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Metabolite Profiling of Sesquiterpene Lactones from Lactuca Species

MAJOR LATEX COMPONENTS ARE NOVEL OXALATE AND SULFATE CONJUGATES OF LACTUCIN AND ITS DERIVATIVES*
Open AccessPublished:September 01, 2000DOI:https://doi.org/10.1016/S0021-9258(19)61456-0
      Wounding leaves or stems of Lactucaspecies 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.
      SL
      sesquiterpene lactone
      ES
      electrospray
      MS
      mass spectrometry
      HPLC
      high pressure liquid chromatography
      RP
      reverse phase
      Sesquiterpene lactones (SLs)1 found in plants are remarkably diverse in terms of their structure, properties, and proposed functions (
      • Rees S.B.
      • Harborne J.B.
      ,
      • Picman A.K.
      ,
      • Fischer N.H.
      ). Among 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 (
      • Picman A.K.
      ,
      • Burnett W.C.
      • Jones S.B.
      • Mabry R.J.
      ,
      • Grayer R.J.
      • Harborne J.J.
      ). The principal constitutive SLs found in species of Lactuca have been reported to be lactucin, lactucopicrin, 8-deoxylactucin (Fig.1), and derivatives such as 11,13 dihydro-analogues (
      • Pyrek J.S.
      ,
      • van Beek T.A.
      • Maas P.
      • King B.M.
      • Leclercq E.
      • Voragen A.G.J.
      • de Groot A.
      ). The SLs accumulate in lactifers, which are closely associated with the vascular tissues of the Compositae (
      • Esau K.
      ). The presence of the SLs in latex released from damaged lactifers is thought to contribute to its analgesic, antitussive, and sedative properties (
      • Mahmoud Z.F.
      • Kassem F.F.
      • Abdel-Salam N.A.
      • Zdero C.
      ,
      • Gromek D.
      • Kisiel W.
      • Klodzinska A.
      • Chojnacka-Wojcik E.
      ). The SLs lactucin, 8-deoxylactucin, and lactucopicrin are also intensely bitter, and their presence within salad lettuce and chicory has considerable economic impact (
      • Price K.R.
      • Dupont M.S.
      • Shepherd R.
      • Chan H.W.-S.
      • Fenwick G.R.
      ). The guaianolide SL phytoalexin from lettuce (Lactuca sativa) lettucenin A (Fig.1) is highly antimicrobial, being one of the most toxic phytoalexins described (
      • Bennett M.H.
      • Gallagher M.D.S.
      • Bestwick C.S.
      • Rossiter J.T.
      • Mansfield J.W.
      ).
      Figure thumbnail gr1
      Figure 1Structures of compounds characterized in lettuce and other members of the Lactuceae.
      In this work we aimed to develop simple methods to profile SLs in lettuce varieties 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 Lactuca tartarica (
      • Kisiel W.
      • Barszcz B.
      • Szneler E.
      ). 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 (
      • Adegawa S.
      • Miyase T.
      • Ueno A.
      • Noro T.
      • Kuroyangi M.
      • Fukushima S.
      ,
      • Nishimura K.
      • Miyase T.
      • Ueno A.
      • Noro T.
      • Kuroyanagi M.
      • Fukushima S.
      ,
      • Seto M.
      • Miyase T.
      • Umehara K .
      • Ueno A.
      • Hirano Y.
      • Otani N.
      ). Identification of these glycosides in aerial parts of L. sativa has been based on structural characterization of the supposed aglycones following digestion of extracts with cellulase or acid hydrolysis (
      • Price K.R.
      • Dupont M.S.
      • Shepherd R.
      • Chan H.W.-S.
      • Fenwick G.R.
      ,
      • Tamaki H.
      • Robinson R.W.
      • Anderson J.L.
      • Stoewsand G.S.
      ). 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, in other species of Lactuca, and also in chicory, are mainly 15-oxalates. These unstable oxalate esters appear to represent a novel class of secondary 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 some compounds that appear to have been mistakenly accepted to be glycosides for over 50 years (
      • Price K.R.
      • Dupont M.S.
      • Shepherd R.
      • Chan H.W.-S.
      • Fenwick G.R.
      ,
      • Schenck G.
      • Graf H.
      • Schreber W.
      ).

      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 (
      • Price K.R.
      • Dupont M.S.
      • Shepherd R.
      • Chan H.W.-S.
      • Fenwick G.R.
      ). 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 (
      • Esau K.
      ). 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 (
      • Kuo Y.H.
      • Lambein F.
      ,
      • Bell E.A.
      • Perera C.K.P.W.
      • Nunn P.B.
      • Simmonds M.S.J.
      • Blaney W.M.
      ), and oxalylalbizzine, which is present in seeds of many Acacia species (
      • Evans C.S.
      • Clardy J.
      • Hughes P.F.
      • Bell E.A.
      ). In mammalian systems, oxalyl thioesters (RSCOCOOH) have been characterized and are implicated in intracellular signaling (
      • Skorszynski S.S.
      • Hamilton G.A.
      ). By contrast, oxalic acid itself is very common, often occurring as crystals of calcium oxalate in cell vacuoles (
      • Hall J.L.
      • Flowers T.J.
      • Roberts R.M.
      ). If both free oxalate and SLs accumulate in the vacuoles of developing lactifers, activity of an oxalate transferase, presumably utilizing 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 (
      • Bones A.M.
      • Rossiter J.T.
      ,
      • Harborne J.B.
      ). 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 (
      • Harborne J.B.
      ). The constitutive SLs, including the sulfated forms from Lactuca species, lacked significant antifungal activity in comparison with the highly toxic phytoalexin lettucenin A. Because they lack antifungal activity, the constitutive SLs in lettuce should not be considered components of defense against fungal pathogens. They may, however, be important in latex as insect antifeedants, as proposed by Rees and Harborne (
      • Rees S.B.
      • Harborne J.B.
      ).
      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 two further 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, C-4, or C-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 (
      • Haslam E.
      ,
      • Gross G.G.
      ). Tannins based on 4-hydroxyphenyl acetyl esters of glucose have not been observed before, although a recent report of the occurrence ofbis (4-hydroyphenylacetyl) esters of inositol inTaraxacum linearizquameum suggests that lettuce may not be the only example (
      • Zidorn C.
      • Ellmerer-Muller E.P.
      • Stuppner H.
      ). 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 reassessed in the light of our results. Differences in the bitterness of leaves ofLactuca 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 (
      • Price K.R.
      • Dupont M.S.
      • Shepherd R.
      • Chan H.W.-S.
      • Fenwick G.R.
      ). Changes in the concentrations of SLs in L. virosa was reported by Gromek (
      • Gromek D.
      ), who found that lactucin and lactucopicrin were present in flowering plants in the second year of growth but not in vegetative, 1-year-old plants. Rees and Harborne (
      • Rees S.B.
      • Harborne J.B.
      ) reported little difference between the ratios of lactucin, lactucopicrin, and 8-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.
      R. A. Sessa, M. H. Bennett, and J. W. 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 andL. virosa. A similar conclusion was reached for resistance to viruses by Tamaki et al. (
      • Tamaki H.
      • Robinson R.W.
      • Anderson J.L.
      • Stoewsand G.S.
      ), 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 ofLactuca, from which it is possible to obtain interspecific hybrids (
      • Pink D.A.C.
      • Keane E.M.
      ), 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 (
      • Giamoustaris A.
      • Mithen R.F.
      ,
      • Giamoustaris A.
      • Mithen R.
      ).
      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 (
      • Chappell J.
      ,
      • Górski P.M.
      • Vickstrom T.E.
      • Pierce M.L.
      • Essenberg M.
      ,
      • Bohlmann J.
      • Meyer-Gauen G.
      • Croteau R.
      ). In chicory, the presence of a terpene cyclase producing germacrene A has been demonstrated (
      • de Kraker J-W.
      • Franssen M.C.R.
      • de Groot A.
      • König W.A.
      • Bouwmeester H.J.
      ). It has been postulated that transformation of germacrene A by oxidation and cyclization could lead to the guaianolide sesquiterpene lactones (
      • de Kraker J-W.
      • Franssen M.C.R.
      • de Groot A.
      • König W.A.
      • Bouwmeester H.J.
      ). Cloning of sesquiterpene cyclases from lettuce would provide more insight into the hydrocarbon precursor of the guaianolides, and we are currently working toward this goal. Furthermore, given the ability to transform lettuce viaAgrobacterium-mediated T-DNA delivery (
      • Curtis I.S.
      • Power J.B.
      • Blackhall N.W.
      • de Laat A.M.M.
      • Davey M.R.
      ), 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 to investigate SL composition in considerable detail (
      • Kisiel W.
      • Stojakowska A.
      • Malarz J.
      • Kohlmünzer S.
      ). The characterization 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.

      Acknowledgments

      We thank Jane Ward (Institute of Arable Crops Research-Long Ashton Research Station) for recording NMR spectra.

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