Significance Cold storage is widely used to extend shelf-life of agriculture products. For tomato, this handling results in reduced flavor quality. Our work provides major insights into the effects of chilling on consumer liking, the flavor metabolome and transcriptome, as well as DNA methylation status. Transcripts for some key volatile synthesis enzymes and the most important ripening-associated transcription factors are greatly reduced in response to chilling. These reductions are accompanied by major changes in the methylation status of promoter regions. Transient increases in DNA methylation occur during chilling. Our analysis provides insight into the molecular mechanisms of tomato fruit flavor loss caused by chilling.

Abstract 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.

The modern commercial tomato is widely perceived as lacking flavor and is a major source of consumer dissatisfaction. Postharvest handling and retail systems are major contributors to poor flavor, particularly the commonly used practice of chilling fruit. Many consumers store purchased fruits in the refrigerator, further contributing to flavor deterioration (1). Tomato flavor is produced by a combination of sugars, acids, and volatiles (2, 3). Production of flavor-associated volatiles is sensitive to temperatures below 12 °C and loss of volatiles has been observed during cold storage (4, 5). In contrast, taste-related chemicals, sugars, and acids, are not significantly affected by cold storage (6, 7).

Flavor-imparting volatiles are derived from amino acids, fatty acids, and carotenoids, and multiple genes essential for their synthesis have been functionally validated (8). For example, C6 volatiles are synthesized by a lipoxygenase, LoxC (9), hydroperoxide lyase (HPL) (10), and ALCOHOL DEHYDROGENASE2 (ADH2) (11). Volatile esters are synthesized by an ALCOHOL ACETYLTRANSFERASE1 (AAT1) (12). The CAROTENOID CLEAVAGE DIOXYGENASE1 (CCD1) contributes to synthesis of volatile apocarotenoids (13). Synthesis of many tomato flavor volatiles increases during fruit ripening. The ripening mutants, Colorless nonripening (Cnr) and nonripening (nor) produce substantially lower levels of lipid-derived volatiles than wild-type (14). One of the main transcription factors (TFs) mediating ripening is RIPENING INHIBITOR (RIN). RIN-binding sites are frequently demethylated upon ripening and binding occurs in concert with demethylation (15), indicating that promoter methylation state influences expression of RIN-dependent genes.

Transcriptomic analysis has identified genes associated with tomato fruit chilling. For example, 14-d chilling of Micro-Tom fruit resulted in differential expression of many genes related to photosynthesis, lipid metabolism, cell wall modification, and antioxidant production (16). Although this work has provided insights into chilling-induced gene expression, it did not directly address the molecular basis for loss of flavor volatiles. Several groups have analyzed changes in LOX activity and C6 volatiles in response to cold storage; reduced production of C6 volatiles cannot be directly explained by LOX activity alone (1, 5).

These observations provide a framework for analysis of the molecular mechanism underlying chilling-induced loss of tomato flavor. In addition to the economic impact of flavor quality loss, the tomato fruit provides an ideal system in which to examine the effects of environmental stress on a genome scale. Here, we provide a comprehensive analysis of the effect of chilling on the transcriptome and flavor metabolome. The large transcriptional reprogramming that occurred in response to chilling and following a recovery period was correlated with alterations in DNA methylation.

Discussion Although modern high-yielding varieties of tomatoes are not as flavorful as older varieties (8), a significant part of the perceived problem in modern commercial tomatoes can be attributed to postharvest chilling (4). Exposure to temperatures as low as 4 °C causes severe damage to flavor quality (1). To determine the underlying molecular basis for that flavor deterioration, we undertook a systematic analysis of fruits exposed to chilling temperature, examining changes in the flavor metabolome, the transcriptome, and alterations in patterns of DNA methylation. Chilling did not alter fruit sugar and acid contents (Fig. 1). However, significant loss of flavor volatiles was observed for fruit stored at 5 °C for 8 d. Even after a 1-d recovery period at 20 °C, volatile composition was still significantly lower than in unchilled fruit, resulting in lower overall consumer liking. Twelve volatiles were significantly altered by cold storage in multiple seasons and cultivars. We observed significant reductions in the contents of volatiles associated with the C5/C6, branched-chain amino acid and ester pathways (Fig. 1C). These reductions were correlated with significantly lower transcript abundance of genes whose products are essential for their synthesis. Although transcript content of some of these genes increased after moving fruits to room temperature for 24 h, most remained significantly lower than in unchilled fruits (Fig. 3). Unlike sugars and acids, volatiles are freely diffusible through the stem scar and must be constantly replenished to maintain appropriate levels within harvested fruit. Because expression of genes encoding essential biosynthetic enzymes is significantly lower at 5 °C, chilling leads to depletion of important flavor volatiles and reduced flavor quality. Although total fruit volatile content was significantly lower in chilled fruits, a subset of flavor volatiles increased during chilling. The contents of two volatiles derived from lycopene cleavage, MHO and geranial, were higher after chilling (Fig. 3D and SI Appendix, Fig. S1B). These volatiles are produced by oxidative cleavage of lycopene, which makes up ∼85% of the carotenoid pool in a ripe fruit (13, 34, 35). Cleavage can be either enzymatic, catalyzed by carotenoid cleavage dioxygenases, or nonenzymatic. The CCD1B transcript is significantly lower in chilled fruit, as are transcripts of multiple carotenoid biosynthetic pathway genes (Fig. 3D and SI Appendix, Fig. S6). The most likely explanation for increased MHO and geranial content is chilling-induced nonenzymatic carotenoid oxidation. Production of reactive oxygen species is one of the main responses of fruit subjected to abiotic stresses, such as high light and cold, and carotenoids were reported to be the main quenchers for singlet oxygen (36, 37). Nonenzymatic oxidation of carotenoids is the main mechanism for production of apocarotenoid volatiles in Arabidopsis exposed to high light stress (38). Compared with ripe fruit on the day of harvest, RNA-Seq analysis detected 5,413 DEGs during cold storage, and 528 DEGs after recovery at ambient temperature (SI Appendix, Fig. S5A), indicating that expression of many genes is sensitive to temperature shift. A global view of transcriptional changes showed that carbohydrate metabolism was significantly down-regulated (Fig. 2A), indicating that energy metabolism is suppressed during cold storage. Our transcriptome data are consistent with previously described proteome changes in chilled tomato fruit, in which proteins belonging to energy metabolism BINs were significantly suppressed (7). Functional classes associated with amino acids, fatty acids, and secondary metabolism were reduced by cold storage followed by a recovery after transfer to 20 °C. In line with the major reprogramming of gene expression during chilling, multiple TFs associated with fruit development exhibited significantly altered transcript abundance in response to chilling (Fig. 3E). In particular, TFs that are essential to ripening are down, including RIN (39), NOR (40), and CNR (41). Reduced expression of these TFs in response to chilling would be expected to globally reduce many ripening-associated processes, permitting the organ to redirect metabolic resources into more suitable stress responses. In addition to these TFs, transcripts of FUL1, a RIN-interacting MADS domain protein affecting aspects of ripening, including volatile synthesis (42), as well as HB-1, a positive regulator of ethylene synthesis (43), are down during chilling. Expression of other TFs that regulate specific aspects of fruit development, including TAGL1 (44, 45) and AP2a (46, 47), increased during chilling. Notably, AP2a is a negative regulator of ethylene synthesis and fruit ripening. Thus, an increase in its expression is consistent with the observed reduction in expression of ethylene synthesis genes. The tomato chilling response also includes CBF transcriptional activators. Transcripts of all three CBF genes (CBF1–3) were significantly elevated in response to chilling and returned to basal levels upon return to ambient temperature (Fig. 2B). Previous comparison of CBF action in tomato indicated that the tomato CBF regulon is considerably smaller than its Arabidopsis counterpart (21). We examined chilling-induced expression of all of the closest homologs of the Arabidopsis regulon. The Arabidopsis regulon consists of 133 up-regulated and 39 down-regulated genes (22). Of the combined 172 genes, 27 exhibited significantly different expression during chilling and that differential expression was often in the opposite direction as in Arabidopsis (SI Appendix, Table S4). Thus, although there is a CBF-associated response to chilling, that response is substantially different from the chilling tolerant Arabidopsis. Finally, tomato fruit ripening is associated with major alterations in DNA (cytosine) methylation, particularly in RIN-binding promoter regions, likely mediated by DML2. This epigenetic reprogramming is essential for ripening (15, 33). We identified 30,918 DMRs in chilled fruits, with 9,951 being in promoter regions. Interestingly, much of the methylation was transient, returning to a prechilled state after 24 h at room temperature. Notably, RIN and many—although not all—of its target genes, displayed an inverse correlation between promoter methylation and transcript abundance (Fig. 5 and SI Appendix, Fig. S6). Our data do not permit us to conclude whether DNA methylation is the cause or an effect of reduced expression. We can conclude that chilling stress causes major changes in the methylation status of the genome and many of these changes occur in promoters of genes known to contribute to ripening, quality, and flavor volatile synthesis, and are altered in response to chilling. It is possible that this methylation associated with reduced expression is an added level of insurance against expression of genes that are not essential to respond to this environmental stress. This point is especially relevant, given that many of the chilling-induced alterations in transcriptome activity appear to operate through broadly acting fruit developmental regulators, such as RIN. It will be interesting to determine whether stress-induced changes in methylation are fruit-specific or generalized to other biotic and abiotic stresses in the tomato plant. In conclusion, we have demonstrated that chilling of ripe tomato fruits results in significantly reduced flavor quality. That reduction is associated with major alterations in contents of volatiles associated with consumer liking. Reduced levels of specific volatiles are associated with greatly reduced levels of transcripts for some key volatile synthesis enzymes. Expression of genes encoding TFs that are essential for ripening, including RIN, NOR, and CNR, are also reduced in response to chilling and may be responsible for reduced transcript levels in the many genes during chilling. Those reductions are accompanied by major changes in the methylation status of promoters, including those of the aforementioned TFs, 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.

Materials and Methods Tomato Fruit Treatment. Tomatoes were grown in a greenhouse on the University of Florida campus. Fruit at the full red ripe stage, free of visual defects, and uniform in size were selected, washed with water, and air dried. Tomatoes were divided into three groups: (i) stored at 5 °C with 92% relative humidity for 7 d, and then transferred to 20 °C for 1-d recovery; (ii) held at 5 °C for 8 d without recovery at ambient temperature; and (iii) fruit on the day of harvest as controls. The first two groups were harvested 8 d before the third group, and fruit were subjected to consumer test on the day of the third harvest. Consumer Tests Analysis. All consumer tests were approved by the University of Florida Institutional Review Board. Taste panels consisted of 76 persons. Panelists rated overall liking of chilled and unchilled Ailsa Craig tomatoes using a hedonic general labeled magnitude scale, as described previously (3). Chilled tomatoes were less liked than unchilled tomatoes when panelists’ liking scores were compared as matched pairs using a one-tailed t test, sign test, or Wilcoxon signed rank test. After measurement of flavor quality, pericarp tissue was frozen in liquid nitrogen and stored at −80 °C until sugar and acid analysis. Ethylene Production Analysis. Fruit were sealed in 500-mL containers for 1 h, and 1 mL of headspace gas samples were analyzed using a HP5890 series II gas chromatograph (Hewlett Packard) equipped with a flame ionization detector. The temperature program was 110 °C for oven, 110 °C for injection port, and 130 °C for detector. Volatile Analysis. Volatile analysis was performed according to the method described previously (48), with three biological replicates of six pooled fruit each. Chopped ripe tomatoes were enclosed in glass tubes flowing with filtered air for 1 h, and volatiles were extracted using a Super Q column. Volatiles were eluted with methylene chloride using nonyl acetate as an internal control, and separated on an Agilent 6890N gas chromatograph equipped with a DB-5 column (Agilent). Retention times were compared with authentic standards, and volatile contents were calculated as ng⋅g−1 fresh weight (FW) h−1. Sugars and Acids Analysis. Contents of glucose, fructose, malic acid, and citric acid were determined as described previously (35). Analysis was performed on three biological replicates, each consisting of six fruit. RNA Isolation and High-Throughput Sequencing. RNA was extracted using an RNeasy Mini kit (Qiagen) following the manufacturer’s instructions, and quality was monitored by gel electrophoresis and A260/A280. Libraries for high-throughput Illumina strand-specific RNA-Seq were prepared as described previously (49). Three biological replicates for each treatment were prepared, each consisting of multiple pooled fruits. The statistics of sequencing quality and correlation data illustrated the global relative relationship between biological replicates and among fruit samples are presented in SI Appendix, Tables S10 and S11. DNA Sequencing. Illumina DNA sequencing was performed on a HiSeq2500 using reagents and protocols provided by Illumina, alignment to the reference tomato genome (v2.40) and determination of expression for each gene were performed as described previously (17). DNA Methylation. Genomic DNA was extracted using a Qiagen DNeasy Plant Mini Kit (https://www.qiagen.com/us), and quality was monitored by gel electrophoresis and a ratio of A260/A280. Bisulfite conversion of tomato genomic DNA and Illumina sequencing were performed as described previously (15). All of the chilling-related methylome data generated for this paper are archived at single-base resolution in the tomato epigenome database at ted.bti.cornell.edu/epigenome/. Statistical Analysis. Volatile emissions were subjected to one-way ANOVA analysis (OriginPro 9.0, Microcal Software). PCA was selected to provide an overview of changes in detected volatiles and global gene-expression patterns in response to cold storage (www.metaboanalyst.ca). DEGs were defined by reads per kilobase per million (RPKM) fold-change > 2 and FDR < 0.05. GO enrichment (geneontology.org/) was performed. DEGs were classified into functional categories defined by MapMan BINs (mapman.gabipd.org). Wilcoxon test and Benjamini–Hochberg correction were performed to provide a statistical-based graphic display of enriched BINs (19).

Acknowledgments We thank Drs. Charles Sims and Asli Odabasi for their help with the consumer panel; Dawn Bies for help with volatile collection; Mark Taylor for help with plant care; and Michael Thomashow for invaluable advice. This work was supported by National Science Foundation Grants IOS-0923312 (to H.J.K., J.J.G., and Z.F.); National Key Research and Development Program 2016YFD0400101; Program of International Science and Technology Cooperation Grant 2011DFB31580; the New Star Program from Zhejiang University; and the China Scholarship Council.