Identification of a Solanum pennellii Chromosome 4 Fruit Flavor and Nutritional Quality-Associated Metabolite QTL

Abstract

A major resource for tomato quality improvement and gene discovery is the collection of introgression lines (ILs) of cultivated Solanum lycopersicum that contain different, defined chromosomal segments derived from the wild tomato relative, S. pennellii. Among these lines, IL4-4, in which the bottom of S. lycopersicum (cv. M82) chromosome 4 is replaced by the corresponding S. pennellii segment, is altered in many primary and secondary metabolites, including many related to fruit flavor and nutritional quality. Here, we provide a comprehensive profile of IL4-4 ripe fruit metabolites, the transcriptome and fine mapping of sub-ILs. Remarkably, out of 327 quantified metabolites, 185 were significantly changed in IL4-4 fruit, compared to the control. These altered metabolites include volatile organic compounds, primary and secondary metabolites. Partial least squares enhanced discriminant analysis of the metabolite levels among sub-ILs indicated that a genome region encompassing 20 putative open reading frames is responsible for most of the metabolic changes in IL4-4 fruit. This work provides comprehensive insights into IL4-4 fruit biochemistry, identifying a small region of the genome that has major effects on a large and diverse set of metabolites.

Keywords: IL4-4; flavor; fruit ripening; metabolome; transcriptome.

FIGURE 1

Heat map of IL4-4 fruit metabolites compared to M82. BCAA, branched-amino acids; FA, fatty acids; AAA, aromatic-amino acids; CAR, carotenoid; ACE, acetate; AA, amino acids; Ssa, Sugars and sugar alcohols; OA, organic acids; Vit, vitamins; Cyc, cyclitol; Alk, alkaloids; DAG, diacylglycerol; DGDG, digalactosyl-diacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TAG, triacylglyceride; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SQDG, sulfoquinovosyldiacylglycerol.

FIGURE 2

Functional classification of DEGs between IL4-4 and M82. Distribution of GO terms for biological process (A) and molecular function (B) conducted with the PANTHER web tool. (C) Overview of the effect of IL4-4 regulated expression changes on metabolism of fruits with MapMan software (Metabolism_overview panel). MapMan functional categories and Log2 fold change values are represented. Significantly increased and decreased transcripts are shown in red and blue, respectively. A dot represents the log2 of the RPKM ratio of a transcript between IL4-4 and control M82.

FIGURE 3

Levels of volatiles and transcripts in the fatty acid pathway in IL4-4 and M82. (A) Schematic representation of the C5 and C6 volatile biosynthetic pathway based on Shen et al. (2014). HPL, hydroperoxide lyase; LOXs, lipoxygenases. (B) Expression of LOX f and HPL genes in ripe tissue of IL4-4 and M82. Ratio (±SE) was calculated by the average RPKM value. An asterisk indicates RPKM ration change >2 and adjusted p < 0.05. (C) levels of C5 and C6 volatiles (±SE) significantly different (p < 0.05, n > 5) between IL4-4 and M82.

FIGURE 4

Changes of metabolites and transcripts in the shikimate-phenylpropanoid pathway in IL4-4 compared to M82. (A) Shikimate and phenylpropanoid pathways. Genes whose transcripts were significantly elevated or reduced in IL4-4 are indicated in red and green, respectively. Dashed arrows represent multiple enzymatic steps. ADT, arogenate dehydratase; ADS, arogenate dehydrogenase; AADC, aromatic amino acid decarboxylase; CM, chorismate mutase; CS, chorismate synthase; C4H, cinnamate-4-hydroxylase; CHS, chalcone synthase; 4CL, 4-coumarate CoA ligase; DAHPS, 3-deoxy-D-arabino-2-heptulosonate 7-phosphate synthase; DHQS, 3-dehydroquinate synthase; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase; EGS, eugenol synthase; IGPS, indole-3-glycerol phosphate synthase; PAI, anthranilate isomerase; PAL, phenylalanine ammonia-lyase; PAR, phenylacetaldehyde reductase; SK, shikimate kinase; SQH, shikimate dehydrogenase; TS, tryptophan synthase. (B) RPKM values of DEGs in the shikimate and phenylpropanoid pathways. (C) Levels (±SE) of significantly altered phenylpropanoid volatiles (p < 0.05). (D) Relative levels of hydrophilic phenylpropanoids. Results are presented as fold change relative to M82 (n > 5).

FIGURE 5

Association of metabolic phenotypes with sub-ILs. (A) Schematic representation of sub-ILs derived from IL4-4. Red and blue lines represent segments of S. pennellii and S. lycopersicum, respectively. Genomic region M was defined as the area between markers C1247 and C200952, indicated by blue lines. The starts and ends of the sub-ILs were defined by the molecular markers listed above (Supplementary Table S6). (B–E) PLS-EDA analysis of metabolite contents of sub-ILs and their parent lines. Phenotypic similarity is indicated by the distance between each point. Blue dots represent IL4-4 and lines with the M region; green dots represent M82 and lines without the M region. IL4-4 and M82 are indicated with arrows. (B) Volatile metabolites; (C) Hydrophilic primary metabolites; (D) Hydrophilic secondary metabolites; (E) Lipophilic metabolites.

FIGURE 6

Metabolites significantly altered in lines containing the M region of IL4-4. Numbers of altered metabolites within a class are indicated in brackets. Putative open reading frames (ORFs) within the M genome region are listed in order.

FIGURE 7

Fruit quality associated features regulated by the M genomic region. (A) C5 volatiles include 1-penten-3-ol, 1-penten-3-one, 3-pentanone, E-2-pentenal, 1-pentanol and Z-2-penten-1-ol. (B) glucose plus fructose. (C) soluble solids. (D) flavonoids. Letters represent significant difference among the five genotypes using ANOVA followed by a Newman–Keuls test (p < 0.01).