High Resolution LC-MS Data Output and Analysis

Transcription

1 High Resolution LC-MS Data Output and Analysis

2 Software for comparing full-scan datasets MetAlign method

3 Software for comparing full-scan datasets MetAlign method Base line correction and peak pickingnew chromatograms with corrected baseline are generated Chromatogram alignment based on RT and nominal mass Statistical analysis

4 MetAlign output, ES(-), Tomato peel, orange, WT vs Mut Y

5 MetAlign Output, Visualization of Differential Peaks

6 Software for comparing full-scan datasets MarkerLynx method

7 Software for comparing full-scan datasets MarkerLynx Method RT correction We don t want to see (e.g. from solvent) RT and mass windows and tolerance parameters if using internal standard peak integration for quantification peak parameters for analysis isotopes in data?

8 Out-put from the MarkerLynx (Waters) Program : Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+) Samples Processed Markers Detected TIC of currently selected sample Response of a marker across all samples Principle Components Analysis Markers responsible for clustering (by PCA)

9 Output Data For High Res. LC-MS Based Metabolomics Nominal Accurate To MS-MS

10 A Case Study in Tomato Small Green Middle Green Big Green Breaker Orange Red

11 Sample Preparation for Tomato Fruit Metabolome Analysis Harvesting tomato fruit at different developmental stages Separation of peel and flesh tissues Immediate freezing in liquid nitrogen Grinding to fine powder Weighing frozen samples Extraction in % Me by vortexing and sonication Analyzing with UPLC-MS [ ES (+) and ES (-) ]

12 Experimental Set-Up for LC-MS Analysis: PEEL and FLESH during tomato fruit development Green stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES- Breaker stage Flesh Peel Orange stage Flesh Peel Red stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES-

13 UPLC-Q-Tof-MS Analysis of Four Stages of Tomato Fruit Development Tom_24_WTa AU 5.e-1 Tom_22_WTa AU AU 5.e-1 Tom_18_WTa AU e Jan-26 3: Diode Array Range: : Diode Array Range: e Tom_2_WTa5 3: Diode Array Range: Red Orange Breaker 3: Diode Array Range: 3.2 Green Time Tom_24_WTa7 % % % % Jan-26 1: TOF MS ES+ TIC 5.e Tom_22_WTa6 1: TOF MS ES+ TIC 5.e Tom_2_WTa5 1: TOF MS ES+ TIC 5.e Tom_18_WTa4 1: TOF MS ES TIC 5.e Red Orange Breaker Green Time UV (24 nm) MS (ES(+),TIC)

14 Out-put from the MarkerLynx (Waters) Program : Analyses of Wild-Type Tomato Peel (Different Stages of Development; ES+)

15 Peel-Flesh Profiles During Fruit Development Red flesh Red peel Orange peel Component 2 Orange flesh Breaker flesh Breaker peel Green flesh Green peel Comp. 1

16 Wild-type Tomato Peel, Different Developmental Stages Selected Ion Chromatograms Red 9-Jan-26 Tom_24_WTa7 DOWN 9-Jan-26 NO CHANGE UP Tom_24_WTa7 1: TOF MS ES+ Tom_24_WTa7 1: TOF MS ES e4 Area 731 Area % Tom_22_WTa6 1: TOF MS ES e4 Area % Tom_22_WTa6 1: TOF MS ES Area % Tom_22_WTa Jan-26 1: TOF MS ES e4 Area 1: TOF MS ES e4 Area Orange Breaker % Tom_2_WTa5 % Tom_18_WTa : TOF MS ES e4 Area 1: TOF MS ES e4 Area % Tom_2_WTa5 1: TOF MS ES Area % Tom_18_WTa4 1: TOF MS ES Area % Tom_2_WTa5 1: TOF MS ES e4 Area % Tom_18_WTa4 1: TOF MS ES e4 Area Green % Time % Time % Time Tomatine Rutin & Quercetin trisacharide Naringenin chalcone

17 Tomato (var. Ailsa Craig) and the Mutant Y, at different ripening stages Green Breaker Orange Red Wild-Type Mutant Y Tomato peel of Mutant Y and Wild-type

18 Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway Phenylalanine Cinnamate Coumarate Coumaroyl - CoA Chalcone synthase (CHS) Naringenin chalcone Naringenin Lignins, Coumarins, Chlorogenic-acid Anthocyanines Flavonols Condensed tannins

19 Experimental Set-Up for LC-MS Analysis: PEEL and FLESH during Y fruit development Y Green stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES- Y Breaker stage Flesh Peel Y Orange stage Flesh Peel Y Red stage Flesh Peel ES+ ES- ES+ ES- ES+ ES- ES+ ES-

20 UV and MS Chromatograms of Red Tomato Peel (Wild Type and Mutant Y ) Tom_56_WTa % Mutant Y Jan-26 1: TOF MS ES+ TIC 2.11e4 Tom_56_WTa AU Mutant Y 3.25 IS 9-Jan-26 3: Diode Array Range: Tom_8_WTa3 1: TOF MS ES+.85 TIC 2.12e4.7 Wild Type e Tom_8_WTa3 3: Diode Array.8 Range: Wild Type 4.95 % Time AU e Time UV (24 nm) MS (ES(+),TIC)

21 HPLC-QTOF QTOF-MS (Ultima-PRI) - Wild Type and Y Peels 1 st principal component (X axis) - developmental stage (47.1%) 2 nd principal component (Y axis) mutation (9.4%) WT-Orange All breaker WT- Red Y -Orange All green Y -Red

22 Post Metalign: all WT > Y mass peaks Scan # RT in umin M/z

23 HPLC-qTOF -MarkerLynx HPLC-qTOF-MetAlign HPLC-qTOF-XCMS

24 In-depth Analysis of Red Tomato Peel (ES+) Wild-type vs. the Y Mutant MarkerLynx Differential Markers Filtering in MS Excel Matlab and subsequently peak analysis by eye

25 The Yellow Pigment Absent in the Y Mutant Fruit Peel is Naringenin Chalcone Tom_8_WTa : TOF MS ES+ TIC 1.37e : TOF MS ES+ 2.35e4 % 5.75 Tomato peel % Tomato peel MixAllStand_b1 1: TOF MS ES TIC e % Looking for a known compound % : TOF MS ES+ 7.9e3 Standard s mixture Naringenin Chalcone Standard Time m/z Cinnamic acid 13 Naringenin Chalcone 14 Naringenin 15 Kaempferol Looking for a known compound

26 Unknown Mass peak (RT=3.51 min) Detected Only in Wild Type Tom_72_WTa19 % % % % % Jan-26 1: TOF MS ES Tom_56_WTa15 1: TOF MS ES Tom_4_WTa11 1: TOF MS ES Tom_24_WTa7 Tom_8_WTa : TOF MS ES : TOF MS ES Time Mutant Y Mutant Y Wild Type Wild Type Wild Type Extracted mass chromatogram at m/z Da

27 Unknown Mass peak Detected in Higher Levels in Y Mut. Y Mut. Y Wild-type Wild type Wild type Tom_72_WTa19 % Tom_56_WTa15 % % % % Jan-26 1: TOF MS ES e3 Area Tom_4_WTa11 1: TOF MS ES e3 Area Tom_24_WTa7 1: TOF MS ES e3 Area Tom_8_WTa3 1: TOF MS ES e3 Area : TOF MS ES e3 Area Time Extracted mass chromatogram at m/z Da

28 What are the Differentially Expressed Metabolites between WT and Y? Peak assignment by: - Accurate mass and elemental composition calculation (natural products database) - MS-MS fragmentation - UV spectrum - Literature - UV & MS spectra databases - Isotope pattern

29 Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES- Mode Differential metabolites (ES-) R only O only Both R & O Total WT > "Y" "Y" > WT

30 Differential Metabolites between the "Y" and Wild Type Peel UPLC-QTOF-MS in ES+ Mode Differential metabolites (ES+) R only O only Both R & O Total WT >"Y" "Y" > WT

31 What are the Differentially Expressed Metabolites between WT and Y? Most mass peaks showing higher levels in WT than in the Y mutant are derivatives of Naringenin Chalcone or Naringenin.

32 Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway Phenylalanine Cinnamate Coumarate Coumaroyl - CoA Chalcone synthase (CHS) Naringenin chalcone Naringenin Lignins, Coumarins, Chlorogenic-acid Anthocyanines Flavonols Condensed tannins

33 Hydroxylated Naringenin & Naringenin- Chalcone Ilana wt-tom rp 23 M5983 Y HPLC-QTOF 1: TOF MS ES Da 875 % % M WT The same m/z 1: TOF MS ES Da Time

34 Spectra of differential compounds at RT=35.79 min and 37.5 min RT=35.79 min RT=37.5 min

35 UV Spectra of Differential Peaks (35.7 min. and 37.5 min.) AU 1.e-2 8.e-3 6.e-3 4.e Naringenin 1.126e-2 AU 6.e-1 4.e-1 2.e e-1 Naringenin Chalcone 2.e-3.. M (35.667) Cm (2131: :216) e-3 1.5e-3 1.e-3 5.e-4 AU 3: Diode Array 2.195e-3 Peak at 35.7 min M (36.9) Cm (229-( )) AU 4.e-3 2.e-3 3: Diode Array 5.444e-3 Peak at 37.5 min nm. nm HO HO A O C B O Naringenin Chalcone O Naringenin

36 Elemental Composition ( ) Elemental composition: C 15 H 12 O 6 for neutral compound Elemental composition of Naringenin: C 15 H 12 O 5 Difference: group instead of H

37 Suggested Metabolite MS-MS Fragmentation of m/z ESI % MS-MS (ES+) fragmentation of m/z Eriodictyol an Hydroxylated Naringenin (C 15 H 12 O 6 ) Main fragmentations in reference: I. J. M. S. 247 (25) 93 m/z Injection of Eriodictyol standard or NMR

38 Naringenin and Naringenin Chalcone Glycosides HPLC-QTOF Extracted Ion Chromatograms at Da Ilana Y-tom rp 55 M5983 1: TOF MS ES Da 1.41e3 % % M Y WT 1: TOF MS ES Da 1.41e Time

39 MS spectra at RT=28, 3 32 and 33 min RT=28.7 min For MS-MS RT=3.5 min RT=32.8 min Exact mass of shows elemental composition of C 21 H 22 O 1 m/z is an hexose derivative of Naringenin or NarChalc RT=33.4 min

40 MS-MS Fragmentation of m/z for Peak at RT=32.8 Peak at 32.8 M67 47 (33.469) Cm (45:411) Naringenin Chalcone (according to UV spectrum) : TOF MSMS ES+ 1.59e3 Hexose (m/z 162) Naringenin Chalcone-hexose % m/z

41 UV Spectra of Peak at 32.8 min. and Naringenin Chalcone M (42.483) Cm (2544-( )) AU 6.e e-1 3: Diode Array 6.774e-1 Peak at 32.8 min 2.e-1. M (32.65) Cm (1954-( )) : Diode Array 3.933e-3 AU 3.e-3 2.e-3 Naringenin Chalcone 1.e nm

42 Methyl ether of Hydroxylated Naringenin and Naringenin Chalcone TIC chromatogram Extracted Ion Chromatogram m/z 33.8 Da MS spectra at RT=43.4min Mut Mut Mut WT WT WT

43 Methyl ether of Hydroxylated Naringenin and Naringenin Chalcone Peaks at 43.4 min and 44.6 min Calculated formula is C 16 H 14 O 6 based on exact mass Proposed structure for the peak at 43.4 min: Hesperetin or a B-ring positional isomer Hesperetin is a Methoxylated Naringnein

44 MS-MS Fragmentation of m/z 33.8 (43.4 min.) Ilana wt-tom rp 23 msms M67 55 (43.325) Cm (55:56) : TOF MSMS 33.8ES [M+H] + % [M+H-H 2 O] m/z Hesperetin

45 Proposed Scheme of Naringenin & Naringenin Chalcone Hydroxylation, Methylation and Glycosylation Glycosylation HO O Naringenin Chalcone HO O B A C O Naringenin O O Glycosylation e.g. Naringenin glucoside O O Glycosylation HO HO O Glycosylation O Hydroxylated Naringenin Chalcone O Hydroxylated Naringenin e.g. Eriodyctyol or a B-ring isomer OMe OMe Glycosylation HO HO O Glycosylation O O Methyl ether of Hydroxylated Naringenin Chalcone Methyl ether of Hydroxylated Naringenin e.g.hesperetin or a B-ring isomer

46 Biosynthesis of Naringenin Chalcone in the Flavonoid Pathway Phenylalanine Cinnamate Coumarate Coumaroyl - CoA Chalcone synthase (CHS) Naringenin chalcone Naringenin Lignins, Coumarins, Chlorogenic-acid Anthocyanines Flavonols Condensed tannins

47 FTMS metabolic phenotyping Crude biological extract Direct injection into FTMS via HPLC autosampler +/- ESI and +/- APCI ionization High resolution separation and detection of metabolites Instrument acquisition time: (2-5 min/sample/mode) Off-line spectral processing Exportation of processed data to database

48 Analytical Instrumentation (FTMS) Fourier Transform Ion Cyclotron Mass Spectrometry

49 Non-targeted metabolic profiling in strawberry Green Red White Turning

50 Positive ion ESI of strawberry fruit Red Strawberry Green Strawberry 8 amu Window

51 High resolution separation X = Y = X m/z X Y Resolution = 135,,.1 AMU Window

52 Empirical formula determination Internal Mass Calibration Of Sample Calibration Results: Ref. Masses Exp. Masses Diff (ppm) Calculate Accurate Mass Of Metabolite Assign Empirical Formula Based Upon Calibration Errors Calculate Possible Empirical Formulas Mass Analysis for mass # 12C 1H 16O 14N 32S mass error = C 21 H 2 O 1 [+H] + Error =.52ppm e e e e e e e e e e e e e e e e e e e e e e e e e e e-6

53 Number of mass peaks and metabolites observed Number of metabolites observed across stages of fruit development, ionization modes and extractions Overall mass peaks Unique mass peaks Green White Turning Red Total Only Both (%) Total 5 AN 5 AN 5 AN 5 AN 5 + AN 5 AN 5 + AN ESI (25%) 1472 ESI (16%) 127 APCI (36%) 1499 APCI (19%) 163 Mean (24%) 1461 Total , (24%) 5844

54 Accurate empirical formula determination % Empirical Formula Assignment Statistics 8% 6% 4% 2% % ESI + ESI - APCI + APCI - Only 1 Formula 2 Formulas More than 2

55 Quantitative determination of change between samples Metabolite Changes Observed In Strawberry Development 15 RS/GS RS/WS 2X 2X RS/TS TS/GS 2X TS/WS 5 2X WS/GS 5X 1X 2X 5X 1X 5X 1X 2X 5X 1X 5X 1X 5X 1X Metabolite Increases Metabolite Decreases

56 Metabolic profiling of strawberry fruit development Metabolite CG EF NM IM EX R/G T/G W/G Group 1: Early expression Profile A Ellagic acid* P. Acid C 14 H 6 O AP- AN Hydroxybenzoic acid* P. Acid C 7 H 6 O AP- AN Arginine A. acid C 6 H 14 N 4 O ES Vanillic acid* P. Acid C 8 H 8 O AP- AN Malic acid O. acid C 4 H 6 O ES- AN Catechin (epi)/leucopelargonidin* Phenolic C 15 H 14 O ES Caffeate* P. Acid C 9 H 8 O AP Ethyl cinnamate* Ester C 11 H 12 O AP- AN Quercitin glucoside* Flavonoid C 21 H 2 O ES Methyl cinnamate* Ester C 1 H 1 O AP- AN Dihydrokaempferol* Flavonoid C 15 H 12 O AP Leucocyanidin* Flavonoid C 15 H 14 O AP- AN N-Alanylcysteine A. Acid d. C 6 H 12 N 2 O 3 S ES+ AN Gentisic/Protocatechuic acid* P. Acid C 7 H 6 O AP Malusfuran Furan C 12 H 8 O AP (E)2-hexenol / hexanal Alcohol C 6 H 12 O.888 AP+ AN Glucogallin* Phenolic C 13 H 16 O AP Pantothenic acid Vitamin C 9 H 17 NO ES+ AN Dihydroxybenzoquinone Phenolic C 6 H 4 O ES+ AN Dihydroquercitin* Flavonoid C 15 H 12 O AP

57 Metabolic profiling of strawberry fruit development Metabolite CG EF NM IM EX R/G T/G W/G Profile I Linoleic acid F. Acid C 18 H 32 O AP+ AN Palmitoleic acid F. Acid C 16 H 3 O AP+ AN Oleic acid F. Acid C 18 H 34 O AP+ AN Pentadecanoic acid F. Acid C 15 H 3 O AP+ AN Palmitic acid F. Acid C 16 H 32 O AP+ AN methylbutyl nonanoate Ester C 14 H 28 O AP+ AN Stearic acid F. acid C 18 H 36 O AP+ AN Furaneol (R=D-glucopyranose) Furan C 12 H 18 O ES+ AN Furaneol (R=D-glucopyranose, malonyl) Furan C 15 H 2 O ES Nonanoic acid F. Acid C 9 H 18 O AP+ AN Capric acid F. Acid C 1 H 2 O AP+ AN Eicosanoic acid F. Acid C 2 H 4 O AP+ AN Linolenic acid F. Acid C 18 H 3 O AP+ AN Cinnamate* P. Acid C 9 H 8 O AP octyl butanoate Ester C 12 H 24 O AP+ AN methyhexyl hexanoate Ester C 13 H 26 O AP+ AN Cis-hex-3-enyl octanoate Ester C 14 H 26 O AP+ AN Methylstearate Ester C 19 H 38 O AP+ AN Sinapyl alcohol* Phenolic C 11 H 14 O AP- AN Scopoletin* Benzopyranoid C 1 H 8 O AP Isopropyl-3-methoxypyrazine Alkaloid C 8 H 12 N 2 O AP+ AN Farnesyl acetone Terpene C 18 H 3 O AP+ AN Hydroxycoumarine* Benzopyranoid C 9 H 6 O AP+ AN Coumarine* Benzopyranoid C 9 H 6 O AP+ AN Pelargonidin* Flavonoid C 15 H 1 O ES Smipine Alkaloid C 1 H 16 N 2 O AP+ AN

58 Group 1: Early expression Catechin (APCI +) Heptylamine (ESI+) Citrate/Isocitrate (APCI+)

59 Group 2: Intermediate expression Phenylalanine (ESI+) Glutamine (ESI+) Benzoate (APCI+)

60 Group 3: Late expression Pelargonidin glucoside (ESI+) Coumaroyl glucoside (ESI-) Furaneol glucoside (ESI+)

61 Glucogallin (hydrolyzable tannins)(-4.3) Gallic acid (+2.) Ellagic acid (-33.3) Hydroxycoumarin (+5.44) Quercetin Quercetin glucoside (-7.7) Catechol (+2.) Protocatechoic acid (-3.2) Vanillic acid (-12.5) Kaempferol glucoside (+2.1) Dihydroquercetin (-2.4) DFR (1.9) Leucocyanidin (-2.7) Cyanidin ANS (+6.7) GT1 (+8.3) Cyanidin glucoside (+2.1) GST (+2.8) Anthranilate (-2.3) Coumarin (+5.5) Hydroxybenzoic acid (-11.1) Kaempferol Catechin (condensed tannins) (-25) vacuole Coumarate glucose (+9.6) Gentisic acid (-3.2) Benzoic acid (+2.) CHS (+3.3) Naringenin chalcone CHI (+4.3) Naringenin F3H (+3.2) Dihydrokaempferol (-9.1) DFR (1.9) Leucopelargonidin (-25) ANS (+6.7) Pelargonidin (+9.46) GT1 (+8.3) Pelargonidin glucoside (+255) GST (+2.8) Phenylalanine (-5.) PAL Cinnamate (+5.8) Coumarate (+3.2) Coumaroyl - CoA CCR (+3.6) GT2 (+3.7) Caffeate Ferulate (+3.9) Hydroxyferulate (+2.4) Sinapate (+3.8) Sinapoyl CoA OMT (+2.6) OMT (+2.6) Caffeoyl CoA Scopoletin (+5.31) CCR (+3.6) Cinnamate glucose (+2.) Ethyl cinnamate (-7.7) Methyl cinnamate (-5.3) + Quinic acid (-4.2) Chlorogenic acid Feruloyl CoA Coniferaldehyde Coniferyl alcohol CCR (+3.6) CAD (+8.8) Sinapaldehyde (+2.4) CAD (+8.8) Sinapyl alcohol CAD (+5.8) Coumaraldehyde (+8.8) (+5.86) Coumaryl alcohol (+3.29)

62 Phenylalanine and changes in gene and metabolite expression?

63 Glucogallin (hydrolyzable tannins)(-4.3) Gallic acid (+2.) Ellagic acid (-33.3) Hydroxycoumarin (+5.44) Quercetin Quercetin glucoside (-7.7) Catechol (+2.) Protocatechoic acid (-3.2) Vanillic acid (-12.5) Kaempferol glucoside (+2.1) Dihydroquercetin (-2.4) Cyanidin DFR (1.9) Leucocyanidin (-2.7) ANS (+6.7) GT1 (+8.3) Cyanidin glucoside (+2.1) GST (+2.8) Anthranilate (-2.3) Coumarin (+5.5) Hydroxybenzoic acid (-11.1) Kaempferol Catechin (condensed tannins) (-25) vacuole Coumarate glucose (+9.6) Gentisic acid (-3.2) Benzoic acid (+2.) CHS (+3.3) Naringenin chalcone CHI (+4.3) Naringenin F3H (+3.2) Dihydrokaempferol (-9.1) DFR (1.9) Leucopelargonidin (-25) ANS (+6.7) Pelargonidin (+9.46) GT1 (+8.3) Pelargonidin glucoside (+255) GST (+2.8) Phenylalanine (-5.) PAL Cinnamate (+5.8) Coumarate (+3.2) Coumaroyl - CoA CCR (+3.6) GT2 (+3.7) Caffeate Ferulate (+3.9) Hydroxyferulate (+2.4) Sinapate (+3.8) Sinapoyl CoA OMT (+2.6) OMT (+2.6) Caffeoyl CoA Scopoletin (+5.31) CCR (+3.6) Cinnamate glucose (+2.) Ethyl cinnamate (-7.7) Methyl cinnamate (-5.3) + Quinic acid (-4.2) Chlorogenic acid Feruloyl CoA Coniferaldehyde Coniferyl alcohol CCR (+3.6) CAD (+8.8) Sinapaldehyde (+2.4) CAD (+8.8) Sinapyl alcohol CAD (+5.8) Coumaraldehyde (+8.8) (+5.86) Coumaryl alcohol (+3.29)

64 Phenylalanine and changes in gene and metabolite expression?

65 Glucogallin (hydrolyzable tannins)(-4.3) Gallic acid (+2.) Ellagic acid (-33.3) Hydroxycoumarin (+5.44) Quercetin Quercetin glucoside (-7.7) Catechol (+2.) Protocatechoic acid (-3.2) Vanillic acid (-12.5) Kaempferol glucoside (+2.1) Dihydroquercetin (-2.4) Cyanidin DFR (1.9) Leucocyanidin (-2.7) Coumarin (+5.5) ANS (+6.7) GT1 (+8.3) Cyanidin glucoside (+2.1) GST (+2.8) Anthranilate (-2.3) Hydroxybenzoic acid (-11.1) Kaempferol Catechin (condensed tannins) (-25) vacuole Coumarate glucose (+9.6) Gentisic acid (-3.2) Benzoic acid (+2.) CHS (+3.3) Naringenin chalcone Naringenin F3H (+3.2) Dihydrokaempferol (-9.1) DFR (1.9) Leucopelargonidin (-25) ANS (+6.7) Pelargonidin (+9.46) GT1 (+8.3) Pelargonidin glucoside (+255) GST (+2.8) CHI (+4.3) Phenylalanine (-5.) PAL Cinnamate (+5.8) Coumarate (+3.2) Coumaroyl - CoA CCR (+3.6) GT2 (+3.7) Caffeate Ferulate (+3.9) Hydroxyferulate (+2.4) Sinapate (+3.8) Sinapoyl CoA OMT (+2.6) Caffeoyl CoA Scopoletin (+5.31) OMT (+2.6) CCR (+3.6) Cinnamate glucose (+2.) Ethyl cinnamate (-7.7) Methyl cinnamate (-5.3) + Quinic acid (-4.2) Chlorogenic acid Feruloyl CoA Coniferaldehyde Coniferyl alcohol CCR (+3.6) CAD (+8.8) Sinapaldehyde (+2.4) CAD (+8.8) Sinapyl alcohol CAD (+5.8) Coumaraldehyde (+8.8) (+5.86) Coumaryl alcohol (+3.29)

66 Over-expression of FaMYB1 in tobacco Tr-m Tr-w Tr-bcred Tr-bcwhite Tr-s Nt A B C D

67 The phenylpropanoid pathway in strawberry & tobacco H O Lignin Phenylalanine Kaempferol Quercetin Dihydroquercetin O + FLS Cyanidin CAD DFR ANS PAL C4H 4 - Coumarate 4 - Coumaroyl - CoA Malonyl - CoA FLS F3 H Cinnamate 4CL CHS Naringenin chalcone HO CHI Naringenin F3H Dihydrokaempferol O + DFR ANS Pelargonidin GT Cyanidin -3-glucoside (tobacco) GT Pelargonidin -3-glucoside (strawberry) RT GST GST vacuole

68 Metabolite changes in the FaMYB1 overexpressing tobacco lines Lignin Quercetin Phenylalanine Dihydroquercetin Cyanidin CAD Kaempferol FLS DFR ANS PAL C4H 4 - Coumarate 4 - Coumaroyl - CoA Malonyl - CoA FLS F3 H Cinnamate 4CL CHS Naringenin chalcone CHI Naringenin F3H Dihydrokaempferol H O O + GT Cyanidin -3-glucoside RT GST vacuole

69 Anthocyanin and flavonol (quercetin) levels are reduced Anthocyanins (total) Flavonols (aglycons) 16 quercetin kaempferol Tr-w2 Tr-w1 Tr-w6 Tr-w5 Tr-w9 Tr-w8 Tr-bcwhite Tr-bcred Tr-w Tr-m Tr-s Nt Flavonol aglycons levels (relative to Nt, %) Tr-w Tr-m Tr-s Nt Total anthocyanins (relative to Nt, %) Tr-w2 Tr-w1 Tr-w6 Tr-w5 Tr-w9 Tr-w8 Tr-bcwhite Tr-bcred Tobacco line Tobacco line

70 Gene expression and enzymatic activity changes in the FaMYB1 over-expressing tobacco lines Lignin Quercetin Phenylalanine CAD Kaempferol FLS Dihydroquercetin DFR ANS PAL 4 - Coumarate 4 - Coumaroyl - CoA Malonyl - CoA FLS F3 H Cinnamate C4H 4CL CHS Naringenin chalcone CHI Naringenin F3H Dihydrokaempferol Cyanidin H O O + GT Cyanidin -3-glucoside RT GST vacuole

71 Anthocyanidin synthase (ANS) expression is down regulated ANS RIBO. Tr-s Tr-m Tr-w Nt Transcript Ratio Tr-s Tr-m Tr-w Nt Tobacco line Tobacco line

72 Flavonoid glucosyl transferase enzyme activity is down regulated Enzyme activity (% of Nt) Nt Tr-s Quercetin Kaempferol Cyanidin Flavonoid substrate

73 FaMYB1 is possibly involved in the regulation of flavonoid biosynthesis in late strawberry fruit ripening It may act as a repressor of late flavonoid biosynthesis genes H O Lignin Phenylalanine Dihydroquercetin Cyanidin O + CAD Kaempferol Quercetin FLS DFR ANS PAL C4H 4 - Coumarate 4 - Coumaroyl - CoA Malonyl - CoA FLS F3 H Cinnamate 4CL CHS Naringenin chalcone HO CHI Naringenin F3H Dihydrokaempferol O + DFR ANS Pelargonidin GT Cyanidin -3-glucoside (tobacco) RT GST GT Pelargonidin -3-glucoside (strawberry) GST vacuole

74 Wild type vs. white mutant tobacco Observed mass of unknown: Empirical formula search result: C 27 H 3 O 15 [+H]+ Mass: Mass error:.4 ppm m/z Proposed metabolite: Cyanidin Rhamnoglucoside

75 See You Next Week