Gain and loss of fruit flavor compounds produced by wild and cultivated strawberry species

Abstract

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.

Figure 1.

Compartmentation of Isoprenoid Biosynthesis in the Plant Cell. The mevalonate pathway is active in the cytosol (and supplies IPP to mitochondria), whereas the methylerythritol 4-phosphate (MEP) pathway is active in plastids. Enzymatic steps similar in both the cytosolic and plastidic pathways are represented in the common area. GGPP, geranylgeranyl diphosphate.

Figure 2.

Terpenoid Production in Wild and Cultivated Strawberry Species. (A) Terpenoids detected by headspace analysis of ripe fruits. GC-MS chromatograms (selected mass-to-charge ratio 93) after headspace Tenax trapping (see Methods) showing the different terpenes emitted by cultivated (top) and wild (bottom) ripe strawberry fruit. A trace of the monoterpene alcohol myrtenol was also detected in wild strawberry (data not shown). (B) Reactions catalyzed by terpene synthases (TPS) for the formation of the monoterpene alcohol linalool and the sesquiterpene alcohol nerolidol. (C) Reactions catalyzed by a terpene synthase (TPS) enzyme for the formation of the monoterpene α-pinene, a cytochrome P450 enzyme catalyzing a subsequent hydroxylation step at C10 forming myrtenol and an alcohol acyltransferase (AAT) forming myrtenyl acetate.

Figure 3.

Expression of the Terpene Synthase and Hydroxylase Genes in Wild and Cultivated Strawberry Species. (A) Detection of FaNES1 expression in ripe strawberry receptacle tissue using cDNA microarrays. Red and green signals represent higher gene expression in the receptacle and achene (seeds) tissues, respectively (yellow signal indicates similar levels in both tissues). (B) FaNES expression in tissues of the cultivated strawberry detected by RNA gel blots. (C) and (D) Mirror images of NES and Pinene Synthase (PINS) expression in ripe fruits of wild and cultivated strawberry species detected by RNA gel blots. The numbers (1) and (2) mark two different wild species or cultivated varieties of strawberry (see Methods). (E) Expression analysis of the F. ananassa Pinene Hydroxylase (FaPINH) gene in different tissues of cultivated (top pair of blots) and the wild strawberry in leaf, root, and red ripe fruit tissues (bottom pair of blots). The entire FaNES1, FvPINS, and FaPINH cDNAs were used to hybridize the RNA gel blots. An rRNA probe was used as a control for equal loading.

Figure 4.

Protein Sequence Alignment of the Strawberry Terpene Synthases. The protein sequences of FaNES1, FaNES2, FvNES1, and FvPINS derived from cultivated (CU) and wild (W) strawberry species were aligned to the sequences of the Citrus limon limonene synthase (Cl_LIMS; GenBank accession number AF514287). The stop codon in the FaNES1 gene sequence, between Met1 (shaded green in all sequences except FaNES1) and Met2 (shaded green only in FaNES1) immediately follows the red shaded Phe residue (F). The RR, L/M-I-D, and DDXXD motifs conserved in monoterpene synthases are shaded yellow. Black, gray, and light gray shading represent 100, 80, and 60% conserved identity between residues, respectively. A 2-bp insertion (CC insertion; see also Figure 11) in the cultivated strawberry species results in a frameshift and an immediate stop codon in the middle of the FaPINS gene-coding region (instead of the orange shaded Leu residue [L] in FvPINS). Substitution (W6R) and removal (I16Δ) of the residues shaded blue in FvNES1 resulted in a change of targeting from plastid localization to localization in mitochondria (mainly) as well as to plastids (see also C12 in Figure 9). The residues present at the ninth position succeeding the twin Arg of the RR(x)₈W motif normally found in the N-terminal part of class III TPS proteins are boxed. The motif is not entirely conserved in FaNES2 and FvNES1 because it contains a Pro (P) instead of a Trp (W) residue.

Figure 5.

Strawberry NES Genes as Members of a New Family of Terpene Synthases (TPS-g). (A) Phylogenetic analysis after ClustalX alignment of terpene synthases representing the seven different TPS families described to date (TPS-a to TPS-g). The alignment used PAM 350 and the neighbor-joining method. The ent-kaurene synthase (syn.) protein was defined as an out-group when rooting the tree. In the scale, bar 0.1 is equal to 10% sequence divergence. So, Salvia officinalis; Pf, Perilla frutescens; Pc, Perilla citridora; Ms, Mentha spicata; Cl, Citrus limon; At, Arabidopsis thaliana; Aa, Artemisia annua; Ga, Gossypium arboreum; Le, Lycopersicon esculentum; St, Solanum tuberosum; Sc, Solidago canadensis; Mp, Mentha piperita; Ag, Abies grandis; Cb, Clarkia breweri; Cm, Cucurbita maxima; Zm, Zea mays; Am, Antirrhinum majus; Ci, Cichorium intybus; Nt, Nicotiana tabacum. GenBank accession numbers are shown in brackets. (B) FaNES1 contains only five introns, compared with the six normally present in class III terpene synthases. Numbers of amino acid residues encoded by each exon are depicted. Intron numbers were derived from Trapp and Croteau (2001). Ex, exon; In, intron; NP, not present.

Figure 6.

Correlation of the Presence and Expression of NES Genes and Volatile Product Formation in Wild and Cultivated Strawberry Species. (A) PCR on genomic DNA of seven cultivated (CU1 to CU7) and seven wild strawberry species (WI1 to WI7) using oligonucleotides flanking the Met1 to Met2 regions, which should amplify all known gene fragments (see Figure 4). The arrow indicates a fragment of ∼150 bp corresponding to the FaNES1 gene. The larger fragments correspond to other NES genes, including those with a proper targeting signal. (B) RT-PCR (left) using RNA derived from red, ripe fruit of cultivated varieties using the same oligonucleotides used in (A). The single band corresponds to the 150-bp fragment detected in (A). Larger fragments corresponding to the other NES transcripts were not detected under these RT-PCR conditions. A larger amount of cDNA and more cycles of amplification were used to clone the fragment corresponding to the FaNES2 gene because of its low abundance. The strawberry alcohol acyltransferase gene (SAAT) (Aharoni et al., 2000) was used as a control (right). (C) Headspace analysis of fruits from two wild and two cultivated strawberry lines (tested molecularly in [A] and [B]) showing the presence or absence of monoterpenes (1, α-pinene; 2, sabinene; 3, β-myrcene; 4, linalool) and the sesquiterpene nerolidol (5).

Figure 7.

Enzymatic Activity Assays Using the FaNES1 and FaNES2 Recombinant Enzymes Produced in E. coli Cells. (A) Seven different constructs (C1 to C7) used for the production of recombinant proteins and tested for enzyme activity. In C3, the stop codon present between the two Met residues (Met1 and Met2) in FaNES1 was removed by site-directed mutagenesis, allowing translation from Met1. All proteins were produced as fusions with a His-tag at their N termini (marked in gray). Results of enzyme activity assays with GPP and FPP are shown on the right-hand side of the construct schemes. lin, linalool; ner, nerolidol; npf, no product formed. (B) Radio-GC analyses of radiolabeled products formed from 20 μM [³H]-geranyl diphosphate (top two panels) or [³H]-farnesyl diphosphate (bottom two panels) by heterologously produced, His-tag purified FaNES1 protein. Panel 1, flame ionization detector (F.I.D.) signal of coinjected, unlabeled linalool. Panel 3, unlabeled standards of (Z)-nerolidol and (E)-nerolidol. Panels 2 and 4, radio traces showing radiolabeled products. For further details, see Methods.

Figure 8.

Chiral Analysis of Linalool and Nerolidol Produced by the Recombinant FaNES1 Protein and Ripe Fruit of the Cultivated Strawberry. (A) and (D) References of R and S linalool enantiomers and of nerolidol enantiomers; 1 and 2, (3R) and (3S) (Z)-nerolidol (elution order not known); 3, (3R)(E)-nerolidol; 4, (3S)(E)-nerolidol. (B) and (E) Enzyme activity assays with the recombinant FaNES1 enzyme and either GPP or FPP as substrate. (C) and (F) Linalool and nerolidol produced by ripe, cultivated strawberry fruit.

Figure 9.

Transient Expression of GFP Fusions in Tobacco Protoplasts Detected by Confocal Laser Scanning Microscopy. pOL-LT-GFP is the original vector used to fuse the different strawberry gene fragments to GFP, which directs GFP expression to the cytosol and nucleoplasm (see Methods). Chloroplasts (chl; ∼5 μm in size), mitochondria (mito; ∼1 μm in size), cytosol (cyto), and nucleoplasm (nuc) are indicated by arrows. If several images are shown for a single construct, they are derived from the same protoplast. ca, chlorophyll autofluorescence detected in the red channel; gfp, green fluorescent protein fluorescence detected in the green channel; ca/gfp, combined red and green channels; mt, MitoTracker mitochondrial stain detected in the orange channel or merged with the green and red channels (ca/gfp/mt). Bottom panel, schematic and localization results for each fusion construct. Met1 and Met2, Met residues at the N termini of the proteins (Figure 4); L/MID, conserved motif in terpene synthases (Figure 4); Sc, stop codon; C, cytosol; P, plastids; M > P, more in mitochondria than in plastids. CLMTS, GFP fusion of the 5′ region of a typical monoterpene synthase from Citrus limon (Lucker et al., 2002).

Figure 10.

Analysis of the Monoterpene Products Formed from GPP in Assays with the Recombinant FvPINS Enzyme. (A) Compounds detected in the volatile profile of the wild strawberry species are marked by a star (cf. Figure 2A). 1, α-pinene; 2, β-pinene; 3, sabinene; 4, β-myrcene; 5, α-phellandrene; 6, β-phellandrene; 7, dihydromyrcenol (tentative); 8, α-terpinolene (tentative); 9, α-terpineol (tentative). (B) Multidimensional GC-MS analysis of the enantiomeric composition of the α-pinene formed from GPP in an assay with the recombinant FvPINS enzyme and the α-pinene present in the headspace of wild strawberry.

Figure 11.

Insertion in the Middle of the FaPINS Gene-Coding Region. A 2-bp insertion (CC, indicated by arrows) results in a frameshift and an immediate stop codon in the middle of the cultivated strawberry FaPINS gene-coding region (see also Figure 4). The insertion was not detected in the FvPINS gene derived from the wild strawberry.

Figure 12.

Protein Sequence Alignment of the Strawberry FaPINH and Cytochrome P450s from Mint (Mentha) and Catharanthus roseus. Mint (Mp, Mentha × piperita; Ms, Mentha spicata) proteins are menthofuran synthase (MFS) (GenBank accession number AF346833; Bertea et al., 2001), (−)-4S-limonene-3-hydroxylase (L3H; GenBank accession number AF124817), and (−)-4S-limonene-6-hydroxylase (L6H; GenBank accession number AF124815) (Lupien et al., 1999); the Catharanthus roseus (Cr) protein is geraniol 10 hydroxylase (G10H) (GenBank accession number AJ251269; Collu et al., 2001). The conserved oxygen binding and heme binding domains (Schuler, 1996) are marked a and b, respectively. Black, gray, and light gray shading represent 100, 80, and 60% conserved identity between residues, respectively.

Figure 13.

Production of Myrtenol by the Recombinant FaPINH Enzyme. Recombinant protein extracted from yeast microsomes and harboring either the empty vector or the vector containing the FaPINH coding region was used for enzymatic assays using (−)-α-pinene as a substrate. Total ion chromatograms from the GC-MS analysis of the products are shown.

Figure 14.

Gain and Loss of Terpenoids in Ripe Strawberry Fruit. Schematic depiction of mechanisms causing the gain and loss of flavor and aroma compounds in strawberry during evolution, which may have been specifically selected for in domesticated species.