Multi-omics analysis unravels chemical roadmap and genetic basis for peach fruit aroma improvement.

Selection of fruits with enhanced health benefits and superior flavor is an important aspect of peach breeding. Understanding the genetic interplay between appearance and flavor chemicals remains a major challenge. We identify the most important volatiles contributing to consumer preferences for peach, thus establishing priorities for improving flavor quality. We quantify volatiles of a peach population consisting of 184 accessions and demonstrate major reductions in the important flavor volatiles linalool and Z-3-hexenyl acetate in red-fleshed accessions. We identify 474 functional gene regulatory networks (GRNs), among which GRN05 plays a crucial role in controlling both red flesh and volatile content through the NAM/ATAF1/2/CUC (NAC) transcription factor PpBL. Overexpressing PpBL results in reduced expression of PpNAC1, a positive regulator for Z-3-hexenyl acetate and linalool synthesis. Additionally, we identify haplotypes for three tandem PpAATs that are significantly correlated with reduced gene expression and ester content. We develop genetic resources for improvement of fruit quality.

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.

The role and interaction between transcription factor NAC-NOR and DNA demethylase SlDML2 in the biosynthesis of tomato fruit flavor volatiles.

Flavor-imparting volatile chemicals accumulate as fruits ripen, making major contributions to taste. The NAC transcription factor nonripening (NAC-NOR) and DNA demethylase 2 (SlDML2) are essential for tomato fruit ripening, but details of the potential roles and the relationship between these two regulators in the synthesis of volatiles are lacking. Here, we show substantial reductions in fatty acid and carotenoid-derived volatiles in tomato slnor and sldml2 mutants. An unexpected finding is the redundancy and divergence in volatile profiles, biosynthetic gene expression, and DNA methylation in slnor and sldml2 mutants relative to wild-type tomato fruit. Reduced transcript levels are accompanied by hypermethylation of promoters, including the NAC-NOR target gene lipoxygenase (SlLOXC) that is involved in fatty acid-derived volatile synthesis. Interestingly, NAC-NOR activates SlDML2 expression by directly binding to its promoter both in vitro and in vivo. Meanwhile, reduced NAC-NOR expression in the sldml2 mutant is accompanied by hypermethylation of its promoter. These results reveal a relationship between SlDML2-mediated DNA demethylation and NAC-NOR during tomato fruit ripening. In addition to providing new insights into the metabolic modulation of flavor volatiles, the outcome of our study contributes to understanding the genetics and control of fruit ripening and quality attributes in tomato.

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.

High-Throughput Chlorophyll and Carotenoid Profiling Reveals Positive Associations with Sugar and Apocarotenoid Volatile Content in Fruits of Tomato Varieties in Modern and Wild Accessions.

Flavor and nutritional quality has been negatively impacted during the course of domestication and improvement of the cultivated tomato (Solanum lycopersicum). Recent emphasis on consumers has emphasized breeding strategies that focus on flavor-associated chemicals, including sugars, acids, and aroma compounds. Carotenoids indirectly affect flavor as precursors of aroma compounds, while chlorophylls contribute to sugar production through photosynthesis. However, the relationships between these pigments and flavor content are still unclear. In this study, we developed a simple and high-throughput method to quantify chlorophylls and carotenoids. This method was applied to over one hundred tomato varieties, including S. lycopersicum and its wild relatives (S. l. var. cerasiforme and S. pimpinellifolium), for quantification of these pigments in fruits. The results obtained by integrating data of the pigments, soluble solids, sugars, and aroma compounds indicate that (i) chlorophyll-abundant varieties have relatively higher sugar accumulations and (ii) prolycopene is associated with an abundance of linear carotenoid-derived aroma compounds in one of the orange-fruited varieties, "Dixie Golden Giant". Our results suggest the importance of these pigments not only as components of fruit color but also as factors influencing flavor traits, such as sugars and aroma.

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.

Transcriptional and epigenetic analysis reveals that NAC transcription factors regulate fruit flavor ester biosynthesis.

Flavor-associated volatile chemicals make major contributions to consumers' perception of fruits. Although great progress has been made in establishing the metabolic pathways associated with volatile synthesis, much less is known about the regulation of those pathways. Knowledge of how those pathways are regulated would greatly facilitate efforts to improve flavor. Volatile esters are major contributors to fruity flavor notes in many species, providing a good model to investigate the regulation of volatile synthesis pathways. Here we initiated a study of peach (Prunus persica L. Batsch) fruits, and identified that the alcohol acyltransferase PpAAT1 contributes to ester formation. We next identified the transcription factor (TF) PpNAC1 as an activator of PpAAT1 expression and ester production. These conclusions were based on in vivo and in vitro experiments and validated by correlation in a panel of 30 different peach cultivars. Based on homology between PpNAC1 and the tomato (Solanum lycopersicum) TF NONRIPENING (NOR), we identified a parallel regulatory pathway in tomato. Overexpression of PpNAC1 enhances ripening in a nor mutant and restores synthesis of volatile esters in tomato fruits. Furthermore, in the NOR-deficient mutant tomatoes generated by CRISPR/Cas9, lower transcript levels of SlAAT1 were detected. The apple (Malus domestica) homolog MdNAC5 also stimulates MdAAT1 expression via binding to this gene's promoter. In addition to transcriptional control, epigenetic analysis showed that increased expression of NACs and AATs is associated with removal of the repressive mark H3K27me3 during fruit ripening. Our results support a conserved molecular mechanism in which NAC TFs activate ripening-related AAT expression, which in turn catalyzes volatile ester formation in multiple fruit species.

Functional divergence of principal alcohol o-acyltransferase for biosynthesis of volatile acetate esters among tomato wild species (Solanum Sect. Lycopersicon).

Volatile esters are the chemicals that have multiple physiological functions including plant defense responses and reproduction. From a human perspective, the esters largely contribute to the fruity aroma of freshy fruits. Composition of volatile esters show a significant diversity among the wild tomato species (Solanum sect. Lycopersicon). To address the basis for this divergence, here we conducted functional analysis of a gene encoding major alcohol o-acyltransferase (AAT1) that catalyzes volatile ester formation. Although AAT1 transcripts were highly expressed in the ripe fruits of all the wild species examined, their enzymatic properties significantly differed due to amino acid sequence variations. Notably, AAT1s from S. pennellii showed the highest ability to produce acetate esters whereas AAT1s from S. neorickii, S. chmielewskii and S. habrochaites had the lowest activities. Further, screenings using domain-swapped or point-mutated AAT1s allowed us to identify Met/Thr352 as one of the critical residues related to the transferase activity with acetyl-CoA. This finding is potentially applied to aroma engineering in which a site-directed mutagenesis at this position in alcohol o-acyltransferases could enable to manipulate volatile ester levels in ripe fruits.

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.

Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato.

Structural variants (SVs) underlie important crop improvement and domestication traits. However, resolving the extent, diversity, and quantitative impact of SVs has been challenging. We used long-read nanopore sequencing to capture 238,490 SVs in 100 diverse tomato lines. This panSV genome, along with 14 new reference assemblies, revealed large-scale intermixing of diverse genotypes, as well as thousands of SVs intersecting genes and cis-regulatory regions. Hundreds of SV-gene pairs exhibit subtle and significant expression changes, which could broadly influence quantitative trait variation. By combining quantitative genetics with genome editing, we show how multiple SVs that changed gene dosage and expression levels modified fruit flavor, size, and production. In the last example, higher order epistasis among four SVs affecting three related transcription factors allowed introduction of an important harvesting trait in modern tomato. Our findings highlight the underexplored role of SVs in genotype-to-phenotype relationships and their widespread importance and utility in crop improvement.

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.

Peach Carboxylesterase PpCXE1 Is Associated with Catabolism of Volatile Esters.

Peach fruit volatile acetate esters impact consumer sensory preference and contribute to defense against biotic stresses. Previous studies showed that alcohol acyltransferase (AAT) family PpAAT1 is correlated with volatile ester formation in peach fruits. However, fruits also contain carboxylesterase (CXE) enzymes that hydrolyze esters. The functions of this family with regard to volatile ester content has not been explored. Here, we observed that content of acetate ester was negatively correlated with expression of PpCXE1. Recombinant PpCXE1 protein exhibited hydrolytic activity toward acetate esters present in peach fruit. Kinetic analysis showed that PpCXE1 showed the highest catalytic activity toward E-2-hexenyl acetate. Subcellular localization demonstrated that PpCXE1 is present in the cytoplasm. Transient expression in peach fruit and stable overexpression in tomato fruit resulted in significant reduction of volatile esters in vivo. Taken together, the results indicate that PpCXE1 expression is associated with catabolism of volatile acetate esters in peach fruit.

Next-Gen Approaches to Flavor-Related Metabolism.

Although flavor is an essential element for consumer acceptance of food, breeding programs have focused primarily on yield, leading to significant declines in flavor for many vegetables. The deterioration of flavor quality has concerned breeders; however, the complexity of this trait has hindered efforts to improve or even maintain it. Recently, the integration of flavor-associated metabolic profiling with other omics methodologies derived from big data has become a prominent trend in this research field. Here, we provide an overview of known metabolites contributing to flavor in the major vegetables as well as genetic analyses of the relevant metabolic pathways based on different approaches, especially multi-omics. We present examples demonstrating how omics analyses can help us to understand the accomplishments of historical flavor breeding practices and implement further improvements. The integration of genetics, cultivation, and postharvest practices with genome-scale data analyses will create enormous potential for further flavor quality improvements.

UDP-glucosyltransferase PpUGT85A2 controls volatile glycosylation in peach.

The monoterpene linalool is a major contributor to aroma and flavor in peach (Prunus persica) fruit. It accumulates during fruit ripening, where up to ~40% of the compound is present in a non-volatile glycosylated form, which affects flavor quality and consumer perception by retronasal perception during tasting. Despite the importance of this sequestration to flavor, the UDP-glycosyltransferase (UGT) responsible for linalool glycosylation has not been identified in peach. UGT gene expression during peach fruit ripening and among different peach cultivars was analyzed using RNA sequencing, and transcripts correlated with linalyl-ß-d-glucoside were selected as candidates for functional analysis. Kinetic resolution of a racemic mixture of R,S-linalool was shown for PpUGT85A2, with a slight preference for S-(+)-linalool. PpUGT85A2 was shown to catalyze synthesis of linalyl-ß-d-glucoside in vitro, although it did not exhibit the highest enzyme activity between tested substrates. Subcellular localization of PpUGT85A2 in the cytoplasm and nucleus was detected. Application of linalool to peach leaf disks promoted PpUGT85A2 expression and linalyl-ß-d-glucoside generation. Transient expression in peach fruit and stable overexpression in tobacco and Arabidopsis resulted in significant accumulation of linalyl-ß-d-glucoside in vivo. Taken together, the results indicate that PpUGT85A2 expression is a major control point predicting linalyl-ß-d-glucoside content.