The m6A reader SlYTH2 negatively regulates tomato fruit aroma by impeding the translation process.

N6-methyladenosine (m6A) is a fundamentally important RNA modification for gene regulation, whose function is achieved through m6A readers. However, whether and how m6A readers play regulatory roles during fruit ripening and quality formation remains unclear. Here, we characterized SlYTH2 as a tomato m6A reader protein and profiled the binding sites of SlYTH2 at the transcriptome-wide level. SlYTH2 undergoes liquid-liquid phase separation and promotes RNA-protein condensate formation. The target mRNAs of SlYTH2, namely m6A-modified SlHPL and SlCCD1B associated with volatile synthesis, are enriched in SlYTH2-induced condensates. Through polysome profiling assays and proteomic analysis, we demonstrate that knockout of SlYTH2 expedites the translation process of SlHPL and SlCCD1B, resulting in augmented production of aroma-associated volatiles. This aroma enrichment significantly increased consumer preferences for CRISPR-edited fruit over wild type. These findings shed light on the underlying mechanisms of m6A in plant RNA metabolism and provided a promising strategy to generate fruits that are more attractive to consumers.

Natural variations in the Sl-AKR9 aldo/keto reductase gene impact fruit flavor volatile and sugar contents.

The unique flavors of different fruits depend upon complex blends of soluble sugars, organic acids, and volatile organic compounds. 2-Phenylethanol and phenylacetaldehyde are major contributors to flavor in many foods, including tomato. In the tomato fruit, glucose, and fructose are the chemicals that most positively contribute to human flavor preferences. We identified a gene encoding a tomato aldo/keto reductase, Sl-AKR9, that is associated with phenylacetaldehyde and 2-phenylethanol contents in fruits. Two distinct haplotypes were identified; one encodes a chloroplast-targeted protein while the other encodes a transit peptide-less protein that accumulates in the cytoplasm. Sl-AKR9 effectively catalyzes reduction of phenylacetaldehyde to 2-phenylethanol. The enzyme can also metabolize sugar-derived reactive carbonyls, including glyceraldehyde and methylglyoxal. CRISPR-Cas9-induced loss-of-function mutations in Sl-AKR9 significantly increased phenylacetaldehyde and lowered 2-phenylethanol content in ripe fruit. Reduced fruit weight and increased soluble solids, glucose, and fructose contents were observed in the loss-of-function fruits. These results reveal a previously unidentified mechanism affecting two flavor-associated phenylalanine-derived volatile organic compounds, sugar content, and fruit weight. Modern varieties of tomato almost universally contain the haplotype associated with larger fruit, lower sugar content, and lower phenylacetaldehyde and 2-phenylethanol, likely leading to flavor deterioration in modern varieties.

A family of methyl esterases converts methyl salicylate to salicylic acid in ripening tomato fruit.

Methyl salicylate imparts a potent flavor and aroma described as medicinal and wintergreen that is undesirable in tomato (Solanum lycopersicum) fruit. Plants control the quantities of methyl salicylate through a variety of biosynthetic pathways, including the methylation of salicylic acid to form methyl salicylate and subsequent glycosylation to prevent methyl salicylate emission. Here, we identified a subclade of tomato methyl esterases, SALICYLIC ACID METHYL ESTERASE1-4, responsible for demethylation of methyl salicylate to form salicylic acid in fruits. This family was identified by proximity to a highly significant methyl salicylate genome-wide association study locus on chromosome 2. Genetic mapping studies in a biparental population confirmed a major methyl salicylate locus on chromosome 2. Fruits from SlMES1 knockout lines emitted significantly (P < 0,05, t test) higher amounts of methyl salicylate than wild-type fruits. Double and triple mutants of SlMES2, SlMES3, and SlMES4 emitted even more methyl salicylate than SlMES1 single knockouts-but not at statistically distinguishable levels-compared to the single mutant. Heterologously expressed SlMES1 and SlMES3 acted on methyl salicylate in vitro, with SlMES1 having a higher affinity for methyl salicylate than SlMES3. The SlMES locus has undergone major rearrangement, as demonstrated by genome structure analysis in the parents of the biparental population. Analysis of accessions that produce high or low levels of methyl salicylate showed that SlMES1 and SlMES3 genes expressed the highest in the low methyl salicylate lines. None of the MES genes were appreciably expressed in the high methyl salicylate-producing lines. We concluded that the SlMES gene family encodes tomato methyl esterases that convert methyl salicylate to salicylic acid in ripe tomato fruit. Their ability to decrease methyl salicylate levels by conversion to salicylic acid is an attractive breeding target to lower the level of a negative contributor to flavor.

Heteromeric interactions of ripening-related ethylene receptors in tomato fruit.

Ripening of climacteric fruits is initiated when the gaseous plant hormone ethylene is perceived by the cell. Ethylene binding to membrane-associated ethylene receptors (ETRs) triggers a series of biochemical events through multiple components, resulting in the induction of numerous ripening-related genes. In tomato (Solanum lycopersicum L.), there are seven members of the ETR family, which each contribute to the regulation of fruit ripening. However, the relative contribution of each individual receptor to ethylene signaling remains unknown. Here, we demonstrated the formation of heteromeric receptor complexes across the two ETR subfamilies in tomato fruit. Immunoprecipitation of subfamily II SlETR4 resulted in co-purification of subfamily I (SlETR1, SlETR2, and SlETR3), but not subfamily II members (SlETR5, SlETR6, and SlETR7). Such biased interactions were verified in yeast two-hybrid assays, and in transgenic Arabidopsis plants, in which heterologous SlETR4 interacts with subfamily I ETRs. Our analysis also revealed that the receptor complexes engage the Raf-like protein kinases SlCTR1 and SlCTR3, which are potential regulators of signaling. Here, we suggest that tomato receptor members form heteromeric complexes to fine-tune signal output to the downstream pathway, which is similar to that of the Arabidopsis system but appears to be partially diverged.

Metabolomic selection for enhanced fruit flavor.

Although they are staple foods in cuisines globally, many commercial fruit varieties have become progressively less flavorful over time. Due to the cost and difficulty associated with flavor phenotyping, breeding programs have long been challenged in selecting for this complex trait. To address this issue, we leveraged targeted metabolomics of diverse tomato and blueberry accessions and their corresponding consumer panel ratings to create statistical and machine learning models that can predict sensory perceptions of fruit flavor. Using these models, a breeding program can assess flavor ratings for a large number of genotypes, previously limited by the low throughput of consumer sensory panels. The ability to predict consumer ratings of liking, sweet, sour, umami, and flavor intensity was evaluated by a 10-fold cross-validation, and the accuracies of 18 different models were assessed. The prediction accuracies were high for most attributes and ranged from 0.87 for sourness intensity in blueberry using XGBoost to 0.46 for overall liking in tomato using linear regression. Further, the best-performing models were used to infer the flavor compounds (sugars, acids, and volatiles) that contribute most to each flavor attribute. We found that the variance decomposition of overall liking score estimates that 42% and 56% of the variance was explained by volatile organic compounds in tomato and blueberry, respectively. We expect that these models will enable an earlier incorporation of flavor as breeding targets and encourage selection and release of more flavorful fruit varieties.

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

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.

Domestication of Crop Metabolomes: Desired and Unintended Consequences.

The majority of the crops and vegetables of today were domesticated from their wild progenitors within the past 12 000 years. Considerable research effort has been expended on characterizing the genes undergoing positive and negative selection during the processes of crop domestication and improvement. Many studies have also documented how the contents of a handful of metabolites have been altered during human selection, but we are only beginning to unravel the true extent of the metabolic consequences of breeding. We highlight how crop metabolomes have been wittingly or unwittingly shaped by the processes of domestication, and highlight how we can identify new targets for metabolite engineering for the purpose of de novo domestication of crop wild relatives.

Identification of a lipase gene with a role in tomato fruit short-chain fatty acid-derived flavor volatiles by genome-wide association.

Fatty acid-derived volatile organic compounds (FA-VOCs) make significant contributions to tomato (Solanum lycopersicum) fruit flavor and human preferences. Short-chain FA-VOCs (C5 and C6) are among the most abundant and important volatile compounds in tomato fruits. The precursors of these volatiles, linoleic acid (18:2) and linolenic acid (18:3), are derived from cleavage of glycerolipids. However, the initial step in synthesis of these FA-VOCs has not been established. A metabolite-based genome-wide association study combined with genetic mapping and functional analysis identified a gene encoding a novel class III lipase family member, Sl-LIP8, that is associated with accumulation of short-chain FA-VOCs in tomato fruit. In vitro assays indicated that Sl-LIP8 can cleave 18:2 and 18:3 acyl groups from glycerolipids. A CRISPR/Cas9 gene edited Sl-LIP8 mutant had much lower content of multiple fruit short-chain FA-VOCs, validating an important role for this enzyme in the pathway. Sl-LIP8 RNA abundance was correlated with FA-VOC content, consistent with transcriptional regulation of the first step in the pathway. Taken together, our work indicates that glycerolipid turnover by Sl-LIP8 is an important early step in the synthesis of multiple short-chain FA-VOCs.

Plant Domestication: Reconstructing the Route to Modern Tomatoes.

An explosion of genome sequencing has facilitated a deep understanding of the tomato's domestication history. Although the species emerged in Ecuador, domestication is likely to have occurred in Mexico whence it was transported to Europe and ultimately improved into one of our favorite foods.

The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor.

Modern tomatoes have narrow genetic diversity limiting their improvement potential. We present a tomato pan-genome constructed using genome sequences of 725 phylogenetically and geographically representative accessions, revealing 4,873 genes absent from the reference genome. Presence/absence variation analyses reveal substantial gene loss and intense negative selection of genes and promoters during tomato domestication and improvement. Lost or negatively selected genes are enriched for important traits, especially disease resistance. We identify a rare allele in the TomLoxC promoter selected against during domestication. Quantitative trait locus mapping and analysis of transgenic plants reveal a role for TomLoxC in apocarotenoid production, which contributes to desirable tomato flavor. In orange-stage fruit, accessions harboring both the rare and common TomLoxC alleles (heterozygotes) have higher TomLoxC expression than those homozygous for either and are resurgent in modern tomatoes. The tomato pan-genome adds depth and completeness to the reference genome, and is useful for future biological discovery and breeding.

Meta-analysis of genome-wide association studies provides insights into genetic control of tomato flavor.

Tomato flavor has changed over the course of long-term domestication and intensive breeding. To understand the genetic control of flavor, we report the meta-analysis of genome-wide association studies (GWAS) using 775 tomato accessions and 2,316,117 SNPs from three GWAS panels. We discover 305 significant associations for the contents of sugars, acids, amino acids, and flavor-related volatiles. We demonstrate that fruit citrate and malate contents have been impacted by selection during domestication and improvement, while sugar content has undergone less stringent selection. We suggest that it may be possible to significantly increase volatiles that positively contribute to consumer preferences while reducing unpleasant volatiles, by selection of the relevant allele combinations. Our results provide genetic insights into the influence of human selection on tomato flavor and demonstrate the benefits obtained from meta-analysis.

Quantitative Trait Loci Analysis Identifies a Prominent Gene Involved in the Production of Fatty Acid-Derived Flavor Volatiles in Tomato.

To gain insight into the genetic regulation of lipid metabolism in tomato, we conducted metabolic trait loci (mQTL) analysis following the lipidomic profiling of fruit pericarp and leaf tissue of the Solanum pennellii introgression lines (IL). To enhance mapping resolution for selected fruit-specific mQTL, we profiled the lipids in a subset of independently derived S. pennellii backcross inbred lines, as well as in a near-isogenic sub-IL population. We identified a putative lecithin:cholesterol acyltransferase that controls the levels of several lipids, and two members of the class III lipase family, LIP1 and LIP2, that were associated with decreased levels of diacylglycerols (DAGs) and triacylglycerols (TAGs). Lipases of this class cleave fatty acids from the glycerol backbone of acylglycerols. The released fatty acids serve as precursors of flavor volatiles. We show that LIP1 expression correlates with fatty acid-derived volatile levels. We further confirm the function of LIP1 in TAG and DAG breakdown and volatile synthesis using transgenic plants. Taken together, our study extensively characterized the genetic architecture of lipophilic compounds in tomato and demonstrated at molecular level that release of free fatty acids from the glycerol backbone can have a major impact on downstream volatile synthesis.

An ETHYLENE RESPONSE FACTOR-MYB Transcription Complex Regulates Furaneol Biosynthesis by Activating QUINONE OXIDOREDUCTASE Expression in Strawberry.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone is a major contributor to the aroma of strawberry (Fragaria × ananassa) fruit, and the last step in its biosynthesis is catalyzed by strawberry quinone oxidoreductase (FaQR). Here, an ethylene response factor (FaERF#9) was characterized as a positive regulator of the FaQR promoter. Linear regression analysis indicated that FaERF#9 transcript levels were correlated significantly with both FaQR transcripts and furanone content in different strawberry cultivars. Transient overexpression of FaERF#9 in strawberry fruit significantly increased FaQR expression and furaneol production. Yeast one-hybrid assays, however, indicated that FaERF#9 by itself did not bind to the FaQR promoter. An MYB transcription factor (FaMYB98) identified in yeast one-hybrid screening of the strawberry cDNA library was capable of both binding to the promoter and activating the transcription of FaQR by ∼5.6-fold. Yeast two-hybrid assay and bimolecular fluorescence complementation confirmed a direct protein-protein interaction between FaERF#9 and FaMYB98, and in combination, they activated the FaQR promoter 14-fold in transactivation assays. These results indicate that an ERF-MYB complex containing FaERF#9 and FaMYB98 activates the FaQR promoter and up-regulates 4-hydroxy-2,5-dimethyl-3(2H)-furanone biosynthesis in strawberry.

Development and characterization of a high quality plum tomato essence.

A tomato essence was developed for potential flavor enhancement of juices and other tomato products. The changes in the flavor profile that occur during tomato processing in plum tomatoes were characterized and compared using purge and trap gas chromatography-mass spectrometry (PT-GC-MS). The pilot tomato juices mostly suffered losses in green and fruity note-related compounds. Of the four essence fractions produced, Fractions 1 and 2 were most complementary to the undesirable flavor changes encountered during thermal processing. Fraction 1 was characterized as a "green tomato note", with substantially higher amounts of 2-isobutylthiazole, α-citral, hexanal, 3-hexen-1-ol, and 6-methyl-5-hepten-2-one; Fraction 2 was described as a "fruity tomato note" with relatively percentage change of compounds; and Fractions 3 and 4 were described as "cooked tomato note" and "faint tomato note", respectively. These results demonstrate the feasibility of adding tomato essence to impart desirable flavor attributes to processed tomato products.

A Comparison of the Effects of FATTY ACID DESATURASE 7 and HYDROPEROXIDE LYASE on Plant-Aphid Interactions.

The spr2 mutation in tomato (Solanum lycopersicum), which disrupts function of FATTY ACID DESATURASE 7 (FAD7), confers resistance to the potato aphid (Macrosiphum euphorbiae) and modifies the plant’s C6 volatile profiles. To investigate whether C6 volatiles play a role in resistance, HYDROPEROXIDE LYASE (HPL), which encodes a critical enzyme in C6 volatile synthesis, was silenced in wild-type tomato plants and spr2 mutants. Silencing HPL in wild-type tomato increased potato aphid host preference and reproduction on 5-week old plants but had no influence on 3-week old plants. The spr2 mutation, in contrast, conferred strong aphid resistance at both 3 and 5 weeks, and silencing HPL in spr2 did not compromise this aphid resistance. Moreover, a mutation in the FAD7 gene in Arabidopsis thaliana also conferred resistance to the green peach aphid (Myzus persicae) in a genetic background that carries a null mutation in HPL. These results indicate that HPL contributes to certain forms of aphid resistance in tomato, but that the effects of FAD7 on aphids in tomato and Arabidopsis are distinct from and independent of HPL.

The genetics of fruit flavour preferences.

Intensively bred fruit crops, including tomatoes and strawberries, are widely viewed as lacking flavour. The lack of breeder focus on the consumer is largely due to the genetic complexity of the flavour phenotype as well as a lack of a simple assay that can define consumer preferences. Rapid advances in genomics have opened up new opportunities to understand the chemistry and genetics of flavour. Here, we describe the underlying causes for the loss of flavour in fruits over time and delineate a blueprint for defining the chemistry of consumer liking, reducing that knowledge into a molecular roadmap for flavour improvement.

Sensory and Flavor Characteristics of Tomato Juice from Garden Gem and Roma Tomatoes with Comparison to Commercial Tomato Juice.

The objective of this study was to characterize the flavor of a premium Florida tomato variety that has significant potential for producing a high quality processed juice product. A high-quality Florida plum tomato variety (Garden Gem), and a typical grocery-store plum tomato variety (Roma) were thermally processed into tomato juices without any additives. The 2 pilot products and a popular commercially available tomato juice (low sodium with sugar and flavor added) were compared using sensory evaluation and instrumental analysis. Flavor compounds in these products were identified using dynamic headspace purge and trap-gas chromatography-mass spectrometry (PT-GC-MS) by MS library match and retention index and were semi-quantitated using internal standards. Color, uniformity, overall liking, tomato flavor, sweetness and texture were rated using a hedonic scale. Analysis of variance, correlation and principal component analysis were used to analyze both sensory and flavor data. Among the 3 products, Garden Gem juice was rated significantly (P < 0.05) higher for overall liking, tomato flavor, and sweetness by the 119 consumer panelists in both seasons. Garden Gem juice was found to contain higher levels of 3 sweet/fruity related aroma compounds: 6-methyl-5-hepten-2-one, linalool, and ß-ionone. The commercial tomato juice contained a high level of the Maillard reaction-related notes furfural, dimethyl sulfide, and the least amount of green-related notes (hexanal, E-2-hexenal and Z-2-heptenal). The flavor profile of the Roma tomato juice was similar to Garden Gem juice except it contained substantially lower amounts of hexanal and 2-isobutylthiazole. The compound ß-ionone (fruity note) was not detected in either the commercial or Roma juice. PRACTICAL APPLICATION: This proof of concept study demonstrates that high flavor quality tomatoes can be used to create better tasting processed tomato products. The study also demonstrates how sensory preference can confer a potential market advantage over existing commercial products. The Garden Gem variety has potential to add desirable flavor attributes to processed tomato products. This research may also provide insights for product developers to which flavor volatiles best reflect sensory observations for different aspects of tomato flavor.

Genetic Control of Floral Architecture: Insights into Improving Crop Yield.

Selection of plants with traits that are beneficial for cultivation and consumption has been a common practice for thousands of years; however, combination of these traits can be detrimental too, for instance by leading to undesirable branching and yield loss in tomato. The findings from Soyk et al. in this issue of Cell help understand how to bypass such negative effects and improve crop productivity.

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

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.

Chilling-induced tomato flavor loss is associated with altered volatile synthesis and transient changes in DNA methylation.

Commercial tomatoes are widely perceived by consumers as lacking flavor. A major part of that problem is a postharvest handling system that chills fruit. Low-temperature storage is widely used to slow ripening and reduce decay. However, chilling results in loss of flavor. Flavor-associated volatiles are sensitive to temperatures below 12 °C, and their loss greatly reduces flavor quality. Here, we provide a comprehensive view of the effects of chilling on flavor and volatiles associated with consumer liking. Reduced levels of specific volatiles are associated with significant reductions in transcripts encoding key volatile synthesis enzymes. Although expression of some genes critical to volatile synthesis recovers after a return to 20 °C, some genes do not. RNAs encoding transcription factors essential for ripening, including RIPENING INHIBITOR (RIN), NONRIPENING, and COLORLESS NONRIPENING are reduced in response to chilling and may be responsible for reduced transcript levels in many downstream genes during chilling. Those reductions are accompanied by major changes in the methylation status of promoters, including RIN Methylation changes are transient and may contribute to the fidelity of gene expression required to provide maximal beneficial environmental response with minimal tangential influence on broader fruit developmental biology.