Unfinished business from April 4!Unfinished business from April 4!Metabolomics, spring 06

Hans BohnertERML 196

bohnerth@life.uiuc.edu

265-5475333-5574

http://www.life.uiuc.edu/bohnert/

Metabolite profiling = a static picture, a snapshot!

Does it matter?*

*Fernie AR et al. (2005) Flux an important, but neglected, component of functional genomics. Curr. Opin. Plant Biology 8, 174.

*Fell DA (2005) Enzymes, metabolites and fluxes. J. Exptl Botany 56, 267.

…. and two case studies.

class April 6

One could use the contraption for other experimentsOne could use the contraption for other experiments Maize

WesternCorn rootworm

Nematode

Trimorphic interaction involving a entomopathogenic nematode

Rasmann et al. (2005)Nature 434, 731.

trap

Experiments similar to the waspExperiments similar to the wasppredation experimentpredation experiment

• Identification of attractant

• Why is US maize not protected

• Does it work in the field

• Isoprenoids in the soil?

2 – β-caryophyllene

Attraction to / by authentic Attraction to / by authentic

ββ-caryophyllene-caryophyllene

OlfactometerOlfactometer arms spiked with arms spiked with

authentic authentic ββ-caryophyllne-caryophyllne

Absence of β-Car.

in some (mostly US)

maize lines

Reproductive success and Reproductive success and ββ-caryophyllene-caryophyllene

Pactol – low amountsGraf – high amounts

healthy

fungal infections

nematode presence

All six containers receivedAll six containers receivedthe same number of nematodesthe same number of nematodes

added β-caryophllene

Emergence of adults is reduced Emergence of adults is reduced

when nematodes are attractedwhen nematodes are attracted

(pactol minus).(pactol minus).

ββ-caryophylline diffuses readily (at least in and out of sand)-caryophylline diffuses readily (at least in and out of sand)

a - Detection in a column of wet sand 10 cm from release point

b – detection in air space above a column of sand

(note the scale differences)

in sand out of medium

Sesquiterpene hydrocarbons in maizeSesquiterpene hydrocarbons in maize

A – leaf herbivore inducible; B – ubiquitous (maize self); C – root specific

high - volatility - low

Terpene synthases in maize Terpene synthases in maize

• Heterologous expression• GC-MS with isotopic tracers• GC-MS of different lines

• Mutational analysis of the “bottom” of the active site region

Sesquiterpene spectrum as affected by mutational analysis of the TPS gene

Tri-trophic interactions - ecological studiesphenotypic behaviorexperimental designmetabolite profilingmolecular analysesbiochemical studiesintraspecific variation - geneticstransgenic approachesbreeding objectives

Systems biology -multi-disciplinaritycollaborationintegration

Experimental complexity in biology approaches what is common in astronomy and, especially, physics.

Metabolite profiling = a static picture, a snapshot! Does it matter?

Static (steady-state) “knowledge units” - genome sequence, microarray profile, proteome composition

How to understand cellular dynamics?

Flux – where to measure, how and what is the most important “link”?

Metabolites – intermediates in pathways to end-products (starch, cellulose, proteins, fats, lipids, second. products)

Enzyme activity changes: steady-state of intermediates or flux?What is affected?yeast metabolomics (mutants) metabolites do change.

Plants – metabolites +/- constant, flux altered photosynthesis – Calvin cycle – [NAD(P)H] – [ATP] – sucrose to starch [ADP-glucose pyrophosporylase]

Steady state alone can be misleadingpool size constant but coordinated increase in flux (activities altered)

Monitoring fluxRate of depletion of an initial substrate

Rate of accumulation of an end product

Isotope labeling of (a) metabolite(s) (complete or in certain atoms)

radioactive or stable isotopes (2H, 3H, 13C, 14C, 15N, 18O, 32P, 35S)

Can we infer flux from changes in intermediates? think allosteric effects of metabolites measuring regulated steps in a pathway is intermediates [conc](consider the Mark Stitt lecture)

Pathways branch (label lost)Different pathway(s) provide(s) intermediate (label diluted by unknown)Tracer addition may change the equilibrium of the systemPlants: where, and how, to introduce the tracerPool size – dilution of labelIs end-product transported – loss of labelDo we know the pathway, or assume we know, and are we right

Need certainty about pathway structures – (MapMan, TAIR, KEGG) – do we?Need certainty about pathway structures – (MapMan, TAIR, KEGG) – do we?

More pitfalls and traps!

Measuring (labeled) substrate consumption – insensitive, inaccurate

Measuring end-product – stable, transported or metabolized (e.g., disappear in cell wall; does CO2 production and glycolysis)

Branched pathways – do we know

Linear relationship between product level and time (growth!)

Experimental material – entire plant, organ (or part of organ), tissue slice, cells, organelles

How “big” is the flux, the pathway – can we actually measure it? NMR (stable isot.), GC-MS, LC-MS - sensitivity and accuracy

Positional information of tracer substrate modification may be important

Long-term feeding expt, or pulse labeling, or pulse/chase expts

Schwender et al. (2004) Rubisco without the Calvin cycle improves the carbon efficiency of developing green seeds. Nature 432, 779. (on web as: Shachar-Hill-Nature-2004)

Figure 1a

Figure 1b

Figure 1 Metabolic transformation of sugars into fatty acids. a, Conversion of hexose phosphate to pentose phosphate through the non-oxidative steps of the pentose phosphate pathway and the subsequent formation of PGA by Rubisco bypasses the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase while recycling half of the CO2 released by PDH. PGA is then further processed to pyruvate, acetyl-CoA and fatty acids.

b, Part of a expanded to indicate carbon skeletons and to define relationships between

V PDH (flux through PDH complex); V X (additional CO2 production by the OPPP, the TCA, and so on); V Rub (refixation by Rubisco). Metabolites: Ac-CoA, acetyl coenzyme-A; DHAP, dihydroxyacetone-3-phosphate; E4P, erythrose-4-phosphate; Fru-6P, fructose-6-phosphate; GAP, glyceraldehydes-3-phosphate; Glc-6P, glucose-6-phosphate; PGA, 3-phosphoglyceric acid; Pyr, pyruvate; R-5P, ribose-5-phosphate; Ru-1,5-P2, ribulose-1,5-bisphosphate; Ru-5P, ribulose-5-phosphate; S-7P, sedoheptulose-7-phosphate; Xu-5P, xylulose-5-phosphate. Enzymes: Aldo, fructose bisphosphate aldolase; Eno, 2-phosphoglycerate enolase; Xepi, xylulose-5-phosphate epimerase; FAS, fatty-acid synthase, PGM, phosphoglyceromutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, phosphoglucose isomerase; Riso, ribose-5-phosphate isomerase; PDH, pyruvate dehydrogenase; PFK, phosphofructokinase; PK, pyruvate kinase, PGK, phosphoglycerate kinase; PRK, phosphoribulokinase; TA, transaldolase; TK, transketolase; TPI, triose phosphate isomerase.

Conclusions Rubisco operates as part of a previously undescribed metabolic routebetween carbohydrate and oil (Fig. 1a). Three stages:(1) conversion of hexose phosphates to ribulose-1,5-bisphosphate by the non-oxidative reactions of the OPPP together with phosphoribulokinase.

(2) conversion of ribulose-1,5-bisphosphate and CO2 (most produced by PDH3) to PGA by Rubisco

(3) metabolism of PGA to pyruvate and then to fatty acids (Fig. 1a).

The net carbon stoichiometry of this conversion: 5 hexose phosphate > 6 pentose phosphate > 12 acetyl-CoA + 6 CO2

The conversion of the same amount of hexose phosphates by glycolysis:5 hexose phosphate >10 Acetyl-CoA + 10 CO2

Roessner-Tunali et al. (2004) Kinetics of labeling of organic and amino acids in potato tubers by gas chromatography-mass spectrometry following incubation in (13)C labelled isotopes. Plant J. 39, 668.

Where does the label go?

• Primary metabolism

• potato tubers

• wild type and transgenics

• EI GC-MS

• U-13C/14C glucose feeding

• pathway verification

Possible reaction ratesto measure

What is U-13C or U-14C glucose?

Wt

INV-2-30

SP-29

Amounts over time (up to 12h)

(P < 0.05)

bold - transgenic difference to wild type

important – watch differences in rates of synthesis (Δf = >100)

A different experiment

Arabidopsis ecotypes in high CO2 in FACE rings

Attempts at correlating

gene expression and

metabolite concentrations

Raffinose

SucroseStarch

Galactose

Glucose Fructose Melibiose

3-Phosphoglycerate

Pyruvate

Acetyl-CoA

Citrate

Isocitrate

alpha-Ketoglutarate

Succinate

Fumarate

Malate

Oxaloacetate

Chorismate

Serine

Cysteine

Tyrosine

Phenylalanine

Prephenate

PEP

Tryptophan

Glycine

Leucine

Valine

Aspartate

Asparagine

Aspartate-4-semialdehyde

Lysine

Homoserine-4-phosphate

Threonine

Isoleucine

Methionine

Glutamate

Glutamine

Proline

2.3.3.1

4.2.1.3

1.1.1.42

6.2.1.41.3.5.1

4.2.1.2

1.1.99.16

5.4.2.1

4.2.1.11

2.7.1.40

1.1.1.952.6.1.523.1.3.32.1.2.1

2.3.1.30

4.2.99.8

3.2.1.26Neutral

Invertase

Invertase, cell wall

Invertase, vacuole

5.3.1.9

2.7.1.1

1.2.1.12

2.7.2.3

Maltose

Oxaloacetate

4.1.1.49

4.1.1.31

4. 1.3.8

2.2.1.61.1.1.862.6.1.42

4.1.3.124.2.1.331.1.1.852.6.1.42

2.6.1.1

6.3.5.4

2.7.2.4

1.2.1.11

4.2.1.52

1.3.1.26

2.6.1.17

3.5.1.18

5.1.1.7

1.1.1.3

2.7.1.39

4.2.3.1

2.2.1.6

1.1.1.86

2.6.1.42

4.2.99

4.4.1.8

2.1.1.142.1.1.10

1.4.7.1

6.3.1.2

4.1.3.27

4.2.1.10

2.7.1.71

2.5.1.194.2.3.5

2.4.2.18

5.3.1.24

4.1.1.48

5.4.99.5

1.3.1.12

4.2.1.51

2.6.1.5

4.2.3.4

3.2.1.1

3.2.1.22.4.1.25MEX1

DEP2

Galactinol2.4.1.123

2.4.1.82

AT5G65750

At4g02610At4g27070

At5g14800At5g62530

Figure 7.

Co

l 21

Transcripts

Metabolites

-0.6 0 0.6

(log2 - fold change)C

ol

27

Cvi

21

Cvi

27

isoforms