Variation in the Acylsucrose Biosynthetic Pathway in Wild Tomato SpeciesAbigail M. Miller1, Pengxiang Fan1, and Robert L. Last1,2

1Department of Biochemistry and Molecular Biology, 2Department of Plant Biology, Michigan State Univeristy, East Lansing, MI 48824, United States of America

Abstract: Acylsugars are specialized metabolites secreted from the tip cells of the type I/IV trichomes on leaves and stems of plants in the Solanaceae family. These specialized sugars serve as an insect defense compound for the plant. The cultivated tomato, Solanum lycopersicum, has a pathway that involves four acylsugar acyltransferase (ASAT) enzymes, which produce a tri- and tetra-acylated sucrose sugars (F-type), whereas in the wild relative, Solanum pennellii, the pathway has diverged, producing a different type of acylsugar with all acyl chains attached to the six membered sucrose pyranose ring (P-type). This divergence in enzyme function potentially correlates to an evolutionary loss or gain of function in the acylsugar biosynthesis pathway. Through alignment of sequences of ASAT2 and ASAT3 from the wild relatives, amino acid regions were found to correlate to specific function differences in the pathway divergence. Using protein homology modeling, mutagenesis, and quantitative measurement of the acylsugar products through mass spectroscopy, specific amino acids were found that can change the in vitro function of the ASAT2 and ASAT3 from both S. lycopersicum and S. pennellii.

1. Background

Acylsucrose molecule found in S. lycopersicum, the cultivated tomato.

Hornworms eat trichomes as one of their first meals, thus attracting them to predators.

3. Acyl-donor specificity

Using multiple sequence alignment, and protein homology modeling, certain residues were found to influence acyl-donor specificity of ASAT2 in S. lycopersicum. The Phe408Val residue close to the putative binding pocket of acyl-CoA is responsible for allowing SlASAT2 to use iC5-CoA as an acyl-donor.

4. ASAT2 activities and acyl-acceptor specificities

The ability of SlASAT2 to use an S1:5(R4) as a substrate correlates to the amino acid residue at position 136; whereas, the ability for SlASAT2 to use an S2:10(R2,R4) as a substrate correlates to the plants having amino acid residue Gly at position 304.

Sl-ASAT2

With site-directed mutagenesis of Cys Gly in SlASAT2, the enzyme can now use S2:10(R2,R4) as a substrate along with S1:5(R2).

SlASAT2

SlASAT2_C304G

1777ASAT2Control

S2:10(R2,R4)

S3:22(R2,R3,R4)

S3:22(R2,R3,R4)

SlASAT2_C304G

iC5-CoA

aiC5-CoA

nC12-CoA

The site-directed mutagenesis of the S. pennellii ASAT2 HisCysGluTyr allows this enzyme to use S1:5 as a substrate to make S2:10(5,5), and S2:17(5,12).

SpASAT2_HC-QY + iC5-CoA, aiC5-CoA, and nC12-CoA + S1:5(R2)

5. Summary

6. ReferencesFan P, Miller AM, Schilmiller AL, Liu X, Ofner I, Jones AD, Zamir D, Last RL, (2016) In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network. Proc Natl Acad Sci USA 113:2 E239-E248.Schilmiller AL, Charbonneau AL, Last RL (2012a) Identification of a BAHD acetyltransferase that produces protective acyl sugars in tomato trichomes . Proc Natl Acad Sci USA 109(40):16377-82.Weinhold A, Baldwin IT (2011) Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation . Proc Natl Acad USA 108: 7855-7859.Kim J, Kang K, Gonzales-Vigil E. Shi F, Jones AD, Barry CS, Last RL (2012) Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the Acyltransferase2 locus in the wild tomato Solanum habrochaites. Plant Physiol 160:1854-70.Schillmiller A, Shi F, Kim J, Charbonneau AL, Holmes D, Jones AD, Last RL (2010) Mass spectrometry screening reveals widespread diversity in trichome specialized metabolites of tomato chromosomal substitution lines . Plant J 62: 391-403.Schilmiller AL, Moghe GD, Fan P, Ghosh B, Ning J, Jones AD, Last RL (2015) Functionally Divergent Alleles and Duplicated Loci Encoding an Acyltransferase Contribute to Acylsugar Metabolite Diversity in Solanum Trichomes . Plant Cell. Doi: 10.1105/tpc.15.00087 27(4):1002-17.Ghosh B, Westbrook T, Jones AD. 2013. Comparative structural profiling of trichome specialized metabolites in tomato (Solanum lycopersicum) and S. habrochaites: acylsugar profiles revealed by UHPLC/MS and NMR . Metabolomics (2014) 10(3): 496-507.Thank you to the NSF, ASPB, Michigan State University’s Biochemistry Department, and Lyman Briggs College for the funding and opportunity to do this research over the last few years.

SpASAT3

SlASAT3_YCT-HSV

SpASAT2SlASAT3

SlASAT2

The summary of the pathway manipulation conducted through multiple mutagenesis experiments. The explanation of the pathway divergence can be seen through the corresponding mutations in vitro.

SlASAT2_C-G

SpASAT2_HC-QY

SpASAT3_V-L_S-HP

2. Acylsugar Acyltransferases (BAHD enzymes) plays an important role in acylsugar diversity

ASATVariation in the acyl acceptor

Variation in the acyl donor

4. ASAT2 activities and acyl-acceptor specificities cont.3. Acyl-donor specificity cont.

S. lycopersicum ASAT3 homology model

Acyl-CoA

Y-41

F(Furanose)-Type P(Pyranose)-Type

Acylsugar variation in different species of tomato. The diversity of the acylsugar structures is from variation in ASAT activity.

S. lycopersicum

S. habrochaites

S. pennellii

SlASAT3ShASAT3

Acyl-donor specificity from Tyr41Cys mutation allows SlASAT3 to add the long chain acyl-donor on the furanose ring.

Weinhold and Baldwin et al. 2011.Schilmiller et al. 2010.

SlASAT3_Y-C

SlASAT3_Y-C

SlASAT2

SlASAT2_F-V

Fan et al. 2016

S1:5

ASAT2 + nC12-CoA + S2:10(R2, R4)

Poster Number: 500-052-Y