Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles

Abstract

The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents.

Keywords: Apocarotenoids; CCD, carotenoid cleavage dioxygenase; Carotenoid cleavage dioxygenase; Carotenoids; Isoprenoids; Plants; Tomato.

Fig. 1

HPLC analysis of incubations with apolycopenals. The four HPLC chromatograms (A) represent product profiles obtained with the different apolycopenals shown in (B, compound I–IV). The structures of the products formed are depicted in (C). SlCCD1B converted apo-8′-lycopenal (I, C₃₀) into 14, 8′-diapocarotene-14, 8′-dial (1, C₁₂, s. Fig. 3), pseudoionone (2, C₁₃), 8′, 10-diapocarotene-8′, 10-dial (3, C₁₇) and 8′, 8-diapocarotene-8′, 8-dial (5, C₂₀). SlCCD1A converted substrate I into products 2, 3, 5, in addition to 6, 8′-diapocarotene-6, 8′-dial (6, C₂₂). SlCCD1B converted apo-10′-lycopenal (II, C27) into pseudoionone (2, C₁₃), 8′, 10-diapocarotene-8′, 10-dial (3, C₁₇) and 10′, 6-diapocarotene-10′, 6-dial (4, C₁₉). SlCCD1A converted substrate II into products 2, 4. SlCCD1B converted apo-12′-lycopenal (III, C₂₅) into 12′, 6-diapocarotene-12′, 6-dial (3^∗, C₁₇) and 12′, 8-diapocarotene-12′, 8-dial (7, C₁₅). SlCCD1A converted substrate III into 12′, 6-diapocarotene-12′, 6-dial (3^∗, C₁₇). SlCCD1B converted apo-15′-lycopenal (IV, C20) into tentatively identified apo-12-lycopenal (8, C₁₅) and 15′, 6-diapocarotene-12′, 6-dial (1^∗, C₁₂). SlCCD1A converted substrate IV into apo-12-lycopenal (8, C₁₅). Numbering of C atoms in apo-8′-lycopenal and deduced cleavage sites for both enzymes are depicted in B.

Fig. 2

GC–MS analysis of SlCCD1B incubation with apo-8′-lycopenal. Volatiles were collected by SPME and analyzed. The enzyme released MHO (A), geranial and neral, corresponding to trans- and cis-citral (B), respectively, and two isomers of pseudoionone. Compounds were identified based on authentic standards and spectral comparison with NIST library.

Fig. 3

HPLC analysis of incubations with β-apo-8′-carotenal. (A) Incubation of SlCCD1B with β-apo-8′-carotenal led to β-ionone (2, C₁₃), 10′,8-diapocarotene-10′,8-dial (3, C₁₇) and two minor peaks (1, 4) corresponding to 14, 8′-diapocarotene-14, 8′-dial (1, C₁₂) and β-apo-14-carotenal (4, C₂₂). SlCCD1A converted β-apo-8′-carotenal into β-ionone (2, C₁₃), 10′,8-diapocarotene-10′,8-dial (3, C₁₇). (B) The identity of product 4 was confirmed by LC–MS analysis that unraveled the expected [M + H]⁺ value of 311.34 co-eluting with the purified product (UV–Vis spectrum depicted in the inset). (C) Deduced cleavage sites of SlCCD1B sites in β-apo-8′-carotenal.

Fig. 4

GC–MS identification of farnesylacetone produced by SlCCD1A and 1B. Volatiles were collected by SPME from the gas space of carotene-accumulating E. coli cultures expressing thioredoxin-SlCCD1A, -1B or thioredoxin (Con) and analyzed by GC–MS. SlCCD1B produced farnesylacetone identified based on standard and by comparing the detected molecule ion of 262.2 and detected fragments with the NIST library. Farnesylacetone was also produced by SlCCD1A, but at a much lower level. (B) Farnesylacetone can be produced from either phytoene (I) or phytofluene (II) through the cleavage of the C13–C14 double bond.

Fig. 5

HPLC analysis of activity in a ζ-carotene-accumulating E. coli strain. (A) Expression of thioredoxin-SlCCD1B in ζ-carotene (Z) producing E. coli cells that also accumulate phytoene (P) and phytofluene (PF) led to two products (1, 2), that also appeared upon the expression of SlCCD1A, but in traces. (B) LC–MS analysis showed [M + H]⁺ values of 261 and 313 co-eluting with apocarotenoids with UV–Vis spectra depicted in the insets, identifying product 1 and 2 as apo-13-ζ-carotenone (C₁₈) and apo-14′-ζ-carotenal (C₂₂), respectively. (C) Structure of ζ-carotene indicating the cleavage site leading to product 1 and 2.

Fig. 6

HPLC analysis of incubations with prolycopene. (A) Incubation with SlCCD1B led to pseudoionone (1, C₁₃), a tentative apo-12-lycopenal (C₁₅, product 2), two new compounds (4 and 5), apo-12′- (6, C₂₅) and apo-10′-lycopenal (7, C₂₇). SlCCD1A converted prolycopene (structure depicted in C) into pseudoionone (1), the products pseudoionone, a tentative 6′,10-diapocarotene-6′,10-dial (C₁₉, 3), the two new compounds (4, 5), and apo-12′- and apo-10′-lycopenal (6, 7). (B) LC–MS analysis of purified product 4 and 5 unraveled [M + H]⁺ ions of 259.26 and 311.22 and UV–Vis spectra (insets) expected for cis-configured apo-13-lycopenone (C₁₈) and apo-14′-lycopenal (C₂₂). (C) Structure of prolycopene indicating the cleavage sites deduced from the products of both enzymes.

Fig. 7

HPLC analysis of incubations with mono- and bicyclic carotenoids. (A) Incubation of SlCCD1A with 3-OH-γ-carotene (structure I in D) led to 3-OH-β-ionone (1, C₁₃), pseudoionone (2, cis-trans-isomers, C₁₃), 10, 8′-diapocarotene-10, 8′-dial (3, C₁₇) and 10, 6′-diapocarotene-10, 6′-dial (5, C₁₉) SlCCD1B converted 3-OH-γ-carotene into 3-OH-β-ionone (1), pseudoionone (2, cis-trans-isomers), 10, 8′-diapocarotene-10, 8′-dial (3, C₁₇) and traces of 10, 6′-diapocarotene-10, 6′-dial (5, C19), and two products (4, 6) tentatively identified as apo-13-lycopenone (C₁₈) and 3-OH-β-apo-14′-carotenal (C₂₂), respectively. (B) SlCCD1A converted 9-cis-β-carotene into β-ionone (1, C₁₃) and presumably 9-cis-configured β-apo-10′-carotenal (6, C₂₇). Incubation with SlCCD1B yielded 6 products, including 1, 6 the two β-apo-11-carotenal (2, C₁₅) and the tentatively identified 9-cis-β-apo-13-carotenone (C₁₈, product 3), β-apo-14′-carotenal (C₂₂, product 4) and 9-cis-β-apo-12′-carotenal (C₂₅, product 5). (C) LC–MS-analysis of purified product 3 and 4 unraveled [M + H]⁺ values of 259.21 and 311.28 and UV–Vis spectra (insets) corresponding to those expected for β-apo-13-carotenone and β-apo-14′-carotenal. (D) Structure of 3-OH-γ-carotene (I) and 9-cis-β-carotene (II) showing the cleavage sites deduced from the products of both enzymes.