Plant Sunscreen: The Formation of Sinapoylmalate by O-Methyl Transferases

Beth KauffmanUnder 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

HOR

ORH3C

RNH2

R

HN

CH3

Methyltransferase

Methyltransferase

Methyl Donor

Methyl Donor

O-Methyltransferases are involved in the formation of flavor and scent compounds

OOCH3

OH

Vanillin

O-Methyltransferases are involved in the formation of pharmaceuticals

O

ONCH3

OCH3

H

O

H

O

H3COOCH3

Noscapine

O-Methyltransferases are involved in the formation of pigments

OH

HO O

OH

OCH3

OH

OCH3

Malvidin

H3CO OCH3

OCH3

OH OOCH3

OH

OH

HO O

OH

OCH3

OH

OCH3

O

ONCH3

OCH3

H

O

H

O

H3COOCH3

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

O

OO OH

HO O

O

CH3

OH

OH3C

White Light Ultra Violet Light

A Caffeic Acid O-Methyltransferase (COMT), ATOMT1 participates in the phenylpropanoid pathway.

H2N O

OH HO

HO

H3CO

O

H

ATOMT1H3CO

HO

H3CO

O

H

phenylalanine5-OH coniferaldehyde

sinapylaldehyde

sinapoylmalate

S-lignin

O

OO OH

HO O

O

CH3

OH

OH3C

O

OO OH

HO O

OH

OH

OH3C

5-OH feruloylmalate

atomt1

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

Gene Disruption

atomt1

atomt1

atomt1

atomt1

atomt1

comt1

comt2

comt3

comt4

comt5

atomt1

atomt1

atomt1

atomt1

atomt1 atomt1

atomt1

atomt1

comt6

comt7

comt8

comt9

comt10

comt11

comt12

comt13

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.

Minutes

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mAu

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mAu

-20

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1205-OH feruloylmalate sinapoylmalate

Candidate COMT’s not consistent with redundant ATOMT1 activity

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Sinapoylmalate

5-O

H Fe

rulo

ylm

alat

e

atomt1

WT

Candidate COMT’s not consistent with redundant ATOMT1 activity

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Sinapoylmalate

5-O

H Fe

rulo

ylm

alat

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comt/atomt1

WT

atomt1

Future Directions

• It may be that a combination of several COMT’s are responsible for the redundant activity

• Triple mutants generated

• Same method of study conducted with triple mutants. If a triple mutant does not form sinapoylmalate, then combination is responsible

atomt1 comt1 comt2

Acknowledgements

Nick AndersonClint Chapple

Jo CusumanoNick BonawitzJeong-Im KimPeng WangYi LiWhitney DolanWenjie ZengMarwa El-HindawyAlexandra SmithNicholas PogranichniyMisha Remy

Chapple Lab

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 O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy. Plant Phys. 159, 618-631.

O-Methyltransferases Have Various Functional Properties

H3CO OCH3

3,5-dimethoxytoluene

O-Methyltransferases Have Various Functional Properties

OCH3

OH

Guaicol

Several Candidate OMT’s are closely related to OMT’s that participate in flavone biosynthesis

Flavone OMT’sPutative OMTArabidopsis COMTLignin OMT’sPhyscomitrella OMT

Figure 1: Evolutionary relationship of 15 putative OMT’s of Arabidopsis thaliana to other known OMT’s. A pink glow indicates that a putative