Plant Sunscreen: The Formation of
Sinapoylmalate by O-Methyl
Transferases
Beth Kauffman
Under the Guidance of:
Nick Anderson and Clint Chapple
Outline
•
•
•
•
•
Why are methyltransferases important?
What is the phenotype of an atomt1 mutant
How is a candidate COMT tested for redundant ATOMT1 activity?
Do any of the candidate COMT’s have redundant activity?
Future Directions?
Methylation is a common reaction in nature
Methyl Donor
Methyltransferase
Methyl Donor
Methyltransferase
O-Methyltransferases are involved in the formation of flavor
and scent compounds
Vanillin
O-Methyltransferases are involved in the formation of
pharmaceuticals
Noscapine
O-Methyltransferases are involved in the formation of
pigments
Malvidin
O-Methyltransferases have various functional properties
A short Life cycle and small genome make Arabidopsis a
model organism
• Model organism
A short Life cycle and small genome make Arabidopsis a
model organism
•
•
•
•
Model organism
Small genome which has been sequenced
Short life cycle compared to other plants
Relatively easy to make a mutation
Sinapoylmalate acts as a UV protectant in Arabidopsis
White Light
Ultra Violet Light
A Caffeic Acid O-Methyltransferase (COMT), ATOMT1
participates in the phenylpropanoid pathway.
sinapoylmalate
atomt1
ATOMT1
phenylalanine
sinapylaldehyde
5-OH coniferaldehyde
5-OH feruloylmalate
S-lignin
ATOMT1 is required for S-Lignin and sinapoylmalate
formation
WT
atomt1
O-methyltransferases may be responsible Sinapoylmalate
formation in an atomt1 mutant
• S-lignin falls below level of detection
• Sinapoylmalate is still formed.
• Arabidopsis has many candidate COMT genes
Candidate COMT’s are investigated using a reverse genetic
approach
Disruption
Gene
comt1
atomt1
comt2
atomt1
comt8
atomt1
comt3
atomt1
comt9
atomt1
comt4
atomt1
comt10
atomt1
comt5
atomt1
comt11
atomt1
comt6
atomt1
comt12
atomt1
comt7
atomt1
comt13
atomt1
HPLC analysis is used to quantify soluble metabolite levels
• Determined metabolic Profile of HPLC Analysis
• If an COMT was responsible, then the mutant’s metabolic
profile would contain no sinapoylmalate.
HPLC analysis is used to quantify soluble metabolite levels
• Determined metabolic Profile of HPLC Analysis
• If an COMT was responsible, then the mutant’s metabolic
profile would contain no sinapoylmalate.
5-OH feruloylmalate
120
sinapoylmalate
120
80
80
60
60
40
40
20
20
0
0
mAu
100
mAu
100
-20
-20
0
5
10
15
20
Minutes
25
30
35
Candidate COMT’s not consistent with redundant ATOMT1 activity
1800000
1400000
WT
1200000
atomt1
1000000
800000
600000
400000
200000
Sinapoylmalate
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5-OH Feruloylmalate
1600000
Candidate COMT’s not consistent with redundant ATOMT1 activity
1800000
1400000
WT
1200000
atomt1
comt/atomt1
1000000
800000
600000
400000
200000
Sinapoylmalate
5000000
4500000
4000000
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
0
5-OH Feruloylmalate
1600000
Future Directions
• It may be that a combination of several COMT’s are
responsible for the redundant activity
• Triple mutants generated
atomt1
comt1
comt2
• Same method of study conducted with triple mutants. If a
triple mutant does not form sinapoylmalate, then
combination is responsible
Acknowledgements
Nick Anderson
Clint Chapple
Chapple Lab
Jo Cusumano
Nick Bonawitz
Jeong-Im Kim
Peng Wang
Yi Li
Whitney Dolan
Wenjie Zeng
Marwa El-Hindawy
Alexandra Smith
Nicholas Pogranichniy
Misha Remy
References
• Besse, P., Da Silva, D., Bory, S., Noirot, M., and Grissoni, M.(2009).
Variation in intron length in caffeic acid O-methyltransferase
(COMT) in Vanilla species (Orchidaceae). Plant Sci. 176, 452-460.
• Goujon, T., Sibout, R., Pollet, B., Maba, B., Nussaume, L., et. al.
(2003). A new Arabidopsis thaliana mutant deficient in the
expression of O-methyltransferase impacts lignins and sinapoyl
esters.Plant. Mol. Bio. 51, 973-989.
• Do, C., Pollet, B., Thevenin, J., Sibout, R., Denoue, D., et. al. (2007).
Both caffeoul Coenzyme A 3-O-methyltransferase 1 and caffeic acid
O-methyltransferase 1 are involved in redundant functions for
lignin, flavonoids and sinapoyl malate biosynthesis in Arabidopsis.
Planta.226, 1117-1129.
• Dang, T. T., and Facchini, P. J. (2012). Characterization of Three OMethyltransferases Involved in Noscapine Biosynthesis in Opium
Poppy. Plant Phys. 159, 618-631.
O-Methyltransferases Have Various Functional Properties
3,5-dimethoxytoluene
O-Methyltransferases Have Various Functional Properties
Guaicol
Several Candidate OMT’s are closely related to OMT’s that participate in flavone biosynthesis
Flavone OMT’s
Putative OMT
Arabidopsis COMT
Lignin OMT’s
Physcomitrella OMT