A flavin-dependent monooxygenase produces nitrogenous tomato aroma volatiles using cysteine as a nitrogen source

Abstract

Tomato (Solanum lycopersicum) produces a wide range of volatile chemicals during fruit ripening, generating a distinct aroma and contributing to the overall flavor. Among these volatiles are several aromatic and aliphatic nitrogen-containing compounds for which the biosynthetic pathways are not known. While nitrogenous volatiles are abundant in tomato fruit, their content in fruits of the closely related species of the tomato clade is highly variable. For example, the green-fruited species Solanum pennellii are nearly devoid, while the red-fruited species S. lycopersicum and Solanum pimpinellifolium accumulate high amounts. Using an introgression population derived from S. pennellii, we identified a locus essential for the production of all the detectable nitrogenous volatiles in tomato fruit. Silencing of the underlying gene (SlTNH1;Solyc12g013690) in transgenic plants abolished production of aliphatic and aromatic nitrogenous volatiles in ripe fruit, and metabolomic analysis of these fruit revealed the accumulation of 2-isobutyl-tetrahydrothiazolidine-4-carboxylic acid, a known conjugate of cysteine and 3-methylbutanal. Biosynthetic incorporation of stable isotope-labeled precursors into 2-isobutylthiazole and 2-phenylacetonitrile confirmed that cysteine provides the nitrogen atom for all nitrogenous volatiles in tomato fruit. Nicotiana benthamiana plants expressing SlTNH1 readily transformed synthetic 2-substituted tetrahydrothiazolidine-4-carboxylic acid substrates into a mixture of the corresponding 2-substituted oxime, nitro, and nitrile volatiles. Distinct from other known flavin-dependent monooxygenase enzymes in plants, this tetrahydrothiazolidine-4-carboxylic acid N-hydroxylase catalyzes sequential hydroxylations. Elucidation of this pathway is a major step forward in understanding and ultimately improving tomato flavor quality.

Keywords: N-hydroxylation; aroma volatile biosynthesis; flavor chemistry; organosulfur metabolism.

Fig. 1.

A gene on Chr12 (Solyc12g013690) is responsible for nitrogenous volatiles biosynthesis in tomato fruit. (A) Structures of main nitrogenous volatiles detected in tomato fruit. (B) Emissions of nitrogenous volatiles from cut ripe fruits of ILs and the tomato parent, cv. M82 (±SE, n = 6). The amount of each nitrogenous volatile is significantly different between M82 and the ILs (P < 0.01). n.d., not detectable. (C) Fine mapping of a nitrogenous volatile-related QTL on chromosome 12. A set of recombinants with low or high (wild type) content of nitrogenous volatiles was derived from a cross between M82 and IL12-2. The illustration shows a small segment of chromosome 12 where the QTL is located. White and dark-gray sections represent genomic segments from S. lycopersicum and S. pennellii, respectively. Arrows indicate the positions of markers used to define recombinants. The light-gray sections represent the regions where a recombination occurred, as delimited by molecular markers. The color inside the diamonds indicates whether the ILs contain the S. lycopersicum (white) or S. pennellii (dark-gray) version of Solyc12g013690. The QTL region as defined by volatile content is shown by dashed lines and enlarged below to show details (assembly SL2.50).

Fig. 2.

Antisense-mediated silencing of Solyc12g013690 (SlTNH1) results in significant reductions in nitrogenous flavor volatiles in fruit. (A) Transcript levels in fruits of four independent transgenic antisense lines relative to the control, cv. Large Red Cherry (±SE, n = 4). The transgenic lines were all significantly different from the control (*P < 0.01). (B) Sum of the emissions of detected nitrogenous volatiles from cut ripe fruits of control and the transgenic lines (±SE, n = 4) (*P < 0.01) (see SI Appendix, Table S1 for emission of each individual compound).

Fig. 3.

Ripening-related accumulation of nitrogenous volatiles and Solyc12g013690 (SlTNH1) expression in tomato. (A) Emissions of nitrogenous volatiles from cut fruits of cv. M82 during the ripening process (±SE, n = 4). n.d., not detectable. (B) Solyc12g013690 transcript levels in leaves, flowers, and throughout the fruit ripening (immature green, breaker, turning, and ripe) of tomato cv. M82 (±SE, n = 4). (C) Solyc12g013690 transcript level in response to ethylene (ET) (±SE, n = 3). Mature green fruits of cv. Pearson and Never-ripe were treated with ethylene for 16 h. Alternatively, fruits at ripe stage were treated with the inhibitor of ethylene perception, 1-MCP, for 12 h.

Fig. 4.

Impairment of SlTNH1 activity leads to accumulation of 2-isobutyltetrahydrothiazolidine-4-carboxylic acid (IT4C) in tomato fruit. (A) IT4C can be easily synthesized from L-Cys and 3-methylbutanal. (B) Untargeted metabolomics by UPLC-QTOF-MS revealed that IT4C accumulates in the SlTNH1 antisense lines and stop codon variant (n = 3, ±SE, *P < 0.05). (C) Extracted ion chromatogram for m/z 190.0902 shows that the metabolic feature accumulating in SlTNH1 antisense lines was unambiguously identified as IT4C. Synthetic IT4C coelutes with the same feature in tomato fruit. MS/MS and NMR spectra of IT4C are provided in the SI Appendix.

Fig. 5.

Biosynthetic incorporation of stable isotope-labeled cysteine and leucine into 2-isobutylthiazole when fed in situ to Tomatoes-on-the-vine at the orange stage. Incorporation ratios were calculated from peak integration areas (PIAs) from APGC-MS/MS analysis where incorporation ratio = [(PIA isotopolog)/(PIA unlabeled + PIA isotopolog) × 100]. n ≥ 3 for all treatments. Expected m/z for 2-isobutylthiazole [M+H] (unlabeled) = 142.0690, [M+H+3] isotopolog = 145.0728, [M+H+5] isotopolog = 147.0856, and [M+H+8] isotopolog = 150.0896.

Fig. 6.

Solyc12g013690 encodes a tetrahydrothiazolidine N-hydroxylase (TNH), and converts 2-tetrahydrothizolidines to nitrogenous flavor volatiles. (A) Model for TNH-dependent nitrogenous volatile biosynthesis in tomato fruit. (B and C) Nitrogenous flavor volatiles are produced in N. benthamiana leaves with transient expression of SlTNH1 and supplementation with the corresponding thiazolidines made from 2-phenylacetaldehyde and L-cysteine (B), and 3-methylbutanal and L-cysteine (C). (±SE, n = 4, *P < 0.05).