Author response: Evolution of a plant gene cluster in Solanaceae and emergence of metabolic diversity (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity. eLife digest Plants produce a vast variety of different molecules known as secondary or specialized metabolites to attract pollinating insects, such as bees, or protect themselves against herbivores and pests. The secondary metabolites are made from simple building blocks that are readily available in plants, including amino acids, fatty acids and sugars. Different species of plant, and even different parts of the same plant, produce their own sets of secondary metabolites. For example, the hairs on the surface of tomatoes and other members of the nightshade family of plants make metabolites known as acylsugars. These chemicals deter herbivores and pests from damaging the plants. To make acylsugars, the plants attach long chains known as fatty acyl groups to molecules of sugar, such as sucrose. Some members of the nightshade family produce acylsugars with longer chains than others. In particular, acylsugars with long chains are only found in tomatoes and other closely-related species. It remained unclear how the nightshade family evolved to produce acylsugars with chains of different lengths. To address this question, Fan et al. used genetic and biochemical approaches to study tomato plants and other members of the nightshade family. The experiments identified two genes known as AACS and AECH in tomatoes that produce acylsugars with long chains. These two genes originated from the genes of older enzymes that metabolize fatty acids – the building blocks of fats – in plant cells. Unlike the older genes, AACS and AECH were only active at the tips of the hairs on the plant’s surface. Fan et al. then investigated the evolutionary relationship between 11 members of the nightshade family and two other plant species. This revealed that AACS and AECH emerged in the nightshade family around the same time that longer chains of acylsugars started appearing. These findings provide insights into how plants evolved to be able to produce a variety of secondary metabolites that may protect them from a broader range of pests. The gene cluster identified in this work could be used to engineer other species of crop plants to start producing acylsugars as natural pesticides. Introduction Despite the enormous structural diversity of plant specialized metabolites, they are derived from a relatively small number of primary metabolites, such as sugars, amino acids, nucleotides, and fatty acids (Maeda, 2019). These lineage-, tissue- or cell- type specific specialized metabolites mediate environmental interactions, such as herbivore and pathogen deterrence or pollinator and symbiont attraction (Mithöfer and Boland, 2012; Pichersky and Lewinsohn, 2011). Specialized metabolism evolution is primarily driven by gene duplication (Moghe and Last, 2015; Panchy et al., 2016), and relaxed selection of the resulting gene pairs allows modification of cell- and tissue-specific gene expression and changes in enzymatic activity. This results in expanded substrate recognition and/or diversified product formation (Khersonsky and Tawfik, 2010; Leong and Last, 2017). The neofunctionalized enzymes can prime the origin and diversification of specialized metabolic pathways (Schenck and Last, 2020; Weng et al., 2012; Weng, 2014). There are many examples of mechanisms that lead to novel enzymatic activities in specialized cell- or tissue-types, however, the principles that govern assembly of multi-enzyme specialized metabolic pathways are less well established. One appealing hypothesis involves the stepwise recruitment of pathway enzymes (Noda-Garcia et al., 2018). In rare cases, non-homologous specialized metabolic enzyme genes occur in proximity to each other in a genomic region, forming a biosynthetic gene cluster (Nützmann et al., 2016; Nützmann and Osbourn, 2014; Rokas et al., 2018). In recent years, an increasing number of specialized metabolic gene clusters (SMGCs) were experimentally identified or bioinformatically predicted in plants (Boutanaev et al., 2015; Castillo et al., 2013; Schläpfer et al., 2017). However, although most experimentally characterized plant SMGCs are co-expressed, the majority of the bioinformatically predicted ones do not show coexpression under global network analysis (Wisecaver et al., 2017). While examples of SMGCs are still relatively rare in plants, experimentally validated cases were reported for a surprisingly diverse group of pathways. These include terpenes (Chae et al., 2014; Prisic et al., 2004; Qi et al., 2004; Wilderman et al., 2004), cyclic hydroxamic acids (Frey et al., 1997), biosynthetically unrelated alkaloids (Itkin et al., 2013; Winzer et al., 2012), polyketides (Schneider et al., 2016), cyanogenic glucosides (Takos et al., 2011), and modified fatty acids (Jeon et al., 2020). However, whereas each cluster encodes multiple non-homologous enzymes of a biosynthetic pathway, evolution of their assembly is not well understood. Acylsugars are a group of insecticidal (Leckie et al., 2016) and anti-inflammatory (Herrera-Salgado et al., 2005) chemicals mainly observed in glandular trichomes of Solanaceae species (Fan et al., 2019; Schuurink and Tissier, 2020). These specialized metabolites are sugar aliphatic esters with three levels of structural diversity across the Solanaceae family: acyl chain length, acylation position, and sugar core (Fan et al., 2019). The primary metabolites sucrose and aliphatic acyl-CoAs are the biosynthetic precursors of acylsucroses in plants as evolutionarily divergent as the cultivated tomato Solanum lycopersicum (Fan et al., 2016; Figure 1), Petunia axillaris (Nadakuduti et al., 2017) and Salpiglossis sinuata (Moghe et al., 2017). The core tomato acylsucrose biosynthetic pathway involves four BAHD [BEAT, AHCT, HCBT, DAT (D'Auria, 2006) family acylsucrose acyltransferases (Sl-ASAT1 through Sl-ASAT4), which are specifically expressed in the type I/IV trichome tip cells (Fan et al., 2016; Schilmiller et al., 2015; Schilmiller et al., 2012). These enzymes catalyze consecutive reactions utilizing sucrose and acyl-CoA substrates to produce the full set of cultivated tomato acylsucroses in vitro (Fan et al., 2016). Figure 1 Download asset Open asset Primary metabolites are biosynthetic precursors of tomato trichome acylsugars. In cultivated tomatoes, the trichome acylsucroses are synthesized by four Sl-ASATs using the primary metabolites – sucrose and different types of acyl-CoAs – as substrates. In this study we provide evidence that medium chain fatty acids are converted to acyl-CoAs by an acyl-CoA synthetase for medium chain acylsugar biosynthesis. Co-option of primary metabolic enzymes contributed to the evolution of acylsugar biosynthesis and led to interspecific structural diversification across the Solanum tomato clade. One example is an invertase-like enzyme originating from carbohydrate metabolism that generates acylglucoses in the wild tomato S. pennellii through cleavage of the acylsucrose glycosidic bond (Leong et al., 2019). In another case, allelic variation of a truncated isopropylmalate synthase-like enzyme (IPMS3) – from branched chain amino acid metabolism – leads to acylsugar iC4/iC5 (2-methylpropanoic/3-methylbutanoic acid) acyl chain diversity in S. pennellii and S. lycopersicum (Ning et al., 2015). Acylsugar structural diversity is even more striking across the family. Previous studies revealed variation in acyl chain length (Ghosh et al., 2014; Liu et al., 2017; Moghe et al., 2017): Nicotiana, Petunia and Salpiglossis species were reported to accumulate acylsugars containing only short acyl chains (carbon number, C ≤ 8). In contrast, some species in Solanum and other closely related genera produce acylsugars with medium acyl chains (C ≥ 10). These results are consistent with the hypothesis that the capability to produce medium chain acylsugars varies across the Solanaceae family. In this study, we identify a metabolic gene cluster on tomato chromosome 7 containing two non-homologous genes – acylsugar acyl-CoA synthetase (AACS) and acylsugar enoyl-CoA hydratase (AECH) – affecting medium chain acylsugar biosynthesis. Genetic and biochemical results show that the trichome enriched AACS and AECH are involved in generating medium chain acyl-CoAs, which are donor substrates for acylsugar biosynthesis. Genomic analysis revealed a syntenic region on chromosome 12, where the acylsucrose biosynthetic Sl-ASAT1 is located (Fan et al., 2016). Phylogenetic analysis of the syntenic regions in Solanaceae and beyond led to evolutionary reconstruction of the origin of the acylsugar gene cluster. We infer that sequential gene insertion facilitated emergence of this gene cluster in tomato. These results provide insights into specialized metabolic evolution through emergence of cell-type specific gene expression, the formation of metabolic gene clusters and illuminates additional examples of primary metabolic enzymes being co-opted into specialized metabolism. Results Identification of a metabolic gene cluster that affects tomato trichome medium chain acylsugar biosynthesis S. pennellii natural accessions (Mandal et al., 2020), as well as the S. lycopersicum M82 ×S. pennellii LA0716 chromosomal substitution introgression lines (ILs) (Eshed and Zamir, 1995), offer convenient resources to investigate interspecific genetic variation that affects acylsugar metabolic diversity (Mandal et al., 2020; Schilmiller et al., 2010). In a rescreen of ILs for S. pennellii genetic regions that alter trichome acylsugar profiles (Schilmiller et al., 2010), IL7-4 was found to accumulate increased C10 medium chain containing acylsugars compared with M82 (Figure 2, A and B). The genetic locus that contributes to the acylsugar phenotype was narrowed down to a 685 kb region through screening selected backcross inbred lines (BILs) (Ofner et al., 2016) that have recombination breakpoints on chromosome 7 (Figure 2C). Because tomato acylsucrose biosynthesis occurs in trichomes, candidate genes in this region were filtered based on their trichome-specific expression patterns. This analysis identified a locus containing multiple tandemly duplicated genes of three families – an acyl-CoA synthetase (ACS), enoyl-CoA hydratase (ECH), and BAHD acyltransferase. Our analysis (Moore et al., 2020) revealed co-expression of four Sl-ASATs (Fan et al., 2019) and three genes at the locus – Solyc07g043630, Solyc07g043660, and Solyc07g043680 (Supplementary file 1 and Figure 2—figure supplement 1). Expression of these three genes was trichome enriched (Figure 2D), and thus they were selected for further analysis. Figure 2 with 1 supplement see all Download asset Open asset Mapping of a genetic locus related to acylsugar variations in tomato interspecific introgression lines. (A) Electrospray ionization negative (ESI-) mode, base-peak intensity (BPI) LC/MS chromatogram of trichome metabolites from cultivated tomato S. lycopersicum M82 and introgression line IL7-4. The orange bars highlight two acylsugars that have higher abundance in IL7-4 than in M82. For the acylsucrose nomenclature, ‘S’ refers to a sucrose backbone, ‘3:22’ means three acyl chains with twenty-two carbons in total. The length of each acyl chain is shown in the parentheses. (B) Peak area percentage of seven major trichome acylsugars in M82 and IL7-4. The sum of the peak area percentage of each acylsugar is equal to 100% in each sample. The data is shown for three plants ± SEM. **p<0.01, Welch two-sample t test. Figure 2—source data 1 includes values for the analysis. (C) Mapping the genetic locus contributing to the IL7-4 acylsugar phenotype using selected backcross inbred lines (BILs) that have recombination break points within the introgression region of IL7-4. (D) Narrowing down candidate genes in the locus using trichome/stem RNA-seq datasets generated from previous study (Ning et al., 2015). A region with duplicated genes of three types – acyl-CoA synthetase (ACS), BAHD acyltransferase, and enoyl-CoA hydratase (ECH) – is shown. The red-blue color gradient provides a visual marker to rank the expression levels represented by Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Coexpression analysis of tomato ACS, ECH, and BAHD acyltransferase family genes is shown in Figure 2—figure supplement 1. Figure 2—source data 1 Data used to make Figure 2B. Peak area percentage of seven major trichome acylsugars in M82 and IL7-4. https://cdn.elifesciences.org/articles/56717/elife-56717-fig2-data1-v2.xlsx Download elife-56717-fig2-data1-v2.xlsx The three candidate genes were tested for involvement in tomato acylsugar biosynthesis by making loss of function mutations using the CRISPR-Cas9 gene editing system. Two guide RNAs (gRNAs) were designed to target one or two exons of each gene to assist site-specific DNA cleavage by hCas (Brooks et al., 2014; Figure 3—figure supplement 1, A–C). In the self-crossed T1 progeny of stably transformed M82 plants, at least two homozygous mutants were obtained in Solyc07g043630, Solyc07g043660, and Solyc07g043680 (Figure 3—figure supplement 1, A–C), and these were analyzed for leaf trichome acylsugar changes. Altered acylsugar profiles were observed in the ACS-annotated Solyc07g043630 or ECH-annotated Solyc07g043680 mutants (Figure 3, A and B), but not in the ACS-annotated Solyc07g043660 mutant (Figure 3—figure supplement 1D). Despite carrying mutations in distinctly annotated genes (ACS or ECH), the two mutants exhibited the same phenotype – no detectable medium acyl chain (C10 or C12) containing acylsugars (Figure 3, A and B). We renamed Solyc07g043630 as acylsugar acyl-CoA synthetase 1 (Sl-AACS1) and Solyc07g043680 as acylsugar enoyl-CoA hydratase 1 (Sl-AECH1) based on this analysis. Figure 3 with 2 supplements see all Download asset Open asset CRISPR/Cas9-mediated gene knockout of tomato Sl-AACS1 or Sl-AECH1 eliminates detectable medium chain containing acylsugars. (A) Combined LC/MS extracted ion chromatograms of trichome metabolites from CRISPR mutants sl-aacs1 and sl-aech1. The medium chain acylsugars that are not detected in the two mutants are denoted by pairs of vertical dotted lines. Figure 3—figure supplement 1 describes the design of the gRNAs and details of the gene edits. (B) Quantification of seven major trichome acylsugars in sl-aacs1 and sl-aech1 mutants. Two independent T2 generation transgenic lines for each mutant were used for analysis. The peak area/internal standard (IS) normalized by leaf dry weight (DW) is shown from six plants ± SEM. Figure 3—source data 1 includes values for the analysis. (C) Confocal fluorescence images showing that GFP fluorescence driven by Sl-AACS1 or Sl-AECH1 is located in the tip cells of type I/IV trichomes. Their tissue specific expressions are similar to Sl-ASAT1 (Fan et al., 2016), which locates in a chromosome 12 region that is syntenic to the locus containing Sl-AACS1 and Sl-AECH1. Figure 3—figure supplement 2 provides the detailed information of the syntenic region. Sl-AACS1, Sl-AECH1, and Sl-ASAT1 are the only gene models with demonstrated functions in acylsugar biosynthesis. Figure 3—source data 1 Data used to make Figure 3B. Quantification of seven major trichome acylsugars in the CRISPR mutants sl-aacs1 and sl-aech1, as well as the parent M82. https://cdn.elifesciences.org/articles/56717/elife-56717-fig3-data1-v2.xlsx Download elife-56717-fig3-data1-v2.xlsx Further genomic analysis revealed that Sl-AACS1 and Sl-AECH1 belong to a syntenic region shared with a locus on chromosome 12, where Sl-ASAT1 is located (Figure 3C and Figure 3—figure supplement 2). Sl-ASAT1 is specifically expressed in trichome tip cells and encodes the enzyme catalyzing the first step of tomato acylsucrose biosynthesis (Fan et al., 2016). This led us to test the cell-type expression pattern of Sl-AACS1 and Sl-AECH1. Like Sl-ASAT1, the promoters of both genes drove GFP expression in the trichome tip cells of stably transformed M82 plants (Figure 3C). This supports our hypothesis that Sl-AACS1 and Sl-AECH1 are involved in tomato trichome acylsugar biosynthesis. Taken together, we identified a metabolic gene cluster involved in medium chain acylsugar biosynthesis, which is composed of two cell-type specific genes. In vitro analysis of Sl-AACS1 and Sl-AECH1 implicates their roles in medium chain acyl-CoA metabolism ACS and ECH are established to function in multiple cell compartments for the metabolism of acyl-CoA (Buchanan et al., 2015), the acyl donor substrates for ASAT enzymes. We sought to understand the organelle targeting of Sl-AACS1 and Sl-AECH1, to advance our knowledge of acylsugar machinery at the subcellular level. We constructed expression cassettes of Sl-AACS1, Sl-AECH1 and Solyc07g043660 with C-terminal cyan fluorescent protein (CFP), hypothesizing that the targeting peptides reside at the N-terminus of precursor proteins. When co-expressed in tobacco leaf epidermal cells, three CFP-tagged recombinant proteins co-localized with the mitochondrial marker MT-RFP (Nelson et al., 2007; Figure 4A and Figure 4—figure supplement 1A). To rule out the possibility of peroxisomal localization, we fused Sl-AACS1, Sl-AECH1, or Solyc07g043660 with N-terminus fused yellow fluorescent protein (YFP), considering that potential peroxisomal targeting peptides are usually located on the C-terminus (Brocard and Hartig, 2006). The expressed YFP-recombinant proteins were not co-localized with the peroxisomal marker RFP-PTS (Nelson et al., 2007; Figure 4—figure supplement 1B). Instead, they appeared distributed in the cytosol (Figure 4—figure supplement 1B), presumably because the N-terminal YFP blocked the mitochondria targeting signal. Taken together, protein expression and co-localization analyses suggest that Sl-AACS1, Sl-AECH1, and Solyc07g043660 encode enzymes targeted to mitochondria. Figure 4 with 2 supplements see all Download asset Open asset Functional analysis of Sl-AACS1 and Sl-AECH1 in N. benthamiana and recombinant Sl-AACS1 enzyme analysis. (A) Confocal images of co-expression analysis in tobacco leaf epidermal cells using C-terminal CFP-tagged either Sl-AACS1 or Sl-AECH1 and the mitochondrial marker MT-RFP. Arrowheads point to mitochondria that are indicated by MT-RFP fluorescent signals. Scale bar equals 10 μm. Figure 4—figure supplement 1B describes that the expressed YFP-recombinant proteins were not co-localized with the peroxisomal marker RFP-PTS (B) Aliphatic fatty acids of different chain lengths were used as the substrates to test Sl-AACS1 acyl-CoA synthetase activity. Mean amount of acyl-CoAs generated (nmol min−1 mg−1 proteins) was used to represent enzyme activities. The results are from three measurements ± SEM. Figure 4—source data 1 includes values for the measurements. (C) Enzyme activity of Sl-AACS1 for six fatty acid substrates. (D) Identification of membrane lipid phosphatidylcholine (PC), which contains medium acyl chains, following transient expression of Sl-AECH1 in N. benthamiana leaves. The results from expressing Sl-AACS1 and co-expressing both Sl-AECH1 and Sl-AACS1 are also shown. Mole percentage (Mol %) of the acyl chains from membrane lipids with carbon number 12, 14, 16, and 18 are shown for three biological replicates ± SEM. *p<0.05, **p<0.01. Welch two-sample t test was performed comparing with the empty vector control. Figure 4—source data 2 includes values for the lipid analysis. Acyl groups of the same chain lengths with saturated and unsaturated bonds were combined in the calculation. Figure 4—figure supplement 2 shows that the putative Sl-AECH1 orthologs from S. pennellii and S. quitoense generated medium chain lipids in the infiltrated leaves. Figure 4—source data 1 Data used to make Figure 4B. Aliphatic fatty acids of different chain lengths were used as the substrates to test Sl-AACS1 acyl-CoA synthetase activity. https://cdn.elifesciences.org/articles/56717/elife-56717-fig4-data1-v2.xlsx Download elife-56717-fig4-data1-v2.xlsx Figure 4—source data 2 Data used to make Figure 4D and Figure 4—figure supplement 1C. N. benthamiana leaf membrane lipid acyl chain composition. https://cdn.elifesciences.org/articles/56717/elife-56717-fig4-data2-v2.xlsx Download elife-56717-fig4-data2-v2.xlsx Sl-AACS1 belongs to a group of enzymes that diverse acid substrates to produce We that Sl-AACS1 medium chain fatty acids as because of Sl-AACS1 acylsugars with medium acyl chains. To the in vitro activity of Sl-AACS1, we recombinant proteins from Enzyme were performed by fatty acid substrates with even carbon from through (Figure The results that Sl-AACS1 fatty acid substrates with lengths from to including with a branched carbon or an unsaturated bond (Figure and However, no activity was observed with the and fatty acids as substrates (Figure These results our hypothesis that Sl-AACS1 medium chain acyl-CoAs, which are in substrates for acylsugar biosynthesis. To test Sl-AACS1 and Sl-AECH1 can produce medium chain acyl-CoAs in we expressed these genes in benthamiana using et al., It is to plant acyl-CoAs, due to their and We used an and characterized membrane which are from acyl-CoA We of the that N. benthamiana membrane lipids do not accumulate detectable acyl chains of 12 carbons or N. benthamiana were infiltrated with containing Sl-AACS1 or Sl-AECH1 or (Figure In to the empty vector of Sl-AECH1 led to detectable levels of acyl chains in the leaf membrane lipid phosphatidylcholine (Figure We also observed increased acyl chains in and in Sl-AECH1 infiltrated plants (Figure 4D and Figure 4—figure supplement These results suggest that Sl-AECH1 in generation of medium chain acyl-CoAs in which are into lipid biosynthesis. medium chain acylsugars were presumably due to the of core acylsugar biosynthetic machinery in N. benthamiana cells. We the known of Sl-AECH1 from Solanum species can medium chain lipids expressed in N. Two with chains were by LC/MS as of lipids containing medium chain fatty acids (Figure 4—figure supplement 2, A and B). The results that only the putative Sl-AECH1 orthologs and – from S. pennellii and S. quitoense – generated medium chain lipids in the infiltrated (Figure 4—figure supplement 2C). This that not all can produce medium chain lipids and that the function of Sl-AECH1 evolved presumably as a of gene duplication (Figure 4—figure supplement 2C). and are evolutionarily in the Solanum chain acylsugars were in Solanum species cultivated including S. pennellii (Leong et al., S. (Moghe et al., as well as the more related S. quitoense (Leong et al., 2020; and S. (Herrera-Salgado et al., We that evolution of and contributed to medium chain acylsugar biosynthesis in a we analyzed the of Solanum species other than cultivated tomato. the acylsugar related containing ACS and ECH was found in both S. pennellii and S. that the cluster assembly evolved of the tomato and (Figure Figure with 2 supplements see all Download asset Open asset and are evolutionarily in Solanum plants. (A) A syntenic genomic region containing and was found in three selected Solanum species. the et al., shown on the The of and in Solanum quitoense are shown genomic because the genes were identified from RNA-seq and are not The lines genes putative orthologs across the four species. The trichome/stem RNA-seq data of two biological S. pennellii replicates are (Supplementary file for genes in the syntenic region. The red-blue color gradient provides a visual marker to rank the expression levels in of medium chain acylsugars from S. quitoense and S. pennellii (Leong et al., 2019) are on the Figure supplement 1 shows that of the M82 CRISPR mutant sl-aacs1 containing acylsugars (B) CRISPR/Cas9-mediated gene knockout of or in S. pennellii produce no detectable medium chain containing acylsugars. The LC/MS extracted ion chromatograms of trichome metabolites are shown for each The to the that both acylsucroses and acylglucoses under The and to the from acylsugars with chain length of C10 and Figure supplement describes the design of the gRNAs and the detailed information of gene edits. (C) or in S. quitoense using leads to of acylsugars. The peak area/internal standard (IS) normalized by leaf dry weight was shown from plants ± SEM. Welch two-sample t test. Figure supplement and describes the design and the LC/MS extracted ion chromatograms of S. quitoense major acylsugars. (D) gene expression of or correlates with acylsugar levels in S. The gene expression data are with acylsugar levels of the same leaf as in Figure supplement Figure data 1 includes data for the S. quitoense Figure data 1 Data used to make Figure and or in S. quitoense using leads to of acylsugars. Download We gene expression and genetic approaches to test the in functions of ACS and ECH in selected Solanum species. To the expression pattern of S. pennellii ACS and ECH cluster genes, we performed RNA-seq analysis on trichomes and to identify acylsugar biosynthetic (Supplementary file 2). The expression pattern of S. pennellii cluster genes is similar to S. one ECH and two ACS genes are enriched in trichomes, including the orthologs of Sl-AACS1 and Sl-AECH1. function was first tested by can the cultivated tomato sl-aacs1 mutant acylsugar containing acylsugars in the stably transformed sl-aacs1 plants (Figure supplement 1). To test and we used CRISPR-Cas9 to make mutants in S. pennellii medium chain acylsugars were detected in generation mutants with for each gene (Figure and Figure supplement 2, A and to the ACS-annotated Solyc07g043660 cultivated tomato mutant (Figure 3—figure supplement of S. pennellii no observed on S. pennellii trichome acylsugars (Figure supplement The medium chain acylsugar S. quitoense was used for and