Genetic mapping and characterization of the globe artichoke (+)-germacrene A synthase gene, encoding the first dedicated enzyme for biosynthesis of the bitter sesquiterpene lactone cynaropicrin
Menin, B. ; Comino, C. ; Portis, E. ; Moglia, A. ; Cankar, K. ; Bouwmeester, H.J. ; Lanteri, S. ; Beekwilder, M.J. - \ 2012
Plant Science 190 (2012). - ISSN 0168-9452 - p. 1 - 8.
germacrene-a synthase - cynara-cardunculus l - functional-characterization - artemisia-annua - cdna isolation - leaf extract - scolymus l. - chicory - hydroxycinnamoyltransferase - identification
Globe artichoke (Cynara cardunculus var. scolymus L., Asteraceae) is a perennial crop traditionally consumed as a vegetable in the Mediterranean countries and rich in nutraceutically and pharmaceutically active compounds, including phenolic and terpenoid compounds. Its bitter taste is caused by its high content of sesquiterpene lactones (STLs), such as cynaropicrin. The biosynthetic pathway responsible for STL biosynthesis in globe artichoke is unknown, but likely proceeds through germacrene A, as has been shown for other Asteraceae species. Here, we investigated the accumulation of cynaropicrin in different tissues of globe artichoke, and compared it to accumulation of phenolic compounds. Cynaropicrin concentration was highest in old leaves. A putative germacrene A synthase (GAS) gene was identified in a set of ~19,000 globe artichoke unigenes. When heterologously expressed in Escherichia coli, the putative globe artichoke GAS converted farnesyl diphosphate (FPP) into (+)-germacrene A. Among various tissues assayed, the level of globe artichoke GAS expression was highest in mature (six week old) leaves. A sequence polymorphism within a mapping population parent allowed the corresponding GAS gene to be positioned on a genetic map. This study reports the isolation, expression and mapping of a key gene involved in STL biosynthesis in C. cardunculus. This is a good basis for further investigation of this pathway
Enantiospecific (+)-and (-)germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif
Prosser, I. ; Altug, I.G. ; Philips, A.L. ; König, W.A. ; Bouwmeester, H.J. ; Beale, M. - \ 2004
Archives of Biochemistry and Biophysics 432 (2004)2. - ISSN 0003-9861 - p. 136 - 144.
site-directed mutagenesis - gamma-humulene synthase - delta-selinene synthase - artemisia-annua l - bacterial expression - germacrene-d - trichodiene synthase - cdna isolation - sesquiterpene cyclase - solidago-canadensis
The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
Gain and loss of fruit flavour compounds produced by wild and cultivated strawberry species
Aharoni, A. ; Giri, A.P. ; Verstappen, F.W.A. ; Bertea, C.M. ; Sevenier, R.E. ; Sun, Z. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2004
The Plant Cell 16 (2004). - ISSN 1040-4651 - p. 3110 - 3131.
messenger-rna accumulation - arabidopsis-thaliana - fragaria-vesca - functional expression - alkaloid biosynthesis - gene-expression - cdna isolation - lithospermum-erythrorhizon - alcohol-dehydrogenase - secondary metabolites
The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as alpha-pinene and beta-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of alpha-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of alpha-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.
Metabolic engineering of monoterpende biosysnthesis: two step production of (+)-trans-Isopiperitenol by tobacco
Lücker, J. ; Schwab, W. ; Franssen, M.C.R. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
The Plant Journal 39 (2004)1. - ISSN 0960-7412 - p. 135 - 145.
peppermint mentha-piperita - functional expression - limonene enantiomers - linalool synthase - cdna isolation - s-linalool - plants - mint - (-)-limonene - cloning
Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.
Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants
Aharoni, A. ; Giri, A.P. ; Deuerlein, S. ; Griepink, F.C. ; Kogel, W.J. de; Verstappen, F.W.A. ; Verhoeven, H.A. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2003
The Plant Cell 15 (2003)12. - ISSN 1040-4651 - p. 2866 - 2884.
sesquiterpene cyclase gene - linalool synthase gene - functional expression - monoterpene biosynthesis - isoprenoid biosynthesis - diphosphate synthase - cdna isolation - s-linalool - glandular trichomes - thaliana
Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants