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Progress Report for the 2010AtSABATH Group Meeting

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:

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Progress Report for the 2010AtSABATH Group Meeting

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

  2. 1. Management 2. Progress and Resources on 5 Genes 3. High-throughput Biochemical Assays

  3. Part I: Report on Management

  4.  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

  5. Database

  6. ccdB 2X35S Knr Hygr RB CmR LB Nos T attR1 attR2 ccdB 35S Specr nptII RB CmR LB T ccdB attR1 attR2 Gentr CmR attP1 attP2 TOPO TOPO ccdB gus Hygr RB CmR Nos T LB attR2 attR1 ccdB Gentr gus nptII RB CmR Nos T LB attR1 attR2 ccdB nptII Gentr CmR gus Nos T RB LB attR2 attR1 ccdB T7 Promoter MK9His RBS CmR ccdB attR1 attR2 gfp6his Hygr RB CmR Nos T LB attR2 attR1 ccdB Thrombin T7 Promoter MK8His RBS CmR attR1 attR2 Gateway Entry and Destination Vectors GW Entry Vectors CCCTT AAGGG GGGAAGTGG TTCCC pENTR/D-TOPO Knr attL2 attL1 pDONR207 GW Destination Vectors for E.coli Expression Knr pH9GW pH8GW Knr GW Destination Binary Vectors for Plant Over-expression pMDC32 pCHF3-GW1 ccdB pCHF3-GW3 35S Specr CmR nptII RB T LB attR2 attR1 GW Destination Binary Vectors for Reporter Assays pMDC162 Knr pDW137-GW1 pDW137-GW2 pMDC107 Knr

  7. Material Archiving 1. DNA Construct Stocks 2. DNA Oligo Stocks 3. Transgenic Seed Stocks

  8. Part II: Resources and Progress on MT6, MT7, MT8 MT10 and MT19

  9. Expression Analysis and cDNA Cloning 6 7 8 10 19

  10. GUS reporter Analysis: MT7

  11. GUS reporter Analysis: MT8

  12. GUS reporter Analysis: MT10

  13. 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 T-DNA Knock-out Lines

  14. Gene Binary Construct Plant Transformation Transgenic Lines MT6 X X MT7 X X MT8 X X MT10 X X MT19 X X X Plant Over-expressiors

  15. Part III: High-throughput Biochemical Assays

  16. Phenolic Acids and Phenylpropanoids BA SA 3-OH BA Anthranilic acid 4-OH BA 4-amino BA 3-hydroxy-4-methoxy Cinnamic acid Cinnamic acid P-coumaric acid Caffeic acid Ferulic acid Rosmarinic acid Vanillic acid 3,5-dimethoxy-4-hydroxy Cinnamic acid chlorogenic acid Shikimic acid Chorismic aicd Gallic acid 4-hydroxy Phenyllactic acid 4-hydroxy Phenylpyruvic acid Phenyllactic acid Phenylpyruvic acid

  17. Phytohormones and Related Compounds ABA GA Indole IAA IBA 2,4-D Jasmonic acid Zeatin Kinetin N6-benzyladenine OPDA GABA (gama-amino butyric acid) DOPA (3,4-Dihydroxyphenylalanine)

  18. Acidic Sugars, Pectin and Vitamin C Galacturonic acid Tri-galacturonic acid n pectin Ascorbic acid

  19. Fatty Acids 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) 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)

  20. Nitrogen-containing Compounds 7-methyl-Xanthine 7-methyl-Xanthosine Xanthosine paraxanthine Theobromine -alanine Camalexin

  21. Amino Acids Tryptophan Phenylalanine Tyrosine Lysine Arginine Alanine Serine Aspartic acid Glutamic acid Valine Threonine Leucine Isoleucine Cysteine Methionine Glycine Proline Glutamine Asparagine Histidine

  22. Substrate Grouping (1 to 3) Benzoic acid Caffeic acid 3,5-dimethoxy-4-hydroxy-cinnamic acid * Group 1 (EtOH) Chlorogenic acid Phenylpyvuvic acid Salicylic acid Ferulic acid Vanillic acid Group 2 (EtOH) Gallic acid 4-hydroxy-phenylpyvuvic acid 3-hydroxy-benzoic acid Anthranillic aicd p-coumaric acid Group 3 (EtOH) Jasmonic acid Shikimic aicd

  23. Substrate Grouping (4 to 6) 4-hydroxy-benzoic acid 4-amino benzoic acid Cinnamic acid 3-hydroxy-4-methoxy-cinnamic acid * Group 4 (EtOH) Rosmarinic acid 4-hydroxy-phenyllactic acid ABA GA IAA Group 5 (EtOH)IBA 2,4-D Indole Zeatin Kinetin 6-benzylaminopurice Group 6 (DMSO) GABA * DOPA * Tryptophan

  24. Substrate Grouping (7 to 10) Trigalacturonic acid Muranic acid Group 7 (DMSO) Octanoic acid Decanoic acid Lauric acid Myristic acid Group 8 (EtOH:Chloroform=1:1) Palmitic acid Stearic acid Xanthosine 7-methyl xanthine theobromine Group 9 (DMSO) paraxanthine beta-alanine (Camalexin) Glutamic acid Valine Alanine Phenylalanine Group 10 (H2O) Aspartic acid Asparagine

  25. MT7 showed activity with Lauric acid

  26. Fully expended leaves of Col at growth stage 3.9

  27. Plant Defense Response (I)

  28. Plant Defense Response (II)

  29. Plant Defense Response (III)

  30. Lauric Acid Traumatic Acid

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