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
Design and Construction of the Project Webpage
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
Database
Gateway Entry and Destination Vectors
GW Entry Vectors
TOPO
pENTR/D-TOPO
attL1
CCCTT
GGGAAGTGG
AAGGG
TTCCC
Knr
attL2
TOPO
ccdB
pDONR207
Gentr
CmR
attP1
attP2
GW Destination Vectors for E.coli Expression
ccdB
T7 Promoter
pH9GW
RBS
MK9His
Knr
CmR
attR1
attR2
ccdB
pH8GW
T7 Promoter
RBS
Thrombin
MK8His
Knr
CmR
attR1
attR2
GW Destination Binary Vectors for Plant Over-expression
pMDC32
RB
ccdB
2X35S
CmR
pCHF3-GW1
35S
RB
CmR
nptII
T
Specr
LB
attR2
ccdB
35S
RB
Knr
LB
attR2
ccdB
attR1
pCHF3-GW3
Hygr
Nos T
attR1
CmR
attR2
nptII
T
Specr
LB
attR1
GW Destination Binary Vectors for Reporter Assays
ccdB
pMDC162
CmR
RB
gus
Nos T
Hygr
LB
Knr
gus
Nos T
nptII
LB
Gentr
LB
Gentr
LB
Knr
attR2
attR1
ccdB
pDW137-GW1
CmR
RB
attR1
attR2
ccdB
pDW137-GW2
CmR
RB
attR2
gus
Nos T
nptII
attR1
ccdB
pMDC107
CmR
RB
attR1
gfp6his
attR2
Nos T
Hygr
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
Homozygou
s lines
MT6
SALK
Intron
X
MT7
SALK
5’-UTR
X
Garlic
Intron
X
GABI
Exon
X
SALK
Exon
X
SALK
Intron
X
Garlic
Exon
X
MT8
MT10
MT19
Plant Over-expressiors
Gene
Binary
Construct
Plant
Transformation
MT6
X
X
MT7
X
X
MT8
X
X
MT10
X
X
MT19
X
X
Transgenic Lines
X
Part III:
High-throughput Biochemical Assays
Phenolic Acids and Phenylpropanoids
COOH
COOH
COOH
COOH
NH2
OH
BA
COOH
COOH
OH
SA
OH
3-OH BA
4-OH BA
NH2
Anthranilic acid
4-amino BA
COOH
COOH
COOH
COOH
COOH
OH
OMe
OH
OMe
OH
OH
Caffeic acid
OH
P-coumaric acid
Ferulic acid
3-hydroxy-4-methoxy
Cinnamic acid
Cinnamic acid
COOH
O
COOH
HO
MeO
HOOC
HO
OMe
OMe
O
OH
OH
OH
OH
COOH
O
HO
Rosmarinic acid
Vanillic acid
3,5-dimethoxy-4-hydroxy
Cinnamic acid
COOH
COOH
O
HO
OH
HO
COOH
HO
MeO
HO
OMe
OH
O
OH
chlorogenic acid
COOH
O
Phenylpyruvic
acid
Gallic acid
Shikimic acid
COOH
HO
Chorismic aicd
COOH
O
OH
4-hydroxy
Phenylpyruvic acid
COOH
OH
OMe
Phenyllactic
acid
HO
COOH
OH
4-hydroxy
Phenyllactic acid
Phytohormones and Related Compounds
OH
H
OH
O
O
COOH
H
O
H
HO
ABA
N
H
COOH
H
GA
Indole
COOH
COOH
N
H
Cl
N
H
O
COOH
Cl
IAA
IBA
2,4-D
H3C
H2
C
CH2
C
H2
OH
N
N
H2
C
HN
HN
HN
O
H2
C
N
HN
N
H
N
H
N
Kinetin
COOH
N
HN
N
Zeatin
HN
O
N
H
Jasmonic acid
N6-benzyladenine
O
OH
OH
OH
NH3
COOH
CH2
O
OH
CH2
GABA
(gama-amino butyric acid)
O
DOPA
(3,4-Dihydroxyphenylalanine)
OPDA
Acidic Sugars, Pectin and Vitamin C
HOOC
HOOC
O
O
HO
HO
OH
HO
OH
HO
OH
Galacturonic acid
Tri-galacturonic acid
HOOC
HOOC
O
O
HO
O
HO
OH
OH
HO
OH
HO
O
OH
n
pectin
O
HO
HO
OH
Ascorbic acid
Fatty Acids
COOH
HOOC
norBixin
Short-chain (2-4) fatty acids
Butanoic acid (4:0)
Medium-chain (6-10) fatty acids
Pentanoic acid (5:0)
Hexanoic acid (6:0)
Octanoic acid (8:0)
Decanoic acid (10:0)
Long-chain (>12) fatty acids
Lauric acid (12:0)
Myristic acid (14:0)
Palmitic acid (16:0)
Stearic acid (18:0)
Arachidic acid (20:0)
Behenic acid (22:0)
Lignoceric acid (24:0)
Oleic acid (18:1)
Petroseenic acid (18:1)
a-Linoleic acid (18:2)
r-linoleic acid (18:3)
Roughanic acid (16:3)
Erucic acid (22:1)
Nitrogen-containing Compounds
O
O
H
N
HN
O
N
H
N
HN
O
NH
Ribose
O
CH3
N
H
NH
O
7-methyl-Xanthine
O
O
H3C
O
N
N
N
H
CH3
N
paraxanthine
N
HN
O
CH3
N
N
CH3
Theobromine
N
S
NH3
C C COOH
H2 H2
-alanine
CH3
N
N
H
Ribose
7-methylXanthosine
Xanthosine
N
HN
N
H
Camalexin
Amino Acids
COOH
COOH
COOH
N
H
COOH
H2N
COOH
H2N
NH2
NH3
HO
Tryptophan
Phenylalanine
H2N
H
CH2
CH2
CH2
CH2
CH2
CH2
NH
CH2
Tyrosine
NH
NH2
NH2
Lysine
Arginine
COOH
COOH
H2N
H
COOH
COOH
H2N
CH3
H2N
H
CH2OH
Alanine
Serine
H2N
H
H
CH2
CH2
CH2
COOH
COOH
Aspartic acid
Glutamic acid
COOH
COOH
COOH
H2N
COOH
H
H2N
H3C CH
H
CH3
H2N
H
H2N
COOH
H
H2N
HC CH3
C
H3C H CH3
CH3
H2N
COOH
H
CH2
OH
CH2
CH3
Valine
Threonine
Leucine
Isoleucine
COOH
H
CH2
H2N
CH2
CH2
O
NH2
Glutamine
H
O
COOH
H2N
H
CH2H
N
CH2
CH2
S
SH
CH3
Cysteine
Methionine
NH2
COOH
C
H2C H NH
C C
H2 H2
N
Asparagine
Histidine
Proline
H
CH2
H
COOH
H2N
H
COOH
H2N
H
H
Glycine
Substrate Grouping (1 to 3)
Benzoic acid
Caffeic acid
3,5-dimethoxy-4-hydroxy-cinnamic acid *
Chlorogenic acid
Phenylpyvuvic acid
Group 1 (EtOH)
Salicylic acid
Ferulic acid
Vanillic acid
Gallic acid
4-hydroxy-phenylpyvuvic acid
Group 2 (EtOH)
3-hydroxy-benzoic acid
Anthranillic aicd
p-coumaric acid
Jasmonic acid
Shikimic aicd
Group 3 (EtOH)
Substrate Grouping (4 to 6)
4-hydroxy-benzoic acid
4-amino benzoic acid
Cinnamic acid
3-hydroxy-4-methoxy-cinnamic acid *
Rosmarinic acid
4-hydroxy-phenyllactic acid
ABA
GA
IAA
IBA
2,4-D
Indole
Group 4 (EtOH)
Group 5 (EtOH)
Zeatin
Kinetin
6-benzylaminopurice
GABA *
DOPA *
Tryptophan
Group 6 (DMSO)
Substrate Grouping (7 to 10)
Trigalacturonic acid
Muranic acid
Octanoic acid
Decanoic acid
Lauric acid
Myristic acid
Palmitic acid
Stearic acid
Xanthosine
7-methyl xanthine
theobromine
paraxanthine
beta-alanine
(Camalexin)
Glutamic acid
Valine
Alanine
Phenylalanine
Aspartic acid
Asparagine
Group 7 (DMSO)
Group 8 (EtOH:Chloroform=1:1)
Group 9 (DMSO)
Group 10 (H2O)
MT7 showed activity with Lauric acid
COOH
Fully expended leaves of Col at growth stage 3.9
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
50
100
150
200
250
300
350
400
450
Plant Defense Response (I)
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
HrpA
CTR
0
200
400
600
800
1000
1200
1400
Plant Defense Response (II)
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
DC3000
avrRpm1
HrpA
CTR
0
500
1000
1500
2000
250
Plant Defense Response (III)
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
DC3000
HrpA
0
500
1000
1500
2000
2500
COOH
Lauric Acid
COOH Traumatic Acid
HOOC