progress report for the 2010atsabath group meeting
DESCRIPTION
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: - PowerPoint PPT PresentationTRANSCRIPT
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, [email protected], 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