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RESUBMITTED – JUNE 6, 2000
Metabolite profiling of sesquiterpene lactones from Lactuca species: major latexcomponents are novel oxalate and sulfate conjugates of lactucin and itsderivatives.
Reuben A. Sessa∗ , Mark H. Bennett∗ , Mervyn J. Lewis§, John W. Mansfield¶ and
Michael H. Beale§¶.
From the Department of Biological Sciences, Wye College, University of London, Ashford,
Kent. TN25 5AH, UK and §IACR-Long Ashton Research Station, Department of Agricultural
Sciences, University of Bristol, Long Ashton, Bristol, BS41 9AF, UK.
Running Title:- Sesquiterpene lactone conjugates in Lactuca
∗ Joint first authors¶ To whom correspondence should be addressed at Dept. Biological Sciences, Wye College, Universityof London, Ashford, Kent TN25 5AH, UK. Tel 44-1233-812401; Fax 44-1233-813140; [email protected], or at IACR-Long Ashton Research Station, University of Bristol, LongAshton, Bristol, BS41 9AF, UK. Tel. 44-1275-549289; Fax. 44-1275-394281; E-mail:[email protected]
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SUMMARY
Wounding leaves or stems of Lactuca species releases a milky latex onto the plant
surface. We have examined the constituents of latex from Lactuca sativa (lettuce) cv.
Diana. The major components were shown to be novel 15-oxalyl and 8-sulfate
conjugates of the guaianolide sesquiterpene lactones, lactucin, deoxylactucin and
lactucopicrin. The oxalates were unstable, reverting to the parent sesquiterpene lactone
on hydrolysis. Oxalyl derivatives have been reported rarely from natural sources. The
sulfates were stable and are the first reported sesquiterpene sulfates from plants.
Unusual tannins based on 4-hydroxyphenylacetyl conjugates of glucose were also
identified. Significant qualitative and quantitative variation was found in sesquiterpene
lactone profiles in different lettuce varieties and in other Lactuca spp. The proportions
of each conjugate in latex also changed depending on the stage of plant development. A
similar profile was found in chicory, in which oxalyl conjugates were identified, but the
8-sulfate conjugates were notably absent. The presence of the constitutive sesquiterpene
lactones was not correlated with resistance to pathogens but may have a significant
bearing on the molecular basis of the bitter taste of lettuce and related species. The
induced sesquiterpene lactone phytoalexin, lettucenin A, was found in the Lactuca spp.
but not in chicory.
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INTRODUCTION
Sesquiterpene lactones (SLs)1 found in plants are remarkably diverse in terms of their
structure, properties and proposed functions (1-3). Amongst the Compositae, over 500
different members of the SL family have been described including not only constitutively
produced secondary metabolites but also phytoalexins which are only synthesized following
microbial challenge (2,4,5). The principal constitutive SLs found in species of Lactuca have
been reported to be lactucin, lactucopicrin, 8-deoxylactucin (Figure 1) and derivatives such as
11,13 dihydro-analogues (6,7). The SLs accumulate in lactifers, which are closely associated
with the vascular tissues of the Compositae (8). The presence of the SLs in latex released
from damaged lactifers is thought to contribute to its analgesic, antitussive and sedative
properties (9,10). The SLs lactucin, 8-deoxylactucin and lactucopicrin are also intensely bitter
and their presence within salad lettuce and chicory has considerable economic impact (11).
The guaianolide SL phytoalexin from lettuce (L. sativa ) lettucenin A (Figure 1), is highly
antimicrobial being one of the most toxic phytoalexins described (12).
In this work we aimed to develop simple methods to profile SLs in lettuce varieties in
order to examine correlations between fungal resistance and the presence of constitutive and
induced SLs. Previous isolations have indicated that glycosides of SLs may also be important
components. For example, the 15-glycososyl conjugate of 11,13-dihydrolactucopicrin has
been identified in roots of L. tartarica (13). This and other related guaianolide SL lactone
glycosides, such as picriside A (lactucin 15-glycoside) and crepidiaside A (8-deoxylactucin-
15-glycoside) have also been identified in other members of the Lactuceae tribe (14-16).
Identification of these glycosides in aerial parts of L. sativa has been based on structural
characterisation of the supposed aglycones following digestion of extracts with cellulase, or
acid hydrolysis (11,17). However, using extraction and profiling techniques that reveal SLs
and SL conjugates we show that the conjugates of lactucin, 8-deoxylactucin and lactucopicrin,
present in the latex of lettuce, other species of Lactuca and also in chicory are mainly 15-
oxalates. These unstable oxalate esters appear to represent a novel class of secondary
1 Abbreviations used are: Amu., atomic mass units; CC, column chromatography; ; ES-MS,electrospray-mass spectrometry; HPLC-DAD, high pressure liquid chromatography with diode arraydetection; Rt, retention time; RP, reverse phase; SL, sesquiterpene lactone; TLC, thin layerchromatography.
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metabolites in plants. We also report the identification of 8-sulfates as additional major SL-
conjugates in lettuce. To our knowledge no sulfated forms of SLs have previously been
described. Our results address compounds some of which appear to have been mistakenly
accepted to be glycosides for over 50 years (11,18).
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EXPERIMENTAL PROCEDURES
Plants
Seeds of Lactuca sativa cvs. Diana and Cobham Green were purchased from A.L.Tozer Ltd.
(Cobham, Surrey,UK) and Cichorium intybus from Chiltern Seeds (Ulverston, Cumbria, UK).
L. sativa cvs. Benita, Sabine, Capitan and Salinas, L. serriola, L. virosa and lines with
ingressed resistance to B. lactucae arising from interspecific L. sativa x L. virosa crosses
were kindly supplied by Dr. David Pink (HRI, Wellesbourne, UK). The plants were raised
under glasshouse conditions with supplementary lighting (80 W m-2) to ensure a minimum
day length of 16 h.
Chromatography and spectroscopy
Routine methods and standard equipment were used for HPLC, HPLC-DAD, TLC and CC
and the determination of UV spectra. 1H and 13C NMR spectra were recorded on a JEOL-
GX400 instrument using solvents indicated in Tables 1 and 2. Electrospray MS and MS-MS
were carried out on a Thermoquest LCQ instrument. Samples were introduced directly as
solutions in acetonitrile : acetic acid (99:1) or methanol : acetic acid (99:1) or by means of a
coupled HPLC using acetonitrile : water(1% acetic acid) programmed solvent mixtures at a
flow rate of 0.5ml min-1.
Isolation of latex and analysis by HPLC
Small, downward pointing, V-shaped incisions were cut into the base of the lettuce stem. The
droplets of latex that exuded were collected and 10• l samples immediately mixed with 1ml
methanol containing1% phosphoric acid. Without further extraction the sample was
centrifuged at 16,000g for 10min and the supernatant filtered through a 0.45• m membrane.
HPLC profiles of the constitutive sesquiterpenes were generated using 15• l injections of the
supernatant onto a reverse phase (RP) C18 column (5• m, 250 x 4.6mm) with a solvent
gradient of 99:1(A:B) to 48:52 (A:B), over 60 min at 1ml min-1 at 350C. A = (H2O, 0.1%
H3PO4); B = (90%CH3CN, 10%H2O). A typical profile is shown in Figure 2. The SLs were
quantified using calibration curves, based on UV detection at 200 and/or 264 nm, prepared for
lactucin.
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Isolation of lactucin and lactucopicrin (Peaks 3 and 10 in Figure 2)
Bolted lettuce stems (cv. Diana, 900g) were homogenized in 85% MeOH (2L) and steeped
overnight. After filtration through miracloth, the residue was further extracted (x3) with 70%
MeOH (1L) and filtered. The filtrates were pooled, concentrated to 800 ml in vacuo, filtered
and then defatted by extraction with 200 ml of hexane. The aqueous methanolic liquor was
extracted with dichloromethane (3 x 250 ml), and the combined dichloromethane extracts
were evaporated. The residue was chromatographed over 25 g silica gel (40-63 • m BDH,
Poole, UK) eluting with hexane : ethyl acetate (1 : 1) to give lactucopicrin (40mg) and
lactucin (11mg), identified by NMR.
Isolation of oxalate-conjugated sesquiterpenes from lettuce (peaks 1 and 7 in Figure 2)
Bolted lettuces (cv Diana, 250g) were homogenized in 70% MeOH (1L) and the extract
filtered through miracloth. The filtrate was reduced to ca. 400 ml and adjusted to pH 2.0
(HCl). After extraction with petroleum ether (bp 60-80, 300ml), the SLs were extracted into
ethyl acetate (3x300ml), which was then dried (MgSO4) and evaporated in vacuo. Preliminary
purification of the residue was by chromatography over 25g of silica gel eluting with a 10%
step gradient from acetone : dichloromethane (1 : 9) to acetone (total vol. 1.2L) followed by
elution with acetone : formic acid (99.8 : 0.2, 500 ml). The acetone : formic acid fractions
were pooled, dried and chromatographed on a 50 x 2.5cm column of Sephadex LH20
(Pharmacia) eluting isocratically with H2O : EtOH : CH3OOH (49.99 : 50 : 0.01). Two
compounds eluting in 114-122 ml and 138-140ml were further purified individually by
passage of the pooled fractions through two, coupled Sep-pak C18 cartridges. The eluates were
dried in vacuo to give lactucin oxalate (7 mg) and lactucopicrin oxalate (10mg), respectively.
For NMR analysis see Tables 1 and 2.
Synthesis of lactucopicrin oxalate
Lactucopicrin (10mg) was treated with a solution of oxalyl chloride (2.0M, 2ml, in
dichloromethane) at 20oC for 16 h. After addition of H
2O (15ml) the reaction product was
extracted with ethyl acetate (3x10ml). The organic phase was evaporated in vacuo and
lactucopicrin oxalate (8mg) purified by chromatography over 5g LiChroprep RP-18, 40-60mm
(Merck), eluting with water : methanol : formic acid (59.9 : 40 : 0.1).
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Isolation and characterisation of sulfate conjugates (peaks 2 and 5 in Figure 2)
The conjugates (c. 1mg) were isolated from latex extracted with CH3CN:H2O (4:1) by
collection of the compounds from RP-semi preparative HPLC. The solvent conditions were
85:15 (A:B) to 55:45 (A:B) over 50 min at 350C and a flow rate of 2 ml min-1. A = (99.9%
H2O : 0.1% CH3OOH); B = (90% CH3CN : 10% H2O). 15-deoxylactucin-8-sulfate
(recovered at Rt 15.8 min) was further purified on the same column using an isocratic solvent
composition of 86.5% H2O: 13.5% CH3CN containing 0.05% formic acid. 15-p-
hydroxyphenyl-acetyllactucin-8-sulfate (Rt = 24.4min) was re-chromatographed on a gradient
of H2O : CH3CN (80% : 20% to 45% : 55%) over 15 min.
Hydrolysis and re-sulfonation of 15-deoxylactucin-8-sulfate
15-deoxylactucin-8-sulfate (c. 200 • g) was hydrolysed with 1M HCl at 65oC for 4h,
extracted with CH2Cl2, which was then evaporated in vacuo. The major hydrolysis product
was isolated by semi-preparative RP-HPLC using 65% H2O : 45% CH3CN containing 0.05%
CH3OOH at a flow rate of 2 ml min-1 (Rt =12min). The product, 15-deoxylactucin (MW 260)
was a single peak which eluted, under analytical conditions, just before 8-deoxylactucin and
11,13-dihydro-8-deoxylactucin (kindly provided by Dr Toshio Miyase, University of
Shizuoka, Japan). The product was treated with an excess of sulfur trioxide.N,N -
dimethylformamide complex (Aldrich) in CH2Cl2 for 2h at 250C. The solution was extracted
with H2O (x2) and the aqueous fractions pooled and applied to a Sep-pak C18 cartridge
(Waters) that had been pre-washed with H2O : CH3OOH (99.9 : 0.1). The cartridge was
washed with 5 ml of the same solvent and then the product was eluted with MeOH. After
drying in vacuo analysis by HPLC and ES-MS demonstrated that the re-sulfonation gave a
single product, which co-migrated with the initially isolated compound.
Isolation of Peaks 4, 6, 8, 9 (see Figure 2)
Peaks 4,6,8 and 9 were collected from semi-preparative RP-HPLC of extracts of latex as
described for the sulfate conjugates, but using a linear gradient to 60%A : 40%B. Peaks 8 and
9 were further purified on the same column using an isocratic solvent composition of 64%A :
36%B.
NMR data for major component of peak 9 (2,3,4-tri-O-4-hydroxyphenylacetyl-glucopyranose)13C-NMR (deuteroacetone) • 40.3, 40.4, 40.5 (3x ArCH2COO), glucose carbons - 62.0 (C-6);
70.0; 70.5; 70.7; 72.7 and 90.5 (C-1), aromatics 116.1 (C-3, C-5); 125.6 (C-1); 131.2 (C-2, C-
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6) and 157.3 (C-4), carbonyls – 171.4; 171.5, 171.8. 1H-NMR (deuteroacetone) • 3.25 (s, 1x
CH2CO); 3.37 (m, 2x CH2CO) glucose hydrogens – 3.39 (m, H2-6); 3.45(br,d, H-5); 4.75(dd,
J=10,3.5Hz, H-2); 5.05(t, J=10Hz, H-4); 5.34(d, J=3.5Hz, H-1) and 5.59 (t, J=10Hz, H-3);
aromatics – 6.78 (d, J=8Hz) and 7.03 (m)
Localisation of SLs in seedlings and developmental study.
Seedlings (7 days after emergence) were dissected into hypocotyls, petioles, and cotyledon
lamina. Tissue samples were extracted and analysed as above. Latex samples from plants at
different developmental stages were examined by analytical HPLC as described above. Latex
was collected from cuts in the stem base of plants at the following stages; rosette, bolted but
before flowering, and seeded. At least five plants were analysed at each growth stage.
Analysis and quantification of lettucenin A and constitutive sesquiterpenoids in CuSO4
elicited leaf discs, cotyledons and culutured cells.
Leaf discs, punched out using an 11mm diameter cork borer, or whole cotyledons were floated
on a solution of CuSO4 (0.5% w/v) containing Tween 20 (0.0001%) for 30 min, rinsed in H2O
and incubated with constant illumination at 160C for 3 days. Callus and cell suspension
cultures were prepared as described by Fagg et al (19).The preparation of methanolic extracts
of tissuse and the analysis and quantification of lettucenin A were as described in Bennett et
al. (12). Constitutive SLs were quantified in extracts using the HPLC conditions routinely
applied to latex as above.
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RESULTS
Identification of sesquiterpene lactones profiled from latex of Lactuca sativa
Chromatographic separation of latex components of L. sativa cv. Diana was achieved by
reversed phase HPLC (Figure 2), initially using UV absorption at 200 nm to detect all
components. Methanolic extracts of latex were injected directly onto the HPLC, in order to
minimise degradation or losses of component SLs before analysis. UV absorption at 264 nm
and diodearray detection was used to identify lactucin derivatives by their characteristic UV
spectra. Electrospray mass spectral data for each HPLC peak were obtained from coupled
HPLC-MS runs or from analysis of components of individual peaks collected from
preparative HPLC. Where possible 1H and 13C-NMR spectra were also recorded from
samples isolated by HPLC. The compounds identified from peaks numbered 1-10 in Figure 2
were as follows.
Lactucin and lactucopicrin Peak 3 gave a molecular ion at m/z 277 in positive ion
ES- MS and was identified as lactucin (MW 276) by comparison of 1H and 13C-NMR data
(Tables 1 and 2) with published spectra (6, 14-16, 20). Similarly, Peak 10 gave a molecular
ion at m/z 411 and a daughter ion at m/z 259 (loss of 4-hydroxyphenylacetic acid) in positive
ion electrospray MS and was identified as lactucopicrin (MW 410) by comparison with
published NMR data (6).
Lactucin-15-oxalate Peak 1 had a molecular weight of 348, revealed by ES-MS
molecular ions at m/z 349 (positive ion mode) and 347 (negative ion mode). Analysis of the
1H-NMR spectrum (Table 1) indicated that this compound was a derivative of lactucin,
retaining all of the signals of lactucin with no additional 1H signals. The most obvious feature
of the 1H-NMR of peak 1 was a significant downfield shift of the 15-hydrogens, suggesting
esterification of the 15-hydroxy group of lactucin. The molecular weight difference between
peak 1 and lactucin (72 amu) indicated the addition of an oxaloyl moiety. This accounts for
the absence of additional 1H-NMR signals. Furthermore, the 13C-NMR spectrum (Table 2)
was very similar to that of lactucin with the addition of two carbonyl resonances at • 162.0 and
165.9. Further evidence was apparent from MS-MS fragmentation spectra. Negative ion ES-
MS-MS gave the following parent-daughter ion relationships :- m/z 347• 275 • 257 (minus
oxaloyl and oxalic acid respectively), which were mirrored in the positive ion spectrum m/z
349• 277• 259. Hydrolysis in water with or without cellulase (Sigma, 10mg ml-1) at pH 5.0
gave lactucin. Final confirmation of the structure to be latucin-15-oxalate was achieved by
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treatment of lactucin with oxalyl chloride which gave a product which, on HPLC, co-
chromatographed with and gave identical MS data to the natural compound.
Lactucopicrin-15-oxalate The major component of latex of cv. Diana (peak 7) had a
molecular weight of 482 revealed by molecular ions at m/z 483(positive ion mode) and 481
(negative ion mode). Analysis of the 1H- and 13C-NMR spectra (Tables 1 and 2) as above
indicated that this compound was the 15-oxalate derivative of lactucopicrin. Further
confirmation was apparent from MS-MS fragmentaion of the negative molecular ion (m/z
481) which gave the following parent-daughter sequences:- 481• 409 and 481• 257 ,
consistent with losses of CO2CO (72 amu) and 4-hydroxyphenylacetyl (134 amu) plus oxalic
acid (90 amu). Similarly, fragmentation of the positive molecular ion (m/z 483) gave rise to
daughter ions at 349 (-134amu, 4-hydroxyphenylacetyl) • 331 (-H2O) • 241 (-90 amu, oxalic
acid). As above, the oxalate was sensitive to hydrolysis and when incubated in water +/-
cellulase returned lactucopicrin. Treatment of lactucopicrin with oxalyl chloride gave a
product that co-chromatographed with and gave identical MS data to the material in peak 7.
8-deoxylactucin-15-oxalate. The mass spectra obtained from peak 4 were suggestive
of a 8-deoxylactucin 15-oxalate structure (MW 332) with the positive ion spectrum exhibiting
333• 261• 243 fragmentation, consistent with losses of oxaloyl and oxalic acid as observed
for lactucin 15-oxalate. This compound was not characterised by NMR and thus the
identification must be regarded as tentative.
15-deoxylactucin-8-sulfate. Peak 2 gave 1H-NMR and 13C-NMR spectra consistent
with a derivative of 15-deoxylactucin. This was evident from the presence of two methyl
groups (C-14 and 15), and the absence of the distinctive 15-CH2OH signals of lactucin and
lactucopicrin. The chemical shifts of H-8, H-9• and particularly H-13a (Table 1) indicated
that the molecule contained a polar substituent at C-8. The absence of 1H or 13C signals
additional to those expected for 15-deoxylactucin indicated that the substituent at C-8 was
inorganic. The molecular weight, obtained by both positive and negative ion electrospray MS
was 340, 80 amu higher than that expected for 15-deoxylactucin (260). This and parent-
daughter ion relationships in the positive ion spectrum [341• 261• 243 (loss of SO3 and
H2SO4 )] are totally consistent with a sulfate substituent at C-8 and thus the structure was
assigned to be 15-deoxylactucin 8-sulfate. The compound was resistant to cellulase digestion,
but could be hydrolysed by warm acid treatment to give 15-deoxylactucin, as demonstrated by
a molecular ion at m/z 261, in the positive ion mass spectrum. Furthermore, treatment of this
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hydrolysis product with N,N-dimethylformamide-sulfur trioxide complex returned 15-
deoxylactucin 8-sulfate, identical to the natural product by HPLC and MS. The alternative
structure, 8-deoxylactucin-15-sulfate, was ruled out by synthetic sulfonation of 8-
deoxylactucin. This isomeric compound elutes just after the natural 15-deoxylactucin-8-
sulfate by HPLC.
15-p-hydroxyphenyl-acetyllactucin-8-sulfate. Peak 5 had a molecular weight of 490.
The major fragmentation in the positive ion electrospray MS was 491• 339, corresponding to
loss of 4-hydroxyphenylacetic acid (152 amu.), suggesting that this compound is an analogue
of lactucopicrin which shows the same feature in its MS (see above). The molecular weight
difference of 80 amu between this compound and lactucopicrin suggests that this compound
also contains a sulfate function. Other parent-daughter fragments m/z 491 • 411• 393 are
consistent with losses of SO3 and H2SO4. Examination of the 1H- and 13C-NMR spectra
(Tables 1 and 2) revealed, unexpectedly, that in this compound the 4-hydroxyphenylacetyl
group is attached to C-15 and the sulfate group is at C-8. Of particular significance are the
chemical shifts of the protons at H-8, H-9 and H-13a which are indicative of the 8-SO3H
substructure (cf . 15-deoxylactucin sulfate), and the chemical shifts of H2-15 which are
consistent with an acyl derivative.
Peak 6 could not be conclusively identified. On the basis of molecular weight
suggested by ES-MS (424) and the presence of both carbohydrate and SL signals in the 1H -
NMR spectrum, the structure was tentatively assigned as 11,13 dihydro-8-deoxylactucin -15-
glycoside (jacquinellin glycoside).
Additional components of the profile Peaks 8 and 9 did not contain
sesquiterpenes and were not completely resolved in preparative HPLC runs. The 1H
and 13C –NMR spectra of the two peaks were similar and indicated that both were
mixtures of multi-(4-hydroxyphenylacetyl) derivatives of glucose and had structures
resembling the gallotannins (21,22). The aromatic regions of the 13C-spectra were
simple, and identical with that in latucopicrin, indicating that all of the aromatic
moieties within the compounds consisted of 4-hydroxyphenylacetyl groups.
Comparison of the chemical shifts of the glucosyl hydrogens in the 1H-spectra with
those of authentic glucose pentabenzoate allowed assignment of the core substitution
pattern. This indicated that the major component of these lettuce tannins was a 2,3,4-
tri-O-4-hydroxyphenylacetyl-• -glucopyranose. Examination of the samples by
negative ion electrospray MS yielded candidate molecular ions at m/z 581, consistent
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with a tris-(4-hydroxyphenylacetyl) monosaccharide (MW 582). Furthermore, MS-MS
fragmentation m/z 581 was consistent with the proposed structure and contained
strong fragments due to sequential losses of 4-hydroxyphenylacetyl (134 amu) and 4-
hydroxyphenylacetic acid (152 amu). However, The MS spectra of both peaks 8 and 9
were not clean and contained ions at higher m/z values, which could not be attributed
to tetra- or penta-(4-hydroxyphenylacetyl) homologues. Integration of the downfield
hydrogens in the 1-H NMR spectra indicated that additional aromatic residues may be
present. Nevertheless, if present, they were not attached to vacant C-1 and C-6
hydroxy groups of the glucopyranose.
Localisation of SLs in seedlings and changes in concentrations during plant
development.
Simple dissection experiments using young lettuce seedlings showed that the
concentrations of SLs extracted from tissues were closely associated with the presence
of vascular elements and in particular the lactifer network. Profiles of the SLs
recovered from seedlings were qualitatively very similar to those found in latex. The
concentrations found in various tissues after dissection were , however, very different.
For example concentration of the major components 15-deoxylactucin-8-sulfate and
lactucopicrin oxalate were respectively (in µg g-1 fresh weight ± SEM), in hypocotyls,
44.8±4.0 and 124±8.4; petioles, 7.9±1.2 and 27.8±1.8, and cotyledon lamina 1.7±0.4
and 8.8±0.7. Samples dissected from whole plants inevitably contained some lactifers
or vascular tissues. However, tissues grown in vitro were undifferentiated, lacking
any vascular elements and none of the constitutive SL conjugates found in latex were
detectable in callus or cells from suspension culture.
Latex was recovered from cuts into the stem base at different stages of plant
growth and SL profiles compared by HPLC. Quantitative data obtained are
summarized in Figure 3. Significant increases in the major SLs, 15-deoxylactucin-8-
sulfate (peak 2) and lactucopicrin oxalate (peak 7) occurred during bolting. Similar
trends were visible for the lower abundance SLs, for example, 15-p-
hydroxyphenylacetyl-lactucin-8-sulfate (peak 5) and lactucin-15-oxalate (peak 1). In
general the increases in concentrations of the SL-sulfates observed during bolting
were maintained during flowering and seed set, whereas the increased levels of
oxalated SLs present at bolting were not sustained during later development. The
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combined mean total concentrations of all SLs measured within latex at the rosette,
bolting, in flower and seeded stages of development were 61.7, 147.1, 110.7 and
111.7 mg ml-1 respectively. Thus the general trend indicated a burst of SL formation
on bolting.
Profiles of SLs in lettuce cultivars, other Lactuca species and chicory
The profiles of SLs in different varieties of lettuce and species of Lactuca were examined by
HPLC. Plants were examined at the rosette stage, prior to bolting. Wild species such as L.
serriola and L. virosa have proved valuable sources of genes for resistance to the downy
mildew fungus Bremia lactucae (23). By comparative analysis of breeding lines we have
examined the correlation between SL profile and resistance. The composition of latex was
also studied in the related species Cichorium intybus (chicory), in which SLs have been
studied as insect antifeedants and potential causes of bitterness (1,11,24).
The HPLC profiles for constitutive SLs, detected by UV at 200 nm, were compared in
latex from L. sativa, L. serriola, L. virosa and the watery exudate recovered from Cichorium
intybus (Figures 2 and 4). The major components in all species were lactucopicrin 15-oxalate
and lactucin 15-oxalate which were notably identified for the first time in chicory. Levels of
lactucin-15-oxalate were particularly high in L. virosa and chicory. In general, when compared
to L. sativa, the sulfated SLs were absent or present at very low levels in latex of chicory, L.
serriola and L. virosa. An exception to this was a high level of 15-p-hydroxyphenylacetyl-
lactucin-8-sulfate in L. serriola. The novel tannins (peaks 8 and 9) were absent from chicory
and L. virosa. There were also quantitative differences amongst the minor components of the
SL profile in other Lactuca species. Comparing L. sativa cultivars, SL sulfates were present
at much lower concentrations in cv. Benita than in the routinely used cv. Diana. In the other
cultivars that were analysed, Capitan, Cobham Green, and Salinas, profiles were overall
similar to Diana (data not shown).
The possibility that the SL profile for L. virosa might be inherited in crosses
with L. sativa cv. Salinas was examined in breeding lines that retained the race non-
specific resistance to B. lactucae found in the wild relative of lettuce. The profiles
recorded for progeny were, however, very similar to those observed in the lettuce
cultivars. Concentrations of the SLs which clearly differentiated the parental
phenotypes, i.e. lactucin oxalate and 15 deoxylactucin-8-sulfate were respectively (in
mg ml-1 of latex ±SEM), in cv. Salinas, 4.75±1.6 and 4.26±1.9 and in L. virosa
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40.7±0.9 and 1.13±0.05. In the F8 breeding line originating from the cross the levels
of these compounds were 10.3±5.6 and 6.6±0.7 mg ml-1, not significantly different
from L. sativa. Thus, although the profile appeared to be an inheritable trait it was
not correlated with resistance. An additional feature was that the progeny retained the
tannins (peaks 8 and 9) which were absent from L. virosa.
Elicitation of lettucenin A and other SLs, and antifungal activities
The SL phytoalexin lettucenin A was first identified in L. sativa and is induced by fungal and
bacterial challenge as well as by treatment with CuSO4 (12, 25). Accumulation of the
phytoalexin was examined in species of Lactuca and in chicory following induction with
copper salts. Detection of the compound utilized its strong fluorescence under UV.
Maximum yields, recorded after incubation for 3 days, were L. sativa, 1.0 • g g-1; L. serriola,
4.0 • g g-1 and L.virosa, 1.2 • g g-1 fresh weight. The higher apparent yield from L. serriola
may have been due to additional damage done to tissue during preparation of leaf discs, owing
to the characteristic morphology of the leaf lamina. Lettucenin A was not identified in
chicory. Changes in concentrations of lettucenin A and other SLs were also examined in
lettuce cotyledons after their treatment with CuSO4. Although concentrations of lettucenin A
increased rapidly from undetectable levels, no reproducible changes were found in the levels
of the lactucin derivatives present before elicitation as illustrated for lactucopicrin in Figure 5.
The antifungal activities of the phytoalexin, lettucenin A and the constitutive SLs from
lettuce were compared. In sensitive TLC plate spot assay tests against Cladosporium
herbarum (26) clear zones of inhibition were caused by 100 ng of lettucenin A, but only weak
activity was detected in the other SLs with samples of 200 • g. The difference in activity
between lettucenin A and the constitutive SLs was, therefore, estimated to be at least 2000
fold.
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DISCUSSION
In studies of the taste of varieties of chicory and lettuce, correlative analyses have shown that
levels of lactucin and lactucopicrin conjugates provide the best indicators of bitterness (11).
Our work has shown that the principal conjugates are not glycosides as frequently reported in
earlier studies, but oxalates. In earlier research these conjugates may not have been recovered
because of their polar nature and also because of their degradation during isolation. The SL
oxalates are unstable and may on decomposition lead to the accumulation of oxalic acid in the
latex released by leaf damage: the oxalate itself may contribute significantly to the sensory
and antifeedant properties of Lactuca latex. An intriguing feature of the SLs is their high
concentration within latex. In lettuce and other Lactuca spp the system of reticulate
anastomosing lactifers provides a continuous interconnecting network of latex-filled cells
throughout the plant (8). The coordinated cellular activities by which SLs and their
conjugates are synthesized and/or are accumulated within the lactifers are important targets
for further research.
The presence of oxalate esters of secondary metabolites appears to be extremely rare.
However, N-oxalates of non-protein amino acids are not uncommon. Examples are N-oxalates
of diamino-propionic and butyric acids which are well known neurotoxins from the legumes
Lathyrus sativus and Lathyrus latifolius (27,28), and oxalylalbizzine which is present in seeds
of many Acacia species (29). In mammalian systems, oxalyl thioesters (RSCOCOOH) have
been characterised and are implicated in intracellular signalling (30). By contrast, oxalic acid
itself is very common, often occurring as crystals of calcium oxalate in cell vacuoles (31). If
both free oxalate and SLs accumulate in the vacuoles of developing lactifers, activity of an
oxalate transferase, presumably utilising oxalyl-CoA, would be expected to allow conjugates
to be generated.
Sulfate conjugates of SLs such as the lactucopicrin-15-sulfate, and 15-deoxy-8-
lactucin sulfate identified in lettuce, to our knowledge, have not been previously reported.
Sulfated compounds are, however, much more common in plants than oxalates. Notable
groups of sulfates are the glucosinolates from crucifers and sulfated flavonoids (32,33). It has
been suggested that, as in animals, conjugation with sulfate may represent a type of
detoxification mechanism in which the sulfate takes the role of the glycoside in solubilizing,
and thereby inactivating, a potentially harmful product (33). The constitutive SLs including
the sulfated forms from Lactuca species lacked significant antifungal activity in comparison
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with the highly toxic phytoalexin lettucenin A. As they lack antifungal activity, the
constitutive SLs in lettuce should not be considered components of defence against fungal
pathogens. They may, however, be important in latex as insect antifeedants, as proposed by
Rees and Harborne (1).
The study also revealed interesting positional variation in the structure of the
conjugates. For example, oxalates were always found on the C-15 position of the lactucin
backbone, whereas sulfates were located at C-8. However, 4-hydroxyphenylacetates were
found at both positions. The identification of the unusual tannins also containing 4-
hydroxyphenylacetyl groups suggests that the acyl transferase responsible for the formation of
these esters may not be highly specific. Although the core component of the tannin was
identified as 2,4,6-tri-(4-hydroxyphenylacetyl)-glucopyranose, there were indications of a
further two aromatic residues. If they are part of the major tannin component, then these
residues are not attached to the glucose but are presumably depsidically linked to the C-2,4 or
6 aromatic substituents. Generally, the majority of hydrolysable tannins in plants are
gallotannins, which have structures based on galloyl (tri-hydroxybenzoyl) groups linked to a
monosaccharide nucleus (21,22). Tannins based on 4-hydroxyphenyl acetyl esters of glucose
have not been observed before, although a recent report of the occurrence of bis (4-
hydroyphenylacetyl) esters of inositol in Taraxacum linearisquameum suggests that lettuce
may not be the only example (34). The significance of the novel structures in lettuce is not
clear, but the astringent properties of tannins are considered to give them a role as predator
repellents.
In latex of Lactuca species, the formation of oxalates, sulfates and 4-
hydroxyphenylacetates generates a diverse profile of sesquiterpene lactone conjugates. One
sesquiterpene glycoside (jacquinellin glycoside, peak 6) was tentatively identified, but the
majority of polar sesquiterpene conjugates in lettuce do not contain carbohydrate as has been
assumed in other studies. Given the different stabilities of the conjugates discovered, the
molecular nature of bitterness of lettuce, and also chicory, needs to re-assessed in the light of
our results. Differences in the bitterness of leaves of Lactuca spp. and chicory have often
been reported. In part, the variability of samples may be associated with leaf age and the stage
of plant growth at the time of sampling (11). Changes in the concentrations of SLs in L.
virosa was reported by Gromek (35) who found that lactucin and lactucopicrin were present in
flowering plants in the second year of growth but not in vegetative, one year old plants. Rees
and Harborne (1) reported little difference between the ratios of lactucin, lactucopicrin and 8-
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deoxylactucin in either roots or leaves of chicory throughout the growing season but they did
not examine flowering plants. The changes in levels of SL-oxalates and sulfates that were
found in lettuce during bolting might be expected to alter the biological properties of latex.
Older plants presumably contain more latex than rosette plants. Although measurement of the
total amount of latex per plant was not feasible, it is obvious that increases in SL
concentration observed in bolting are amplified by an increase in latex per se. The overall
increase in SL content of older plants may be responsible for increased bitterness, as found in
taste tests (Sessa, Bennett and Mansfield, unpublished observation).
Although resistance to downy mildew disease was not correlated with the SL profile,
the inheritability of the profile was indicated from our analysis of progeny from the crosses
between L. sativa and L. virosa. A similar conclusion was reached for resistance to viruses by
Tamaki et al. (17) who studied virus resistance in progeny from a L. saligna and L. sativa
crosses. The cultivar of L. sativa used in their work, cv. Montello, had low levels of lactucin
and lactucopicrin and was devoid of 8-deoxylactucin. In common with L. saligna, progeny
that are resistant to cauliflower mosaic virus, lettuce mosaic virus and broad bean wilt virus
contained low levels of SLs, similar to those found in cv. Montello. The differences apparent
in SL profiles between cultivars of L. sativa and species of Lactuca from which it is possible
to obtain interspecific hybrids (23), indicate that it may be possible to identify genes
controlling SL composition and create isogenic lines differing only in the presence or absense
of certain SLs. Such an approach has allowed a critical, genetically-based, assessment of the
role of glucosinolates in the interactions between Brassica spp., and their pests and pathogens
(36,37)
There has been considerable progress in understanding the biosynthesis of isoprenoids
such as the sesquiterpenes, and in cloning genes encoding key enzymes, most notably terpene
cyclases (38-40). In chicory, the presence of a terpene cyclase producing germacrene A has
been demonstrated (41). It has been postulated that transformation of germacrene A by
oxidation and cyclisation could lead to the guaianolide sesquiterpene lactones (41). Cloning of
sesquiterpene cyclases from lettuce would provide more insight into the hydrocarbon
precursor of the guaianolides, and we are currently working towards this goal. Furthermore,
given the ability to transform lettuce via Agrobacterium-mediated T-DNA delivery (42), it
should soon prove possible to manipulate its terpenoid constituents. An alternative usage of
transformed tissues is to search for new forms of SLs, for example hairy root cultures of L.
virosa produced after transformation with Agrobacterium rhizogenes, have recently been used
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to investigate SL composition in considerable detail (43). The characterisation of SLs
reported here provides the accurate information required to select targets for, and to assess the
results of, future genetic engineering of terpenoid metabolism in lettuce.
Acknowledgements – We thank the BBSRC for a studentship to Reuben Sessa and Jane Ward
(IACR-LARS) for recording NMR spectra.
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Figure Legends
Figure 1. Structures of compounds characterized in lettuce and other members of the
Lactuceae.
Figure 2. Separation of sesquiterpenoid lactones from latex of L. sativa cv. Diana. The
HPLC profile includes peaks identified as: 1, latucin-15-oxalate; 2, 15-deoxylactucin-8-
sulfate; 3, lactucin; 4, 8-deoxylactucin 15-oxalate; 5, 15-p-hydroxyphenyl-acetyllactucin-8-
sulfate; 6, 11,13 dihydro-8-deoxylactucin -15-glycoside (jacquinellin glycoside); 7,
lactucopicrin oxalate; 8 and 9, 2,3,4-tri-O-(4-hydroxyphenylacetyl)glucopyranose and
homologues; 10, lactucopicrin.
Figure 3. Changes in the composition of latex analysed from L. sativa cv. Diana at different
stages of plant development. Columns, from left to right, present data from plants at stages;
vegetative rosette, bolting, in flower and seeded. Key to HPLC peak numbers is given in
Figure 2
Figure 4. Comparison of the HPLC profiles of sesquiterpenoid lactones from latex of Lactuca
sativa cv. Benita, L. virosa, L. serriola and Cichorium intybus (chicory). Key to peak
numbers as in Figure 2.
Figure 5. Elicitation of accumulation of the phytoalexin lettucenin A, but not the constitutive
sesquiterpenoid lactone lactucopicrin, after treatment of leaf discs of L. sativa cv. Diana with
CuSO4.
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multiplicity/couplinga
Lactucopicrin lactucopicrin 15-oxalate
Lactucin lactucin-15-oxalate
15-deoxylactucin-8-sulfate
15-p-hydroxyphenyl-acetyllactucin-8-sulfate
R1 OH OCOCOOH OH OCOCOOH H OCOCH2PhpOHb
R2 OCOCH2PhpOHb OCOCH2PhpOHb OH OH OSO3H OSO3H
H-3 br,s 6.31 6.31 6.28 6.21 6.19 6.13H-5α br,d, 10Hz 4.03 4.02 3.82 3.94 3.79 3.91H-6β t, 10Hz 3.90 4.02 3.74 3.80 3.82 3.85H-7α dt, 10, 3Hz 3.52 3.55 3.08 3.10 3.26 3.25H-8β dt, 10, 3Hz 4.85 4.88 3.73 3.74 4.21 4.22H-9α dd, 13, 10Hz 2.84 2.88 2.76 2.78 2.65 2.67H-9β dd, 13, 1.5Hz 2.27 2.30 2.28 2.35 2.90 2.94H-13a d, 3Hz 5.37 5.39 6.13 6.15 6.38 6.37H-13b d, 3Hz 5.89 5.91 6.01 6.02 6.01 6.01H3-14 s 2.32 2.33 2.34 2.35 2.30 2.30H2-15 2d, 18Hz 4.24, 4.66 4.98, 5.33 4.26, 4.67 4.82, 5.22 2.21(s) 4.88, 5.24
CH2(bz) 2d, 15Hz 3.62, 3.68 3.66, 3.67 - - - 3.65 (s)ArH 2d, 8Hz 6.72, 7.04 6.72, 7.10 - - - 6.70, 7.09
All spectra were recorded at 400MHz in d6-DMSO. Signals are reported on the δ scale, downfield of internaltetramethylsilane; a multiplicities and coupling constants refer to compounds with R1 and R2 substituents; for 15-deoxylactucin-8-sulfate (R1=H) H3-15 is a singlet as shown; br = broad, s = singlet, d = doublet, t = triplet, dt =doubletriplet, dd = doubledoublet, 2d = two doublets. b PhpOH = para-hydroxyphenyl.
Table 1 1H-NMR Spectra of Guaianolide Sesquiterpene Lactones Isolated from Latex of Lactuca sativa
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lactucopicrina lactucopicrin 15-oxalate
lactucinc lactucin-15-oxalate
15-deoxylactucin-8-sulfate
15-p-hydroxyphenyl-acetyllactucin-8-sulfate
R1 OH OCOCOOH OH OCOCOOH H OCOCH2PhpOHb
R2 OCOCH2PhpOHb OCOCH2PhpOHb OH OH OSO3H OSO3H
C-1 133.0 (s) 132.9d 133.2d 132.4d 134.9d 132.0d
C-2 194.1 (s) 193.5 194.8 193.8 194.7 194.5C-3 132.0 (d) 132.2d 133.2d 131.6d 133.3d 132.5d
C-4 174.9 (s) 174.8 175.0 174.9 168.8 168.5C-5 47.5 (d) 47.7 49.6e 48.4e 50.2 47.7C-6 80.3 (d) 79.9 81.6 80.5 81.3 80.4C-7 52.9 (s) 52.7 58.2 58.7 54.7 54.4C-8 69.2 (d) 69.1 67.7 66.3 71.4 71.2C-9 43.3 (t) 43.4 49.1e 48.1e 44.2 44.4
C-10 144.6 (s) 145.9 146.4 147.7 145.6 147.4C-11 136.5 (s) 136.2 138.9 137.9 136.5 136.4C-12 168.1 (s) 167.9 169.1 168.6 170.2 168.5C-13 120.9 (t) 121.1 121.9 121.6 122.0 122.2C-14 20.6 (q) 20.8 21.4 21.2 20.6 20.8C-15 61.2 (t) 63.3 62.5 61.7 19.4 (q) 63.0
oxalate - 162.9, 166.3 - 162.0, 165.9 - -
-OCO- 170.7 (s) 170.7 171.0CH2(bz) 39.6 (t) obsc* obsc*Ph-C1 123.8 (s) 123.8 123.1
Ph-C2, C6 130.4 (d) 130.4 130.3Ph-C3, C5 115.2 (d) 115.2 115.1
Ph-C4 156.4 (s) 156.4 156.3
Solvent d6-DMSO d6-DMSO d5-pyridine d6-DMSO d6-DMSO d6-DMSO
All spectra were recorded at 100MHz . Signals are reported on the δ scale, downfield of internal tetramethylsilane; a
assignments made on the basis of off resonance decoupled spectra; bPhpOH = para-hydroxyphenyl. c literature spectrum(Nishimura et al., 1986). d,e assignments may be interchanged in each column; *obsc = signal obscured by solvent signal.
Table 2. 13C-NMR Spectra of Guaianolide Sesquiterpene Lactones Isolated from Latex of Lactuca sativa cv.Diana
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BealeReuben A. Sessa, Mark H. Bennett, Mervyn J. Lewis, John W. Mansfield and Michael H.
derivativeslatexcomponents are novel oxalate and sulfate conjugates of lactucin and its Metabolite profiling of sesquiterpene lactones from Lactuca species: Major
published online June 16, 2000J. Biol. Chem.
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