Download Unfinished business from April 4!

Metabolomics, spring 06
Unfinished business from April 4!
Hans Bohnert
ERML 196
[email protected]
265-5475
333-5574
http://www.life.uiuc.edu/bohnert/
class April 6
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.
One could use the contraption for other experiments
Maize
Western
Corn rootworm
Nematode
trap
Rasmann et al. (2005)
Nature 434, 731.
Trimorphic interaction involving a entomopathogenic nematode
Experiments similar to the wasp
predation 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
β-caryophyllene
Olfactometer arms spiked with
authentic β-caryophyllne
Absence of β-Car.
in some (mostly US)
maize lines
Reproductive success and
β-caryophyllene
Pactol – low amounts
Graf – high amounts
healthy
fungal infections
nematode presence
All six containers received
the same number of nematodes
added β-caryophllene
Emergence of adults is reduced
when nematodes are attracted
(pactol minus).
in sand
out of medium
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)
β-caryophylline diffuses readily (at least in and out of sand)
Sesquiterpene hydrocarbons in maize
A – leaf herbivore inducible; B – ubiquitous (maize self); C – root specific
high - volatility - low
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 studies
phenotypic behavior
experimental design
metabolite profiling
molecular analyses
biochemical studies
intraspecific variation - genetics
transgenic approaches
breeding objectives
Systems biology multi-disciplinarity
collaboration
integration
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 misleading
pool size constant but coordinated increase in flux (activities altered)
Monitoring flux
Rate 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 system
Plants: where, and how, to introduce the tracer
Pool size – dilution of label
Is end-product transported – loss of label
Do we know the pathway, or assume we know, and are we right
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
Figure 1a
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 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 coenzymeA; DHAP, dihydroxyacetone-3-phosphate; E4P, erythrose-4-phosphate; Fru-6P,
fructose-6-phosphate; GAP, glyceraldehydes-3-phosphate; Glc-6P, glucose-6phosphate; PGA, 3-phosphoglyceric acid; Pyr, pyruvate; R-5P, ribose-5-phosphate; Ru1,5-P2, ribulose-1,5-bisphosphate; Ru-5P, ribulose-5-phosphate; S-7P, sedoheptulose7-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-3phosphate 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 route
between 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
Where does the label go?
• Primary metabolism
• potato tubers
• wild type and transgenics
• EI GC-MS
• U-13C/14C glucose feeding
• pathway verification
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.
Possible reaction rates
to measure
Wt
INV-2-30
SP-29
What is U-13C or U-14C glucose?
Amounts over time (up to 12h)
bold - transgenic
difference to wild type
(P < 0.05)
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
Transcripts
-0.6
0
2.4.1.123
Galactose
0.6
Galactinol
Raffinose
2.4.1.82
Starch
(log2 - fold change)
Sucrose
3.2.1.1
Metabolites
Neutral
Invertase
Cvi 27
Cvi 21
Col 27
Col 21
3.2.1.2
2.4.1.25
MEX1
Cysteine
Maltose
3.2.1.26
Invertase, cell wall
Invertase, vacuole
Fructose
Glucose
DEP2
4.2.99.8
Melibiose
5.3.1.9
Tryptophan
isoforms
2.7.1.1
At4g02610
At4g27070
4.1.1.48
2.3.1.30
1.2.1.12
5.3.1.24
2.7.2.3
2.1.2.1
3.1.3.3
Serine
Glycine
2.6.1.52
1.1.1.95
2.4.2.18
3-Phosphoglycerate
5.4.2.1
Leucine
2.6.1.42
1.1.1.85
4.2.1.33
4.1.3.12
4.1.3.27
4.2.1.11
Phenylalanine
4.1.1.49
PEP
4.2.3.4
2.7.1.40
4.1.1.31
2.6.1.42
Valine
4.2.1.10
1.1.1.86
2.2.1.6
2.6.1.5
Pyruvate
2.7.1.71
Oxaloacetate
Asparagine
4.2.1.51
2.5.1.19
4.2.3.5
Acetyl-CoA
6.3.5.4
Chorismate
2.6.1.1
Aspartate
Oxaloacetate
1.3.1.12
Citrate
Tyrosine
1.1.99.16
1.2.1.11
4.2.1.3
Aspartate-4-semialdehyde
Malate
Isocitrate
1.1.1.3
Proline
4.2.1.52
2.7.1.39
1.3.1.26
Homoserine-4-phosphate
1.1.1.42
4.2.1.2
At5g14800
At5g62530
2.6.1.17
4.2.99
3.5.1.18
alpha-Ketoglutarate
Fumarate
4.4.1.8
1.4.7.1
Glutamate
AT5G65750
5.1.1.7
6.3.1.2
Threonine
1.3.5.1
2.2.1.6
1.1.1.86
2.6.1.42
Isoleucine
2.1.1.14
2.1.1.10
Methionine
Prephenate
2.3.3.1
2.7.2.4
4.2.3.1
5.4.99.5
4. 1.3.8
Lysine
6.2.1.4
Succinate
Glutamine
Figure 7.