Variation of the acylsucrose biosynthesis pathway catalyzed by ASAT2 and 3 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

Background: The acylsucrose biosynthesis pathway is composed of a sucrose backbone with multiple acyl-CoA donors added onto the pyranose and furanose rings. This research shows the variation and manipulation of acyl donors and acyl acceptors between S. lycopersicum, the cultivated tomato, and other wild tomato accessions. Through multiple enzyme assays, and mutagenesis, this research helps develop the framework for manipulation of a secondary pathway and illustrates the natural diversity and evolution of acylsugars in tomato plants.

1

7

4

3

2

S. lycopersicum S. pennellii

S1:5 (R4)

S2:10 (R2, R4)

S2:10 (R3,R4)

S3:22 (R2,R3,R4)

SpASAT3SlASAT2

SpASAT2

6

5 8

7

Enzyme S1:5 + iC5 S2:10 + iC5 S2:10 + nC12

SpASAT3 S2:10 X X

SlASAT3 X S3:15 S3:22

1376SpASAT3 S2:10 X X

1278SpeASAT3_1 S2:10 S3:15 S3:22

1718ShASAT3 S2:10 S3:15 S3:22

1969SchASAT3 S2:10 S3:15 S3:22

1278SpeASAT3_2 S2:10 S3:15 S3:22

1364ShuASAT3 S2:10 S3:15 S3:22

2172SaASAT3 X S3:15 X

Common acylsugar structure found in S. lycopersicum. Acyl chains added by ASAT2 on the R3 position, and by ASAT3 on the R2 position of the pyranose ring (Kim 2010).

Acylsugars are a natural pesticide for the tomato plant. Hornworm larvae on tobacco plants eat acylsugars as a first meal, and are tagged with volatile components which attract predators(Weinhold 2011).

Protein homology model used to find amino acids for mutagenesis to test ASAT2 activity. Phe408Val results in a gain of activity shown in the chromatogram below.

Pathway difference between S. lycopersicum and S. pennellii. S. pennellii acylsugars have a completely acylated pyranose ring, and no acyl chains on the furanose ring (Schilmiller 2015).

Multiple sequence alignment (MSA) of ASAT3 in wild tomatoes, G to C aligns with the activity of S. lycopersicum differing from S. pennellii when using S1:5 as a substrate.

SlASAT2

SlASAT2_C304G

1777ASAT2

Mutagenesis of C304G ASAT2 which gains activity to use S2:10 as a substrate.

iC5-CoA

aiC5-CoA

nC12-CoA

SpASAT2_HC-QY

SlASAT3_YCT-HSV

S1:5(R4) +iC5-CoA

Mutagenesis of SlASAT3 gains the activity to use S1:5(R4) as a substrate. Mutations found using the same method as demonstrated in section 4.

What about S. pennellii ASAT3 using S2:10?

SlASAT2_C304G

SpASAT2_HC-QY

SlASAT3_YCT-HSV

Activity of ASAT3 in wild tomatoes and S. lycopersicum was tested in order to find a correlation for SpASAT3 using S2:10. Species were selected based on sequence alignment resembling both S. lycopersicum and S. pennellii activity.

Future Work

References and Acknowledgements

Retention time for S3:22 is shifted for 1718ShASAT3 which indicates that the nC12-CoA is added on at a different position on the furanose ring. The original position is on the R3’, but this shows that it is somewhere new.

1969SpASAT3

1278SpeASAT3

1777ShASAT3

The NSF for funding the Trichome project, Lyman Briggs College for Spring 2015 research scholarships, and ASPB for funding Summer 2015 research.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 109:16377-82.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.15.00087Ghosh 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: 1-12.

Alignments of the S. pennellii and the wild tomato species will help us understand the evolutionary significance of this pathway divergence.

Analyzing the retention time differences in section 8 will need to be conducted and NMR structures will also need to be developed.

Summary of the three mutations

Acyl-acceptor mutagenesis

Proposed acylsucrose pathway

Mutagenesis with three mutations

ASAT2 acyl-donor mutagenesis

What about SpASAT3 using S2:10?Mutagenesis HC135QY

S3:15

S4:17

2172ASAT2_V408F

2172ASAT2

SlASAT2_F408V

SlASAT2

-----+++++----

ASAT2ASAT3

S1:5

S1:5

S1:5 as substrate

Mutations found through a similar MSA method as in section 4.