 year at 20C. 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
4C, 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,
 Metabolic proling of isoprenoids 553

Table 1. Isoprenoids separated on a reverse-phase C30 HPLC

system and spectral characteristics (in the eluting solvent)
used in identication from photodiode array detection

Retention time Spectral characteristics

Isoprenoid (min) (nm at lmax)
Carotenoids
15-cis-phytoene 24.69 275.8, 285.6, 297.3
All-trans-phytoene 25.62 278.0, 286.0, 301.6
Phytouene-1 27.05 330.8, 346.3, 366.4
Phytouene-2 27.70 330.1, 346.3, 366.4
Phytouene-3 28.23 330.1, 347.5, 366.4
z-carotene-1 28.96 374.8, 397.7, 421.8
z-carotene-2 29.61 371.2, 396.4, 418.2
z-carotene-3 32.03 379.6, 400.1, 425.4
z-carotene-4 32.50 379.6, 400.1, 425.4
z-carotene-5 34.70 379.6, 400.1, 425.4
Neurosporene 33.98 416.2, 440.2, 469.3
cis-lycopene-1 33.88 439.9, 465.3, 496.9
cis-lycopene-2 36.42 441.2, 467.8, 496.9
All-trans-lycopene 40.69 446.0, 472.6, 504.2
Poly-cis-lycopene 30.90 403.0, 426.6
d-carotene 32.40 424.0, 448.0, 482.5
a-carotene 26.79 423.0, 446.3, 474.1
b-carotene 28.22 , 453.2, 478.0
b-cryptoxanthin 22.78 430.0, 451.2, 477.8
Zeaxanthin 16.79 425.0, 451.2, 477.8
Lutein 15.00 421.0, 443.9, 477.8
Violaxanthin 11.17 419.0, 439.1, 469.3
Neoxanthin 10.04 415.0, 440.3, 466.9
Astaxanthin 13.50 , 474.1,
Canthaxanthin 18.56 , 477.8,
Figure 2. HPLC prole of isoprenoid standards recorded at (a) 287 nm Tocopherols
and (b) 460 nm.
In both (a) and (b), the left and right panels are the chromatogram and a-Tocopherol 11.81 290.0
derived spectra, respectively. The compounds in (a) are (1) vitamin K 3; a-Tocopherol acetate 13.83 285.0
(2) plastoquinone; (3) g-tocopherol; (4) d-tocopherol; (5) a-tocopherol; (6) d-Tocopherol 9.23 295.0
a-tocopherol acetate; (7) vitamin K 1; (8) ubiquinone-7; (9) ubiquinone-9. g-Tocopherol 10.68 298.0
In (b), the compounds are (1) astaxanthin; (2) lutein; (3) zeaxanthin; (4)
canthaxanthin; (5) b-cryptoxanthin; (6) a-carotene; (7) b-carotene; (8) Quinones
13-cis-lycopene; (8) 9-cis-lycopene; (8) all-trans lycopene. The solvent
system is described in Experimental procedures. Ubiquinone-7 17.55 274.5
Ubiquinone-9 23.75 274.5
Ubiquinone-10 28.20 278.0
Plastoquinone 5.60 278.1
1992; Fraser et al., 1993; Indyk, 1987; Weber, 1987) and 3.77 339.8
Vitamin K3
tocopherols (Abidi and Mounts, 1997; Indyk, 1990). While 14.83 329.1
Vitamin K1
evaluating these systems for potential use in isoprenoid Chlorophylls
proling, it became clear that they had been tailored to a
Chlorophyll a 21.33 432.6, 657
specic group of compounds or isoprenoid class.
Chlorophyll b 16.69 467.8, 656
Simultaneous carotenoid and tocopherol separations do
exist, but their application is principally related to clinical
Data provided are for all-trans isomers unless otherwise stated.
samples (Barua and Olsen, 1998; Schuep et al., 1995), and Isomer congurations are assigned from spectral and chromato-
the number of compounds that can be resolved makes their graphic characteristics (Britton, 1985). In cases where no isomer
use restrictive. An alternative, C30 reverse-phase matrix can be assigned (e.g. z-carotene), numbers have been used.
(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, istic spectral properties of these molecules. The range of
tocopherols, plastoquinones, vitamin K and ubi-quinone isoprenoids separated and identied using this protocol is
(Figure 2a) as well as carotenoids (Figure 2b) were given in Table 1. Since astaxanthin and canthaxanthin are
separated in one chromatographic run. The online PDA not found in higher plant tissues, they were used as internal
detector facilitates identication from the character- standards to determine recoveries, relative reten-

Blackwell Science Ltd, The Plant Journal, (2000), 24, 551558

554 Paul D. Fraser et al.

tion times and for quantication. Calibration curves

2
revealed linearity, with R 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 210 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-toco-
pherol (n = 3), 5% for plastoquinone (n = 3), 8% for
lutein (n = 3) and 5% for b-carotene (n = 6).

Application of isoprenoid proling by HPLCPDA

Arabidopsis thaliana. Wild-type extracts were analysed

using the C30 HPLC system. Four xanthophylls, lutein (200
1 1
6 40 mg g dry weight, n = 3), violaxanthin (86 6 13 mg g
1
dry weight, n = 3), neoxanthin (4 6 0.6 mg g dry weight, n
1
= 3) and zeaxanthin (48 6 6 mg g dry weight, n = 3) were
1
detected. b-carotene (160 6 8.4 mg g dry weight, n = 3)
was the principal carotene present. Other isoprenoids
1
determined were a-tocopherol (170 6 18 mg g dry weight,
1
n = 3), plastoquinone (140 6 13 mg g dry weight, n = 3),
1
ubiquinone (200 6 8.6 mg g dry weight, n = 3) and
1
chlorophylls (677 6 23 mg g 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 leaet (about
10 mm in length). Thus, the method is suitable for the
analysis of Arabidopsis lines grown on agar for 23 weeks
in Petri dishes (140 mm in diameter; approximately 200
plants per dish) and frozen at 20C. 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 proles, Arabidopsis was grown on four
inhibitors of isoprenoid biosynthesis with different modes of
action. Norurazon inhibits carotenoid forma-tion by
inhibiting phytoene desaturation (Sandmann, 1993).
Sulcotrione is an inhibitor of 4-hydroxyphenylpyr-uvate
dioxygenase, an enzyme involved in plastoquinone Figure 3. HPLC chromatograms recorded at 287 nm of (a) Arabidopsis
wild-type, (b) Arabidopsis following Norurazon treatment, and (c)
formation (Schulz et al., 1993). Inhibition of plastoquinone Arabidopsis following Sulcotrione treatment.
formation has the secondary effect of inhibiting carotenoid The peaks are (1) plastoquinone; (2) a-tocopherol; (3) 15-cis-phytoene;
synthesis by preventing the electron transfer necessary for (4) all-trans phytoene; (5) ubiquinone-9. Approximately 2 mg of freeze-
dried material was extracted as described in Experimental procedures.
desaturation to proceed (Norris et al., 1995). Both
Sulcotrione and Norurazon-treated plants are bleached in
appearance. HPLCPDA analyses using the C 30 system
show the accumulation of cis- and trans-phytoene in the treatment with Sulcotrione, but not Norurazon, results in a
norurazon and Sulcotrione-treated Arabidopsis extracts reduction of plastoquinone (Figure 3b,c). The ratio of
(Figure 3b,c). No phytoene was detectable in the wild-type plastoquinone:phytoene was approximately 1 following
grown under identical conditions (Figure 3a). Therefore, the noruazon treatment but approximately 5 with Sulcotrione.
analysis conrms that the phytoene desaturation is inhibited Thus, the inhibition of phytoene desaturation has been
in both cases. More signicantly, however, the ability to identied and the indirect mechanism causing the
identify simultaneously other isoprenoids (plastoquinone, Sulcotrione-phenotype elucidated. Therefore, this sys-tem
ubiquinone and tocopherols) shows that may be used to differentiate between the different

Blackwell Science Ltd, The Plant Journal, (2000), 24, 551558


modes of action of bleaching herbicides. Such an
approach will also facilitate the identication 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 proles
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 specic accumula-tion of a
carotenoid precursor (Figure 4c,d). However, the reduction
in plastid-synthesized isoprenoids (chlorophyll, carotenoids,
tocopherols) provides a characteristic prole 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 signicant increase in
Blackwell Science Ltd, The Plant Journal, (2000), 24, 551558
Metabolic proling of isoprenoids 555
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) b-
carotene; (6) cis-b-carotene; (7) a-carotene; (8) d-carotene; (9) all-trans
lycopene; (9) cis-lycopene; (10) zeaxanthin; (11) plastoquinone; (12) a-
tocopherol; (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.
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 C 18 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 toco-
pherols and quinones (Figure 5c) can be analysed simul-
taneously, 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 plastoqui-none 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.
556 Paul D. Fraser et al.

1%
for A /1 cm

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-toco-pherol; (7)
15-cis-phytoene; (7), all-trans phytoene; (8) ubiquinone-9.
Spectrophotometric analyses cannot detect these changes
accurately. Indeed, quantication of coloured carotenoids
by the spectrophotometric procedure of Lichtenthaler and
Wellburn (1983) or using a general absorption coefcient
of 2500 (Britton, 1985) results in under-
estimations of 51 6 2% (n = 4) and 56 6 2% (n = 4),
respectively, compared to HPLC determinations.
Estimating the carotenoid content from the spectrophoto-
metric analysis using the percentage composition
obtained from HPLC also under-estimated the amount
by 56% 6 4 (n = 4), while quantication by comparison
to a known amount of external standard under-estimated
the amount by 30 6 4% (n = 4).
Blackwell Science Ltd, The Plant Journal, (2000), 24, 551558
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,
phytouene; ZC, z-carotene.
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 identication and quantication 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
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, b-
cryptoxanthin, 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, phytouene and z-carotene
were puried from the Phycomyces blakesleeanus S442 mutant, as
described by Fraser et al. (1991). Neurosporene was obtained from
Escherichia coli harbouring the plasmid

pACCAR-EBI (Albrecht et al., 1995) containing the phytoene
desaturase gene from Rhodobacter capsulatus. d-carotene was
puried from ripe fruit of the del tomato. Violaxanthin, neox-anthin
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 herbi-cides
Norurazon (4-chloro-5-(methylamino)-2-(3-(triuoro-methyl)phenyl-
3(2H)-pyridazinone) and Sulcotrione (2-[2-chloro-4-
methanesulphonyl benzol] cyclohexane-1,3-dione), CPTA (2-(4-
chlorophenylthio)-triethylamine) and Clomazone (2-[2-chloro-
phenyl]-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 sur-
faced-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
con-trolled environmental growth chambers with a 16 h
photoperiod provided by uorescent lights (250 mmol m
2 mm or 150 3 4.6 mm) column (Jones Chromatography) coupled to a
1 5 mm (10 3 4.6 mm) Phase Sep C 18 guard column (Phase
sec ). Day and night temperatures were 23C and 19C,
respectively. Relative humidity was maintained above 70%.
Inhibitors were prepared in methanol (typically as 3 100 stock
solutions) and stored at 20C. Media containing the inhibitors
were prepared by adding an aliquot from the stock until a nal
concentration of typically 100 m M 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
20C. 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 supple-mentary lighting. Fruit
was typically harvested at 7 days post-breaker (i.e. red ripe).
Fruit were cut in half, seeds removed and then frozen at 20C.
Extraction of isoprenoids
Freeze-dried material of Arabidopsis (110 mg) or tomato fruit
(10200 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 screw-capped Pyrex
test tubes (15 ml). Whenever possible, all subse-quent
manipulations were carried out on ice and shielded from strong light.
For extraction of 12 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 4C. TrisHCl (50 m M, pH 7.5) (contain-ing 1 M NaCl) was
then added (100 ml) and a further incubation at 4C 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 4C. The hypophase was
removed with a Pasteur pipette and the aqueous phase re-extracted
with chloroform (400 ml). The pooled chloro-form extracts were
dried under a stream of nitrogen or by centrifugal evaporation. Dried
residues were stored under an atmosphere of nitrogen at 20C
prior to HPLC. In order to efciently extract larger quantities (e.g.
fresh tissue or 50500 mg dry powder), the volume of buffer (50 m M
TrisHCl pH 7.5) and
Blackwell Science Ltd, The Plant Journal, (2000), 24, 551558
Metabolic proling of isoprenoids 557
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, saponication 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
60C for 30 min in darkness. Buffer (50 m M TrisHCl pH 7.5) was
then added and the extraction performed as described above.
Isoprenoid separation and detection by HPLCPDA
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
32
analysed using the Waters Millennium software supplied.
Throughout chromatography, the eluate was monitored continuously
from 200 to 600 nm. Column tempera-ture was maintained at 25C
by a no. 7955 column oven (Jones Chromatography, Hengoed, Mid-
Glamorgan, UK). A reverse-phase C 18 Nucleosil 5 mm (250 3 4.6
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 C 30,
5 mm column (250 3 4.6 mm) coupled to a 20 3 4.6 mm C 30 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 (3060 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
1
acetate for a further 10 min. In all cases, ow rates of 1 ml min
were used.
Quantication
Peak areas of the standards were determined at the wavelength
32
providing maximum absorbance using the Waters Millennium
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
efcient cultivation of tomato plants.
558 Paul D. Fraser et al.
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