Progress Report for the 2010AtSABATH Group Meeting Feng Chen Ann Arbor, June 2004

Progress Report for the 2010AtSABATH Group Meeting

Feng Chen

Ann Arbor, June 2004

1. Management

2. Progress and Resources on 5 Genes

3. High-throughput Biochemical Assays

Part I: Report on Management

 NSF Arabidopsis 2010: Functional Analysis of the

SABATH Family of Methyltransferases                                            

Project Summary The Arabidopsis thaliana genome contains 24 related genes that encode

methyltransferase enzymes (MTs) distinct from any other known MTs. One MT from this group has been shown to convert jasmonic acid, an important plant hormone, into the jasmonate methyl ester, thereby changing the activity of the hormone in significant ways. Preliminary experiments suggest that the other 23 MTs of this group convert several important hormones and other plant constituents into the methyl esters, thereby exerting important effects on the biological activity of these molecules and consequently on a myriad of important physiological processes. The aim of the project is to identify the function of all the MTs of this group (i.e., which compound each of them methylates) by a combination of methods that involve genetics, enzymology, protein structure determination, and analytical chemistry. The consequences of the methylation of such hormonal molecules on the physiology of the plant will be examined in selected cases, which may include processes involving plant response to pathogens, drought conditions, and herbivory. The results are expected to provide a better understanding of plant responses to environmental conditions, thus helping improve crop yield and nutritional value. In addition, by developing methodologies for determining which Arabidopsis genes are involved in the synthesis of the plant’s diverse repetoire of small molecules, the project will contribute to the elucidation of the function of other Arabidopsis genes involved in hitherto unknown biochemical pathways. The project will also provide interdisciplinary opportunities for training undergraduates, graduates, and post-docs.

Created by Feng Chen, fengc@umich.edu, last updated on January 26, 2004

Design and Construction of the Project Webpage

Database

attL1 attL2

CCCTT AAGGGGGGAAGTGG TTCCC

TOPO

TOPO

pENTR/D-TOPO

pDONR207

Knr

attP1 attP2

CmR

ccdBGentr

attR1 attR2CmR

ccdBT7 Promoter RBS MK9HispH9GW

attR1 attR2CmR

ccdBT7 Promoter RBS MK8His ThrombinpH8GW

Knr

Knr

GW Entry Vectors

GW Destination Vectors for E.coli Expression

pMDC32

pCHF3-GW1

pCHF3-GW3

GW Destination Binary Vectors for Plant Over-expression

attR1 attR2

CmR

ccdBHygr LBRB Nos T

2X35SKnr

attR1 attR2

CmR

ccdBnptII LBRB T

35S Specr

RB nptII LBattR2 attR1

CmR

ccdBT

35S Specr

pMDC162

pDW137-GW1

pDW137-GW2

pMDC107

attR1 attR2

CmR

ccdBgus LBRB Nos T Hygr

attR1 attR2

CmR

ccdBgfp6his LBRB Nos T Hygr

Knr

attR1 attR2

CmR

ccdBgus LBRB Nos T nptII Gentr

RB gus LBattR2 attR1

CmR

ccdBNos T nptII Gentr

Knr

GW Destination Binary Vectors for Reporter Assays

Gateway Entry and Destination Vectors

Material Archiving

1. DNA Construct Stocks

2. DNA Oligo Stocks

3. Transgenic Seed Stocks

Part II: Resources and Progress on

MT6, MT7, MT8 MT10 and MT19

Expression Analysis and cDNA Cloning

6 7 8 10 19

GUS reporter Analysis: MT7

GUS reporter Analysis: MT8

GUS reporter Analysis: MT10

T-DNA Knock-out Lines

Gene Source Position of insertion

Homozygous lines

MT6 SALK Intron X

MT7 SALK 5’-UTR X

 MT8

Garlic Intron X

GABI Exon X

MT10 SALK Exon X

 MT19

SALK Intron X

Garlic Exon X

Plant Over-expressiors

Gene Binary Construct

Plant Transformation

Transgenic Lines

MT6 X X  

MT7 X X  

MT8 X X  

MT10 X X  

MT19 X X X

Part III: High-throughput Biochemical

Assays

COOH

OH

COOH

OH

COOHCOOH

OH

OH

COOH

OMe

COOH

OH

COOH

OH

OH

COOH

BA SA

Phenolic Acids and Phenylpropanoids

3-OH BA 4-OH BA

Cinnamic acid P-coumaric acid

Vanillic acid

Caffeic acid

OH

COOH

OMe

Ferulic acid

OH

OH

O

O

OH

OH

COOH

OH

chlorogenic acid

NH2

COOH

Anthranilic acid

OH

O COOH

COOH

Chorismic aicd

OH

O

OOH

OH

OH

HOOC

Rosmarinic acid

COOH

OMe

OMe

MeO

Gallic acid

COOH

NH2

4-amino BA

COOH

OH

OMe

3-hydroxy-4-methoxyCinnamic acid

COOH

OMeMeO

OH

3,5-dimethoxy-4-hydroxy Cinnamic acid

Shikimic acid

Phenylpyruvicacid

4-hydroxyPhenylpyruvic acid

Phenyllacticacid

4-hydroxyPhenyllactic acid

COOH

OH

OH OH

COOH

O

COOH

O

OHCOOH

OH

OH

COOH

OH

NH

COOH

H

H

H

H

OO

COOHOH

OH

ABA GA

IAA

Phytohormones and Related Compounds

O

OHCOOH

NH3O

OH

GABA(gama-amino butyric acid) DOPA

(3,4-Dihydroxyphenylalanine)

CH2

CH2OH

O

OH

OH

NH

COOH

IBA

Cl

Cl

O COOH

2,4-D

N NH

NNH

NHCH2

CH2

CH3

CH2

OH

Zeatin

N NH

NNH

NHCH2

O

Kinetin

N NH

NNH

NHCH2

N6-benzyladenine

NH

Indole

O

COOH

Jasmonic acid

O

COOH

OPDA

O

OH

OHOH

OH

HOOC

OO

O

OHOH

OH

HOOC HOOC

OH OH

OH

Galacturonic acid

Acidic Sugars, Pectin and Vitamin C

npectin

OH

OH

O O

OH OH

Ascorbic acid

Tri-galacturonic acid

O

OH

OHOH

OH

HOOC

Fatty Acids

COOHHOOC

norBixin

Short-chain (2-4) fatty acids

Medium-chain (6-10) fatty acids

Long-chain (>12) fatty acidsLauric acid (12:0) Oleic acid (18:1)Myristic acid (14:0)Palmitic acid (16:0) Petroseenic acid (18:1)Stearic acid (18:0) a-Linoleic acid (18:2)Arachidic acid (20:0) r-linoleic acid (18:3)Behenic acid (22:0) Roughanic acid (16:3)Lignoceric acid (24:0) Erucic acid (22:1)

Butanoic acid (4:0)

Pentanoic acid (5:0)Hexanoic acid (6:0)Octanoic acid (8:0)Decanoic acid (10:0)

N

NNH

N

O

O

CH3

CH3

Theobromine

Nitrogen-containing Compounds

-alanine

NH3 CH2

CH2

COOH

NH

NH

NH

NH

O

ORibose

Xanthosine

NH

NNH

NH

O

O

CH3

Ribose

7-methyl-Xanthosine

N

NNH

NH

O

O

CH3

7-methyl-Xanthine

N

NN

NH

O

O

CH3CH3

paraxanthine

S

N

NH

Camalexin

NH

COOH

NH3

Tryptophan

Amino Acids

NH2

COOH

Phenylalanine

OH

NH2 COOH

Tyrosine

Methionine

NH2CH3

COOH

H

Alanine

NH2

COOH

H

CH2OH

Serine

NH2

COOH

H

CH2COOH

Aspartic acid

NH2

COOH

H

CH2CH2COOH

Glutamic acid

NH2

COOH

H

CH

CH3

CH3

Valine

NH2

COOH

H

CH3

OHH

Threonine

NH2

COOH

H

CH2CHCH3 CH3

Leucine

NH2

COOH

H

CH

CH2CH3

CH3

Isoleucine

NH2

COOH

H

CH2SH

Cysteine

NH2

COOH

H

CH2CH2CH2NH

NH

NH2

Arginine

NH2

COOH

H

CH2CH2CH2CH2NH2

Lysine

NH2

COOH

H

CH2CH2S

CH3

NH2

COOH

H

CH2CH2

O NH2

Glutamine

CH2

NH2

COOH

H

NH2O

Asparagine

CH2

NH2

COOH

H

N

NH

Histidine

CH

CH2

CH2

NHCH2

COOH

Proline

NH2H

H

COOH

Glycine

Benzoic acid Caffeic acid3,5-dimethoxy-4-hydroxy-cinnamic acid * Group 1 (EtOH)Chlorogenic acidPhenylpyvuvic acid Salicylic acidFerulic acid Vanillic acid Group 2 (EtOH)Gallic acid4-hydroxy-phenylpyvuvic acid  3-hydroxy-benzoic acidAnthranillic aicdp-coumaric acid Group 3 (EtOH)Jasmonic acidShikimic aicd

Substrate Grouping (1 to 3)

4-hydroxy-benzoic acid4-amino benzoic acidCinnamic acid3-hydroxy-4-methoxy-cinnamic acid * Group 4 (EtOH)Rosmarinic acid4-hydroxy-phenyllactic acid   ABAGAIAA Group 5 (EtOH)IBA2,4-DIndole   ZeatinKinetin6-benzylaminopurice Group 6 (DMSO)GABA *DOPA *Tryptophan

Substrate Grouping (4 to 6)

Trigalacturonic acidMuranic acid Group 7 (DMSO)

Octanoic acidDecanoic acidLauric acidMyristic acid Group 8 (EtOH:Chloroform=1:1) Palmitic acidStearic acid  Xanthosine 7-methyl xanthinetheobromine Group 9 (DMSO)paraxanthinebeta-alanine(Camalexin) Glutamic acidValine AlaninePhenylalanine Group 10 (H2O)

Aspartic acidAsparagine

Substrate Grouping (7 to 10)

MT7 showed activity with Lauric acid

COOH

Fully expended leaves of Col at growth stage 3.9

Plant Defense Response (I)

Plant Defense Response (II)

Plant Defense Response (III)

COOH

COOHHOOC

Lauric Acid

Traumatic Acid