1. Variation in the Acylsucrose Biosynthetic Pathway in Wild Tomato Species
Abigail 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
1777ASAT2
Control
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
HisCysGluTyr 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. References
Fan 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
ASAT
Variation 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
SlASAT3
ShASAT3
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