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

Abstract

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.

Figure 1

Overview of the methyl salicylate and salicylic acid biosynthesis pathway. Pathway components located in the chloroplast are found within the green circle. Enzymes are in red. PAL, phenyalanine ammonia lyase; BA2H, benzoic acid 2-hydroxylase; SlMES, methyl esterase; SlUGT5, UDP glycosyltransferase; NSGT1, non-smoky glycosyltransferase 1.

Figure 2

Identification of SlMES candidate genes by a GWAS. The X-axis represents different chromosomes in different colors and the Y-axis represents the –log10(P-value) of the variants used for analysis. Two major loci on chromosomes 2 and 9 are associated with methyl salicylate production. The locus on chromosome 2 (green) overlaps with a cluster of methyl esterases, whereas the locus on chromosome 9 (purple) corresponds to the NSGT1 locus (Tikunov et al., 2013). The green horizontal solid and dashed lines represent significance thresholds for the association a significant level of 5% and 1%, respectively. The zoomed in region from most significant peak on chromsome 2 represents the gene models in MES locus. The green and purple bars represents the introns and exons of the genes, respectively, with each gene’s name and Solyc number below the models. The scale above the gene model represents the scale of physical position of the genes in the tomato genome.

Figure 3

Mapping of MeSA2.1 (QTL associated with methyl salicylate on chromosome 2) and MeSA9.1 (QTL associated with methyl salicylate on chromosome 9) in a biparental population. A, Frequency histogram of the number of plants (Y-axis) and methyl salicylate levels in nanogram per gram fruit weight per hour (X-axis). X-axis has a scale break from 15 to 85 to accommodate the high level of skewness for low levels of methyl salicylate and present the low counts of accessions having high level of methyl salicylate. B, Composite interval mapping of methyl salicylate with the markers indicated by the black triangles. logarithm of the odds (LOD) score (blue) and confidence interval at 95% (gray) and 99% (black) are represented. The percentage of trait variation accounted by each significant QTL is also shown. LOD is calculated as –log10(P-value) and is the measure of strength of evidence for the presence of a QTL at a particular location. C, Strong synergistic interaction between MES and NSGT1 loci on methyl salicylate levels. The boxplots represents the interaction between MES and NSGT1 loci using the most significant marker (position provided in the parenthesis below the locus name) from each locus identified from QTL mapping. The Y-axis represents the methyl salicylate levels in red ripe fruits (in nanogram per gram fruit weight per hour) of F₂ plants. The primary X-axis represents different alleles at MES (BGV006931, BGV014508, and Heterozygous alleles) represented by the most significant KASP marker on chromosome 2 from QTL mapping and secondary X-axis represents different alleles of NSGT1 (BGV006931, BGV014508, and Heterozygous alleles) represented by the most significant marker on chromosome 9. The center line is the median, with individual measurements represented as dots, and the upper and lower quartiles shown in boxes. D, A representation of the parental haplotypes demonstrate that the four SlMES genes are at different spacing from one another and an insertion in SlMES3 leading to a truncated version of the protein.

Figure 4

SlMES affects methyl salicylate levels independently of characterized methyl salicylate synthetic genes. Methyl salicylate levels in BGV008218 and BGV006779, which contain the same NSGT1 haplotype. Methyl salicylate was collected from the headspace of chopped tomato fruit for 1 h, then eluted and run on an Agilent LC–MS. Values are reported as the nanograms of methyl salicylate per gram of fresh tomato weight per hour of collection and represent the average of at least three technical and biological replicates collected throughout the growing season; bars are standard error. Each replicate contained five to six plants.

Figure 5

Conservation between MES locus genes. A, Phylogeny of the MES locus genes. MES1 from S. lycopersicoides (Solyd02g058110) was selected as the outgroup. Solyd02g058110 is methylesterase in S. lycopersicoides. Scale bar indicates nucleotide substitutions per site. B, Conserved protein motifs in methyl esterases from the tomato reference genome Heinz1706 and other plant species. Motifs were numbered in descending size order. Predicted active site and pocket residues are highlighted in yellow, and show that SlMES3 in the reference variety is missing Motif 5, which is expected to decrease protein activity. C, Alignment of SlMES3 to SlMES2, using the Heinz reference sequence for both and the Needleman–Wunsch algorithm. A line connecting two residues indicates they are identical, two dots connecting residues indicates they are similar but not identical, and a single dot means the residues are not similar. Coloring corresponds with the Motifs 1–8 in (B). Predicted active site and pocket residues are indicated with stars.

Figure 6

Relative expression of methyl esterase (MES) genes in different tomato accessions in ripe tomato and their corresponding methyl salicylate levels are also presented besides the accessions name. The methyl salicylate levels are in nanogram per gram fruit weight per hour). The MES genes expression is normalized using the expression of housekeeping gene, ACT4 (Solyc04g011500). Error bars show the standard error of two technical replicates.

Figure 7

Methyl salicylate levels in single, double, and triple slmes mutants. Methyl salicylate was collected from chopped ripe fruit for an hour and measured on an Agilent GC–MS, reported as nanograms of volatile per gram of ripe tomato fruit. Results are from three individual mutation lines with two to three reps per line and three to five plants per rep. A, Levels of methyl salicylate in ripe fruit from Brandywine (wild-type) and the single slmes1 loss-of-function mutants grown in field conditions. B, Levels of methyl salicylate in ripe fruit from wild-type and single slmes1 loss-of-function mutants grown in greenhouse conditions. For all graphs, numbers above each bar are the mean volatile content, and error bars represent the standard error. *P < 0.05, **P < 0.01, ***P < 0.001 ****P < 0.0001 by Student’s t test to the wild-type. C, Methyl salicylate levels in the fruit of Moneymaker and Brandywine and in double and triple slmes loss of function mutants. The letter above the standard error bars show significance using Tukey’s significance test (P < 0.05).

Figure 8

SlMES1 is a methyl esterase acting on methyl salicylate and methyl benzoate. A, Purified His-tagged SlMES1 and SlMES3 were incubated with methyl salicylate for up to 30 min before the reaction was stopped with HCl and volatile starting material remaining quantified on a mass spectrometer. Methyl salicylate was undetectable after 30 min incubation with SlMES1 and decreased by ∼80% after incubation with SlMES3 for the same length of time. B, Salicylic acid inhibits the conversion of methyl salicylate to salicylic acid by SlMES1. When salicylic acid is added to the reaction mix, more methyl salicylate remains at the end of the reaction. C, Predicted reaction and K_m’s of SlMES1 and SlMES3 based on action on methyl salicylate, and observations from other species that methyl salicylate is converted to salicylic acid and methanol by the action of similar methyl esterases. Results for SlMES1 are from four replicates; results for SlMES3 are from three replicates.