In vitro reconstruction and analysis of evolutionary PNAS PLUS variation of the acylsucrose metabolic network

Pengxiang Fana, Abigail M. Millera, Anthony L. Schilmillera, Xiaoxiao Liub, Itai Ofnerc, A. Daniel Jonesa,b, Dani Zamirc, and Robert L. Lasta,d,1

aDepartment of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824; bDepartment of Chemistry, Michigan State University, East Lansing, MI 48824; cThe Institute of Sciences and Genetics in Agriculture, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel; and dDepartment of Plant Biology, Michigan State University, East Lansing, MI 48824

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved December 7, 2015 (received for review September 9, 2015) Plant glandular secreting are epidermal protuberances that metabolized to volatile fatty acids by Manduca sexta larvae, and these produce structurally diverse specialized metabolites, including medi- airborne products attract predatory ants (6). Protective properties cally important compounds. Trichomes of many in the night- against have made increasing total acylsugars or altering shade family () produce O-acylsugars, and in cultivated and acyl chain types a target for breeding insect-resistant cultivated to- wild tomatoes these are mixtures of aliphatic esters of sucrose and matoes ( lycopersicum)(16–18). In addition, synthetic su- glucose of varying structures and quantities documented to contribute crose esters mimicking natural acylsugars have been applied as safe, to insect defense. We characterized the first two enzymes of acylsu- biodegradable insecticidal compounds (19–21)andalsohavecom- crose biosynthesis in the cultivated tomato Solanum lycopersicum. mercial value in the food, cosmetic, and pharmaceutical industries These are type I/IV -expressed BAHD acyltransferases encoded (22, 23). ─ by Solyc12g006330 or S. lycopersicum acylsucrose acyltransferase 1 Recent work on acylsucrose biosynthesis in trichomes of the to- ─ (Sl-ASAT1) and Solyc04g012020 (Sl-ASAT2). These enzymes were mato clade revealed that this relatively closely related group of — used in concert with two previously identified BAHD acyltrans- plants produces a surprisingly diverse group of acylsucroses in — ferases to reconstruct the entire cultivated tomato acylsucrose bio- tip cells of the long hair-like type I/IV trichomes (14, 24–26). The synthetic pathway in vitro using sucrose and acyl-CoA substrates. genetic and biochemical mechanisms underlying some of this phe- Comparative genomics and biochemical analysis of ASAT enzymes notypic diversity have begun to be revealed. For example, 2-meth- were combined with in vitro mutagenesis to identify amino acids that ylpropanoic acid (iC4) and 3-methylbutanoic acid (iC5) acyl chain influence CoA ester substrate specificity and contribute to differences variation in Solanum pennellii accessions is influenced by varia- in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic en- tionintheactivityofasetoftruncated isopropylmalate synthase gineering of these insecticidal metabolites in plants and microbes. 3 (IPMS3) enzymes (26). Differences in patterns of Solanum hab- rochaites acylsucrose acetylation (24) and acyl chain length and Solanum | glandular trichomes | acylsugar | specialized metabolism | position variation (25) are due to genetic variation in two BAHD B A H D genotype to phenotype [ EAT, HCT, CBT, AT (27, 28)] acyltransferases. Solanum lycopersicum AcylSugar AcylTransferase 3 (Sl-ASAT3) catalyzes acylation of diacylsucroses on the five-membered (furanose) ring to lants are masters of metabolism, producing hundreds of make triacylsucroses (25). Variant forms of ASAT3 were described Pthousands of small molecules known as specialized metabolites, which vary widely in structure, abundance, and physical and biological in wild tomato accessions: these use different chain length acyl-CoA properties. These metabolites tend to be produced by enzymes that esters with diacylsucrose, or acylate the six-membered (pyranose) evolve faster than those that produce “central” metabolites such as ring of monoacylated sucrose, demonstrating recent evolutionary amino acids, nucleotides, sugars, and cofactors (1–3), and the path- ways and metabolic intermediates involved in biosynthesis of many Significance specialized metabolites remain mysterious. Despite the growing availability of genomic DNA sequences, understanding the genetic Throughout the course of human history, plant-derived natural and biochemical mechanisms that contribute to this phenotypic di- products have been used in medicines, in cooking, as pest control versity and plasticity presents enduring and major challenges in plant agents, and in rituals of cultural importance. Plants produce rap- biochemistry. It is of great interest to understand and manipulate the idly diversifying specialized metabolites as protective agents and biosynthesis of these biologically active molecules. to mediate interactions with beneficial organisms. In vitro re-

Specialized metabolites typically are produced in a cell- or construction of the cultivated tomato insect protective acylsucrose PLANT BIOLOGY tissue-specific manner and are generally limited in their taxonomic biosynthetic network showed that four acyltransferase enzymes distribution. Glandular secreting trichomes provide an example of are sufficient to produce the full set of naturally occurring com- such a differentiated structure; these epidermal “hairs” produce a pounds. This system enabled identification of simple changes in variety of metabolites of importancetohumans,includingaromatic enzyme structure leading to much of the acylsucrose diversity flavor components (e.g., in hops for beer and Mediterranean herbs produced in epidermal trichomes of wild tomato. These findings for cooking), psychoactive cannabinoids in Cannabis, and the an- will enable analysis of trichome specialized metabolites through- timalarial drug artemisinin in Artemisia annua (4, 5). out the Solanaceae and demonstrate the feasibility of engineering Some trichome-produced metabolites have documented direct these metabolites in plants and microorganisms. and indirect antiherbivore activities (4, 6–8). For example, acylsugars Author contributions: P.F., A.L.S., and R.L.L. designed research; P.F., A.M.M., A.L.S., and are a group of structurally related specialized metabolites produced X.L. performed research; I.O. and D.Z. contributed new reagents/analytic tools; P.F., A.L.S., in plants of the nightshade family—the Solanaceae (9, 10). Char- X.L., I.O., A.D.J., D.Z., and R.L.L. analyzed data; and P.F. and R.L.L. wrote the paper. acterized examples in the tomato group of Solanum consist of either The authors declare no conflict of interest. a glucose or a sucrose backbone with three to four aliphatic acyl This article is a PNAS Direct Submission. groups of varying carbon numbers ranging from 2 to 12 esterified to 1To whom correspondence should be addressed. Email: [email protected]. – the sugar hydroxyl groups (11 15). Nicotiana attenuata acylsucroses This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. are at the center of a multitrophic defense interaction where they are 1073/pnas.1517930113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1517930113 PNAS | Published online December 29, 2015 | E239–E248 Downloaded by guest on October 1, 2021 changes leading to diversification of enzymatic activity. The phylo- Positive-ion mode MS fragment ion masses revealed that these genetically related ASAT4 enzyme (formerly AT2) is responsible triacylsucroses—S3:15-P (5, 5, 5) and S3:22-P (5, 5, 12)— for making tetraacylsucroses in S. lycopersicum and S. habrochaites presumably have all three acyl chains on the pyranose ring (SI trichomes by acetylating triacylsucroses using acetyl-CoA (C2-CoA) Appendix, Fig. S2A). This is in contrast to the acylsucroses S3:15 (29). A variety of loss-of-function alleles of this enzyme are found in populations of S. habrochaites from northern Peru and Ecuador (24), reinforcing the idea that ASAT diversification plays an im- portant role in shaping the strong phenotypic diversity in trichomes 10 of wild tomato. Although variation in these enzymes influences the A acylsugar phenotypes seen in cultivated and wild tomatoes, much of the diversity remains to be explained at a molecular level, including 8 35S:ASAT1-intron-1TASA (RNAi) in M82 the order of acysugar assembly. In addition, identification of the enzymes that produce the ASAT3 substrates is needed to un- 6 derstand cultivated and wild tomato acylsucrose biosynthesis in greater detail. In this study, two trichome-specific S. lycopersicum BAHD acyl- 4 transferases that catalyze consecutive reactions to produce these Acylsugars Total diacylsucrose intermediates were identified. Sl-ASAT1 was found to Peak area/Internal standard 2 use sucrose and various acyl-CoAs to make monoacylsucroses with pyranose R4 acylation. Sl-ASAT2 adds a second acyl chain to the R3 R4 position of the ASAT1 S1:5 (iC5 ) product (note that “S” refers 0 “ ” to a sucrose backbone, 1:5 indicates the presence of a single iC5 -10 -04 -13 -26 -06 -02 -33 -01 -34 -19 -20 -07 -32 -16 -17 -29 -03 -08 -15 -11 -22 -24 -31 -05 -21 -28 -25 -23 -27 -18 -09 -30 -12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 M82 T ester decoration on sucrose, and the superscript “R4” describes the T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T acylation position) to make the R3,4 diacylsucrose Sl-ASAT3 sub- B 100 1: TOF MS ES- strates. With the four ASAT enzymes in hand, we reconstructed the BPI IL4-1 5.00e4 cultivated tomato acylsucrose biosynthetic network using sucrose and acyl-CoA substrates. Comparative functional analysis of ASAT2 variants from nine wild tomato relatives led to identification of two S3:22 (5,5,12)

residues affecting ASAT2 iso-C5-CoA (iC5-CoA) and anteiso-C5- S4:24 (2,5,5,12) CoA (aiC5-CoA; 2-methylbutyryl-CoA) substrate preference. Using S4:17 (2,5,5,5) S3:22-P (5,5,12) S3:22-P S3:15-P (5,5,5) S3:15-P S3:15 (5,5,5) S4:16 (2,4,5,5) Relative abundance % a similar approach, a residue controlling the ability of ASAT3 to use 0 long chain acyl-CoAs was identified. The in vitro reconstruction 100 1: TOF MS ES- system allowed us to test the impact of these variant enzymes and BPI M82 5.00e4 changes in acyl-CoA substrate concentrations on the S. lycopersicum acylsucrose biosynthetic network, demonstrating the value of using the in vitro pathway to understand acylsugar evolution. These results S4:17 (2,5,5,5) S4:24 (2,5,5,12)

provide a model for understanding how small changes in enzyme S3:22 (5,5,12) sequence lead to large changes in metabolic diversity. S4:16 (2,4,5,5) S3:15 (5,5,5) Relative abundance % 0 Results 2.00 4.00 6.00 8.00 10.00 12.00 14.00 Retention time (min) Identification of Two Trichome-Specific BAHD Acyltransferases Involved C 0.3 in Tomato Acylsugar Biosynthesis. Cultivated tomato produces pri- marily tri- and tetraacylated sucrose esters in the tip cell of the long Sl-ASAT2:ASAT2 in IL4-1 multicellular “type I/IV” trichomes (14, 29). Although two trichome 0.2 apical cell-expressed BAHD acyltransferases that produce tri- and tetraacylsucroses from diacylsucroses were recently described (25, 29), the enzymes that convert sucrose to diacylated sucrose have not 0.1 been previously reported. We used functional genomics approaches S3:22-P/S3:22 Peak area ratio to identify candidate enzymes for these earlier steps in the pathway. Bioinformatic analysis revealed 92 genes predicted to encode BAHD acyltransferase sequences in the S. lycopersicum genome 0.0 (SI Appendix,Fig.S1A). Twenty-two genes were selected as targets -19 -17 -23 -20 -24 -21 -26 -22 -11 -13 -25 -27 -10 -14 -15 -12 -04 -09 -06 -01 -02 -07 -08 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 M82 T T T T T T T T T T T T T T T T T T T T T T T for RNAi suppression in M82; these were predicted to encode full- IL4-1 length proteins and had evidence of trichome expression based on a Fig. 1. The S. lycopersicum M82 trichome acylsugar profile is affected by S. lycopersicum trichome EST database (30) (see the proteins in- changes in two BAHD acyltransferases. (A) RNAi suppression of Sl-ASAT1 causes dicatedwitharrowsinSI Appendix,Fig.S1A). Acylsugars in RNAi reduction of M82 tomato total acylsugar peak areas. Summed extracted ion T0 primary transgenic plants were analyzed, and lines targeting chromatogram peak areas divided by internal standard peak areas for all de-

Solyc12g006330, which we renamed Sl-ASAT1, showed reduction of tectable acylsugars are shown for each independent T0 primary transformant. total acylsugar levels compared with the M82 parental line (Fig. 1A) M82dataarefromfivebiologicalreplicates± SD. (B) Introgression of S. pen- and no detectable acylsucrose intermediates, pointing to a role for nellii chromosome 4 region IL4-1 causes accumulation of acylsucroses S3:15-P Sl-ASAT1 in acylsugar biosynthesis in M82. (5, 5, 5) and S3:22-P (5, 5, 12), which are not seen in M82 extracts. Negative-ion- Published studies revealed major differences in trichome metab- mode base-peak intensity LC/MS chromatograms are shown for IL4-1 and M82. Fragment ion masses in positive-ion-mode mass spectra (SI Appendix,Fig.S2A) olite accumulation (15, 25, 26, 29) in several introgression lines (ILs), “ ” S. pennellii indicate that these metabolites contain all three acyl chains on one ring. -P which have regions of the LA0716 genome substituted in means that three acyl chains are presumably on the pyranose ring. (C)Sl-ASAT2 place of the S. lycopersicum M82 genome (31). Rescreening the ILs – – transgenic expression causes reversal of the IL4-1 mutant phenotype. LC/MS by liquid chromatography time of flight mass spectrometry (LC ToF peak area ratios for the IL4-1–specific S3:22-P and S3:22 acylsugars are shown

MS) identified a more subtle phenotype: accumulation of two low- for each independent T0 primary transgenic line. Data for IL4-1 and M82 are abundance species in IL4-1 not seen in the M82 parent (Fig. 1B). each from five biological replicates ± SD.

E240 | www.pnas.org/cgi/doi/10.1073/pnas.1517930113 Fan et al. Downloaded by guest on October 1, 2021 PNAS PLUS AB [M+Cl]- 1: TOF MS ES- 6 S1:5 387.1+461.1 0.2000Da 1’ 6’ 100 4 4.78e4

- 3 3’ 4’ % [M+formate] 2 Sucrose Sl-ASAT1 or Sp-ASAT1

S1:5 isomer Relative abundance

0 2.00 3.00 4.00 5.00 6.00 7.00 8.00 R4= iC4, iC5, aiC5, nC10, nC12 Retention time (min)

O S2:9 (iC5,iC4) 100 S1:5 (iC5R4) 1: TOF MS ES- C S-CoA 471.1+541.2 0.3000Da iC4-CoA % 7.90e4 0

O 100 S2:10 (iC5,iC5) 1: TOF MS ES- S-CoA 471.1+555.2 0.3000Da iC5-CoA % 7.90e4 0

R4 R3 O S2:10 (iC5 ,aiC5 ) Sl-ASAT2 100 1: TOF MS ES- + S-CoA 471.1+555.2 0.3000Da % 7.90e4 0 S1:5 (iC5R4) aiC5-CoA 100 S2:15 (iC5,nC10) 1: TOF MS ES- O 471.1+625.3 0.3000Da S-CoA % 7.90e4 nC10-CoA 0 S2:17 (iC5,nC12) 100 1: TOF MS ES- 471.1+653.3 0.3000Da

O % 7.90e4 PLANT BIOLOGY S-CoA 0 1.00 2.00 3.00 4.00 5.00 nC12-CoA Retention time (min)

Fig. 2. Consecutive in vitro reactions with Sl-ASAT1 and Sl-ASAT2 proteins produce diacylsucroses from sucrose. (A) Result of Sl-ASAT1 enzyme activity assay using sucrose − and iC5-CoA as substrates. Negative-ion-mode LC/MS extracted ion chromatograms for m/z 387.1 (sucrose; [M+formate] ) and the corresponding ion for reaction product − m/z 461.1 (S1:5; [M+Cl] ) are shown. “S” represents an acylsucrose backbone, “1” indicates the number of acyl chains, and “5” corresponds to the total number of carbons in the acyl chain. The minor peak is a S1:5 (iC5R6) isomer produced by nonenzymatic rearrangement. (B) Summary of the reactions catalyzed by Sl-ASAT1 or Sp-ASAT1

(Sopen12g002290) with sucrose and acyl-CoA substrates of different chain lengths. The R4 acylation of sucrose by Sl-ASAT1 was verified by NMR for the monoacylsucroses containing an iC5 acyl chain as shown in table S4 of Schilmiller et al. (25) or the nC12 chain (SI Appendix, Table S1). (C) In vitro production of R3-acylated diacylsucroses by Sl- ASAT2 using S1:5 (iC5R4) and different acyl-CoAs (iC4-, iC5-, aiC5-, nC10-, and nC12-CoA) as substrates. Negative-ion-mode LC/MS extracted ion chromatograms for S1:5 and different diacylsucrose products are shown. The S2:10 (iC5R4,aiC5R3) structure was verified by NMR as shown in table S3 of Schilmiller et al. (25).

(5, 5, 5) and S3:22 (5, 5, 12), which are typically seen in the M82 one on the furanose ring (SI Appendix, Fig. S2A). The locus parent and which have two acyl chains on the pyranose ring and controlling this acylsugar phenotype was narrowed down to a

Fan et al. PNAS | Published online December 29, 2015 | E241 Downloaded by guest on October 1, 2021 region containing 64 genes by screening selected backcross in- the product of Sl-ASAT1—and the structurally diverse acyl-CoA bred lines (BILs) that have recombination breakpoints on chro- donor substrates iC4-CoA, aiC5-CoA, nC10-CoA, and nC12-CoA to mosome 4 (SI Appendix,Fig.S2B). Among the 64 genes, a strong make the corresponding diacylsucroses (Fig. 2C).Theenzymepro- candidate gene—Solyc04g012020 (Sl-ASAT2)—and its putative duced only a small amount of product with iC5-CoA compared with orthologous gene Sopen04g006140 (Sp-ASAT2) in LA0716 were other substrates, which indicates that iC5-CoA is not a preferred identified based on the prediction that they encode enzymes substrate for Sl-ASAT2. NMR analysis was performed on the pu- belonging to the BAHD acyltransferase family. rified compound S2:10 (iC5, aiC5) made by sequential reaction of The in vivo function of Sl-ASAT2 was tested by transgenic sucrose with iC5-CoA catalyzed by Sl-ASAT1 and the subsequent plant experiments. F1 plants generated by crossing IL4-1 to M82 reaction of this product with aiC5-CoA catalyzed by Sl-ASAT2. showed the M82 acylsugar phenotype, consistent with the hy- NMR chemical shift data indicate that Sl-ASAT2 added the aiC5 pothesis that Sp-ASAT2 is recessive to Sl-ASAT2. Thus, we grouptotheR3 position of the sucrose backbone as shown in table predicted that transformation of IL4-1 with an Sl-ASAT2 trans- S3 of Schilmiller et al. (25). The diacylsucrose S2:17 (iC5, nC12), gene driven by its own promoter would restore IL4-1 acylsugar made by sequential reaction of sucrose with iC5-CoA (catalyzed by profiles to the wild-type M82 phenotype. Indeed, 15 of 23 in- Sl-ASAT1) and its product with nC12-CoA (catalyzed by Sl- dependent IL4-1 T0 transformant plants showed varying levels of ASAT2), has the same chromatographic retention time as S2:17 complementation with the peak area ratio of S3:22-P to S3:22 (iC5R4, nC12R3) purified from IL11-3 (SI Appendix, Fig. S5), restored from 0.23 in IL4-1 to less than 0.05, which is close to the which has NMR-resolved structural information showing the iC5 ratio observed in M82 (Fig. 1C). Sl-ASAT2 RNAi lines were group at the R4 position and the nC12 group at the R3 position generated in M82, and reduced total acylsugar levels were ob- (25). This result suggests that Sl-ASAT2 added the long acyl served for 24 of 29 independent transgenic lines (SI Appendix, chain to the R3 position of the S1:5 monoacylsucrose substrate to Fig. S2C); this is consistent with the hypothesis that Sl-ASAT2 make R3,R4 substituted diacylsucroses. In contrast, Sl-ASAT2 plays an in vivo role in wild-type M82 acylsugar production. The does not efficiently use purified S1:12 (nC12R4) as the acyl ac- T0 transgenic RNAi (SI Appendix, Fig. S3) and complementation ceptor using any of the acyl-CoA donors tested. results (SI Appendix, Fig. S4) were confirmed in T1 progeny lines Kinetic analyses were performed to obtain more detailed in- generated by self-crossing. formation regarding the in vitro properties of the acyltransferases. Deep RNA-seq analysis revealed that both Sl-ASAT1 and Sl- ASAT1 and ASAT2 used short chain acyl-CoA esters with apparent ASAT2 mRNAs are highly enriched in M82 tomato trichomes (26), Km values in the 20- to 50-μM range, and the longer chain nC12- and trichome-enriched expression of Sl-ASAT1 and Sl-ASAT2 in CoA exhibited substrate inhibition of both enzymes (SI Appendix, M82 stem trichomes and Sp-ASAT2 in S. pennellii LA0716 Table S2 and Fig. S6), as was also previously reported for ASAT3 trichomes were confirmed by RT-PCR (SI Appendix,Figs.S1C and (25). An apparent Km of 2.3 mM was measured for ASAT1 and the S2D). These results were further validated and refined by producing acceptor substrate sucrose (SI Appendix, Table S2 and Fig. S6). In M82 transgenic lines expressing a green fluorescent protein– addition, the ASAT1 enzyme showed evidence of substrate pro- β-glucuronidase (GFP–GUS) reporter driven by the promoter of miscuity as was previously documented for other BAHD enzymes Sl-ASAT1 or Sl-ASAT2. Each promoter drove GFP expression in (32). The aromatic benzoyl-CoA was efficiently used as a donor the tip cell of type I/IV trichomes in stably transformed M82 substrate with sucrose, whereas the negatively charged malonyl-CoA plants (SI Appendix, Figs. S1D and S2E). This pattern is identical did not yield detectable product (SI Appendix, Table S3 and Fig. S7). to that of Sl-ASAT3 (25), Sl-ASAT4 (29), and Sl-IPMS3 (26) and Although ASAT1 had no activity with the monosaccharide glucose, supports the hypothesis that Sl-ASAT1 and Sl-ASAT2 have it used the glucose-containing disaccharides cellobiose, lactose, functions in acylsugar biosynthesis. maltose, and trehalose as acceptor substrates when acyl-CoA sub- strates were used, albeit much less efficiently than with sucrose (less Sl-ASAT1 and Sl-ASAT2 Work Sequentially to Produce Diacylsucroses than 3% of the monoacylsucrose products peak areas) (SI Appendix, in Vitro. The combination of lack of accumulation of partially acyl- Fig. S7). Finally, all four ASAT enzymes were found to be readily ated acylsugar pathway intermediates in RNAi plants and type I/IV reversible when incubated with their usual products and 100 μM apical cell expression led to the hypothesis that Sl-ASAT1 catalyzes CoA (SI Appendix,Fig.S8), as reported for other BAHD enzymes the first step of acylsucrose biosynthesis. Indeed, recombinant Sl- (33, 34). In addition, S1:5 (iC5R4) tended to hydrolyze to sucrose ASAT1 protein expressed in Escherichia coli converted sucrose and incubated with CoA in the absence of enzyme. iC5-CoA to make the monoacylsucrose S1:5 (Fig. 2A). Sl-ASAT1 can also use other short chain (iC4-CoA, aiC5-CoA) or long-chain In Vitro Reconstruction of M82 Acylsucrose Biosynthesis. In vitro (nC10-CoA, nC12-CoA) acyl-CoAs as in vitro acyl donors to make reconstruction serves as an excellent approach to validate bio- the respective monoacylsucroses (Fig. 2B). To determine the synthetic pathways and provides the opportunity to explore the Sl-ASAT1 reaction product structures, S1:5 and S1:12 were purified feasibility of metabolic engineering approaches. We asked whether and analyzed using NMR spectroscopy. Acylation at the sucrose R4 the four recombinant enzymes—ASAT1 through ASAT4—could position was observed for both, with the NMR chemical shift data use acyl-CoA substrates to produce the acylsucroses extracted from for S1:12 (nC12R4) shown in SI Appendix,TableS1and for S1:5 cultivated tomato. Reconstruction of the M82 acylsucrose bio- (iC5R4) in table S4 in Schilmiller et al. (25). A chromatographically synthetic network starting with sucrose was performed by sequen- separable S1:5 isomer, seen as a minor later eluting peak (Fig. 2A), tially adding the four enzymes and appropriate acyl-CoA substrates was purified from the original S1:5 product and found to be acylated in a single tube (Fig. 3A). Because M82 tomato trichomes produce at the R6 position; this was demonstrated to be an in vitro artifact acylsucroses with long or short chains at the R3 position (14), caused by acyl chain rearrangement that is promoted by high parallel Sl-ASAT2 reactions were performed using either aiC5- pH. The putative ortholog of ASAT1 in S. pennellii LA0716, CoA or nC12-CoA as the acyl donor substrate and S1:5 (iC5R4), Sopen12g002290, encodes a protein that shares 97.9% amino acid produced by Sl-ASAT1, as the acyl acceptor substrate. The diac- identity with Sl-ASAT1. This protein also produces monoacylsucroses ylsucrose products from the second step were then used as sub- (Fig. 2B), indicating that ASAT1 has a conserved function in the strates for Sl-ASAT3, followed by reaction of these triacylsucrose tomato branch of Solanum. Taken together, our results indicate products with C2-CoA and Sl-ASAT4 (chromatograms “S” and that ASAT1 catalyzes the first step of sucrose acylation and “L,” respectively, in Fig. 3B). The sequential assays produced produces an R4 monoacylated sucrose product. mono, di-, tri-, and tetraacylsucroses, and the resultant S3:15 (5, 5, Sl-ASAT2 in vitro enzyme activity was tested using protein 5), S4:17 (2, 5, 5, 5), S3:22 (5, 5, 12), and S4:24 (2, 5, 5, 12) had expressed in E. coli. We found that Sl-ASAT2 uses S1:5 (iC5R4)— chromatographic retention times identical to the M82 trichome

E242 | www.pnas.org/cgi/doi/10.1073/pnas.1517930113 Fan et al. Downloaded by guest on October 1, 2021 acylsucroses (Fig. 3B). The high-collision-energy negative-ion mode In Vitro System Responds to Changes in Acyl-CoA Precursor Availability. PNAS PLUS mass spectra of these four compounds revealed fragment ion spectra Acylsugar phenotypic diversity was observed for various wild tomato indistinguishable from the tri- and tetraacylsucroses extracted from species and for accessions within S. pennellii and S. habrochaites M82 that were previously documented by Schilmiller et al. (29). (14, 24–26). For example, we recently demonstrated that intro- Although the sequential reconstruction experiments provided gression of a region of S. pennellii LA0716 from the top of chro- strong support that these four enzymes are sufficient to produce the mosome 8 (IL8-1-1) causes increased accumulation of iC4- major products in leaf-surface extracts from sucrose and CoA es- containing acylsucroses due to introduction of a gene encoding a ters, simultaneous presence of enzymes and substrates presumably truncated Leu feedback-insensitive isopropylmalate synthase more closely reflects the in vivo reaction conditions. The four en- (IPMS)-likeenzyme(Sp-IPMS3) (15, 26). Unlike the enzymatically zymes, sucrose, and acyl-CoA substrates were added simultaneously active Sl-IPMS3, this S. pennellii isoform has a defect in in vitro for these mixed assays: iC5-CoA, which is the acyl donor for Sl- IPMS activity, and we hypothesized that this defect blocks pro- ASAT1 and Sl-ASAT3, and C2-CoA, the acyl donor for Sl-ASAT4, duction of iC5-CoA and diverts its precursor to iC4-CoA, leading to accumulation of iC4 acyl chain-containing acylsugars (26). We used were added to the mixed reactions, together with varied types of the in vitro reconstructed S. lycopersicum acylsucrose pathway to acyl-CoA substrates for Sl-ASAT2, either with short (iC4-CoA, test the hypothesis that the IL8-1-1 high-iC4 acylsucrose phenotype aiC5-CoA) or with long acyl chains (nC10-CoA, nC12-CoA). Col- is due to an increase in the ratio of iC4-CoA to iC5-CoA substrates. lectively, the reactions produced the full set of tri- and tetraa- We varied the relative amounts of iC4-CoA and iC5-CoA added to cylsucroses that accumulate in vivo, including the iC4- and nC10- in vitro reactions that contained all substrates and enzymes added containing compounds that are relatively minor components in simultaneously (SI Appendix,Fig.S9C). As shown in Fig. 4, both the tomato plants (SI Appendix,Fig.S9A and B). All in vitro-produced absolute and relative amounts of the four C4-containing metabolites tri- and tetraacylsucroses shared the same m/z and chromatographic increased as the percentage of total iC4-CoA [100 × iC4-CoA/(iC4- retention times with the corresponding acylsucroses extracted from CoA + iC5-CoA)] went from 12.5% to 50%. Increases in S3:20 M82 trichomes (SI Appendix,Fig.S9A and B). The in vitro pro- (4,4,12)andS4:22(2,4,4,12)wereespeciallysensitivetoiC4-CoA duction of the full set of M82 acylsucroses provides strong evidence substrate availability, presumably because their synthesis is com- that Sl-ASAT1, Sl-ASAT2, Sl-ASAT3, and Sl-ASAT4 are the major pletely dependent on this substrate. These in vitro results are con- enzymes in the acylsugar metabolic network in the apical cell of sistent with the hypothesis that provision of acyl-CoA esters cultivated tomato type I/IV trichomes. influences the overall composition of acylsucroses in trichomes of

A

B PLANT BIOLOGY

Fig. 3. In vitro reconstruction of production of the four major M82 acylsugars by sequential addition of ASAT1, ASAT2, ASAT3, and ASAT4 using sucrose and acyl-CoA substrates. (A) Schematic representation of sequential enzyme assays. The first reaction used sucrose and iC5-CoA to make the monoacylsucrose product S1:5 (iC5R4) catalyzed by Sl-ASAT1. After enzyme heat inactivation, the acyl-CoA short-chain aiC5-CoA (S) or long-chain nC12-CoA (L) was added with Sl-ASAT2, and the corre- sponding diacylsucroses were produced. Next, Sl-ASAT3 and iC5-CoA were added followed by Sl-ASAT4 and C2-CoA to produce tri- and tetraacylsucroses, respectively. (B) LC/MS-extracted ion chromatogram analysis of the products of the sequential reactions. “S” and “L” represent the sequential assay for which the short-chain aiC5- CoA or long-chain nC12-CoA, respectively, were used with Sl-ASAT2. Color coding of the product peaks and names corresponds to enzyme names and acyl chains in A. Relative abundance for each chromatogram is based on setting the major peak to 100%; M82: S4:17 (2, 5, 5, 5); L: S3:22 (5, 5, 12); S: S4:17 (2, 5, 5, 5).

Fan et al. PNAS | Published online December 29, 2015 | E243 Downloaded by guest on October 1, 2021 donor specificity. Protein structure homology modeling was per- 40 S3:20 (4,4,12) formed for Sl-ASAT2 using a trichothecene 3-O-acetyltransferase− 30 S4:22 (2,4,4,12) acyl CoA complex (3B2S) (36). This analysis revealed that—of the S3:21 (4,5,12) candidate residues possibly affecting iC5-CoA and aiC5-CoA sub- 20 S4:23 (2,4,5,12) strate specificity identified in the comparative sequence analysis (Fig. 5A)—Phe408 and Ile44 were closest to the putative acyl-CoA–binding 10 pocket (Fig. 5B). Site-directed mutagenesis and in vitro enzyme assays were used to 4 test the hypothesis that these candidate amino acids influence acyl- CoA substrate preference. Consistent with expectation, mutagenesis

3 of Sl-ASAT2 from Phe408 to Val408 increased the ability of the en- Fold change Fold Peak area/Internal standard standard area/Internal Peak 2 zyme to use iC5-CoA to produce S2:10 (5, 5) (Fig. 6): the variant enzyme has an apparent K value for iC5-CoA of 26.3 ± 3.2 μM. 1 m This is similar to the apparent Km value 27.1 ± 5.2 μMforiC5-CoA 0 for Solanum arcanum LA2172 ASAT2, an isoform that efficiently 12.5% 25% 37.5% 50% uses iC5-CoA as a substrate (SI Appendix,Fig.S11). The reciprocal [100 × iC4-CoA/(iC4-CoA + iC5-CoA)] mutagenesis change—with S. arcanum LA2172 ASAT2 mutated 408 408— Fig. 4. Varying the ratio of iC4-CoA and iC5-CoA causes changes in accumu- from Val to Phe ledtoanenzymewithundetectableactivity lation of iC4-containing acylsucroses in vitro. Quantification of LC/MS-extracted for iC5-CoA (Fig. 6) while retaining the ability to use aiC5-CoA and ion chromatogram peak areas divided by internal standard peak areas for the nC12-CoA as substrates. In contrast, mutating position 43 of the in vitro-produced acylsucroses S3:20 (4, 4, 12), S4:22 (2, 4, 4, 12), S3:21 (4, 5, 12), S. habrochaites LA1718 ASAT2 from Leu to Ile conferred the ability and S4:23 (2, 4, 5, 12) showed that the accumulation of iC4-containing acyl- to use aiC5-CoA as a substrate (Fig. 6) with the apparent Km value sucroses increased as the percentage of total iC4-CoA [100 × iC4-CoA/(iC4-CoA + observed of 159 ± 23 μM(SI Appendix,Fig.S11). The conversion of iC5-CoA)] went from 12.5% to 50%. The fold change for each acylsucrose is ± Ile to Leu at amino acid 44 of Sl-ASAT2 abolishes its ability to use shownwithreplicates SE. aiC5-CoA as a substrate (Fig. 6; SI Appendix,Fig.S11) without af- fecting the ability to accept the long-chain nC12-CoA as a substrate. S. lycopersicum. They validate the idea that differences in iC4-con- Taken together, this comparative biochemical analysis identified two taining acylsugars in the IL8-1-1 introgression line and S. pennellii residues affecting ASAT2 acyl-CoA substrate specificity. accessions are due to changes in iC5- and iC4-CoA availability Identification of an ASAT3 Residue Associated with Long-Chain Acylation caused by differences in IPMS3 enzyme structure and function (26). of the Acylsucrose Furanose Ring. In contrast to acylsucroses extracted Comparative Analysis of Natural Variant Enzymes Reveals Amino Acid from S. lycopersicum M82, which have an iC5 acyl chain at the R3′ position of the furanose ring, some S. habrochaites accessions pro- Residues Affecting ASAT2 Acyl-CoA Substrate Specificity. Differ- – ences in ASAT3 and ASAT4 enzyme activities were previously duce acylsucroses with long chains (C10 C12) at this position (14, demonstrated to contribute to metabolite diversity in S. hab- 25). These differences correlate with variation in in vitro ASAT3 activities from those accessions (25). We used the comparative rochaites and S. pennellii (24, 25). Sp-ASAT2, which shares biochemical approach to identify amino acids responsible for these 95.1% protein identity with Sl-ASAT2, showed barely detectable differences in ASAT3 acyl-CoA substrate specificities. As shown in activity with the Sl-ASAT2 acyl acceptor substrates S1:5 (iC5R4) SI Appendix,Fig.S12, 19 residues correlated with activity differences and different acyl-CoAs. This observation suggests that di- between Sl-ASAT3 and the long-chain acyl-CoA−using ASAT3 versification of ASAT2 substrate selectivity could also influence variants from S. habrochaites LA1777 and LA1731. Homology the acylsugar diversity observed in various wild tomato species modeling of Sl-ASAT3 with the trichothecene 3-O-acetyltransferase− (25). To test this prediction, nine putative ASAT2 orthologs acyl CoA complex 3B2S structure suggested that amino acids 35 and were cloned from wild tomato relatives that are phylogenetically 41 are close to the acyl-CoA–binding pocket (Fig. 7A). Indeed, positioned between cultivated tomato and S. pennellii, using mutation of Tyr41—found in short acyl chain-using enzymes—to the primers based on published genomic resequencing data (35). 41 R4 Cys found in the S. habrochaites Sh-ASAT3-F enzymes converted Enzyme activities were then tested using S1:5 (iC5 ) and dif- Sl-ASAT3 into an enzyme that adds nC12 to the furanose ring of ferent acyl-CoAs as substrates (Fig. 5A). The ASAT2 isoforms of S2:10 (5, 5) to produce S3:22 (5, 5, 12), a product not seen in M82 the closest relatives of cultivated tomato showed similar acyl- trichome extracts (Fig. 7 B and C). Sl-ASAT3_Y41C had an ap- CoA substrate preference to that of Sl-ASAT2 when using S1:5 parent Km value of 1.1 ± 0.5 μM for nC12-CoA and also exhibited (iC5R4) as the acyl acceptor substrate: Solanum pimpinellifolium substrate inhibition by nC12-CoA with an apparent Ki value of 4.5 ± LA1578 and Solanum galapagense LA1401 had barely detectable 2.0 μM(SI Appendix,Fig.S11). Again, these results are similar to activities with iC5-CoA as a donor substrate, but used a variety of values observed for LA1777 Sh-ASAT3-F (25). other acyl-CoAs (Fig. 5A). This in vitro activity is consistent with The ability of a single amino acid substitution to change Sl- the lack of iC5 acylation at the R3 position of cultivated tomato ASAT3 into a Sh-ASAT3–type activity suggested that we could acylsucroses (14). In contrast, ASAT2 isoforms from the transform the S. lycopersicum metabolic network into that seen in remaining species used iC5-CoA more efficiently (Fig. 5A). An S. habrochaites LA1777 by substitution of this variant mutant interesting exception is that aiC5-CoA was a poor substrate for enzyme. Indeed, use of this Sl-ASAT3_Y41C variant enzyme in the S. habrochaites LA1718-ASAT2 isoform (Fig. 5A). The rel- the sequential in vitro metabolic network reconstruction system atively high protein sequence identity of ASAT2 variants allowed led to accumulation of four different acylsucroses containing C10 us to recognize a small number of amino acid residues that acylations on the furanose ring (SI Appendix, Fig. S13). These correlate with differences in ASAT2 variant substrate specificity four in vitro-synthesized acylsucroses shared the same chro- (Fig. 5A; SI Appendix, Fig. S10). This analysis led to identifica- matographic retention time with the corresponding acylsugars tion of four candidate residues associated with the preference extracted from LA1777 leaf trichomes (SI Appendix, Fig. S13A), for iC5-CoA and six candidate residues that correlate with and positive-mode high-collision-energy mass spectra revealed aiC5-CoA utilization. long-chain acylation on the furanose ring (SI Appendix, Fig. We reasoned that variant amino acids near to the predicted acyl- S13B). These results are consistent with the hypothesis that a CoA–binding pocket were the strongest candidates for affecting acyl single amino acid change explains some of the major differences

E244 | www.pnas.org/cgi/doi/10.1073/pnas.1517930113 Fan et al. Downloaded by guest on October 1, 2021 PNAS PLUS A Enzyme activity Residue positions S1:5 (iC5R4) as acyl acceptor iC5- aiC5- nC12- 30 104 106 408 44 185 257 295 298 320 ASAT2 CoA CoA CoA 87 S.lycopersicum M82 - + + P V E F I P T L H A 98 S.pimpinellifolium LA1578 - + + P V E F I P T L H A

99 S.galapagense LA1401 - + + P V E F I P T L H A S.neorickii LA2133 + + + H I K V I P T L H A 91 97 S.arcanum LA2172 + + + H I K V I P T L H A S.chilense LA1969 + + + H I K V M P T L H A

71 S.peruvianum LA1278 + + + H I K V M P T L H A 98 S.corneliomulleri LA0107 + + + H I K V M P T L H A S.habrochaites LA1777 + + + H I K V I P T L H A 57 S.habrochaites LA1718 + - + H I K V L A I V N G Sotub04g011000 0.02 B

P-185 H-298

Acyl-CoA L-295 P-30 Acyl-CoA

V-104 I-44

T-257 F-408 E-106 A-320

Fig. 5. ASAT2 amino acid polymorphisms and variation in acyl-CoA substrate specificities. (A) Alignment of amino acid polymorphisms of ASAT2 putative orthologs with acyl-CoA substrate specificities. ASAT2 phylogenetic tree obtained using protein sequences. “+” means good enzyme activities with detectable product peaks; “−” means no or barely detectable enzyme activity. Polymorphisms at amino acid residues 30, 104, 106, and 408 correspond with different ASAT2 activity using iC5-CoA as the substrate. LA1718 ASAT2, which is the only enzyme that does not efficiently use aiC5-CoA, has residues at positions44, 185, 257, 295, 298, and 320 that are unique compared with other ASAT2. (B, Left) The alignment of homology-modeled cartoon structure of Sl-ASAT2 (orange) superimposed upon the crystallographic cartoon structure of a trichothecene 3-O-acetyltransferase−acyl CoA complex (3B2S) (gray) (36), which has acyl-CoA (red) and protein crystallized in a complex. (Right) Highlights of Sl-ASAT2 residues that correlate with differences in acyl-CoA substrate specificity and model near the 3B2S acyl-CoA.

in acylsucrose structures observed among accessions of S. hab- its role in catalyzing the first step of the biosynthetic pathway. rochaites and between S. lycopersicum and S. habrochaites. Second, in contrast to ASAT2-ASAT4, we found no in vivo phe- notypic or in vitro enzyme activity evidence for ASAT1 genetic Discussion variation leading to altered enzyme activities across the tomato During the past 10 y the glandular-secreting trichomes of cultivated clade, which shares a last common ancestor several million years ago and wild tomatoes have emerged as a model for studying the evo- (43). As acylsugar metabolic networks are characterized in Sol- lution of previously uncharacterized specialized metabolic networks, anaceae species outside of the tomato group, it will be interesting to including terpenes in type VI glands (37–41), methylated flavonoids learn whether this enzyme was also conserved as the committing step (42), and acylsugars in type I/IV trichomes (24–26, 29). These studies over tens of millions of years of evolution. revealed strong metabolic diversity both within tomato species and As seen for other BAHD acyltransferases (for example, see ref. PLANT BIOLOGY across the tomato clade of the genus Solanum, leading us to study 32), Sl-ASAT1 showed evidence of promiscuity in vitro, acylating the genetic and biochemical mechanisms associated with this evo- sucrose at the R4 position using acyl-CoAs with different acyl chain lutionarily rapid diversification. In this work we describe the char- lengths (iC4, iC5, nC10, iC12) or branching patterns (subterminally acterization of ASAT1 and ASAT2 BAHD acyltransferases that branched aiC5 and terminally branched iC5). Despite the ability catalyze the first two steps of acylsucrose biosynthesis from sucrose in of the enzyme to use longer chain CoAs, only iC4 and iC5 chains S. lycopersicum. These enzymes were used in combination with the thus far have been reported at the R4 position of acylsucroses of previously described Sl-ASAT3 and Sl-ASAT4 to reconstitute the S. lycopersicum M82 and three different accessions of S. habrochaites synthesis of the major S. lycopersicum tri- and tetraacylsucroses in (14). This seeming conflict likely arises because the next enzyme—Sl- vitro. When combined with phenotypic analyses and demonstration ASAT2—is unable to use the S1:12 (nC12R4) ASAT1 products as that these enzymes are expressed in the acylsucrose-producing type substrates to produce diacylsucroses. This presumably causes pro- I/IV trichome apical cells, these in vitro reconstruction experiments duction of dead-end monoacylated products that may be degraded confirm the roles of these enzymes in acylsugar synthesis. by an acylsucrose acylhydrolase that cleaves the R4 position of ASAT1, which catalyzes the first acylation step in tomato acyl- acylsucroses—or converted to compounds of sufficiently different sucrose biosynthesis, has several features that distinguish it from structure that they are not detected by our analytical methods. other characterized ASAT enzymes. First, its RNAi lines do not In contrast to the uniform composition of the R4 acyl groups accumulate detectable acylsucrose intermediates, consistent with of acylsucroses extracted from S. lycopersicum, the R3 position

Fan et al. PNAS | Published online December 29, 2015 | E245 Downloaded by guest on October 1, 2021 408 ASAT2 positions that impacted substrate utilization. ASAT2 Val/Phe strongly influenced utilization of terminally branched iC5-CoA as a donor substrate. The second ASAT2 residue identified 44 408 in this analysis, Leu43 found in S. habrochaites LA1718 (Ile44 in Sl-ASAT2 Sl-ASAT2 Ile Phe Sl-ASAT2), influences utilization of aiC5-CoA as donor sub- Sl-ASAT2_I44L Leu Sl-ASAT2_F408V Val strate. The ability to facilely identify a single amino acid residue that influences discrimination between such structurally similar LA1718-ASAT2 Leu LA2172-ASAT2 Val substrates (terminally and subterminally branched C5-CoAs) LA1718-ASAT2_L43I Ile LA2172-ASAT2_V408F Phe speaks to the power of this approach. Finally, comparison of Sh- ASAT3 variants that can add a longer chain to the R3′ position to 0.0 0.5 1.0 0.0 0.1 0.2 0.3 Peak area/ Internal standard Peak area/ Internal standard S2:10 (iC5, aiC5) S2:10 (iC5, iC5) A

Fig. 6. Single-residue substitutions affect ASAT2 substrate preference for iC5-CoA or aiC5-CoA. (Left) The amount of S2:10 (iC5,aiC5) produced by ASAT2 of varying structures using S1:5 (iC5R4) and aiC5-CoA as substrates. The ASAT2 variants with Ile44 have higher activity using aiC5-CoA as sub- strate than those with Leu44.(Right) The amount of S2:10 (iC5,iC5) produced by different ASAT2s using S1:5 (iC5R4) and iC5-CoA as substrates. The Val408 ASAT2 variants have higher activity using iC5-CoA as substrate than those with Phe408. Average LC/MS peak areas divided by internal standard peak areas of the S2:10 products with ±SD were calculated from three replicates.

acyl chain length and branching pattern is quite variable, with B SlASAT3_Y41C + S2:10 + nC12-CoA iC4, aiC5, iC10, nC10, and nC12 acyl chains observed (14, 25). 100 This in vivo diversity correlates well with the ability of Sl-ASAT2 1: TOF MS ES- R4 555.2+737.4 0.3000Da to acylate S1:5 (iC5 ) at the R3 position using diverse acyl- 1.84e5 CoAs. The next two enzymes—Sl-ASAT3 and Sl-ASAT4—can use acyl acceptor substrates with varied R3 substitutions in vitro to produce the major acylsucroses found in M82 (Fig. 3 and SI S2:10 (iC5R4, aiC5R3) % Appendix, Fig. S9). Although we have reconstituted a four-enzyme pathway in culti- S3:22 (5, 5, 12) vated tomato, open questions remain regarding the pathway in dif- ferent tomato species. For example, what enzyme(s) are responsible for production of the acylsugars containing all acylchains on the 0 1.00 2.00 3.00 4.00 5.00 6.00 pyranose ring in IL4-1 (SI Appendix,Fig.S2A)? How does IL4-1 Retention time (min) produce most of the same major acylsugars as M82 despite the in- trogression of the putative SpASAT2 ortholog, which does not use C [M+Na]+ S1:5 (iC5R4) as substrate. These suggest that unknown enzymatic 100 715.45 activities remain to be discovered to add to the current acylsugar ESI+ 40V metabolic network. + Understanding the genetic mechanisms leading to changes in the types of specialized metabolites that accumulate over evolutionary

time and the biochemical basis for substrate specificity and enzy- % matic promiscuity is central to plant improvement efforts. Enzyme structure and function analysis depends upon identification of amino 345.26 acids that influence activity, but even using directed evolution and “semi-rational” approaches to protein engineering by in vitro mu- tagenesis can be quite time-consuming, requiring construction and 0 m/z 0 100 200 300 400 500 600 700 800 testing of large numbers of single or multiple amino acid variants (44–47) and the availability of suitable substrates. Fig. 7. ASAT3 position 41 polymorphisms control acyl chain length preference. We took advantage of existing acylsucrose variation within (A) The predicted homology-modeled structure of Sl-ASAT3 superimposed on accessions of S. habrochaites and other tomato species to seek the 3B2S structure. The Sl-ASAT3 structure (green ribbon) and acyl-CoA (red) are ASAT2 and ASAT3 amino acids that are responsible for these shown. The amino acids highlighted as blue or orange are the residues in phenotypic differences. Comparisons of primary sequence vari- Sl-ASAT3 that are not found in two S. habrochaites ASAT3 isoforms that can use long-chain acyl-CoAs as substrates. The residue Y41 is shown in orange. ation with in vitro assays performed using a variety of acyl-CoA R4 R3 substrates revealed relatively small numbers of amino acids as (B) Sl-ASAT3_Y41C uses purified S2:10 (iC5 ,aiC5 ) and nC12-CoA as substrates to produce a triacylsucrose S3:22 (5, 5, 12). LC/MS-extracted ion chromatograms candidates for influencing substrate specificity. These candidate are shown for m/z 555.2 (S2:10) and m/z 737.4 (S3:22). (C) Positive-ion-mode mass residues were screened further for those that might be positioned spectrum with a collision potential of 40 V is shown for S3:22 (5, 5, 12). Presence to interact with the CoA substrates using homology modeling. In of the fragment ion with m/z 345 indicates that the long acyl chain was added to all three cases the approach led to identification of amino acid the furanose ring of sucrose.

E246 | www.pnas.org/cgi/doi/10.1073/pnas.1517930113 Fan et al. Downloaded by guest on October 1, 2021 those with a preference for short-chain acyl-CoAs revealed that LA1777) were obtained from the C. M. Rick Tomato Genetic Resource Center PNAS PLUS substitution of Cys in place of Tyr at position 41 is sufficient (tgrc.ucdavis.edu). ASAT2-coding sequences were amplified using cDNA tran- to transform substrate specificity. The comparative genomic/ scribed from RNA extracted from 5-wk-old plant leaf tissues as the templates biochemical approach coupled with in vitro analysis has also and using primers that were designed based on the wild tomato whole-ge- been applied to identify key residues of enzymes involved in nome resequencing data (35). Details of ASAT2 sequencing and determination of orthology and GenBank accession numbers for different ASAT2 genes are terpene (48) and artemisinin biosynthesis (49). This approach described in SI Appendix, Materials and Methods. has the potential to be generally applicable to structure–function studies of any enzymes that make products that are variable Protein Expression and Enzyme Assay. Recombinant proteins were generated across related populations or related species of plants. using E. coli as the host for enzyme assays. The full-length ORF sequence was The results from this study have implications for engineering of cloned into pET28b. ASAT1 and ASAT2 enzyme assays were performed by acylsugars and related compounds. The ability to test the impact of incubating purified recombinant proteins in 30 μL of 50 mM ammonium combinations of BAHD acyltransferase isoforms on product types acetate (pH 6.0) buffer with 100 μM acyl-CoA and an acylsucrose acceptor. should inform breeding and genome-editing approaches to modify Methods used to determine the apparent Km value for different acyl-CoA biotic stress tolerance of tomato and other Solanaceae plants. It substrates were performed as previously described (25). The detailed steps will also permit regiospecific synthesis of compounds for activity for protein expression and enzyme activity assays are in SI Appendix, Ma- screening or large-scale production by synthetic biology ap- terials and Methods. proaches—for example, novel pesticides, antimicrobials, phar- maceutical excipients, and emulsifiers. LC/MS Analysis of Acylsugars. The trichome acylsugars extracted from were analyzed using a Shimadzu LC-20AD HPLC system connected to a Waters Materials and Methods LCT Premier ToF MS. Enzyme assay samples were analyzed using a Waters Acquity UPLC system connected to Waters Xevo G2-S QToF LC/MS. Detailed Tomato Transformation. Transformation of tomato M82 and IL4-1 was performed LC/MS methods are in SI Appendix, Materials and Methods. using Agrobacterium tumefaciens strain AGL0 (50). BAHD acyltransferase gene suppression was performed by cloning a fragment of each gene from M82 ge- Homology Structural Modeling of ASAT2. The Phyre web-based protein homol- nomic DNA into the pHELLSGATE12 binary vector (51) and transforming M82 ogy/analogy recognition engine (53) was used to predict the tertiary structures of plants. Sl-ASAT2 under the control of its native promoter was cloned into pK7WG Sl-ASAT2 and Sl-ASAT3. The trichothecene 3-O-acetyltransferase structure (Protein (52) and transformed into IL4-1. For in planta reporter gene analysis, the pro- moter regions of Sl-ASAT1 and Sl-ASAT2 were cloned into pKGWFS7 (52) and Data Bank ID: 3B2S) was used as a template to overlay with Sl-ASAT2 and Sl-ASAT3 transformed into M82. Detailed information is in SI Appendix, Materials and modeled structure and displayed using PyMOL (Version 1.7.4 Schrödinger). Methods. Site-Directed Mutagenesis. Site-directed mutations were created by PCR- Plant Trichome Acylsugar Extraction. Trichome acylsugars were extracted from based plasmid amplification using the Q5 Site-Directed Mutagenesis Kit the youngest expanded leaves of 3- to 4-week-old plants (15). A single leaflet (NEB). The primers used to introduce mutations were designed based on the was dipped in 1 mL of extraction solvent, which contained acetonitrile/iso- web-based software NEBaseChanger (Version 1.2.2, NEB) and are listed in SI propanol/water (3:3:2) with 0.1% formic acid and 10 μM propyl 4-hydrox- Appendix, Materials and Methods. The presence of the mutations was ybenzoate as internal standard, and the mixture was gently agitated for confirmed by DNA sequencing. 2 min. ACKNOWLEDGMENTS. We thank members of the Solanum Trichome Project Mapping the IL4-1 Acylsugar Locus Using Backcross Inbred Lines. The BIL for their contributions to this work, especially Kathleen Imre for help with tomato transformation; Jing Ning, Gaurav Moghe, and Bryan Leong for helpful population was constructed and genotyped as previously described (26). BILs that comments on the manuscript; and Banibrata Ghosh for developing the LC have chromosome introgression regions covering the IL4-1 and IL4-2 overlap re- elution methods for the large-scale purification of acylsucroses. We acknowl- gion were selected for acylsugar profile screening using LC/MS. A set of BILs edge the Michigan State University Center for Advanced Microscopy and the with recombination breakpoints in the overlap region were tested for their Research Technology Support Facility (Mass Spectrometry and Metabolomics acylsugar phenotypes. Two SNP markers and one self-designed insertion/deletion Core). Work in the A.D.J. and R.L.L. laboratories was funded by National Science marker (forward primer 5′-TAAAACCTTAGAATCGTTCTCGT-3′ and reverse primer Foundation Grant IOS-1025636; A.D.J. acknowledges support from Michigan 5′-AAATGATCACTGAAGAATTTCCA-3′) were used for further mapping analysis. AgBioResearch Project MICL02143; research in the D.Z. laboratory was sup- ported by the European Research Council advanced grant entitled YIELD. ASAT2 A.M.M. was supported by Plant Genomics at Michigan State University Summer Amplification of Putative Orthologs from Wild Tomatoes. Accessions Research Experience for Undergraduates Program and by an American Society of S. pimpinellifolium (LA1578), S. galapagense (LA1401), Solanum neorickii of Plant Biologists Summer Undergraduate Research Award. Wild tomato spe- (LA2133), S. arcanum (LA2172), Solanum chilense (LA1969), Solanum peruvia- cies seeds used in this study were obtained from the C. M. Rick Tomato Genetics num (LA1278), Solanum corneliomulleri (LA0107), and S. habrochaites (LA1718, Resource Center (University of California, Davis).

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