-- Introduction to Introduction to ‘‘OmicsOmics’’ and Systems Biology

Sixue Chen, Ph.D.

Department of Botany, Plant Molecular and Cellular Biology

Genetics Institute

E-mail: schen@ufl.edu

-- Demonstration of onDemonstration of on--line line ‘‘OmicsOmics’’ resourcesresources

-- Lab demonstration of modern ProteomicsLab demonstration of modern Proteomics

Introduction to Introduction to ‘‘OmicsOmics’’ and Systems Biology

Sixue Chen, Ph.D.

Department of Botany, Plant Molecular and Cellular Biology

Genetics Institute

E-mail: schen@ufl.edu

Large scale biology – ‘Omics’: revolution in screening important traits and creation of ‘in silico’ organisms

Chen lab research towards plant systems biology, an emerging discipline facilitates rational plant engineering

‘Omics’ modules – example of proteomics in addressing the differences in wheat (C3 plant) and corn (C4 plant)

OutlineOutline

What is modern and future biological sciences ?

Central Dogma of Molecular GeneticsCentral Dogma of Molecular Genetics

(The guiding principle that controls trait expression)

DNA(gene)

RNA

Protein Trait(or phenotype)

Transcription

Translation

Plant height

Seed shape

Traits and Phenotypes are Controlled by Molecular NetworksTraits and Phenotypes are Controlled by Molecular Networks

Trying to understand life without knowledge of biochemical network would be like trying to understand Shakespeare without knowledge of English grammar.

Our ProblemsOur Problems

• That one gene encodes one protein, which catalyzes one reaction and determines one phenotype is no longer the case.

• How to capture all molecules and their interactions, dynamics, regulations and turnover … ?

• How to determine the rate-limiting molecule and step ? How to predict ?

• Manipulating one gene can cause pleiotropic effects ?

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.

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Large scale biology – ‘Omics’: revolution in screening important traits and creation of ‘in silico’ organisms

Chen lab research towards plant systems biology, an emerging discipline facilitates rational plant engineering

‘Omics’ modules – example of proteomics in addressing the differences in wheat (C3 plant) and corn (C4 plant)

OutlineOutline

What is modern and future biological sciences ?

‘Omics’ and Systems Biology

• “Omics”– Genomics – the comprehensive

study of whole sets of genes & their interactions (DNA microarrays)

– Proteomics - the study of the full set of proteins encoded by a genome

– Metabolomics - the comprehensive study of the small molecules or metabolites

– Bioinformatics - the application of computer & statistical techniques to the management of biological information

Systems Biology-“An interdisciplinary approach for integrating experimental data with mathematical modeling tools to analyze & predict the behavior of biological systems.” (Henson, 2005)

Starvation: Importance of CassavaStarvation: Importance of Cassava

Cassava (Manihot esculenta) - yucca, manihot, tapioca• cultivated in tropics and sub-tropics for its edible storage root

• a major source of dietary energy for more than 700 million people

• source for a variety of food stuffs, animal feed and industrial products

• major component in micro-economies of more than 150 countries

Cassava tuberous roots developed from Cassava tuberous roots developed from fibrous rootsfibrous roots

• Tuberous roots develop form fibrous roots through massive cell division and differentiation of parenchyma cell of the secondary xylem

• Not all fibrous roots are designated for tuberous root formation

Proteomics is the approachProteomics is the approach

Protein map for comparison with other cultivars or tissue types

Compare physiological functions of the two types of roots

Identify targets for engineering

Fibrous Root

Tuberous Root

Protein Identification2D gel overlay

3D view of Fibrous Gel

3D view of Tuberous Gel

Protein Identification Using Liquid Chromatography Protein Identification Using Liquid Chromatography Mass SpectrometryMass Spectrometry

HPLC coupled online with 4000 QTRAP Mass SpectrometerHPLC coupled online with 4000 QTRAP Mass Spectrometer

Novel Targets for Biotechnological Application Novel Targets for Biotechnological Application

258257

258 257

263 263

Heat shock protein

BioCassava Plus project: it is proposed to increase the yield and protein content of cassava storage roots by four-fold

Galactose utilization in yeast

– Science 292: 929-934 (galactose utilization)Combines: literature knowledge, microarray, proteomics, visualization, and

network techniques to refine what is known about galactose utilization in yeast.

– Genome Res. 13: 244-253 (Genome scale network reconstruction)

Strategy

• For each gene or condition change (i.e. delete the gene) and measure the global effect on both mRNA and protein levels.

• Integrate mRNA and protein responses with the pathway model and with global network of protein interactions.

• Formulate new hypotheses to explain novel observations and refine models.

Galactose metabolism

- Science 292: 929-934

Expression measurements

10000

cDNAs

Hybridizationto microarray

mRNAextraction

Reverse transcriptionand fluorescent labeling of cDNA

WT control Mutant

Expression measurements

Microarray:

• a perturbed stain vs. wt + gal, 4 replicates

• statistics: maximum-likelihood estimation →997 significant genes → 16 clusters by self-organizing maps, each cluster contains genes with similar responses over all perturbations.

Expression measurements

Visualizing the data

gal4Δ

- Blue line (p-p); Yellow line (p-d); node diameter scales with the magnitude of cha

More Systems Biology to follow…

Systems Biology SchemeGenome(gene)

Transcriptome(mRNA)

Proteome(protein)

Metabolome(metabolite)

Phenotype

Representation of data in XML format- molecule identity and structure- quantification- functional status (modification and complex)- localization and dynamics

transcriptomics proteomics metabolomics

Statistical association, networking and modeling

New functions for genes, proteins and metabolites

In Silico Plant, Prediction and Rational Engineering

Simultation and phenotype explanation

Environment

Hypothesis modification

Large scale biology – ‘Omics’: revolution in screening important traits and creation of ‘in silico’ organisms

Chen lab research towards plant systems biology, an emerging discipline facilitates rational plant engineering

‘Omics’ modules – example of proteomics in addressing the differences in wheat (C3 plant) and corn (C4 plant)

OutlineOutline

What is modern and future biological sciences ?

Plants Produce Many Chemicals That Are Related to Our LifePlants Produce Many Chemicals That Are Related to Our Life

CH 2 CH

CH 2

S in ig r in A lly lg lu c o s in o la te

CH 2 CH

CH 2

CH 2

I. G lu c o s in o la te s d e rive d fro m m e th io n in e

G lu c o n a p in B u t-3 -e n ylg lu co s in o la te

CH 2 CH

CH 2

CH 2

CH 2

G lu c o b ra ss ic a n a p in P e n t-4 -e n ylg lu c o s in o la te

CH 2 CH

CH

CH 2O H

P ro g o itr in (2 R )-2 -H yd ro x yb u t-3 -e n ylg lu c o s in o la te

CH 2 CH

CH

CH 2O H

E p ip ro g o itr in (2 S )-2 -H yd ro xyb u t-3 -e n ylg lu co s in o la te

CH 2 CH

CH 2

CH

CH 2O H

N a p o le ife r in (2 R )-2 -H yd ro x yp e n t-4 -e n ylg lu c o s in o la te

CH 3 S CH 2

CH 2

CH 2

G lu co ib e rv ir in 3 -M e th ylth io p ro p ylg lu co s in o la te

CH 3 S CH 2

CH 2

CH 2

CH 2

G lu c o e ru c in 4 -M e th ylth io b u ty lg lu co s in o la te

CH 3 S CH 2

CH 2

CH 2

CH 2

CH 2

G lu co b e rte ro in 5 -M e th ylth io p e n ty lg lu co s in o la te

CH 3 S O CH 2

CH 2

CH 2

G lu co ib e r in 3 -M e th yls u lp h in y lp ro p ylg lu co s in o la te

CH 3 S O CH 2

CH 2

CH 2

CH 2

G lu c o ra p h a n in 4 -M e th yls u lp h in y lb u ty lg lu co s in o la te

CH 3 S O CH 2

CH 2

CH 2

CH 2

CH 2

G lu c o a lvs s in 5 -M e th ylsu lp h in ylp e n tylg lu c o s in o la te

CH 3 S O CH

CH

CH 2

CH 2

G lu co ra p h e n in 4 -M e th yls u lp h in y lb u t-3 -e n ylg lu c o s in o la teCH 3 S O 2 C

H 2

CH 2

CH 2

G lu c o c h e iro lin 3 -M e th ylsu lp h o n ylp ro p ylg lu c o s in o la te

G lu c o e ryso lin 4 -M e th ylsu lp h o n ylb u ty lg lu co s in o la te CH 3 S O 2 CH 2

CH 2

CH 2

CH 2

II. G lu c o s in o la te s d e rive d fro m p h e n y la la n in e

CH 2C

H 2

CH 2

G lu c o n a s tu rtiin P h e n e th ylg lu c o s in o la te

CH

CH 2O H

G lu c o b a rb a r in 2 -H yd ro x y-2 -p h e n yle th ylg lu c o s in o la teCH 2

OH

G lu c o le p ig ra m in m -H yd ro xyb e n zylg lu co s in o la te

CH 2

OHS in a lb in p -H yd ro x yb e n zylg lu c o s in o la teCH 2

C H 3 O

G lu c o lim n a n th in m -M e th o x yb e n zylg lu c o s in o la teCH 2

C H 3 OG lu c o a u b r ie tin p -M e th o x yb e n zylg lu co s in o la te

III. G lu c o s in o la te s d e rive d fro m tryp to p h a nG lu co b ra s s ic in In d o le -3 -ylm e th ylg lu co s in o la te

N

CH 2R 4

R 1

R 1 R 4 = H

N e o g lu co b ra s s ic in n -M e th o xyin d o le -3 -y lm e th ylg lu c o s in o la te R 1 = O C H 3 R 4 = H

S u lp h o g lu co b ra s s ic in n -S u lp h o in d o le -3 -y lm e th ylg lu c o s in o la te R 1 = S O 3- R 4 = H

4 -M e th o xyg lu c o b ra ss ic in 4 -M e th o x yin d o le -3 -ylm e th ylg lu co s in o la te R 4 = O C H 3

R 4 = O HR 1 = H

R 1 = H

-

O

N

S

O SO3

R

OH

OHOHOH

glucosinolate

FlavourPlant-insect

Defense

N/S nutritionN/S nutrition

Growth (IAA)Growth (IAA)

BiofumigationBiofumigation

“ I do not like broccoli. And I haven't liked it since I was a little kid and my mother made me eat it. And I'm President of the United States and I'm not going to eat any more broccoli.” – George Bush

Anticarcinogenicactivities

Goitrogenicproperty

Genetic Engineering Problem: Modulation of glucosinolatesaffects normal plant growth and development

35S::CYP83B1

ControlControlcyp79f1 Control35S::CYP79F2

cyp83a1

WT

atvamugt74WT

IG

A B C D

cyp79f1 cyp79f1

E HF

WT sur1

H

atvam3-4Wild type

thiohydroximate-O-sulfate

IAA

indole-3-acetonitrile

NIT

tryptophan

indole

indole-3-glycerolphosphate

indole glucosinolate

aldoxime

(n)homomethionine

methionine

S-alkylthiohydroximate

syringyllignin

sinapateesters

MAM

CYP79F1/F2

shikimatepathwaychorismateanthranilate

phenylalanine

cinnamic acid

lignin

flavonoids

coniferaldehyde/coniferyl alcohol

CCoAOMT

sinapaldehyde/sinapyl alcohol

COMT

SUR1

thiohydroximate

desulfoglucosinolate

aliphatic glucosinolate

aldoxime

S-alkylthiohydroximate

CYP79B2/B3

SUR1

thiohydroximate

desulfoglucosinolate

CYP83A1

S-GT S-GT

ST ST

dihydrocamalexic acid

CYP83B1

tryptamine

N-hydroxytryptamine

camalexin

CYP71B15

indole-3-pyruvic acid

indole-3-acetaldehyde

indole-3-acetaldehyde

TGG

AtDofMyb51 ATR1 IQD1 Myb28/29SLIM1

Sulfur deficiency

AP2

Wound, pathogen and insect

ET JA SA

Other factorse.g., K+

CYP71A13

- Yan and Chen (2008) Planta 226, 1343.

Proteomics: large scale analysis of proteins Proteomics: large scale analysis of proteins

Control Mutant

Protein expressionProtein expressionPhosphoproteomicsPhosphoproteomics

GlycoproteomicsGlycoproteomics

MetabolomicsMetabolomics: large: large--scale analysis of metabolitesscale analysis of metabolites

Statistical Analysis, Network and ModelingStatistical Analysis, Network and Modeling

Validation of targets and rational engineering.

““Golden RiceGolden Rice””Over 120 million children worldwide are deficient in vitamin A. Rice has been engineered to accumulate β-carotene, Incorporation of this trait into rice cultivars and widespread distribution could prevent 1 to 2 million

deaths each year.

Vitamin A deficiency is a serious problem• Causes blindness• Influences severity of diarrhea, measles

Large scale biology – ‘Omics’: revolution in screening important traits and creation of ‘in silico’ organisms

Chen lab research towards plant systems biology, an emerging discipline facilitates rational plant engineering

‘Omics’ modules – example of proteomics in addressing the

differences in wheat (C3 plant) and corn (C4 plant)

OutlineOutline

What is modern and future biological sciences ?