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.

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.

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.

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.

Sl-AACS1 belongs to a group of enzymes that activate diverse carboxylic acid substrates to produce acyl-CoAs. We hypothesized that Sl-AACS1 uses medium chain fatty acids as substrates, because ablation of Sl-AACS1 eliminated acylsugars with medium acyl chains. To characterize the in vitro activity of Sl-AACS1, we purified recombinant His-tagged proteins from Escherichia coli. Enzyme assays were performed by supplying fatty acid substrates with even carbon numbers from C2 through C18 (Figure 4B). The results showed that Sl-AACS1 utilized fatty acid substrates with lengths ranging from C6 to C12, including those with a terminal branched carbon (iC10:0) or an unsaturated bond (trans-2-decenoic acid, C10:1) (Figure 4, B and C). However, no activity was observed with the 3-hydroxylated C12 and C14 fatty acids as substrates (Figure 4B). These results support our hypothesis that Sl-AACS1 produces medium chain acyl-CoAs, which are in vivo substrates for acylsugar biosynthesis.

To test whether Sl-AACS1 and Sl-AECH1 can produce medium chain acyl-CoAs in planta, we transiently expressed these genes in Nicotiana benthamiana leaves using Agrobacterium-mediated infiltration (Sainsbury et al., 2009). It is challenging to directly measure plant acyl-CoAs, due to their low concentration and separate organellar pools. We used an alternative approach and characterized membrane lipids, which are produced from acyl-CoA intermediates. We took advantage of the observation that N. benthamiana membrane lipids do not accumulate detectable acyl chains of 12 carbons or shorter. N. benthamiana leaves were infiltrated with constructs containing Sl-AACS1 or Sl-AECH1 individually, or together (Figure 4D). In contrast to the empty vector control, infiltration of Sl-AECH1 led to detectable levels of C12 acyl chains in the leaf membrane lipid phosphatidylcholine (PC) (Figure 4D). We also observed increased C14 acyl chains in PC, phosphatidylglycerol (PG), sulfoquinovosyl diacylglycerol (SQDG), and digalactosyldiacylglycerol (DGDG) in Sl-AECH1 infiltrated plants (Figure 4D and Figure 4—figure supplement 1C). These results suggest that Sl-AECH1 participates in generation of medium chain acyl-CoAs in planta, which are channeled into lipid biosynthesis. No medium chain acylsugars were detected, presumably due to the lack of core acylsugar biosynthetic machinery in N. benthamiana mesophyll cells.

We asked whether the closest known homologs of Sl-AECH1 from Solanum species can generate medium chain lipids when transiently expressed in N. benthamiana. Two SQDGs with C12 chains were monitored by LC/MS as peaks diagnostic of lipids containing medium chain fatty acids (Figure 4—figure supplement 2, A and B). The results showed that only the putative Sl-AECH1 orthologs Sopen07g023250 (Sp-AECH1) and Sq_c37194 (Sq-AECH1) – from S. pennellii and S. quitoense respectively – generated medium chain lipids in the infiltrated leaves (Figure 4—figure supplement 2C). This confirms that not all ECHs can produce medium chain lipids and suggests that the function of Sl-AECH1 evolved recently, presumably as a result of neofunctionalization after gene duplication (Figure 4—figure supplement 2C).

Medium chain acylsugars were documented in Solanum species besides cultivated tomato, including S. pennellii (Leong et al., 2019), S. nigrum (Moghe et al., 2017), as well as the more distantly related S. quitoense (Leong et al., 2020; Hurney, 2018) and S. lanceolatum (Herrera-Salgado et al., 2005). We hypothesized that evolution of AACS1 and AECH1 contributed to medium chain acylsugar biosynthesis in Solanum. As a test, we analyzed the genomes of Solanum species other than cultivated tomato. Indeed, the acylsugar related synteny containing ACS and ECH was found in both S. pennellii and S. melongena (eggplant), suggesting that the cluster assembly evolved before divergence of the tomato and eggplant lineage (Figure 5A).

We applied gene expression and genetic approaches to test the in vivo functions of ACS and ECH in selected Solanum species. To explore the expression pattern of S. pennellii ACS and ECH cluster genes, we performed RNA-seq analysis on trichomes and shaved stems to identify acylsugar biosynthetic candidates (Supplementary file 2). The expression pattern of S. pennellii cluster genes is strikingly similar to S. lycopersicum: one ECH and two ACS genes are highly enriched in trichomes, including the orthologs of Sl-AACS1 and Sl-AECH1. Sp-AACS1 function (Sopen07g023200) was first tested by asking whether it can reverse the cultivated tomato sl-aacs1 mutant acylsugar phenotype. Indeed, Sp-AACS1 restored C12 containing acylsugars in the stably transformed sl-aacs1 plants (Figure 5—figure supplement 1). To directly test Sp-AACS1 and Sp-AECH1 function, we used CRISPR-Cas9 to make single mutants in S. pennellii LA0716. No medium chain acylsugars were detected in T0 generation mutants with edits for each gene (Figure 5B and Figure 5—figure supplement 2, A and C). Similar to the ACS-annotated Solyc07g043660 cultivated tomato mutant (Figure 3—figure supplement 1D), deletion of S. pennellii ortholog Sopen07g023220 has no observed effects on S. pennellii trichome acylsugars (Figure 5—figure supplement 2D).

The medium chain acylsugar producer S. quitoense (Hurney, 2018) was used for AACS1 and AECH1 functional analysis because of its phylogenetic distance from the tomato clade - it is in the Solanum Leptostemonum clade (including eggplant) - and the fact that it produces medium chain acylsugars. We found trichome-enriched putative orthologs of AACS1 and AECH1 in the transcriptome dataset of S. quitoense (Moghe et al., 2017), and tested their in vivo function through virus-induced gene silencing (VIGS) (Figure 5—figure supplement 2E). Silencing either gene led to decreased total acylsugars (Figure 5C and Figure 5—figure supplement 2F), which correlated with the degree of expression reduction in each sample (Figure 5D). These results are consistent with the hypothesis that Sq-AACS1 and Sq-AECH1 are involved in medium chain acylsugar biosynthesis, because all acylsugars in S. quitoense carry two medium chains (Leong et al., 2020; Hurney, 2018). The importance of AACS1 and AECH1 in medium chain acylsugar biosynthesis in distinct Solanum clades inspired us to explore the evolutionary origins of the gene cluster.

We sought to understand how the acylsugar gene cluster evolved and whether it correlates with the distribution of medium chain acylsugars across the Solanaceae family. Taking advantage of the available genome sequences of 13 species from Solanaceae and sister families, we analyzed the regions that are syntenic with the tomato acylsugar gene cluster (Figure 6—figure supplement 1). This synteny was found in all these plants, including the most distantly related species analyzed, Coffea canephora (coffee, Rubiaceae) (Figure 6—figure supplement 1). BAHD acyltransferases were the only genes observed in the syntenic regions both inside and outside the Solanaceae, in contrast to ECH and ACS, which are restricted to the family (Figure 6A and Figure 6—figure supplement 1). Within the syntenic regions of the species analyzed, ECH homologs, including pseudogenes, are present in all Solanaceae except for Capsicum species, while ACS is more phylogenetically restricted, being found only in Nicotiana and Solanum (Figure 6A and Figure 6—figure supplement 1).

We then performed phylogenetic analysis to reconstruct the evolutionary history of the ACS, ECH, and BAHD acyltransferase genes in the syntenic region (Figure 6). This analysis revealed a model for the temporal order of emergence for the three types of genes, leading to their presence in the syntenic regions in extant Solanaceae plants (Figure 6B). We propose that the BAHD acyltransferase was the first of three genes that emerged before the divergence between Solanaceae and Rubiaceae, and was likely lost in Convolvulaceae. This hypothesis is based on the discovery of a BAHD acyltransferase pseudogene in the syntenic region of C. canephora (Figure 6A and Figure 6—figure supplement 1), which is one of the closest Coffea sequences sister to the ASAT clade (Figure 6—figure supplement 2 and Figure 6—figure supplement 5). In our model, ECH was likely inserted into the syntenic region before the Solanaceae-specific whole genome duplication (WGD) event (Figure 6B and Figure 6—figure supplement 3).

We propose that ACS was inserted into the synteny through segmental duplication (Bailey et al., 2002; Figure 6—figure supplement 4). However, whether ACS insertion happened before or after the Solanaceae-specific WGD event cannot be resolved by the phylogenetic analysis (Figure 6—figure supplement 4). If the insertion happened before WGD, one ACS gene loss on chromosome 12 in Solanum – as well as two independent gene losses on chromosomes 7 and 12 in both Petunia (Figure 6—figure supplement 4) and in Salpiglossis sinuata (Figure 6—figure supplement 6) – should have happened. However, if the insertion happened after WGD, then only one gene loss in Petunia and Salpiglossis needs to be invoked (Figure 6—figure supplement 6). The latter scenario is more likely based on the principle of parsimony.

Our ancestral state reconstruction inference supports the notion that the medium chain acylsugars co-emerged with the ACS/ECH genes in the syntenic regions in the common ancestor of Solanum (Figure 6—figure supplement 7). This leads us to propose that the emergence of both ACS and ECH genes in the synteny was a prerequisite for the rise of medium chain acylsugars in Solanaceae species (Figure 6B). Consistent with the hypothesis, only short chain acylsugars were observed in Petunia (Liu et al., 2017; Figure 6—figure supplement 8), which correlates with the absence of ACS homolog (Figure 6B). In contrast, medium chain acylsugars were detected throughout Solanum (Figure 6—figure supplement 8), supported by the observation that both ACS and ECH homologs are present in extant Solanum species (Figure 6B). Interestingly, although Nicotiana species collectively have both ACS and ECH genes (Figure 6B), they do not produce medium chain acylsugars (Figure 6—figure supplement 8) presumably due to gene losses. For example, the ECH homologs are pseudogenes in N. benthamiana and N. tomentosiformis (Figure 6A). These results show that the presence of both functional ACS and ECH genes are associated with the accumulation of medium chain acylsugars, supporting our hypothesis above.

Although no medium chain acylsugars were detected in Nicotiana species examined, the ACS and ECH genes may have been present in the syntenic region prior to divergence of Solanum and Nicotiana. This suggests that one or more species that diverged from the common ancestor of Solanum and Nicotiana could have medium chain acylsugars. To test this hypothesis, we extended the phenotypic analysis to six such Solanaceae genera (Figure 6—figure supplement 8). Indeed, we found that species in Jaltomata, Physalis, Iochroma, Atropa, and Hyoscyamus, which diverged from the common ancestor with Nicotiana but before Solanum, have medium chain acylsugars (Figure 6—figure supplement 8).

The seeds of cultivated tomato Solanum lycopersicum M82 were obtained from the C.M. Rick Tomato Genetic Resource Center (https://tgrc.ucdavis.edu/), RRID:SCR_014954. Tomato introgression lines (ILs) and tomato backcross inbred lines (BILs) were from Dr. Dani Zamir (Hebrew University of Jerusalem). The tomato seeds were treated with ½ strength bleach for 30 min and washed with de-ionized water three or more times before placing on wet filter paper in a Petri dish. After germination, the seedlings were transferred to peat-based propagation mix (Sun Gro Horticulture) and transferred to a growth chamber for two or three weeks under 16 hr photoperiod, 28°C day and 22°C night temperatures, 50% relative humidity, and 300 μmol m⁻² s⁻¹ photosynthetic photon flux density. The youngest fully developed leaf was submerged in 1 mL extraction solution in a 1.5 mL screw cap tube and agitated gently for 2 min. The extraction solution contains acetonitrile/isopropanol/water (3:3:2) with 0.1% formic acid and 10 μM propyl-4-hydroxybenzoate as internal standard. The interactive protocol of acylsugar extraction is available in Protocols.io at http://dx.doi.org/10.17504/protocols.io.xj2fkqe.

Assembly of the CRISPR-Cas9 constructs was as described (Brooks et al., 2014; Leong et al., 2019). Two guide RNAs (gRNAs) were designed targeting one or two exons of each gene to be knocked out by the CRISPR-Cas9 system. The gRNAs were obtained from gene blocks (gBlocks) synthesized by IDT (Integrated DNA Technologies, location) (Supplementary file 3). For each CRISPR construct, two gBlocks and four plasmids from Addgene, pICH47742::2 × 35 S-5′UTR-hCas9 (STOP)-NOST (Addgene no. 49771), pICH41780 (Addgene no. 48019), pAGM4723 (Addgene no. 48015), pICSL11024 (Addgene no. 51144), were mixed for DNA assembly using the Golden Gate assembly kit (NEB).

For in planta tissue specific reporter gene analysis, 1.8 kb upstream of the annotated translational start site of Sl-AACS1 and Sl-AECH were amplified using the primer pairs SlAACS1-pro_F/R and SlAECH1-pro_F/R (Supplementary file 3). The amplicon was inserted into the entry vector pENTR/D-TOPO, followed by cloning into the GATEWAY vector pKGWFS7. For ectopically expressing Sp-AACS1 in the cultivated tomato CRISPR mutant sl-aacs1, Sp-AACS1 gene including 1.8 kb upstream of the translational start site of Sp-AACS1 was amplified using the primer pair SpAACS1-pro-gene_F/R (Supplementary file 3). The amplicon was inserted into the entry vector pENTR/D-TOPO, followed by cloning into the GATEWAY vector pK7WG.

The plant transformation of S. lycopersicum M82 and S. pennellii LA0716 was performed using the Agrobacterium tumefaciens strain AGL0 following published protocols (Leong et al., 2019; McCormick, 1997). The primers used for genotyping the S. lycopersicum M82 transgenic plants harboring pK7WG or pKGWFS7 construct are listed in Supplementary file 3. For genotyping the S. lycopersicum M82 CRISPR mutants in the T1 generation, the sequencing primers listed in Supplementary file 3 were used to amplify the genomic regions harboring the gRNAs and the resultant PCR products were sent for Sanger sequencing. For genotyping the S. pennellii LA0716 CRISPR mutants in the T0 generation, the sequencing primers listed in Supplementary file 3 were used to amplify the genomic regions harboring the gRNAs. The resulting PCR products were cloned into the pGEM-T easy vector (Promega) and transformed into E. coli. Plasmids from at least six individual E. coli colonies containing the amplified products were extracted and verified by Sanger sequencing.

For protein subcellular targeting analysis, the open reading frame (ORF) of Sl-AACS1, Sl-AECH1, and Solyc07g043660 were amplified using the primers listed in Supplementary file 3. These amplicons were inserted into pENTR/D-TOPO respectively, followed by subcloning into the GATEWAY vectors pEarleyGate102 (no. CD3-684) and pEarleyGate104 (no. CD3-686), which were obtained from Arabidopsis Biological Resource Center (ABRC). For the pEarleyGate102 constructs, the CFP was fused to the C-terminal of the tested proteins. For the pEarleyGate104 constructs, the YFP was fused to the protein N-terminus. Transient expressing the tested proteins was performed following an established protocol (Batoko et al., 2000) with minor modifications. In brief, cultures of A. tumefaciens (strain GV3101) harboring the expression vectors were washed and resuspended with the infiltration buffer (20 mM acetosyringone, 50 mM MES pH 5.7, 0.5% glucose [w/v] and 2 mM Na₃PO₄) to reach OD_600nm = 0.05. Four-week-old tobacco (Nicotiana tabacum cv. Petit Havana) plants grew in 21°C and 8 hr short-day conditions were infiltrated, and then maintained in the same growth condition for three days before being sampled for imaging. The GV3101 cultures containing the mitochondria marker MT-RFP (Nelson et al., 2007) were co-infiltrated to provide the control signals for mitochondrial targeting. In separate experiments, the GV3101 cultures containing the peroxisome marker RFP-PTS (Nelson et al., 2007) were co-infiltrated to provide the control signals for peroxisomal targeting.

A Nikon A1Rsi laser scanning confocal microscope and Nikon NIS-Elements Advanced Research software were used for image acquisition and handling. For visualizing GFP fluorescence in trichomes of the tomato transformants, the excitation wavelength at 488 nm and a 505- to 525 nm emission filter were used for the acquisition. For visualizing signals of fluorescence proteins in the tobacco mesophyll cells, CFP, YFP and RFP, respectively, were detected by excitation lasers of 443 nm, 513 nm, 561 nm and emission filters of 467–502 nm, 521–554 nm, 580–630 nm.

For N. benthamiana transient expression of Sl-AACS1, Sl-AECH1, and homologs of AECH1, the ORFs of these genes were amplified using primers listed in Supplementary file 3, followed by subcloning into pEAQ-HT vector using the Gibson assembly kit (NEB). Linearization of pEAQ-HT vector was performed by XhoI and AgeI restriction enzyme double digestion. A. tumefaciens (strain GV3101) harboring the pEAQ-HT constructs were grown in LB medium containing 50 µg/mL kanamycin, 30 µg/mL gentamicin, and 50 µg/ml rifampicin at 30 °C. The cells were collected by centrifugation at 5000 g for 5 min and washed once with the resuspension buffer (10 mM MES buffer pH 5.6, 10 mM MgCl₂, 150 µM acetosyringone). The cell pellet was resuspended in the resuspension buffer to reach OD_600nm = 0.5 for each strain and was incubated at room temperature for 1 hr prior to infiltration. Leaves of 4 to 5 week-old N. benthamiana grown under 16 hr photoperiod were used for infiltration. Five days post infiltration, the infiltrated leaves were harvested, ground in liquid nitrogen, and stored at −80 °C for later analysis.

The membrane lipid analysis was performed as previously described (Wang and Benning, 2011). In brief, the N. benthamiana leaf polar lipids were extracted in the organic solvent containing methanol, chloroform, and formic acid (20:10:1, v/v/v), separated by thin layer chromatography (TLC), converted to fatty acyl methylesters (FAMEs), and analyzed by gas-liquid chromatography (GLC) coupled with flame ionization. The TLC plates (TLC Silica gel 60, EMD Chemical) were activated by ammonium sulfate before being used for lipid separation. Iodine vapor was applied to TLC plates after lipid separation for brief reversible staining. Different lipid groups on the TLC plates were marked with a pencil and were scraped for analysis. For LC/MS analysis, lipids were extracted using the buffer containing acetonitrile/isopropanol/water (3:3:2) with 0.1% formic acid and 10 μM propyl-4-hydroxybenzoate as the internal standard.

To express His-tagged recombinant protein Sl-AACS1, the full-length Sl-AACS ORF sequence was amplified using the primer pair SlAACS1-pET28_F/R (Supplementary file 3) and was cloned into pET28b (EMD Millipore) using the Gibson assembly kit (NEB). The pET28b vector was linearized by digesting with BamHI and XhoI to create overhangs compatible for Gibson assembly. The pET28b constructs were transformed into BL21 Rosetta cells (EMD Millipore). The protein expression was induced by adding 0.05 mM isopropyl β-D-1-thiogalactopyranoside to the cultures when the OD_600nm = 0.5. The E. coli cultures were further grown overnight at 16 °C, 120 rpm. The His-tagged proteins were purified by Ni-affinity gravity-flow chromatography using the Ni-NTA agarose (Qiagen) following the product manual.

Measurement of acyl-CoA synthetase activity was performed using minor modifications of the coupled enzyme assay described by Schneider et al., 2005. A multimode plate reader (PerkinElmer, mode EnVision 2104) compatible with the 96-well UV microplate was used for the assays. The fatty acid substrates were dissolved in 5% Triton X-100 (v/v) to make 5 mM stock solutions. The enzyme assay premix was prepared containing 0.1 M Tris-HCl (pH 7.5), 2 mM dithiothreitol, 5 mM ATP, 10 mM MgCl₂, 0.5 mM CoA, 0.8 mM NADH, 250 µM fatty acid substrate, 1 mM phosphoenolpyruvate, 20 units myokinase (Sigma-Aldrich, catalog no. M3003), 10 units pyruvate kinase (Roche, 10128155001), 10 units lactate dehydrogenase (Roche, 10127230001), and was aliquoted 95 µL each to the 96-well microplate. The reaction was started by adding 5 µL (1–2 µg) proteins. The chamber of the plate reader was set to 30 °C and the OD at 340 nm was recorded every 5 min for 40 min. Oxidation of NADH, which is monitored by the decrease of OD_340nm, was calculated using the NADH extinction coefficient 6.22 cm² µmol⁻¹. Every two moles of oxidized NADH is equivalent to one mole of acyl-CoA product generated in the reaction. To measure the parameters of enzyme kinetics, the fatty acid substrate concentration was varied from 0 to 500 µM, with NADH set at 1 mM. The fatty acid substrates, sodium acetate (C2:0), sodium butyrate (C4:0), sodium hexanoate (C6:0), sodium octanoate (C8:0), sodium decanoate (C10:0), sodium laurate (C12:0), sodium myristate (C14:0), sodium palmitate (C16:0), and sodium stearate (C18:0), were purchased from Sigma-Aldrich. Trans-2-decenoic acid (C10:1), 8-methylnonanoic acid (iC10:0), 3-hydroxy lauric acid (C12:OH), and 3-hydroxy myristic acid (C14:OH) were purchased from Cayman Chemical.

Total RNA was extracted from trichomes isolated from stems and shaved stems of 7-week-old S. pennellii LA0716 plants using the RNAeasy Plant Mini kit (Qiagen) and digested with DNase I. A total of four RNA samples extracted from two tissues with two replicates were used for RNA sequencing. The sequencing libraries were prepared using the KAPA Stranded RNA-Seq Library Preparation Kit. Libraries went through quality control and quantification using a combination of Qubit dsDNA high sensitivity (HS), Applied Analytical Fragment Analyzer HS DNA and Kapa Illumina Library Quantification qPCR assays. The libraries were pooled and loaded onto one lane of an Illumina HiSeq 4000 flow cell. Sequencing was done in a 2 × 150 bp paired end format using HiSeq 4000 SBS reagents. Base calling was done by Illumina Real Time Analysis (RTA) v2.7.6 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.

The paired end reads were filtered and trimmed using Trimmomatic v0.32 (Bolger et al., 2014a) with the setting (LLUMINACLIP: TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:30), and then mapped to the S. pennellii LA0716 genome v2.0 (Bolger et al., 2014a) using TopHat v1.4 (Trapnell et al., 2009) with the following parameters: -p (threads) 8, -i (minimum intron length) 50, -I (maximum intron length) 5000, and -g (maximum hits) 20. The FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for the genes were analyzed via Cufflinks v2.2 (Trapnell et al., 2010).For differential expression analysis, the HTseq package (Anders et al., 2015) in Python was used to get raw read counts, then Edge R version 3.22.5 (McCarthy et al., 2012) was used to compare read counts between trichome-only RNA and shaved stem RNA using a generalized linear model (glmQLFit).

For VIGS analysis of Sq-AACS1 and Sq-AECH1 in S. quitoense, the fragments of these two genes, as well as the phytoene desaturase (PDS) gene fragment, were amplified using the primers listed in Supplementary file 3, cloned into pTRV2-LIC (ABRC no. CD3-1044) using the ligation-independent cloning method (Dong et al., 2007), and transformed into A. tumefaciens (strain GV3101). The VIGS experiments were performed as described (Leong et al., 2020). In brief, the Agrobacterium strains harboring pTRV2 constructs, the empty pTRV2, or pTRV1 were grown overnight in separate LB cultures containing 50 µg/mL kanamycin, 10 µg/mL gentamicin, and 50 µg/ml rifampicin at 30 °C. The cultures were re-inoculated in the induction media (50 mM MES pH5.6, 0.5% glucose [w/v], 2 mM NaH₂PO₄, 200 µM acetosyringone) for overnight growth. The cells were harvested, washed, and resuspended in the buffer containing 10 mM MES, pH 5.6, 10 mM MgCl₂, and 200 µM acetosyringone with the OD_600nm = 1. Different cultures containing pTRV2 constructs were mixed with equal volume of pTRV1 cultures prior to infiltration. The 2- to 3-week-old young S. quitoense seedlings grown under 16 hr photoperiod at 24°C were used for infiltration: the two fully expanded cotyledons were infiltrated. Approximately three weeks post inoculation, the fourth true leaf of each infiltrated plant was cut in half for acylsugar quantification and gene expression analysis, respectively. The onset of the albino phenotype of the control group infiltrated with the PDS construct was used as a visual marker to determine the harvest time and leaf selection for the experimental groups. At least fourteen plants were analyzed for each construct. The trichome acylsugars were extracted using the solution containing acetonitrile/isopropanol/water (3:3:2) with 0.1% formic acid and 1 μM telmisartan as internal standard, following the protocol at http://dx.doi.org/10.17504/protocols.io.xj2fkqe.

The leaf RNA was extracted using RNeasy Plant Mini kits (Qiagen) and digested with DNase I. The first-strand cDNA was synthesized by Superscript II (Thermofisher Scientific) using total RNA as templates. Quantitative real-time PCR was performed to analyze the Sq-AACS1 or Sq-AECH1 mRNA in S. quitoense leaves using the primers listed in Supplementary file 3. EF1α was used as a control gene. A QuantStudio 7 Flex Real-Time PCR System with Fast SYBR Green Master Mix (Applied Biosystems) was used for the analysis. The relative quantification method (2^-ΔΔCt) was used to evaluate the relative transcripts levels.

Trichome acylsugars extracted from tomato IL and BILs were analyzed using a Shimadzu LC-20AD HPLC system connected to a Waters LCT Premier ToF mass spectrometer. Ten microliter samples were injected into a fused core Ascentis Express C18 column (2.1 mm ×10 cm, 2.7 μm particle size; Sigma-Aldrich) for reverse-phase separation with column temperature set at 40°C. The starting condition was 90% solvent A (0.15% formic acid in water) and 10% solvent B (acetonitrile) with flow rate set to 0.4 mL/min. A 7 min linear elution gradient was used: ramp to 40% B at 1 min, then to 100% B at 5 min, hold at 100% B to 6 min, return to 90% A at 6.01 min and hold until 7 min.

For analyzing trichome acylsugars extracted from S. pennellii transgenic plants and membrane lipids from N. benthamiana, a Shimadzu LC-20AD HPLC system connected to a Waters Xevo G2-XS QToF mass spectrometer was used. The starting conditions were 95% solvent A (10 mM ammonium formate, pH 2.8) and 5% solvent B (acetonitrile) with flow rate set to 0.3 mL/min. A 7 min linear elution gradient used for acylsugar analysis was: ramp to 40% B at 1 min, then to 100% B at 5 min, hold at 100% B to 6 min, return to 95% A at 6.01 min and hold until 7 min. A 12 min linear elution gradient used for the lipid analysis was: ramp to 40% B at 1 min, then to 100% B at 5 min, hold at 100% B to 11 min, return to 95% A at 11.01 min and hold until 12 min.

For analyzing trichome acylsugars extracted from other plants, a Waters Acquity UPLC was coupled to a Waters Xevo G2-XS QToF mass spectrometer. The starting condition was 95% solvent A (10 mM ammonium formate, pH 2.8) and 5% solvent B (acetonitrile) with flow rate set to 0.3 mL/min. A 7 min linear elution gradient was: ramp to 40% B at 1 min, then to 100% B at 5 min, hold at 100% B to 6 min, return to 95% A at 6.01 min and held until 7 min. A 14 min linear elution gradient was: ramp to 35% B at 1 min, then to 85% B at 12 min, then to 100% B at 12.01 min, hold at 100% B to 13 min, return to 95% A at 13.01 min and held until 14 min.

For Waters LCT Premier ToF mass spectrometer, the MS settings of electrospray ionization in negative mode were: 2.5 kV capillary voltage, 100°C source temperature, 350°C desolvation temperature, 350 liters/h desolvation nitrogen gas flow rate, 10 V cone voltage, and mass range m/z 50 to 1500 with spectra accumulated at 0.1 s/function. Three collision energies (10, 40, and 80 eV) were used in separate acquisition functions to generate both molecular ion adducts and fragments. For Waters Xevo G2-XS QToF mass spectrometer, the MS settings of the negative ion-mode electrospray ionization were as follows: 2.00 kV capillary voltage, 100°C source temperature, 350°C desolvation temperature, 600 liters/h desolvation nitrogen gas flow rate, 35 V cone voltage, mass range of m/z 50 to 1000 with spectra accumulated at 0.1 s/function. Three collision energies (0, 15, and 35 eV) were used in separate acquisition functions. The MS settings for positive ion-mode electrospray ionization were: 3.00 kV capillary voltage, 100°C source temperature, 350°C desolvation temperature, 600 liters/h desolvation nitrogen gas flow rate, 35 V cone voltage, mass range of m/z 50 to 1000 with spectra accumulated at 0.1 s/function. Three collision energies (0, 15, and 45 eV) were used in separate acquisition functions. The Waters QuanLynx software was used to integrate peak areas of the selected ion relative to the internal standard. For quantification purpose, data collected with the lowest collision energy was used in the analysis.

The publicly available tomato RNA-seq datasets and the methods used for normalizing FPKM, gene expression correlation analysis were described in a recent study (Moore et al., 2020). 926 RNA-seq Sequence Read Archive (SRA) files for tomato from 47 studies were downloaded from National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) (Table S6 in Moore et al., 2020). Reads were filtered using Trimmomatic (Bolger et al., 2014b) based on the sequence quality with default settings, and mapped to the tomato NCBI S. lycopersicum genome 2.5 using TopHat (Trapnell et al., 2009). Read files with <70% mapped reads were discarded. Fragments per kilobase of transcript per million mapped reads (FPKM) were calculated using Cufflinks (Trapnell et al., 2010). The pipeline for FPKM calling used in this study was put in https://github.com/ShiuLab/RNAseq_pipeline (Uygun et al., 2020; copy archived at https://github.com/elifesciences-publications/RNAseq_pipeline). The median FPKM of multiple replicates was used for each sample, resulting in FPKM values in 372 samples. To draw the heatmap of gene expression profiles, FPKM values of a gene across all the samples were scaled, where the maximum FPKM was scaled to 1, while the minimum value was 0.

Protein sequences of annotated genes and the corresponding annotation files in General Feature Format (GFF) of 11 Solanaceae species, Ipomoea trifida, and Coffea canephora were downloaded from National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/genome/) or Solanaceae Genomics Network (SGN, https://solgenomics.net/). The GFF files contain the coordinates of annotated genes on assembled chromosomes or scaffolds. The sources and version numbers of sequences and GFF files used are: S. lycopersicum ITAG3.2 (SGN) and V2.5 (NCBI), S. pennellii SPENNV200 (NCBI) and v2.0 (SGN), S. tuberosum V3.4 (SGN), S. melongena r2.5.1 (SGN), Capsicum annuum L. zunla-1 V2.0 (SGN), C. annuum_var. glabriusculum V2.0 (SGN), Nicotiana attenuata NIATTr2 (SGN), N. tomentosiformis V01 (NCBI), N. benthamiana V1.0.1 (SGN), Petunia axillaris V1.6.2 (SGN), P. inflata V1.0.1 (SGN), I. trifida V1.0 (NCBI), and C. canephora Vx (SGN).

To hypothesize the evolutionary history of genes in the acylsugar gene cluster, putative pseudogenes, which are homologs to protein-coding genes but with predicted premature stops/frameshifts and/or protein sequence truncation, were also identified for each species as described (Wang et al., 2018). Protein sequences from Arabidopsis thaliana, Oryza sativa, and S. lycopersicum were used as queries in the searches against the genomic regions of target species using TBLASTN (Altschul et al., 1990). The intergenic genomic sequences were identified as potential pseudogenes using the pipeline from as previously described (Campbell et al., 2014; Zou et al., 2009). If one of the six-frame translated sequences of the intergenic genomic sequences had significant similarity to annotated protein sequences, and had premature stops/frameshifts and/or were truncated (<30% of functional paralogs), the gene was defined as a pseudogene.

Genome-wide syntenic analysis was conducted using annotated protein-coding genes and putative pseudogenes from all the species with MCScanX-transposed (Wang et al., 2013) as described (Wang et al., 2018). The MCScanX-based analysis did not lead to a syntenic block of acylsugar gene cluster on chromosome 7 of S. melongena r2.5.1, which can be due to true absence, issues with genome assembly, or lack of coverage. To verify this, protein sequences of S. lycopersicum genes in genomic blocks on chromosome 7 were searched against an updated S. melongena genome from The Eggplant Genome Project (http://ddlab.dbt.univr.it/eggplant/) that led to the identification of the synteny.

Homologous genes of Sl-AACS1 (ACS), Sl-AECH1 (ECH), and Solyc07g043670 (BAHD acyltransferase) were obtained through BLAST (Altschul et al., 1990) search from the genomes of 11 Solanaceae species, Ipomoea trifida, and Coffea canephora with an Expect value threshold of 1e-5. To simply the phylogenetic tree, sequences which are distantly related to the target genes were removed, and the remained sequences were used to rebuild the phylogenetic trees. The amino acid sequences were aligned using MUSCLE (Edgar, 2004) with the default parameters. The phylogenetic trees were built using the maximum likelihood method with 1000 bootstrap replicates. The trees were generated using RAxML/8.0.6 (Stamatakis, 2014) with the following parameters: -f a -x 12345 p 12345 -# 1000 m PROTGAMMAAUTO --auto-prot=bic, and were shown with midpoint rooting. The final sequence alignments used to generate the phylogenetic trees were provided in Supplementary file 5.

Ancestral trait state reconstruction was conducted using the maximum likelihood model Mk1 in Mesquite 3.6 (Massidon and Maddison, 2018). Four traits were inferred for their ancestral states. They are the presence of medium chain acylsugars, presence of ACS genes in the synteny, presence of ECH genes in the synteny, and presence of both ACS and ECH genes in the synteny. The phylogeny of Solanaceae species was based on a previous study (Särkinen et al., 2013).

Acyl chains were characterized from the corresponding fatty acid ethyl esters following transesterification of acylsugar extractions as previously reported (Ning et al., 2015). Plants were grown for 4–8 weeks and approximately ten leaves were extracted for 3 min in 10 mL of 1:1 isopropanol:acetonitrile with 0.01% formic acid. Extractions were dried to completeness using a rotary evaporator and then 300 µL of 21% (v/v) sodium ethoxide in ethanol (Sigma) was added and incubated for 30 min with gentle rocking and vortexing every five minutes and 400 µL hexane was added and vortexed for 30 s. To the hexane layer, 500 µL of saturated sodium chloride in water was added and vortexed to facilitate a phase separation. After phase separation, the top hexane layer was transferred to a new tube. The phase separation by addition of 500 µL hexane was repeated twice, with the final hexane layer transferred to a 2 mL glass vial with a glass insert.

The fatty acid ethyl esters were analyzed using an Agilent 5975 single quadrupole GC-MS equipped with a 30 m, 0.25 mm internal diameter fused silica column with a 0.25 µm film thickness VF5 stationary phase (Agilent). Injection of 1 µL of each hexane extract was performed using splitless mode. The gas chromatography program was as follows: inlet temperature, 250°C; initial column temperature, 70°C held for 2 min; ramped at 20 °C/min until 270°C, then held at 270°C 3 min. The helium carrier gas was used with 70 eV electron ionization. Acyl chain type was determined through NIST Version 2.3 library matches of the mass spectra of the corresponding ethyl ester and relative abundances were determined through integrating the corresponding peak area over the total acyl chain peak area.