Mark G. Taylor

The chemical interactions underlying tomato flavor preferences.

Although human perception of food flavors involves integration of multiple sensory inputs, the most salient sensations are taste and olfaction. Ortho- and retronasal olfaction are particularly crucial to flavor because they provide the qualitative diversity so important to identify safe versus dangerous foods. Historically, flavor research has prioritized aroma volatiles present at levels exceeding the orthonasally measured odor threshold, ignoring the variation in the rate at which odor intensities grow above threshold. Furthermore, the chemical composition of a food in itself tells us very little about whether or not that food will be liked. Clearly, alternative approaches are needed to elucidate flavor chemistry. Here we use targeted metabolomics and natural variation in flavor-associated sugars, acids, and aroma volatiles to evaluate the chemistry of tomato fruits, creating a predictive and testable model of liking. This nontraditional approach provides novel insights into flavor chemistry, the interactions between taste and retronasal olfaction, and a paradigm for enhancing liking of natural products. Some of the most abundant volatiles do not contribute to consumer liking, whereas other less abundant ones do. Aroma volatiles make contributions to perceived sweetness independent of sugar concentration, suggesting a novel way to increase perception of sweetness without adding sugar.

Flavour compounds in tomato fruits: identification of loci and potential pathways affecting volatile composition

The unique flavour of a tomato fruit is the sum of a complex interaction among sugars, acids, and a large set of volatile compounds. While it is generally acknowledged that the flavour of commercially produced tomatoes is inferior, the biochemical and genetic complexity of the trait has made breeding for improved flavour extremely difficult. The volatiles, in particular, present a major challenge for flavour improvement, being generated from a diverse set of lipid, amino acid, and carotenoid precursors. Very few genes controlling their biosynthesis have been identified. New quantitative trait loci (QTLs) that affect the volatile emissions of red-ripe fruits are described here. A population of introgression lines derived from a cross between the cultivated tomato Solanum lycopersicum and its wild relative, S. habrochaites, was characterized over multiple seasons and locations. A total of 30 QTLs affecting the emission of one or more volatiles were mapped. The data from this mapping project, combined with previously collected data on an IL population derived from a cross between S. lycopersicum and S. pennellii populations, were used to construct a correlational database. A metabolite tree derived from these data provides new insights into the pathways for the synthesis of several of these volatiles. One QTL is a novel locus affecting fruit carotenoid content on chromosome 2. Volatile emissions from this and other lines indicate that the linear and cyclic apocarotenoid volatiles are probably derived from separate carotenoid pools.

Histology of, and physical factors affecting, transient GUS expression in pearl millet (Pennisetum glaucum (L.) R. Br.) embryos following microprojectile bombardment

Transient GUS (β-glucuronidase) expression was visualized in whole and sectioned embryos of Pennisetum glaucum (L.) R. Br. (pearl millet) after microprojectile bombardment with pMON 8678 DNA. Strongest GUS expression occurred in cells located in the center of GUS positive spots with decreasing intensity in surrounding cells. GUS positive cells could be seen up to 12 cell layers beneath the epidermis. Needle-like crystals of the GUS assay product were found throughout the cytoplasm of GUS positive cells. The number of GUS positive spots was correlated to the microprojectile spread pattern on the medium surface. Shorter bombardment distances (6.6 and 9.8 cm) and the standard accelerator speed gave the best results for transient expression but also caused maximum tissue damage. The speed and distance, however, had little influence on the ability of bombarded embryos to form compact callus. The developmental stage of the bombarded immature embryos was the determining factor in the formation of compact callus, from which plants were regenerated.

Enhanced GUS gene expression in cereal/grass cell suspensions and immature embryos using the maize uhiquitin-based plasmid pAHC25

Transient GUS (β-glucuronidase) expression was visualized in cell suspensions of Triticum aestivum, Zea mays, Pennisetum glaucum, Saccharum officinarum, Pennisetum purpureum and Panicum maximum after microprojectile bombardment with pBARGUS and pAHC25 plasmid DNAs. pBARGUS contains the GUS (UidA) gene coding region driven by the Adh1 promoter and the Adh1 intron 1, as well as the BAR gene coding region driven by the CaMV 35S promoter and the Adh1 intron 1. pAHC25 contains the GUS and BAR gene coding regions driven by the maize ubiquitin promoter, first exon and first intron (Ubi1). The effectiveness of the constructs was first compared in cell suspension cultures by counting blue expression units (b.e.u.). The expression of construct pAHC25 ranged from 3 to 50 fold greater than pBARGUS in different species. In addition, the two plasmids were quantitatively compared in Triticum aestivum and Zea mays by using the more sensitive GUS fluorometric assay to determine the amount of methylumbellyferride (MU) produced. There was more than a 30 fold increase in MU production with pAHC25 than with pBARGUS in the wheat suspension, while the maize suspension showed only a 2.5 fold increase with the pAHC25 construct. Transient GUS expression was also visualized in immature embryos of Pennisetum glaucum following bombardment with pBARGUS and pAHC25 DNA. Expression of plasmid pAHC25 was twice as high as pBARGUS. A comparison of two DNA/gold preparation methods, as well as repeated sonications of the DNA/gold mixture, had no effect on the number of b.e.u.

A chemical genetic roadmap to improved tomato flavor

Looking for lost flavor in tomatoes Commercially available tomatoes are renowned these days for sturdiness, but perhaps not for flavor. Heirloom varieties, on the other hand, maintain the richer flavors and sweeter tomatoes of years past. Tieman et al. combined tasting panels with chemical and genomic analyses of nearly 400 varieties of tomatoes. They identified some of the flavorful components that have been lost over time. Identification of the genes that have also gone missing provides a path forward for reinstating flavor to commercially grown tomatoes. Science, this issue p. 391 Genomic analysis shows what genes to put back to reinstate flavor in tomatoes. Modern commercial tomato varieties are substantially less flavorful than heirloom varieties. To understand and ultimately correct this deficiency, we quantified flavor-associated chemicals in 398 modern, heirloom, and wild accessions. A subset of these accessions was evaluated in consumer panels, identifying the chemicals that made the most important contributions to flavor and consumer liking. We found that modern commercial varieties contain significantly lower amounts of many of these important flavor chemicals than older varieties. Whole-genome sequencing and a genome-wide association study permitted identification of genetic loci that affect most of the target flavor chemicals, including sugars, acids, and volatiles. Together, these results provide an understanding of the flavor deficiencies in modern commercial varieties and the information necessary for the recovery of good flavor through molecular breeding.

Functional analysis of a tomato salicylic acid methyl transferase and its role in synthesis of the flavor volatile methyl salicylate

Methyl salicylate (MeSA) is a volatile plant secondary metabolite that is an important contributor to taste and scent of many fruits and flowers. It is synthesized from salicylic acid (SA), a phytohormone that contributes to plant pathogen defense. MeSA is synthesized by members of a family of O-methyltransferases. In order to elaborate the mechanism of MeSA synthesis in tomato, we screened a set of O-methyltransferases for activity against multiple substrates. An enzyme that specifically catalyzes methylation of SA, SlSAMT, as well as enzymes that act upon jasmonic acid and indole-3-acetic acid were identified. Analyses of transgenic over- and under-producing lines validated the function of SlSAMT in vivo. The SlSAMT gene was mapped to a position near the bottom of chromosome 9. Analysis of MeSA emissions from an introgression population derived from a cross with Solanum pennellii revealed a quantitative trait locus (QTL) linked to higher fruit methyl salicylate emissions. The higher MeSA emissions associate with significantly higher SpSAMT expression, consistent with SAMT gene expression being rate limiting for ripening-associated MeSA emissions. Transgenic plants that constitutively over-produce MeSA exhibited only slightly delayed symptom development following infection with the disease-causing bacterial pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). Unexpectedly, pathogen-challenged leaves accumulated significantly higher levels of SA as well as glycosylated forms of SA and MeSA, indicating a disruption in control of the SA-related metabolite pool. Taken together, the results indicate that SlSAMT is critical for methyl salicylate synthesis and methyl salicylate, in turn, likely has an important role in controlling SA synthesis.

Identification of Genes in the Phenylalanine Metabolic Pathway by Ectopic Expression of a MYB Transcription Factor in Tomato Fruit

Ectopic expression of a MYB transcription factor, ODO1, in tomato fruit altered Phe and phenylpropanoid metabolism, as shown by metabolic profiling and flux analysis. This disruption permitted identification of a set of coregulated genes, including prephenate aminotransferase, which converts prephenate to arogenate. The results establish a fruit arogenate pathway for Phe synthesis. Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and 13C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles.

A 13-lipoxygenase, TomloxC, is essential for synthesis of C5 flavour volatiles in tomato

C5 volatile compounds, derived from fatty acids, are among the most important contributors to consumer liking of fresh tomatoes. Despite their important roles in flavour, the genes responsible for C5 volatile synthesis have yet to be identified. This work shows that their synthesis is catalysed in part by a 13-lipoxygenase (LOX), TomloxC, the same enzyme responsible for synthesis of C6 volatiles. C5 synthesis is independent of hydroperoxide lyase (HPL); moreover, HPL knockdown significantly increased C5 volatile synthesis. This LOX-dependent, HPL-independent pathway functions in both fruits and leaves. Synthesis of C5 volatiles increases in leaves following mechanical wounding but does not increase in response to infection with Xanthomonas campestris pv. vesicatoria. Large reductions in C5 and C6 volatiles in antisense TomloxC knockdown plants were observed but those reductions did not alter the development of disease symptoms, indicating that these volatiles do not have an important defensive function against this bacterial pathogen.

Catabolism of Branched Chain Amino Acids Supports Respiration but Not Volatile Synthesis in Tomato Fruits

The branched-chain amino acid transaminases (BCATs) have a crucial role in metabolism of the branched-chain amino acids leucine, isoleucine, and valine. These enzymes catalyze the last step of synthesis and the initial step of degradation of these amino acids. Although the biosynthetic pathways of branched chain amino acids in plants have been extensively investigated and a number of genes have been characterized, their catabolism in plants is not yet completely understood. We previously characterized the branched chain amino acid transaminase gene family in tomato, revealing both the subcellular localization and kinetic properties of the enzymes encoded by six genes. Here, we examined possible functions of the enzymes during fruit development. We further characterized transgenic plants differing in the expression of branched chain amino acid transaminases 1 and 3, evaluating the rates of respiration in fruits deficient in BCAT1 and the levels of volatiles in lines overexpressing either BCAT1 or BCAT3. We quantitatively tested, via precursor and isotope feeding experiments, the importance of the branched chain amino acids and their corresponding keto acids in the formation of fruit volatiles. Our results not only demonstrate for the first time the importance of branched chain amino acids in fruit respiration, but also reveal that keto acids, rather than amino acids, are the likely precursors for the branched chain flavor volatiles.

Characterization of the Branched-Chain Amino Acid Aminotransferase Enzyme Family in Tomato

Branched-chain amino acids (BCAAs) are synthesized in plants from branched-chain keto acids, but their metabolism is not completely understood. The interface of BCAA metabolism lies with branched-chain aminotransferases (BCAT) that catalyze both the last anabolic step and the first catabolic step. In this study, six BCAT genes from the cultivated tomato (Solanum lycopersicum) were identified and characterized. SlBCAT1, -2, -3, and -4 are expressed in multiple plant tissues, while SlBCAT5 and -6 were undetectable. SlBCAT1 and -2 are located in the mitochondria, SlBCAT3 and -4 are located in chloroplasts, while SlBCAT5 and -6 are located in the cytosol and vacuole, respectively. SlBCAT1, -2, -3, and -4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, but SlBCAT1 and -2 were less effective than SlBCAT3 and -4 in growth restoration. All enzymes were active in the forward (BCAA synthesis) and reverse (branched-chain keto acid synthesis) reactions. SlBCAT3 and -4 exhibited a preference for the forward reaction, while SlBCAT1 and -2 were more active in the reverse reaction. While overexpression of SlBCAT1 or -3 in tomato fruit did not significantly alter amino acid levels, an expression quantitative trait locus on chromosome 3, associated with substantially higher expression of Solanum pennellii BCAT4, did significantly increase BCAA levels. Conversely, antisense-mediated reduction of SlBCAT1 resulted in higher levels of BCAAs. Together, these results support a model in which the mitochondrial SlBCAT1 and -2 function in BCAA catabolism while the chloroplastic SlBCAT3 and -4 function in BCAA synthesis.