biochemistry is a dynamic multi-level reaction …...2-keto-l-gluconic acid diethylene glycerol...

1

Plant Biology Division

Current and Future Directions of LC/MS Current and Future Directions of LC/MS Based Metabolomics of the Model legume Based Metabolomics of the Model legume

Medicago truncatulaMedicago truncatula

Mohamed A. Farag, Corey D. Broeckling, David V. Huhman, Ewa Urbanczyk-Wochniak, Zhentian Lei, Bonnie S. Watson, Satish

Nagaraj, and Lloyd W. Sumner Marina Naoumkina, Bettina Deavours, and Richard A. Dixon


Biochemistry is a Dynamic MultiBiochemistry is a Dynamic Multi--level level Reaction NetworkReaction Network

mRNAυ1 Active

Protein

υ3 υ4υ2Polypeptde

Proteindegradation

υ10 Metabolite BMetabolite A

Protein Sorting

υ8υ5

υ7

Protein-Protein Interaction

υ9υ6

Transcriptome

Proteome

Metabolome

genome

PTMs

2


Proteome Metabolome

Alter Gene Expression

Integrated Functional Genomics & Systems BiologyIntegrated Functional Genomics & Systems BiologyTranscriptomeGenome

Relational DatabasesStore, Compare, Cluster, & Integrate

Correlation of Genes and Gene ProductsIdentification of Gene Function

Gene AnnotationIdentification of Pathways

DNAsequence

databases

Biological Knowledge, Plant Improvement, Genetic and Metabolic Engineering

data preprocessingdata preprocessing


Medicago truncatula Medicago truncatula ((a model legume)a model legume)

*Arumuganathan and Earle, 1991

Small diploid genome,*Self fertile, and short generation

High sequence similarity with alfalfa, pea, and soybean

Transformation methodsNumerous ecotypesFast neutron & transposon mutants Large abundance of EST sequencesGenomic sequencing underway

(~160Mb acquired)

Species MbpMedicago truncatula 454-526Glycine max 1115M. sativa 1510

3


Formononetin (isoflavone)

OHO

OOCH3

Procyanidin trimer(condensed tannins)

•Isoflavonoids (plant defense, phytoestrogen, and anticancer)

•Condensed tannins (antioxidants; improve bypass protein and silage properties)

•Lignin (forage digestibility; fiber)

•Triterpene saponins (antifeedant/bloat, anti-insect, anticholesterol, anticancer)

MedicagoMedicago is a rich source of natural productsis a rich source of natural products

HO

HO

OH

OH

OH

OH

O

OH

OH

OH

OH

O

OH

OH

OH

HO

OH

O

CO2H

HOCO2H

HO

MedicagenicAcid

Dixon & Sumner, Plant Physiol., 2003, 131, 878-885

CH3OH

HO

OHO

OHEstradiol

Equol HOH3CO

COOH

OHOCH3

COOH

Lignin


An integrated approach to functional genomics and An integrated approach to functional genomics and bioinformatics in a model legumebioinformatics in a model legume

• Sample collection at 21 time points post elicitation

• Triplicate controls & elicited• 640 total samples• Generate bioinformatic tools to

determine causal relationships between genes, proteins, and natural products

• Isoflavonoid pathway serves as internal control and validation

• New Discoveries! • Improve knowledge of legume

biology

http://medicago.vbi.vt.eduhttp://www.noble.org/medicago/index.htm

Bioinformatics / analysis

M.t. Cell Cultures

Control Yeastelicited

UVelicited

Jasmonateelicited

cDNAmicroarray

2-DEProteomcis

GC-MS & LC-MS

Samples

Biological knowledge

NSF-PGRP #0109732) Mendes, Dixon, May, Smith, Sumner

4


Salts/IonicCarbohydratesAmino acidsOrganic acids

PhenolicsFlavonoidsSaponins

Polar Organics

HPLC/PDA/MSMALDI-TOFMS

GC/MS

(Solvent Selective Extraction & Parallel Analyses)(Solvent Selective Extraction & Parallel Analyses)The Chemically Complex MetabolomeThe Chemically Complex Metabolome

= Greater visualization of the metabolome= Greater visualization of the metabolome

Plant MaterialCell disruption

FAMEsLipids

Terpeneswaxes

GC-MS HPLC/MSCE-MS

GC/MS

H2O MeOH CHCl3

SPE?


POLAR EXTRACT

LIPOPHILIC EXTRACT

GC/MS Metabolite Profiling GC/MS Metabolite Profiling M. truncatulaM. truncatula

Roessner et al., Plant J. 2000, 23, 131-142Fiehn et al, Nat. Biotechnol. 2000, 18, 1157-1161

LipophilicPolar

H2O/CHCl3

6 mg Dry Tissue

derivatize

5


2-imino Propanoic Acid Citric Acid Gluconic Acid Malonic Acid Pinitol Sucrose2-keto-l-gluconic acid Diethylene Glycerol Ether Glucose Maltose Pipecolic Acid Tartaric Acid4-Aminobutyric Acid Erythritol Glucose-6-Phosphate Mannitol Proline Threonic Acid4-hydroxypyridine Ethanol Amine Glutamic Acid Mannose Propanoic Acid, 2-Methoxyimino ThreonineAconitic Acid Ethyl Amine Glyceric Acid Melibiose Propylamine ThreoninolAdenine Ethyl Phosphoric Acid Glycero-3-Phosphate Melibiose Propylene Glycol ThymineAdenosine Ethylene Glycol Glycerol Methionine Putrescine trans-Aconitic AcidAlanine Ferulic Acid Glycine Methyl Malonic Acid Pyroglutamic Acid TrehaloseAllantoin Fructose Glycolic Acid Methyl-Phosphoric Acid Pyroglutamic Acid(Glutamine) TryptophanAlpha-glycerophosphate ester Fructose-6-Phosphate Homoserine Myo-Inositol Pyruvic Acid TyrosineAlpha-Ketoglutaric Acid Fumaric Acid Hydroxy-Butanoic Acid Nicotinic Acid Raffinose UracilArabinose Galactaric Acid Inositol-Phosphate Nigerose Ribonic Acid UreaAscorbic Acid Galactinol Isoleucine Norleucine Ribose Uric AcidAsparagine Galactitol Isomaltose Ononitol Serine UridineAspartic Acid Galactonic Acid Lactic Acid Ornithine Shikimic Acid Valine/NorvalineAzelaic Acid Galactose Lauryl Alcohol Oxalic Acid Sorbitol XylitolB-Alanine Glucaric Acid Leucine p-Coumaric Acid Sorbitol-6-Phosphate Xylonic Acid gamma lactoneBenzoic Acid Glucaric Acid 1,4 lactone Lysine Phenylalanine Spermine XyloseBoric Acid Glucoheptonic Acid Maleic Acid Phosphoric Acid Stearic AcidCadaverine Glucoheptulose Malic Acid Phosphoric Acid 2,3 oxypSuccinic Acid

Identified MetabolitesIdentified MetabolitesPolar Root Extracts--118 Metabolites


HPLC/PDA/MS/MS is a Highly SpecificHPLC/PDA/MS/MS is a Highly SpecificMultidimensional Metabolic Profiling ToolMultidimensional Metabolic Profiling Tool

•HPLC retention time•On-line photodiode array spectra for

compounds with chromophores•On-line mass selective detection•On-line tandem and MSn capabilities

•Comparative Profiling +Structural Determination!!!

6


Retention time (min) Wavelength (nm)

Abs

orba

nce

(mA

u)

LC/MS with onLC/MS with on--line photodiode array detectionline photodiode array detection

[2M-H]-

0

20

40

60

80

100Abund.

250 500 750135.1

268.9

490.9

[M-H]-

Sumner et al, Recent Advances in Phytochemistry Vol 36, –Phytochemistry in the Genomics and Post-Genomics Era, 2002, pgs 31-61, Pergamon, Oxford, UK..

Plant Biology Division20 25 30 35 40 Time [min]

A

C

Abso

rban

ce (M

AU)

Rel

ativ

e Ab

unda

nce

1

23

6

5

7

11

12

13

14 16

19

20

30

B

20 25 30 35 40 Time [min]

2 3

5 8

10

1213

14

15

1620

23 2425

26

27

2829

32

2

5

8

10

12

14

15 19

34

292427

20 25 30 35 40 Time [min]

200 450325200 360280

15

19

20

23

104.3269

299

461 499

+MS, 28.0min

0

2

4

5x10Intens.

100 200 300 400 500

[M+H]+

162

[M+H-162]+

Retention time (min)

1718 24

2526

272829

32 33

32

25

352126

4

9

10

2123

7

7

4 30 31

8

9

31

34

35

22

16

22

1 2

3

20 25 30 35 40 Time [min]

A

C

Abso

rban

ce (M

AU)

Rel

ativ

e Ab

unda

nce

1

23

6

5

7

11

12

13

14 16

19

20

30

B

20 25 30 35 40 Time [min]

2 3

5 8

10

1213

14

15

1620

23 2425

26

27

2829

32

2

5

8

10

12

14

15 19

34

292427

20 25 30 35 40 Time [min]

200 450325200 360280

15

19

20

23

104.3269

299

461 499

+MS, 28.0min

0

2

4

5x10Intens.

100 200 300 400 500

[M+H]+

162

[M+H-162]+

Retention time (min)

1718 24

2526

272829

32 33

32

25

352126

4

9

10

2123

7

7

4 30 31

8

9

31

34

35

22

16

22

1 2

3

LC/MS with onLC/MS with on--line photodiode array detectionline photodiode array detection

7


HighHigh--throughput Data Alignmentthroughput Data Alignment

Time

Inte

nsity

11.092 11.099 11.106 11.113 11.119 11.126 11.133 11.140 11.147 11.153 11.160 11.167R 56805 55915 61141 63768 68918 74303 75993 77945 76107 73481 70509 65665R 54235 52330 52469 55392 58845 58717 59674 59995 60782 56135 52522 49649R 54721 52564 52834 54662 56277 55905 57464 56240 55839 52313 48172 45756R 53293 55980 55069 58025 57325 57610 58378 55694 53920 49773 47605 43780R 51416 51240 50360 51368 53886 53484 54004 54802 54365 52252 49582 48464

TIC: 6.03mg root 1ml polar 160/160Peak# Ret Time Type Width Area 1 19.197 PV 0.043 451979 2 19.531 VV 0.034 1396954 3 19.585 VV 0.030 250189 4 19.803 VV 0.035 294593 5 20.087 VV 0.036 226863 TIC: 6.03mg root 1ml polar 160/160Peak# Ret Time Type Width Area 1 19.194 BV 0.043 497196 2 19.530 VV 0.035 1361651 3 19.584 VV 0.031 237827 4 19.802 VV 0.043 367820 5 20.084 VV 0.042 297422

• Coelution of metabolites• Retention Time Shifts• Interferences from

solvents & derivatization reagents

• Noise• Variance in the # of

variables


Ethanolamine

Phosphate

TIC

Glycerol

Selected ion Peak Extraction & IntegrationSelected ion Peak Extraction & Integration

(mainlib) Silanamine, 1,1,1-trimethyl-N-(trimethylsilyl)-N-[2-[(trimethylsilyl50 130 210 290 370 450 530 610

0

50

100

59

73

100 147

174

262

NSiSi

OSi

Etha

nolamine

(replib) Silanol, trimethyl-, phosphate (3:1)50 130 210 290 370 450 530 610

0

50

100

59

73

147207

299

OP

Si

O

O

OSi

Si

Phos

phate

(mainlib) Trimethylsilyl ether of glycerol50 130 210 290 370 450 530 610

0

50

10073

103

147205

293

O

Si

O

Si

O

Si

Glyc

erol

Duran et al,

8


MetMet--IDEA: IDEA: ((MetMetabolomics abolomics IIonon--Based Based DData ata EExtraction xtraction AAlgorithm)lgorithm)

Broeckling et al, Anal. Chem., in press


2D2D--PCAPCA--Cell cultureCell culture--Yeast elicitationYeast elicitation

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 10 20 30 40 50 60

time (h)

relative ion

C(AVR)

YE(AVR)

rt 14.434 (m/z 121)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 10 20 30 40 50 60

time (h)

relative ion

C(AVR)

YE(AVR)

rt 16.5 (m/z 415)

Farag et al.

9

3-Malonyl-CoA + 4-Coumaroyl-CoA

Isoliquirtigenin

LiquirtigeninNaringenin

2’-Hydroxyformononetin (HF)

4’,7-dihydroxyflavone (DHF) 3’,5-dmethoxyLuteolin (L)

DaidzinLG

DHFG

YE Elicited Cell CulturesYE Elicited Cell Cultures

LGM

Biochanin A (B)Formononetin (F)

Vestitione

Medicarpin

Daidzein (D)Genestein (G)

BG

HFG

FGBGG BGGM

MG MGM

FG M

Glucosides

Aglycones

Flavones

HFGM

GGGGM

DGM

VG M

Isoflav-3-ene Isoflav-3-ene GM

Vestitol (V)

4X; 6-48 h

GGG2X; 6-15 h 2X; 30-48 h

-0.5X; 36 h -0.5X; 36 h

Malonated Glucosides

10X; 3-24 7X; 3-18

15X; 2-36 h Farag et al.

VR

I2’OH

CHI

Farag et al.


c

eIFRtc52198

c

eIFStc45136

c

eCHStc51018

0 5 15 30 45 1 2 3 4 6 8 10 12 15 18 21 24 30 36 42 48

Min Hr

ActinTC43462 ce

Time post-elicitation

PALtc51375 ce

YEAST ELICITOR TIME COURSEYEAST ELICITOR TIME COURSE

c-controle-elicited

10


Induction of Induction of chalconechalcone--flavanoneflavanone isormeraseisormerase

Lei et al.(A) YE 36 hr control(A) YE 36 hr control (B) YE 36 hr elicited (B) YE 36 hr elicited

3-Malonyl-CoA + 4-Coumaroyl-CoA

Isoliquirtigenin

LiquirtigeninNaringenin

2’-Hydroxyformononetin (HF)

4’,7-dihydroxyflavone (DHF) 3’,5-dmethoxyLuteolin (L)

DaidzinLG

DHFG

YE Elicited Cell CulturesYE Elicited Cell Cultures

LGM

Biochanin A (B)Formononetin (F)

Vestitione

Medicarpin

Daidzein (D)Genestein (G)

BG

HFG

FGBGG BGGM

MG MGM

FGM

Glucosides

Aglycones

Flavones

HFGM

GGGGM

DGM

VGM

Isoflav-3-ene Isoflav-3-ene GM

Vestitol(V)

4X; 6-48 h

GGG2X; 6-15 h 2X; 30-48 h

-0.5X; 36 h -0.5X; 36 h

Malonated Glucosides

10X; 3-24 7X; 3-18

15X; 2-36 h Farag et al.

VR

I2’OH

CHI

Farag et al.

Irilone (I)IGM

ISGISGM Irisolidone (IS)

AGAGM

AGM

Alfalone

2X; 10-48 h

Afrormosin

20X; 3-48 h

7X; 8-48 h

8X; 3-48 h

???????????????G5X; 3-48 h 40X; 3-48 h

11


22.0 22.5 23.0 23.5 24.0 24.5 Time [min]0

2

4

6

5x10Intens.

15 16

17

18

19

20

22.0 22.5 23.0 23.5 24.0 24.5 Time [min]0

2

4

6

5x10Intens.

YE 12 hCont.12 h

Hex;162255

417

533.5

100 200 300 400 500 m/z

Full scan MS

80.196106121

137182

199 237

MS/MS (255)

nm250 300 350 400 450

mAU

0

5

10

15

20

25

30

35

40

DAD1, 23.745 (113 mAU,Apx) of MF140005.D

395

Yeast Elicitation Induces Novel Yeast Elicitation Induces Novel PhytoalexinPhytoalexin

Farag et al.

4’, 6-dihydroxy aurone(Hispidol-Glucoside)

O

O

HO

O O

OH

OH

OH

OH


89.5282.22 partial (92%)similar to GP|5002342 peroxidase 1 precursor {Phaseolus vulgaris}

86.8876.97 partial (97%)similar to GP|2245683 peroxidase precursor {Glycine max}

88.2783.64 partial (93%)similar to GP|537315 peroxidase {Medicago sativa}

90.7985.71 partial (88%)similar to PIR|JC4781 peroxidase (EC 1.11.1.7) 1C precursor - alfalfa

98.8797.46 completehomologue to PIR|JC4780 peroxidase (EC 1.11.1.7) 1B precursor - alfalfa

% Sim% IDHit coverageDescription

TC 50982, pI=7.4TC 51005, pI=5.81X

TC 50994, pI=7.46

TC 53160, pI=6.97 x

Basic peroxidase cDNA isolated from soybean hypocotyls infected with Phytophthora sojae f.sp. glycines

Identified in elicited cell culture and fungal infected plant

TC 56738, pI=8.9

TC 50982& 50994

Gene Discovery -Peroxidases elicited with Yeast

0-48hr Control 0-48hr Elicited

12


OH

O

OH

HOO

O

HO

OHperoxidaseFunctional analysis of peroxidases in E. coli

Isoliquiritigenin (IL) Hispidol (H)

5 10 15 20 25 30 35 Time [min]

TC56738

H

nm240 260 280 300 320 340 360 380

DAD1, 32.542 (212 mAU,Bln) of MF000026.D

TC50994

nm250 275 300 325 350 375 400 425

DAD1, 33.944 (227 mAU,Apx) of MF000025.D

IL

IL

pET-28a vector

H

H

TC50982

IL

161.6

269.3

32.3min

100 120 140 160 180 200 220 240 260 280

253.3-MS, 33.8min (#1210)

100 120 140 160 180 200 220 240 260 m/z

5 10 15 20 25 30 35 Time [min]

5 10 15 20 25 30 35 40 Time [min]

5 10 15 20 25 30 35 Time [min]

325 nm

50x

45x

OH

O

OO

M+

M+390 nm

Quinol vinyl ether

40x


Need for Increased Metabolomics Depth of Coverage Need for Increased Metabolomics Depth of Coverage

● Current metabolome estimates Bacteria ~ 600 to 900 metabolites Single plant species ~ 15,000 metabolites Plant Kingdom ~200,000 due to secondary metabolism

● Currently profiling approx 600 components by GC/MS + 150 by LC/MS = 750 total

● Therefore, there is a dramatic need for more comprehensive metabolome coverage

● Increased depth of coverage can be achieved by increased resolution in both the chromatography and mass domains

Bino et al, Trends in Plant Science, 9(9): 418-425.

13


Separation Efficiencies & Peak CapacitiesSeparation Efficiencies & Peak Capacities● Separation efficiency – unitless measure of band

broadening per unit time

● Peak capacity - maximum number of theoretical peaks resolvable by the chromatographic system

( ) p

RRR

dLR

wtttN =

−=⎟⎟

⎠

⎞⎜⎜⎝

⎛=⎟

⎟⎠

⎞⎜⎜⎝

⎛=⎟⎟

⎠

⎞⎜⎜⎝

⎛= 2

22

½

'2

b

'2'

11654.5

W16

ασ

1ln4 1

2 +⎟⎟⎠

⎞⎜⎜⎝

⎛=

tt

RNn


Need for Increased Resolving Power (M/Need for Increased Resolving Power (M/∆∆M) M)

natural products genistein (C15H10O5 = 270.2390) Medicarpin (C16H14O4 = 270.2830 ) M/∆M = 6136

rutin (C27H30O16=610.5180) hesperidin (C28H34O15=610.5620) M/∆M = 13,864

14


Increased Comprehensive Coverage using Ultra Increased Comprehensive Coverage using Ultra performance liquid chromatography (UPLC) performance liquid chromatography (UPLC) QtofMSQtofMS

↑ resolution/plate #↑ Peak capacity↑ metabolomics

potential

UPLC-QtofMS base-peak ion chromatograms obtained for the combined methanol extracts from soybean and Medicago truncatula (cv Jemalong A17). Separations were achieved using a Waters Acquity UPLC 2.1 x 100 mm, BEH C18 column with 1.7 µm particles, a flow of 600 µl/min, and a linear gradient of 0.1%acetic acid:acetonitrile (5:95 to 30:70 over 30min). Mass spectra were collected on a Waters QTOFMS Premier.


500,000UPLC (2.1 x 100 mm @ 600 µL/min)

225,000Capillary HPLC (300 µm x 150 mm @ 4 µL/min)

87,000Analytical HPLC (4.6 x 250mm @ 0.8mL/min)

Ave Efficiency (plates per meter)

HPLC system

15


O

O

OCH2OH

HOHO O

O

CH2OH

CO

O

O CH2OH

OH

CH2OH

HOHO OH

COOH

CO

HO CH2OH

OH

HO CH2OH

O

O

HO

COOH

HOHO O

O CH2OH

OHOHHO

O CH2OH

OHOHHO

O

OOH

HO OH

OCH2OH

HO

HO O

O

OH

HOOH

CH2OH

COOH

HO

HO

CH2OH

COOH

HO COOH

COOH

HO

HO

Glycosyltransferases

P450s

E

β-Amyrin

HO

A B

C D

HederageninBayogenin Medicagenic acid Soyasapogenol ESoyasapogenol B

3-Glc, 28Glc-Medicagenic acid3-Glc-Ara, 28-Glc-Hederagenin Soyasaponin I

(3-Rha-Gal-GlcA-Soyasapogenol B)

β-Amyrin synthase (AS)

Squalene epoxidase (SE)

Squalene synthase (SS)

FPP

β-Amyrin

Mevalonic acid

2,3-Oxidosqualene

Squalene

Cycloartenol

Sesquiterpenes

12

3 45

67

8

9

10

1112

13

14

1516

17

18

19 20 21

22

2324

25 26

27

28

2930

HO

O

HO

O

PPO

Cycloartenol synthase (CAS)

Constructing a Constructing a TriterpeneTriterpene Saponin Pathway in Saponin Pathway in Medicago truncatula Medicago truncatula

Achnine et al, Plant J, Mar 2005; 41(6): 875-87. Suzuki et al., Plant J. 2002, 32(6):1033-48.Suzuki et al, Planta, Mar 2005; 220(5): 696-707. Huhman & Sumner, Phytochemistry, 2002, 59, 347-360. Huhman et al, J. Ag Food Chem., 2005, 53(6):1914-20.


Ecotype Profiling•Approx. 120 Ecotypes

(natural mutants, ie genotypicalvariants)

•Elucidate biochemical basis of genotypes/phenotypes using

metabolite profiling

•Establish relationships between genotypes

•Mine ecotypes for saponin enrichment or deficiencies

MogulMogul

16


Time (min)1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

%

5

Jemalong

Mogul

Time (min) %A %B0 95 53:00 70 3013:00 50 5014:00 5 9516:00 5 9516:01 95 518:00 95 5

UPLC/UPLC/QtofMSQtofMS 18 Minute Gradient for High 18 Minute Gradient for High throughthrough--put Profilingput Profiling


Factor1

Factor2

Factor3

A17_1A17_2

GRC_065_3

JEM_1 A17_3

JEM_2

JEM_3

GRC_065_2

MOGUL_3

HARBINGER_1

CRE_006_1

HARBINGER_2

CRE_006_2

HARBINGER_3

CRE_006_3

MOGUL_1

GRC_065_1

MOGUL_2

PCA of Elite CultivarsPCA of Elite Cultivars

17


Factor1

Factor2

Factor3

HexHexRha-Bayogenin

3-GlcA-28-Ara-Rha-Xyl-Medicagenic Acid

3-Glc-Medicagenic AcidHex-Soyasapogenol E

Rha-Hex-Hex-New Aglycone3-Glc-28-Glc-Malonyl-Medicagenic Acid

Hex-New Aglycone

Hederagenin

Hex-Hex-Bayogenin

3Glc-28-Ara-Rha-Xyl-Medicagenic Acid

Rha-Hex-Hex-Hederagenin

Rha-Hex-Hex-Hex-HederageninHex-New Aglycone

3-Glc-Glc-Medicagenic Acid

3-Glc-Malonyl-Medicagenic Acid

3-Glc-28-Glc-Medicagenic AcidRha-Hex-Hex-Soyasapogenol E

Medicagenic Acid

3-Glc-Glc-28-Ara-Rha-Xyl-Api-Medicagenic Acid

Rha-Hex-?

3-Ara-Glc-Ara-Hederagenin

Bayogenin

3-Glc-Glc-28-Ara-Rha-Xyl-Medicagenic Acid

Pent-Hex-Hederagenin

Rha-Hex-?

Rha-Glc-Med or Hex-GlcA-Hed

Hex-Hederagenin

Hex-Hex-Hex-Bayogenin

Rha-Hex-Hex-Bayogenin

GlcA-? Multiple Aglycone in t

GlcA?

Gal-GlcA-Soyasapogenol B

Rha-Ara-GlcA-Soyasapogenol B

3-Rha-Xyl-GlcA-Soyasapogenol E

Soyasapogenol E

Hex-HexA-Soy E

Glc-Gal-GlcA-Soyasapognol B

Ara-GlcA-Soyasapogenol B

Rha-Gal-GlcA-Soyasapogenol B (

PCA Loadings of Elite CultivarsPCA Loadings of Elite Cultivars


3pM_Soy1_13pM_Soy1_10

3pM_Soy1_2

3pM_Soy1_3

3pM_Soy1_43pM_Soy1_53pM_Soy1_63pM_Soy1_7

3pM_Soy1_83pM_Soy1_8

3pM_Soy1_9

BORUNG_2 86

BORUNG_3 29

CALIPH_1 50

CRE_005_1 161

CRE_005_3 187

CRE_006_1 35CRE_006_2 13CRE_006_3 24

CRE_007_1 57

CRE_007_2 71

CRE_007_3 26

CRE_009_1 15CRE_009_2 64

DZA_033_1 33DZA_033_2 91DZA_033_3 40DZA_045_1 99

DZA_045_3 16

DZA_046_1 174

DZA_046_2 219

DZA_046_3 159

DZA_055_1 294DZA_055_2 276

DZA_055_3 260

DZA_058_1 141

DZA_058_2 156

DZA_058_3 175

DZA_059_1 220DZA_059_2 142

DZA_061_1 51

DZA_061_2 90

DZA_061_3 42

DZA_202_2 172DZA_202_3 130

DZA_210_1 176DZA_210_2 214DZA_210_3 213

DZA_219_1 157

DZA_219_2 166DZA_219_3 165

DZA_221_1 125DZA_221_2 211DZA_221_3 112

DZA_230_1 177

DZA_230_2 109

DZA_230_3 124

DZA_231_1 210DZA_231_2 136

DZA_231_3 127

DZA_233_1 222

DZA_233_2 181

DZA_233_3 201

DZA_236_2 120

DZA_236_3 198

DZA_241_1 89DZA_241_2 100DZA_241_3 54

DZA_242_1 268DZA_242_2 169

DZA_242_3 306

DZA_243_1 290DZA_243_2 310

DZA_243_3 162

DZA_244_1 194

DZA_244_2 289

DZA_244_3 315

DZA_246_1 228DZA_246_2 151

DZA_246_3 274

DZA_309_1 245DZA_309_2 262DZA_309_3 267

DZA_312_1 45DZA_312_2 62DZA_312_3 107

DZA_327_1 202

DZA_327_2 167

DZA_327_3 208

ESP_050_1 22ESP_050_3 84

ESP_080_2 248

ESP_080_3 314

ESP_098B_1 158ESP_098B_2 186

ESP_098B_3 173

ESP_101_1 301

ESP_101_2 265

ESP_101_3 285

ESP_105_1 139ESP_105_2 148ESP_105_3 193

ESP_140_1 316

ESP_140_2 229

ESP_155_1 269ESP_155_2 283ESP_155_3 232

ESP_158_1 302ESP_158_2 287

ESP_158_3 266

ESP_161_1 261

ESP_161_2 275ESP_161_3 298

ESP_163_3 247

ESP_170_1 231

ESP_170_2 234ESP_170_3 226

ESP_171_2 20

ESP_172_2 97

F11_007_1 70F11_007_2 81F11_007_3 56

F11_008_1 80F11_008_2 32F11_008_3 88

F20_031_2 44

F20_031_3 48F20_047_1 28F20_047_2 38F20_047_3 36

F20_058_1 12F20_058_2 41F20_058_3 75

F20 081 1 279

F20_086_1 291

F20_086_2 191

F20_086_3 300F20_089_1 150

F20_089_2 131F20_089_3 113

GRC_020_1 278

GRC_020_2 184

GRC_020_3 249

GRC_033_1 138

GRC_033_2 117

GRC_033_3 118

GRC_037_1 21GRC_037_2 61GRC_037_3 37

GRC_040_1 149

GRC_040_2 207

GRC_040_3 216

GRC_043_1B 238GRC_043_2B 303GRC_043_3B 160

GRC_063_1 215GRC_063_2 153

GRC_063_3 126

GRC_064_1 284

GRC_064_2 230

GRC_064_3 288

GRC_093_2 76GRC_093_3 14

GRC_098_1 140GRC_098_2 185GRC_098_3 199

PARABINGA_1 9PARABINGA_2 43

PARABINGA_3 10

PARAGGIO_1 46PARAGGIO_2 103

PARAGGIO_3 87

PRT_179_1 5PRT_179_2 30PRT_179_3 93

SEPHI_1 25SEPHI_2 69SEPHI_3 19

A17_1 49

A17_2 101A17_3 67

CALIPH_2 79

CALIPH_3 53

CRE_009_3 98

CYPRUS_1 55

CYPRUS_2 58

CYPRUS_3 7

DZA_045_2 23

_ _

DZA_059_3 188

DZA_105_1 218

DZA_105_2 135DZA_105_3 225

DZA_202_1 221

DZA_213_1 189DZA_213_2 111DZA_213_3 171

DZA_323_1 217DZA_323_2 212DZA_323_3 116

ESP_031_1 110ESP_031_2 182ESP_031_3 121

ESP_039_1 190ESP_039_2 163ESP_039_3 224

ESP_040_1 251

ESP_040_2 308

ESP_040_3 270

ESP_041_1 206ESP_041_2 152ESP_041_3 195

ESP_043_1 317

ESP_043_2 237

ESP_043_3 273

ESP_045_1 286

ESP_045_2 227ESP_045_3 236

ESP_048_1 281

ESP_048_2 277ESP_048_3 296

ESP_050_2 2

ESP_074_1 313ESP_074_2 299ESP_074_3 253

ESP_096_1 257ESP_096_2 132ESP_096_3 146

ESP_098A_1 168ESP_098A_2 223ESP_098A_3 143

ESP_100_1 115ESP_100_2 133ESP_100_3 154

ESP_103_1 264ESP_103_2 254

ESP_103_3 256

ESP_104_1a 271

ESP_104_2 137ESP_104_1 129

ESP_140_3 240ESP_156_1 242ESP_156_2 252

ESP_156_3 307

ESP_159_1 105ESP_159_2 108ESP_159_3 72

ESP_160_1 196ESP_160_2 204ESP_160_3 164

ESP_162_1 123ESP_162_2 147ESP_162_3 178

ESP_163_1 258ESP_163_2 255

ESP_165_2 292ESP_165_3 243

ESP_169_1 293ESP_169_2 304

ESP_169_3 241

ESP_171_1 85ESP_171_3 78

ESP_172_1 18ESP_172_3 39

ESP_173_1 155ESP_173_2 280ESP_173_3 179

ESP_175_1 17

ESP_175_2 104

ESP_175_3 8

F20_009_1 77F20_009_2 34F20_009_3 96

F20_015_1 233F20_015_2 250F20_015_3 239

F20_025_1 235F20_025_2 309

F20_025_3 259

F20_048_1 144F20_048_2 145F20_048_3 200

F20_061_1 183F20_061_2 114F20_061_3 170

F20_081_1 279

F20_081_2 209

F20_081_3 282

F20_087_1 246

F20_087_2 205

F20_087_3 297

GRC_052_1 203GRC_052_2 192

GRC_052_3 122

GRC_065_1 1GRC_065_2 60GRC_065_3 27

HARBINGER_1 68HARBINGER_2 73HARBINGER_3 82

JEM_1 66JEM_2 83

JEM_3 11

MOGUL_1 59MOGUL_2 63MOGUL_3 3

PRT_176_1 74

PRT_176_2 65

PRT_176_3 4

PRT_177_1 128PRT_177_2 119PRT_177_3 134

PRT_178_1 6PRT_178_2 95

ESP_178_3 106PRT_180_1 295PRT_180_2 311

PRT_180_3 272

SALERNES_1 102SALERNES_2 52SALERNES_3 94

Hex

Hex

Rha

-Bay

ogen

in

Hex

-Hex

-Hex

-Bay

ogen

in

Rha

-Hex

-Hex

-New

Agl

ycon

e

Hex

-Hex

-Bay

ogen

in

Rha

-Hex

-Hex

-Hex

-Hed

erag

enin

3-G

lc-2

8-G

lc-M

edic

agen

ic A

cid

3-G

lc-G

lc-2

8-A

ra-R

ha-X

yl-A

pi-M

edic

agen

ic A

cid

3-G

lc-G

lc-2

8-A

ra-R

ha-X

yl-M

edic

agen

ic A

cid

3-G

lcA

-28-

Ara

-Rha

-Xyl

-Med

icag

enic

Aci

d

3-G

lc-2

8-G

lc-M

alon

yl-M

edic

agen

ic A

cid

3Glc

-28-

Ara

-Rha

-Xyl

-Med

icag

enic

Aci

d

Hex

-New

Agl

ycon

e

Rha

-Hex

-Hex

-Soy

asap

ogen

ol E

Rha

-Hex

-?

Pen

t-Hex

-Hed

erag

enin

Rha

-Hex

-Hex

-Bay

ogen

in

Glc

A?

Rha

-Hex

-Hex

-Hed

erag

enin

3-G

lc-G

lc-M

edic

agen

ic A

cid

Glc

-Gal

-Glc

A-S

oyas

apog

nol B

Rha

-Gal

-Glc

A-S

oyas

apog

enol

B (

Rha

-Hex

-?1

3-G

lc-M

edic

agen

ic A

cid

Gal

-Glc

A-S

oyas

apog

enol

B

Rha

-Ara

-Glc

A-S

oyas

apog

enol

B

3-G

lc-M

alon

yl-M

edic

agen

ic A

cid

Ara

-Glc

A-S

oyas

apog

enol

B

3-A

ra-G

lc-A

ra-H

eder

agen

in

Rha

-Glc

-Med

or H

ex-G

lcA

-Hed

Glc

A-?

Mul

tiple

Agl

ycon

e in

t

Hex

-New

Agl

ycon

e1

3-R

ha-X

yl-G

lcA

-Soy

asap

ogen

ol E

Hex

-Hex

A-S

oy E

Med

icag

enic

Aci

d

Bay

ogen

in

Glc

A-H

eder

agen

in

Hex

-Hed

erag

enin

Hex

-Soy

asap

ogen

ol E

Hed

erag

enin

Soy

asap

ogen

ol E

18


Metabolomics, Proteomics, transcriptomics, and integrated functional genomics are rapidly maturing discovery sciences yielding new:

● Hypothesis: MeJA induces concurrent response in primary metabolism and saponin biosynthesis suggesting a significant repartitioning of carbon from primary to secondary metabolism.

● Gene Validation: Metabolic profiling data validates the presence and induction of a threonine aldolase in plants

● Gene Discovery: Yeast induces novel phytoalexin auroneHispidol (peroxidase)

● Mechanistic Insight: MeJA induces isoflavonoidaccumulation via a different mechanism than Yeast.

SummarySummary


?Fishing??Fishing?

19


AcknowledgementsAcknowledgementsMetabolomics $$$ The Noble FoundationCorey D. Broeckling (Colorado State Univ.) $$$ NSF PGRP #DBI-0109732Mohamed Farag (Univ. Cairo) $$$ NSF 2010 MCB-0520283David V. Huhman $$$ NSF 2010 MCB-0520140Ewa Urbanczyk-Wochniak $$$ State of OklahomaAnthony L. Duran (Cargill) LECOJ. Tim Smith & Brad Williams (SEOSU)

Noble Bioinformatics Proteomics Patrick Zhao, Indira Reddy, & Yuanji Zhang Zhentian Lei Bonnie S. Watson Virginia Bioinformatics InstituteSatish Nagaraj Pedro Mendes

Bharat MehrotraSaponins & Cell CultureRichard Dixon, Lahoucine Achnine Genomics/EST/MicrorrayHideyuki Suzuki, Jack Blount Richard Dixon & Group

Marina Naoumkina, Bettina DeavoursGregory D. May & Group Bruce Roe & International Consortium NCGR/TIGR/VBI

NSF

biochemistry is a dynamic multi-level reaction …...2-keto-l-gluconic acid diethylene glycerol...

Documents