BACKGROUND
chamomile  is one of the oldest and agronomically most important medicinal plant species in europe.
it originates from southeastern europe and western asia, but is nowadays cultivated throughout the world.
the essential oil of chamomile flowers has significant antiphlogistic  <cit> , spasmolytic  <cit>  and antimicrobial  <cit>  activity and is therefore used for several pharmaceutical, nutritional and cosmetic applications.
the pharmaceutically active components of the flower oil are chamazulene, a degradation product spontaneously formed during steam distillation from the sesquiterpene lactone matricine, several bisabolol-type sesquiterpenes -α-bisabolol, bisabolol oxides), flavonoids and two en-in-dicycloethers  <cit>  with chamazulene and the bisabolols being the main active constituents  <cit> .
the qualitative and quantitative terpene composition of the flower oil varies among different chamomile cultivars  <cit>  and is dependent on the developmental stage and the cultivation conditions of the plant  <cit> .
besides flowers, chamomile roots and shoots are also rich in essential oil.
however, in contrast to the flower oil which is mainly produced in glandular trichomes, these oils accumulate in schizogenous oil passages and oil cells and are dominated by sesquiterpene hydrocarbons and alcohols like -β-farnesene and spathulenol, respectively  <cit> .
terpenes are produced by the action of terpene synthases , which convert the ubiquitous prenyl diphosphates, geranyl diphosphate , farnesyl diphosphate  and geranylgeranyl diphosphate  into the respective mono-, sesqui- and diterpene skeletons.
common to all terpene synthases is the formation of highly reactive carbocationic intermediates which can undergo a great variety of rearrangements resulting in a huge number of different terpene structures .
many terpene synthases are multiproduct enzymes producing more than one compound from their substrate.
for example, the recently reported enzyme mttps <dig> from medicago truncatula forms a complex mixture of  <dig> sesquiterpenes  <cit> .
thus, the complex terpene blends of plants are often produced by only a limited number of multiproduct tps enzymes  <cit> .
despite the pharmaceutical and economic importance of chamomile essential oil, little is known about the biosynthesis of its major constituents in chamomile.
thus, we started to investigate enzymes responsible for terpene biosynthesis in this plant species.
here, we report the identification and characterization of five terpene synthases involved in essential oil production.
qrt-pcr analysis revealed organ-specific expression patterns of tps genes which are consistent with the abundance of enzyme products in the respective plant organs.
a -germacrene a synthase  is most likely a key enzyme in the biosynthesis of the pharmaceutical active sequiterpene lactone matricine.
RESULTS
the terpene composition of chamomile essential oils isolated from different plant organs
to study terpene biosynthesis in chamomile, we used the german cultivar ‘bodegold’ which was reported to be rich in the total amount of flower essential oil  <cit> .
since the terpene composition in the different plant organs of this cultivar has not been comprehensively described, a detailed terpene analysis including ray florets, disk florets, leaves, stems and roots was performed .
the ray florets and disk florets showed an identical blend of terpenes .
the total amount of terpenes in the disk florets, however, was approximately four times higher than in the ray florets .
both flower types produced the sesquiterpenes bisabolol oxide a , bisabolol oxide b , α-bisabolol , -β-farnesene  and -germacrene d  as major components.
additionally,  <dig> mono- and sesquiterpenes could be identified as minor compounds.
in contrast to flowers, the leaf terpene blend was dominated by the sesquiterpene hydrocarbons -α-farnesene , -germacrene d , bicyclogermacrene  and β-selinene .
monoterpenes were only found in smaller amounts.
α-bisabolol and the bisabolol oxides could not be detected in the leaves.
the terpene composition of the stems was nearly identical to that of the leaves but the quantity of some of the compounds differed significantly.
the major compound in the stems was identified as the sesquiterpene hydrocarbon -β-farnesene .
in the roots, -β-farnesene also dominated the terpene blend .
besides -β-farnesene, the unusual triquinane-type sesquiterpene, α-isocomene , and the monoterpenoid ester geranyl valerate  were also detected in roots.
in contrast to the above-ground organs of the plant, the roots produced no monoterpenes.
data are given as means ± se .
compounds are listed in order of gc elution time.
*indicates compound identified using authentic standards.
all other compounds were tentatively identified based on their mass spectra in comparison to those in the literature and databases as described in the methods.
results of statistical analysis are given as f-values  of a one-way repeated measures anova tests if variances were equal and errors normally distributed, or as χ2-values  if the nonparametric test was used.
if the anova revealed significant differences, post-hoc tests were conducted and different letters next to the data indicate significant differences  among plant organs.
isolation of terpene synthase genes from chamomile
to identify terpene synthase sequences in chamomile we constructed degenerate primers based on known tps genes from other asteraceae species .
using these primers we could amplify dna fragments of about 200 bp in length.
sequencing of several of these fragments revealed five different partial tps sequences.
the complete open reading frames  of these tps genes were obtained by 5′-race and 3′race.
the orfs with 1638 bp, 1641 bp, 1692 bp, 1791 bp, and 1650 bp were designated as mrtps1mrtps2mrtps3mrtps <dig>  and mrtps <dig>  respectively.
the encoded proteins mrtps <dig>  mrtps <dig>  mrtps <dig>  mrtps <dig> and mrtps <dig> exhibited highly conserved sequence elements of terpene synthases like the ddxxd motif and the nse/dte motif, which were implicated in the binding of the substrate diphosphate group .
another conserved sequence found in chamomile terpene synthases was the rxr motif which plays a role in the complexation of the diphosphate group after ionization of the substrate  <cit> .
one of the proteins, mrtps <dig>  contained an extended n-terminus which was recognized by the program ‘chlorop’  as a signal peptide with a length of  <dig> amino acids .
a blastp analysis with chamomile tps in the ncbi protein database  revealed that mrtps <dig>  mrtps <dig>  mrtps <dig>  mrtps <dig> and mrtps <dig> showed highest amino acid sequence similarity to the -β-caryophyllene synthase qsh <dig>  from artemisia annua  <cit> , epi-cedrol synthase  from a. annua  <cit> , germacrene a synthase cigaslo  from cichorium intybus  <cit> , -β-ocimene synthase  from vitis vinifera  <cit>  and -germacrene d synthase  from solidago canadensis  <cit> , respectively.
a dendrogram analysis was conducted to determine the evolutionary relatedness of chamomile terpene synthases to those of other asteraceae .
mrtps <dig>  mrtps <dig>  mrtps <dig> and mrtps <dig> were found to belong to the tps a subfamily which encompasses sesquiterpene synthases from angiosperms, whereas mrtps <dig> fell into the tps b subfamily covering angiosperm monoterpene synthases  <cit> .
the asteraceae tps a sequences formed two distinct clades .
mrtps <dig> grouped together with different germacrene a synthases and one δ-cadinene synthase in one clade, while mrtps <dig>  mrtps <dig> and mrtps <dig> were found in the second clade comprising sesquiterpene synthases with diverse catalytic activities.
functional characterization of chamomile terpene synthases
for biochemical characterization, the orfs of chamomile terpene synthases were cloned into the expression vector pask-iba <dig> and the proteins were expressed in escherichia coli.
the putative monoterpene synthase mrtps <dig> was expressed as a n-terminal truncated protein lacking the predicted signal peptide .
protein extracts from transformed escherichia coli were tested in assays containing the potential substrates geranyl diphosphate  or farnesyl diphosphate  in the presence of the cosubstrate mg2+.
mrtps <dig>  mrtps <dig>  mrtps <dig> and mrtps <dig> showed highest activity with fpp as substrate and only trace activity with gpp  and were therefore characterized as sesquiterpene synthases.
mrtps <dig> produced -β-caryophyllene as the major product and trace amounts of α-humulene .
in contrast, mrtps <dig> showed a broader sesquiterpene product spectrum with the angular triquinane, α-isocomene, being the main compound.
additionally, the enzyme produced detectable amounts of three other triquinanes, β-isocomene, silphinene and modeph-2-ene.
like mrtps <dig> it also produced -β-caryophyllene and α-humulene, but in low amounts .
the major product of mrtps <dig> detected under standard gc conditions was β-elemene .
since β-elemene can be formed as a thermal rearrangement product from germacrene a in a hot gc injection port  <cit> , mrtps <dig> products were also analyzed using a colder gc injector temperature .
although minor amounts of β-elemene were still present in the gc chromatogram, the major peak could be identified as germacrene a, demonstrating the genuine activity of mrtps <dig> .
the enzyme mrtps <dig> produced mainly germacrene d and trace amounts of a few unidentified sesquiterpenes.
unlike the other tps, mrtps <dig> only accepted gpp as substrate.
it produced acyclic monoterpenes with -β-ocimene being the major product and -β-ocimene as a trace compound .
the activity of mrtps <dig> confirms the sequence evidence indicating that this enzyme is a monoterpene synthase.
a chiral analysis of the enzyme products -β-caryophyllene from mrtps <dig> and mrtps <dig>  germacrene a from mrtps <dig> and germacrene d from mrtps <dig> was performed using chiral gc-ms. both mrtps <dig> and mrtps <dig> produced exclusively --β-caryophyllene .
the thermal rearrangement product of germacrene a was identified as -β-elemene .
since the heat-induced cope rearrangement of germacrene a to β-elemene retains the stereochemical configuration at c <dig>  <cit> , the enzyme product was determined as -germacrene a. mrtps <dig> produced exclusively the -enantiomer of germacrene d. a chiral analysis of -β-caryophyllene, β-elemene and germacrene d in the plant extracts revealed that they contained the same enantiomers as produced by the heterologously-expressed enzymes .
transcript abundance of mrtps genes in different organs of chamomile
to study the tps gene expression in the different plant organs, we measured the accumulation of tps transcripts in the ray florets, disk florets, leaves, stems and roots using qrt-pcr .
mrtps <dig> and mrtps <dig> showed identical expression patterns.
the highest transcript accumulation was in the disk florets, a moderate transcript accumulation in ray florets and trace accumulation in the green parts of the plant.
both genes were not expressed in the roots.
the monoterpene synthase gene mrtps <dig> and the sesquiterpene synthase gene mrtps <dig> revealed a similar expression pattern with highest transcript accumulation in the green plant parts and the disk florets and only trace transcript accumulation in the roots.
in contrast, mrtps <dig> was mainly expressed in roots and only low levels of mrtps <dig> transcripts were also detected in leaves and stems.
mrtps <dig> was not expressed in ray florets and showed only trace expression in disk florets.
discussion
chamomile tpss contribute to terpene biosynthesis in different organs of the plant
our analysis confirmed previous studies that showed an organ-specific production of essential oils in chamomile  <cit> .
such organ-dependent differences in terpene content have also been described for other plant species.
for example, the terpene blend of maize leaves is qualitatively and quantitatively different from that of maize roots and maize husks  <cit> .
the composition of such terpene mixtures is often reflected in the summarized product spectra of a few multi-product terpene synthases  <cit> .
in this study we identified five terpene synthases of chamomile that formed compounds which occur in chamomile oils.
the peak expression of mrtps <dig>  mrtps <dig> and mrtps <dig> in flowers and above-ground tissues corresponded well with the accumulation of their respective enzyme products in these tissues  indicating a direct contribution of mrtps <dig>  mrtps <dig> and mrtps <dig> to essential oil biosynthesis in flowers and leaves.
the multiproduct enzyme mrtps <dig> was mainly expressed in roots and produced some of the sesquiterpenes found in this organ.
while mrtps <dig> was only transcribed in flowers, the major enzyme product -β-caryophyllene was present in all analyzed plant tissues .
since -β-caryophyllene was also formed as a minor product from mrtps <dig> it is likely that both mrtps <dig> and mrtps <dig> are responsible for -β-caryophyllene formation in all plant organs.
however, the concentration of -β-caryophyllene in leaves was higher than expected by the low transcript abundance of mrtps <dig> and mrtps <dig> and could be explained by the presence of another leaf-specific tps capable of producing this sesquiterpene.
multiple -β-caryophyllene synthase genes expressed in different plant organs were also identified in the recently sequenced genome of grapevine  <cit> .
the spatial and temporal production of plant terpenes is often controlled by transcriptional regulation of tps genes.
for example, the maize sesquiterpene synthases tps <dig> and tps <dig> which form the major sesquiterpenes in this plant part are exclusively expressed in the husk covering the maize ears  <cit> .
in shampoo ginger , an α-humulene synthase gene was reported to be specifically expressed in the rhizome where it is probably involved in zerumbone biosynthesis  <cit> .
furthermore, the diurnal emission of the floral monoterpenes myrcene and -β-ocimene from snapdragon flowers is controlled by the tissue-specific and rhythmic expression of two monoterpene synthase genes  <cit> .
posttranscriptional regulation of tps enzymes  <cit>  as well as light-dependent substrate availability  <cit>  are also discussed as regulatory steps in terpene formation.
our data suggest that the qualitative terpene composition of chamomile essential oils is mainly controlled by the organ-specific expression of tps genes.
however, the complete absence of stored monoterpenes in roots could also be explained by the absence of the precursor gpp in that organ.
quantitative differences in terpene content and tps transcript abundance between, for example, ray florets and disk florets could be due to a different density of glandular trichomes, the site of terpene production in these organs.
mrtps <dig> may be the key enzyme in matricine biosynthesis in chamomile flowers
the sesquiterpene lactone matricine is one of the major active compounds of chamomile flowers.
it is very unstable and decomposes during steam distillation to chamazulene, a blue compound causing the characteristic color of chamomile oil.
beside the bisabolols and flavonoids, chamazulene is mainly responsible for the antiphlogistic activity of chamomile extracts and oils  <cit> .
so far, little is known about the molecular basis of sesquiterpene lactone biosynthesis.
the first committed step is the conversion of fpp to the respective sesquiterpene skeletons catalyzed by sesquiterpene synthases.
for example, in the chinese medicinal plant artemisia annua, amorpha- <dig> -diene synthase was described as a key enzyme involved in the biosynthesis of artemisinin, an amorphane-type sesquiterpene endoperoxide with antimalarial activity  <cit> .
germacrene a synthases from lettuce , chicory , ixeris dentata and sunflower  were reported to catalyze the first step in guaianolide, eudesmanolide and germacranolide sesquiterpene lactone formation in these plant species  <cit> .
in this study we identified the -germacrene a synthase mrtps <dig> from chamomile.
the gene was exclusively expressed in disk and ray florets which are known to be the sole accumulation site for the guaianolide sesquiterpene lactone matricine  <cit> .
despite high mrtps <dig> expression levels, only trace amounts of germacrene a were detected in floral tissues as thermally rearranged β-elemene .
most likely -germacrene a is rapidly converted into matricine following the proposed reaction path shown in figure  <dig>  an enzyme that converts germacrene a to germacrene a acid was recently described in lettuce  <cit> .
this protein, a cytochrome p <dig> of the cyp <dig> family, catalyzes a regioselective three-step oxidation of -germacrene a at c <dig>  more recently, a second p <dig> from lettuce, cyp71bl <dig> was reported to catalyze a 6-α-hydroxylation of germacrene a acid.
the resulting 6-α-hydroxy-germacrene a acid spontaneously undergoes a lactonization which yields costunolide, one of the simplest sesquiterpene lactones  <cit> .
the biosynthesis of matricine in chamomile could also follow the route from -germacrene a to costunolide , with further steps including a ring closure forming the guaiane skeleton, two additional hydroxylations, an acetylation and hydrogenation.
although mrtps <dig> is likely involved in matricine biosynthesis, we cannot exclude the presence of a further germacrene a synthase which could also contribute to this key reaction.
the existence of multiple germacrene a synthases in asteraceae was recently described for cichory  <cit>  and sunflower  <cit> .
to elucidate the biosynthesis of matricine in more detail, we started to search for other potential germacrene a synthase candidate genes.
furthermore we are planning to identify p <dig> enzymes in chamomile which might be involved in the formation of costunolide from -germacrene a.
CONCLUSIONS
terpenes are major components of the essential oils of chamomile and contribute to their pharmaceutical activity.
terpene synthases were identified in this study that are involved in essential oil formation in various plant organs of chamomile.
the qualitative terpene composition of the oils seems to be controlled by spatial expression of tps genes.
due to their importance for essential oil production, these genes could be used to generate markers for the breeding of new chamomile cultivars with increased terpene content or with a specific oil composition.

 chamomile is one of the oldest and agronomically most important medicinal plant species in europe. it originates from southeastern europe and western asia, but is nowadays cultivated throughout the world. the essential oil of chamomile flowers has significant antiphlogistic, spasmolytic and antimicrobial activity and is therefore used for several pharmaceutical, nutritional and cosmetic applications. the pharmaceutically active components of the flower oil are chamazulene, a degradation product spontaneously formed during steam distillation from the sesquiterpene lactone matricine, several bisabolol-type sesquiterpenes -α-bisabolol, bisabolol oxides), flavonoids and two en-in-dicycloethers with chamazulene and the bisabolols being the main active constituents. the qualitative and quantitative terpene composition of the flower oil varies among different chamomile cultivars and is dependent on the developmental stage and the cultivation conditions of the plant. besides flowers, chamomile roots and shoots are also rich in essential oil. however, in contrast to the flower oil which is mainly produced in glandular trichomes, these oils accumulate in schizogenous oil passages and oil cells and are dominated by sesquiterpene hydrocarbons and alcohols like -β-farnesene and spathulenol, respectively. terpenes are produced by the action of terpene synthases, which convert the ubiquitous prenyl diphosphates, geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate into the respective mono-, sesqui- and diterpene skeletons. common to all terpene synthases is the formation of highly reactive carbocationic intermediates which can undergo a great variety of rearrangements resulting in a huge number of different terpene structures. many terpene synthases are multiproduct enzymes producing more than one compound from their substrate. for example, the recently reported enzyme mttps from medicago truncatula forms a complex mixture of sesquiterpenes. thus, the complex terpene blends of plants are often produced by only a limited number of multiproduct tps enzymes. despite the pharmaceutical and economic importance of chamomile essential oil, little is known about the biosynthesis of its major constituents in chamomile. thus, we started to investigate enzymes responsible for terpene biosynthesis in this plant species. here, we report the identification and characterization of five terpene synthases involved in essential oil production. qrt-pcr analysis revealed organ-specific expression patterns of tps genes which are consistent with the abundance of enzyme products in the respective plant organs. a -germacrene a synthase is most likely a key enzyme in the biosynthesis of the pharmaceutical active sequiterpene lactone matricine.  the terpene composition of chamomile essential oils isolated from different plant organs to study terpene biosynthesis in chamomile, we used the german cultivar ‘bodegold’ which was reported to be rich in the total amount of flower essential oil. since the terpene composition in the different plant organs of this cultivar has not been comprehensively described, a detailed terpene analysis including ray florets, disk florets, leaves, stems and roots was performed. the ray florets and disk florets showed an identical blend of terpenes. the total amount of terpenes in the disk florets, however, was approximately four times higher than in the ray florets. both flower types produced the sesquiterpenes bisabolol oxide a, bisabolol oxide b, α-bisabolol, -β-farnesene and -germacrene d as major components. additionally, mono- and sesquiterpenes could be identified as minor compounds. in contrast to flowers, the leaf terpene blend was dominated by the sesquiterpene hydrocarbons -α-farnesene, -germacrene d, bicyclogermacrene and β-selinene. monoterpenes were only found in smaller amounts. α-bisabolol and the bisabolol oxides could not be detected in the leaves. the terpene composition of the stems was nearly identical to that of the leaves but the quantity of some of the compounds differed significantly. the major compound in the stems was identified as the sesquiterpene hydrocarbon -β-farnesene. in the roots, -β-farnesene also dominated the terpene blend. besides -β-farnesene, the unusual triquinane-type sesquiterpene, α-isocomene, and the monoterpenoid ester geranyl valerate were also detected in roots. in contrast to the above-ground organs of the plant, the roots produced no monoterpenes. data are given as means ± se. compounds are listed in order of gc elution time. *indicates compound identified using authentic standards. all other compounds were tentatively identified based on their mass spectra in comparison to those in the literature and databases as described in the methods. results of statistical analysis are given as f-values of a one-way repeated measures anova tests if variances were equal and errors normally distributed, or as χ2-values if the nonparametric test was used. if the anova revealed significant differences, post-hoc tests were conducted and different letters next to the data indicate significant differences among plant organs. isolation of terpene synthase genes from chamomile to identify terpene synthase sequences in chamomile we constructed degenerate primers based on known tps genes from other asteraceae species. using these primers we could amplify dna fragments of about 200 bp in length. sequencing of several of these fragments revealed five different partial tps sequences. the complete open reading frames of these tps genes were obtained by 5′-race and 3′race. the orfs with 1638 bp, 1641 bp, 1692 bp, 1791 bp, and 1650 bp were designated as mrtps1mrtps2mrtps3mrtps and mrtps respectively. the encoded proteins mrtps mrtps mrtps mrtps and mrtps exhibited highly conserved sequence elements of terpene synthases like the ddxxd motif and the nse/dte motif, which were implicated in the binding of the substrate diphosphate group. another conserved sequence found in chamomile terpene synthases was the rxr motif which plays a role in the complexation of the diphosphate group after ionization of the substrate. one of the proteins, mrtps contained an extended n-terminus which was recognized by the program ‘chlorop’ as a signal peptide with a length of amino acids. a blastp analysis with chamomile tps in the ncbi protein database revealed that mrtps mrtps mrtps mrtps and mrtps showed highest amino acid sequence similarity to the -β-caryophyllene synthase qsh from artemisia annua, epi-cedrol synthase from a. annua, germacrene a synthase cigaslo from cichorium intybus, -β-ocimene synthase from vitis vinifera and -germacrene d synthase from solidago canadensis, respectively. a dendrogram analysis was conducted to determine the evolutionary relatedness of chamomile terpene synthases to those of other asteraceae. mrtps mrtps mrtps and mrtps were found to belong to the tps a subfamily which encompasses sesquiterpene synthases from angiosperms, whereas mrtps fell into the tps b subfamily covering angiosperm monoterpene synthases. the asteraceae tps a sequences formed two distinct clades. mrtps grouped together with different germacrene a synthases and one δ-cadinene synthase in one clade, while mrtps mrtps and mrtps were found in the second clade comprising sesquiterpene synthases with diverse catalytic activities. functional characterization of chamomile terpene synthases for biochemical characterization, the orfs of chamomile terpene synthases were cloned into the expression vector pask-iba and the proteins were expressed in escherichia coli. the putative monoterpene synthase mrtps was expressed as a n-terminal truncated protein lacking the predicted signal peptide. protein extracts from transformed escherichia coli were tested in assays containing the potential substrates geranyl diphosphate or farnesyl diphosphate in the presence of the cosubstrate mg2+. mrtps mrtps mrtps and mrtps showed highest activity with fpp as substrate and only trace activity with gpp and were therefore characterized as sesquiterpene synthases. mrtps produced -β-caryophyllene as the major product and trace amounts of α-humulene. in contrast, mrtps showed a broader sesquiterpene product spectrum with the angular triquinane, α-isocomene, being the main compound. additionally, the enzyme produced detectable amounts of three other triquinanes, β-isocomene, silphinene and modeph-2-ene. like mrtps it also produced -β-caryophyllene and α-humulene, but in low amounts. the major product of mrtps detected under standard gc conditions was β-elemene. since β-elemene can be formed as a thermal rearrangement product from germacrene a in a hot gc injection port, mrtps products were also analyzed using a colder gc injector temperature. although minor amounts of β-elemene were still present in the gc chromatogram, the major peak could be identified as germacrene a, demonstrating the genuine activity of mrtps. the enzyme mrtps produced mainly germacrene d and trace amounts of a few unidentified sesquiterpenes. unlike the other tps, mrtps only accepted gpp as substrate. it produced acyclic monoterpenes with -β-ocimene being the major product and -β-ocimene as a trace compound. the activity of mrtps confirms the sequence evidence indicating that this enzyme is a monoterpene synthase. a chiral analysis of the enzyme products -β-caryophyllene from mrtps and mrtps germacrene a from mrtps and germacrene d from mrtps was performed using chiral gc-ms. both mrtps and mrtps produced exclusively --β-caryophyllene. the thermal rearrangement product of germacrene a was identified as -β-elemene. since the heat-induced cope rearrangement of germacrene a to β-elemene retains the stereochemical configuration at c, the enzyme product was determined as -germacrene a. mrtps produced exclusively the -enantiomer of germacrene d. a chiral analysis of -β-caryophyllene, β-elemene and germacrene d in the plant extracts revealed that they contained the same enantiomers as produced by the heterologously-expressed enzymes. transcript abundance of mrtps genes in different organs of chamomile to study the tps gene expression in the different plant organs, we measured the accumulation of tps transcripts in the ray florets, disk florets, leaves, stems and roots using qrt-pcr. mrtps and mrtps showed identical expression patterns. the highest transcript accumulation was in the disk florets, a moderate transcript accumulation in ray florets and trace accumulation in the green parts of the plant. both genes were not expressed in the roots. the monoterpene synthase gene mrtps and the sesquiterpene synthase gene mrtps revealed a similar expression pattern with highest transcript accumulation in the green plant parts and the disk florets and only trace transcript accumulation in the roots. in contrast, mrtps was mainly expressed in roots and only low levels of mrtps transcripts were also detected in leaves and stems. mrtps was not expressed in ray florets and showed only trace expression in disk florets. discussion chamomile tpss contribute to terpene biosynthesis in different organs of the plant our analysis confirmed previous studies that showed an organ-specific production of essential oils in chamomile. such organ-dependent differences in terpene content have also been described for other plant species. for example, the terpene blend of maize leaves is qualitatively and quantitatively different from that of maize roots and maize husks. the composition of such terpene mixtures is often reflected in the summarized product spectra of a few multi-product terpene synthases. in this study we identified five terpene synthases of chamomile that formed compounds which occur in chamomile oils. the peak expression of mrtps mrtps and mrtps in flowers and above-ground tissues corresponded well with the accumulation of their respective enzyme products in these tissues indicating a direct contribution of mrtps mrtps and mrtps to essential oil biosynthesis in flowers and leaves. the multiproduct enzyme mrtps was mainly expressed in roots and produced some of the sesquiterpenes found in this organ. while mrtps was only transcribed in flowers, the major enzyme product -β-caryophyllene was present in all analyzed plant tissues. since -β-caryophyllene was also formed as a minor product from mrtps it is likely that both mrtps and mrtps are responsible for -β-caryophyllene formation in all plant organs. however, the concentration of -β-caryophyllene in leaves was higher than expected by the low transcript abundance of mrtps and mrtps and could be explained by the presence of another leaf-specific tps capable of producing this sesquiterpene. multiple -β-caryophyllene synthase genes expressed in different plant organs were also identified in the recently sequenced genome of grapevine. the spatial and temporal production of plant terpenes is often controlled by transcriptional regulation of tps genes. for example, the maize sesquiterpene synthases tps and tps which form the major sesquiterpenes in this plant part are exclusively expressed in the husk covering the maize ears. in shampoo ginger, an α-humulene synthase gene was reported to be specifically expressed in the rhizome where it is probably involved in zerumbone biosynthesis. furthermore, the diurnal emission of the floral monoterpenes myrcene and -β-ocimene from snapdragon flowers is controlled by the tissue-specific and rhythmic expression of two monoterpene synthase genes. posttranscriptional regulation of tps enzymes as well as light-dependent substrate availability are also discussed as regulatory steps in terpene formation. our data suggest that the qualitative terpene composition of chamomile essential oils is mainly controlled by the organ-specific expression of tps genes. however, the complete absence of stored monoterpenes in roots could also be explained by the absence of the precursor gpp in that organ. quantitative differences in terpene content and tps transcript abundance between, for example, ray florets and disk florets could be due to a different density of glandular trichomes, the site of terpene production in these organs. mrtps may be the key enzyme in matricine biosynthesis in chamomile flowers the sesquiterpene lactone matricine is one of the major active compounds of chamomile flowers. it is very unstable and decomposes during steam distillation to chamazulene, a blue compound causing the characteristic color of chamomile oil. beside the bisabolols and flavonoids, chamazulene is mainly responsible for the antiphlogistic activity of chamomile extracts and oils. so far, little is known about the molecular basis of sesquiterpene lactone biosynthesis. the first committed step is the conversion of fpp to the respective sesquiterpene skeletons catalyzed by sesquiterpene synthases. for example, in the chinese medicinal plant artemisia annua, amorpha- -diene synthase was described as a key enzyme involved in the biosynthesis of artemisinin, an amorphane-type sesquiterpene endoperoxide with antimalarial activity. germacrene a synthases from lettuce, chicory, ixeris dentata and sunflower were reported to catalyze the first step in guaianolide, eudesmanolide and germacranolide sesquiterpene lactone formation in these plant species. in this study we identified the -germacrene a synthase mrtps from chamomile. the gene was exclusively expressed in disk and ray florets which are known to be the sole accumulation site for the guaianolide sesquiterpene lactone matricine. despite high mrtps expression levels, only trace amounts of germacrene a were detected in floral tissues as thermally rearranged β-elemene. most likely -germacrene a is rapidly converted into matricine following the proposed reaction path shown in figure an enzyme that converts germacrene a to germacrene a acid was recently described in lettuce. this protein, a cytochrome p of the cyp family, catalyzes a regioselective three-step oxidation of -germacrene a at c more recently, a second p from lettuce, cyp71bl was reported to catalyze a 6-α-hydroxylation of germacrene a acid. the resulting 6-α-hydroxy-germacrene a acid spontaneously undergoes a lactonization which yields costunolide, one of the simplest sesquiterpene lactones. the biosynthesis of matricine in chamomile could also follow the route from -germacrene a to costunolide, with further steps including a ring closure forming the guaiane skeleton, two additional hydroxylations, an acetylation and hydrogenation. although mrtps is likely involved in matricine biosynthesis, we cannot exclude the presence of a further germacrene a synthase which could also contribute to this key reaction. the existence of multiple germacrene a synthases in asteraceae was recently described for cichory and sunflower. to elucidate the biosynthesis of matricine in more detail, we started to search for other potential germacrene a synthase candidate genes. furthermore we are planning to identify p enzymes in chamomile which might be involved in the formation of costunolide from -germacrene a.  terpenes are major components of the essential oils of chamomile and contribute to their pharmaceutical activity. terpene synthases were identified in this study that are involved in essential oil formation in various plant organs of chamomile. the qualitative terpene composition of the oils seems to be controlled by spatial expression of tps genes. due to their importance for essential oil production, these genes could be used to generate markers for the breeding of new chamomile cultivars with increased terpene content or with a specific oil composition. 
the essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. the terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. thus, mrtps but also the other tps genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils. while the products of mrtps mrtps mrtps and mrtps accumulate in the oils without further chemical alterations, -germacrene a produced by mrtps accumulates only in trace amounts, indicating that it is converted into another compound like matricine. the tps transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding tps products isolated from these organs suggesting that the spatial regulation of tps gene expression qualitatively contribute to terpene composition. four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons --β-caryophyllene, -germacrene a, -β-ocimene and -germacrene d. it is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. a fifth tps, the multiproduct enzyme mrtps was mainly expressed in roots and formed several asteraceae-specific tricyclic sesquiterpenes with -α-isocomene being the major product. chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases, the key enzymes in constructing terpene carbon skeletons. here, we present the identification and characterization of five tps enzymes contributing to terpene biosynthesis in chamomile.
 the essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. it is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases, the key enzymes in constructing terpene carbon skeletons.  here, we present the identification and characterization of five tps enzymes contributing to terpene biosynthesis in chamomile. four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons --β-caryophyllene, -germacrene a, -β-ocimene and -germacrene d. a fifth tps, the multiproduct enzyme mrtps was mainly expressed in roots and formed several asteraceae-specific tricyclic sesquiterpenes with -α-isocomene being the major product. the tps transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding tps products isolated from these organs suggesting that the spatial regulation of tps gene expression qualitatively contribute to terpene composition.  the terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. while the products of mrtps mrtps mrtps and mrtps accumulate in the oils without further chemical alterations, -germacrene a produced by mrtps accumulates only in trace amounts, indicating that it is converted into another compound like matricine. thus, mrtps but also the other tps genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils. 