System Response of Metabolic Networks in Chlamydomonas reinhardtii during Nitrogen Starvation

Leading to Lipid Accumulation

Nishikant Wase, P.hD Paul N. Black, Ph.D.

Concetta C. DiRusso, Ph.D.

Department of Biochemistry University of Nebraska-Lincoln

Talk outline

u  Brief overview of systems biology

u  Algal biofuels & Chlamydomonas

u  Quantitative proteomics and metabolomics

experiments

u  Results and Conclusions

Why study systems biology ?

v  Complete understanding of a biological phenomena

v  Understanding complex organization

v  Regulatory mechanisms (homeostasis)

v  Energy utilization

v  Response to the environmental stimuli

v  Reproduction (DNA guaranties exact replication)

v  Evolution (capacity of species to change over time)

Phenotype

Genotype

mRNA expression

Proteomics (iTRAQ)

Metabolomics (GC-MS)

Data Integration

6

iTRAQ Isobaric Tag for relative and absolute quantification

Reacts with NH2 groups

N

NCH3

O

NH

O

RAdds tag of mass 145 to terminal NH2 groups and lysines

N

NCH3

CH2+

Rest of molecule +

Reporter ion

MS/MS Fragmentation

iTRAQ labeling

Decreased in stress

Stress

Control No change

Stress Control

Increased in stress Stress

Control

Algae and Biodiesel

•  Algae Biodiesel is a good replacement for standard crop biodiesels like soy and canola

•  Up to 70% of algae biomass is usable oils

•  Algae does not compete for land and space with other agricultural crops

•  Some algal species survive in water of high salt content and use water that was previously deemed unusable.

•  To study the proteome and metabolome changes, Chlamydomonas is used as a model organism in current study.

0 50 100 1500.0

5.0×106

1.0×107

1.5×107

2.0×107

2.5×107

3.0×107TAP N+ TAP N-

Hours

cells

/ m

L

Analysis of Growth, Starch, photosynthetic pigments and fluorescent microscopy Starting culture at 1.0 x E06 cells / mL

24N+ 24N- 48N- 144 N-

Triplicate cultures of 100 mL suspension was maintained.

24N+ 24N- 48N- 144N-0.00

0.50

1.00

1.50

2.00

* *

***

Hours of N starvation

µg/2

.0 x

107 c

ells

24N+ 24N- 48N- 144N-0.00

2.00

4.00

6.00

******

***

Hours of N starvation

µg / 1

.0 x

107 c

ells

24N+ 24N- 48N- 144N-0.00

5.00

10.00

15.00

******

***

Hours of N starvation

µg / 1

.0 x

107 c

ells

24N+ 24N- 48N- 144N-0.00

5.00

10.00

15.00

****** ***

Hours of N starvation

µg / 1

.0 x

107 c

ells

A B

C D

*""""""p"value(<"0.05**""""p"value"<"0.01***""p"value"<"0.001

Starch Chl A

Chl B Total Carotenoid

24N+ 24N- 48N-

144N- 0

5000

10000

15000

20000

2500024N+24N-48N-144N-

Days

RFUC16

:0

C16

:1(9

)

C16

:2(7

,10)

C16

:3(7

,10,

13)

C16

:4(4

,7,1

0,13

)

C18

:0

C18

:1(9

)

C18

:1(1

1)

C18

:2(9

,12)

C18

:3(5

,9,1

2)C

18:3

(9,1

2,15

)0

50

100

150 24N+ 24N- 48N- 144N-

µg

mg

-1 D

CW

a,b,c

c cb,c

a,b,c

a,b,cc

a,b,ca,b,c

a,b,ca,b,c

a,b,ca,b,c

c

a, b

Changes in Fatty acid methyl esters during N stress

a: signi!cant di"erence between 24N+ vs 24N- b: signi!cant di"erence between 24N+ vs 48N- c: signi!cant di"erence between 24N+ vs 144N-

Chlamydomonas reinhardtii

Protein Extraction

Sample pooling & LC-SCX fractionation followed by MALDI spotting

MALDI TOF-TOF Tandem MS analysis

ProteinPilot V 4.0

Trypsin Digestion

Peptides

24N+ 24N- 48N- 144N-

iTRAQ113

iTRAQ114

iTRAQ115

iTRAQ116

iTRAQ117

iTRAQ118

iTRAQ119

iTRAQ121

iTRAQ peptide labelling

•  Database search (forward and reverse) •  FDR analysis •  Quantification values •  Statistical analysis •  Network anaylsis

Proteomics experimental workflow

Critical(FDR Local(FDR Global&FDR Global(FDR(from(Fit1.0% 735 807 8205.0% 765 904 89510.0% 790 937 951

Number&of&Proteins&Detected

Proteins)Identified)at)Critical)False)Discovery)RatesFDR and Quantitation analysis

0%#

10%#

20%#

30%#

40%#

50%#

60%#

70%#

80%#

90%#

100%#

0%# 10%# 20%# 30%# 40%# 50%# 60%# 70%# 80%# 90%# 100%#

False

#Disc

overy#R

ate#####

Reported#Protein#Confidence#

ProteinPilot#Reported#vs.#EsCmated#FDR#

Global#FDR#Local#FDR#Global#FDR#(Fit)#

0%#

2%#

4%#

6%#

8%#

10%#

12%#

0# 200# 400# 600# 800# 1000# 1200#

False#Discovery#Rate#

Ranked#Proteins#

Es<mated#False#Discovery#Rates#

Global#FDR#Local#FDR#Cri<cal#Values#(Global)#Cri<cal#Values#(Local)#Global#FDR#(Fit)#

765 proteins

820 proteins

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

0

100

200

300

400

500

600

700

10 20 30 40 50 60 70 80 90 100 >100

% cu

mulat

ive fr

eque

ncy

Freq

uenc

y

% variation

24N+

24N-

48N-

144N-

95.0 %

Determination of threshold for protein quantitation analysis

Protein subcellular localization of identified proteins using Wolf-Psort tool

chlo%38%%

chlo_mito%1%%

cysk%1%%

cyto%28%%

cyto_nucl%1%%

E.R.%1%%

E.R._plas%0%%

extr%2%%

mito%15%%

nucl%8%%

pero%1%%

plas%3%%

vacu%1%%

Subcellular%localizaBon%of%idenBfied%proBens%Abbrev& Localiza.on&chlo% chloroplast%cyto% cytosol%cysk% cytoskeleton%E.R.% endoplasmic%extr% extracellular%golg% Golgi%lyso% lysosome%mito% mitochondria%nucl% nuclear%pero% peroxisome%plas% plasma%vacu% vacuolar%

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2

P-Valu

e

Log10 Ratio

Volcano Plot of Log10 ratio vs p-value of all quanti!ed control (N2 repleted) proteins

114:113

-0.2 0.2

Log10 0.2 = 1.58 (linear scale) Log10 -0.2 = 0.63 (linear scale) Replicate Analysis

0.0000001

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2

P-V

alu

e

Log10 Ratio

Volcano Plot of Log10 ratio vs p-value of all quanti!ed N2 stress proteins

115:113 116:113 117:113 118:113 119:113 121:113

0.2 -0.2

A" B"

2" 0"

28"10" 15"

36"

9"

24"hrs"(40)" 48"hrs"(52)"

7" 2"

21"6" 16"

14"

11"

144"hrs"(57)"

24"hrs"(36)" 48"hrs"(50)"

144"hrs"(89)"

Up#regulated,Dn#regulated,

Distribution of changed proteins according to metabolic pathways Venn diagram showing protein changes Replicate analysis of Proteins from

control cultures

Volcano plot of protein changes in N stressed cultures Protein changes in metabolic pathways

Alan

ine, a

spart

ate an

d glut

amate

meta

bolis

m

Amino

acyl-

tRNA

bios

ynthe

sis

Calci

um si

gnali

ng pa

thway

Carb

on fix

ation

in ph

otosy

ntheti

c org

anism

s

Cell c

ycle

Citra

te cy

cle (T

CA cy

cle)

Cyste

ine an

d meth

ionine

meta

bolis

m

Fatty

acid

biosy

nthes

is

Glyc

ine, s

erine

and t

hreo

nine m

etabo

lism

Glyc

olysis

/ Gluc

oneo

gene

sis

Nitro

gen m

etabo

lism

Oxida

tive p

hosp

horyl

ation

Pento

se ph

osph

ate pa

thway

Pero

xisom

e

Phag

osom

e

Photo

synth

esis

Photo

synth

esis

- ante

nna p

rotei

ns

Porp

hyrin

and c

hloro

phyll

meta

bolis

m

Purin

e meta

bolis

m

Pyru

vate

metab

olism

Ribo

some

Starc

h and

sucro

se m

etabo

lism

Unkn

own0

2468

1030

40

50

60

# prot

eins

Up-regulated

Dn-regulated

Metabolomics Experimental workflow

Metabolomic experiment flowchart

m/z 204 m/z

133

m/z 131

m/z 129

m/z 374

m/z 368

Select optimal quantifier ions

metabolites export align annotate

sam

ples

metabolites

sam

ples

Normalize and transform data

classes

sam

ples

Statstical Analysis

Get significance and hidden data structures

hypotheses

data

experiments in

terp

reta

tion Interpret findings

Generate new hypotheses

….

An example of total ion chromatogram 24N+ metabolite sample

Amino acids 13%

other 39%

Lipids 10%

Nucleic acids 5%

carboxylic acid 4%

Phosphate 9%

sugar alcohol 2%

sugar 18%

Classes of compounds profiled using GC-MS

Multivariate analysis:

PLS-DA score plot of all the 36 GC-MS run. It shows how the samples are discriminated. Red: 24N+, blue: 24N-, green: 48N- and dark orange: 144N-

24N+ 24N- 48N- 144N-

Heat map visulaization and Hiercherial cluster tree representation of top 25 significantly changed metabolites.

24N+ 24N- 48N- 144N-

Log2 fold change

-Log

10 (p

-val

ue)

Changes in metabolites at 144 hours

Log2 fold change

-Log

10 (p

-valu

e)

Changes in metabolites at 48 hours Changes in metabolites at 24 hours

Log2 fold change

-Log

10 (p

-valu

e)

Volcano plot of overall metabolite changes during N stress

Effect of exogenous citrate on growth & lipid accumulation while growing in TAP media

0 12 24 36 48 60 72 840.0

0.5

1.0

1.5Neg3mM4mM5mM6mM7mM8mMPos

hours of culture

Bio

mas

s (O

D 5

50 n

m)

A

N+ 3.0 4.0 5.0 6.0 7.0 8.0 N-0

10000

20000

30000

40000

50000

60000

Citrate Conc (mM)

Nile

Red

Sig

nal

Neg

Citrate 5 mM

Citrate 8 mM

Pos

Neg 1 2 3 4 5 6 7 8 9 10 Pos

Citrate conc (mM) Growth with diff. conc. of citrate

Lipid accumulation at diff. conc. of citrate

Membrane(domain((

FMN(

Complex(I(NADH(dehydrogenase(

Flavo(protein(

Fe(protein(

FrdD(FrdC(

Succinate( Fumarate(

SDH2(SDH3(

Complex(II(

UQ(

UQH2(

Cytb(

Cytc(ISP(

Core2(Core1(

Complex(III( Complex(IV(

Va(Vb(

III(

VII(

II(

VIIa(

Cytc(

Complex(V(

Inner(mito(membrane(

Mitochondrial(matrix(

NADH( NAD+(((H+(

F0(

F1(

α( α(β(β(

γ(ε(

Proton(channel(

ATP(Synthase(

Cyt(bc1(complex(

Cyt(c(oxidase( b(

global

- 3 0 3

24N

-/24

N+

48N

-/24

N+

.144

N-/

24N

+

# Accession Protein Peptides(>95% Confidence)1 Cre01.g051900.t1.2 Rieske iron-sulfur protein of mitochondrial ubiquinol-Cytochrome c reductase62 Cre02.g076350.t1.2 Vacuolar ATP synthase subunit B 1 23 Cre02.g100200.t1.2 NADH:ubiquinone oxidoreductase 22 kDa subunit 24 Cre06.g304350.t1.2 Cytochrome c oxidase subunit VIb 65 Cre06.g310950.t1.2 FAD dependent oxidoreductase 46 Cre07.g338050.t1.2 Mitochondrial F1F0 ATP synthase associated 36.3 kDa protein 1 47 Cre09.g405850.t1.1 NADH:ubiquinone oxidoreductase 49 kDa ND7 subunit 58 Cre10.g422600.t1.1 NADH:ubiquinone oxidoreductase 51 kDa subunit 49 Cre10.g434450.t1.2 NADH:ubiquinone oxidoreductase 39 kDa subunit 51 0 Cre10.g459200.t1.2 NADH:ubiquinone oxidoreductase B16.6 subunit 31 1 Cre10.g461050.t1.2 NADH:ubiquinone oxidoreductase 18 kDa subunit 31 2 Cre11.g481450.t1.2 F-type H+-transporting ATPase subunit b 31 3 Cre12.g523850.t1.2 Ubiquinol:Cytochrome c oxidoreductase 50 kDa core 1 subunit 1 11 4 Cre12.g555250.t1.2 NADH:ubiquinone oxidoreductase B14 subunit 21 5 Cre15.g638500.t1.2 NADH dehydrogenase 31 6 Cre16.g664600.t1.2 NADH dehydrogenase (ubiquinone) Fe-S protein 4 41 7 Cre17.g698000.t1.2 P-type ATPase/cation transporter, plasma membrane 61 8 g1098.t1 Vacuolar ATP synthase subunit A 1 81 9 g11598.t1 NADH:ubiquinone oxidoreductase 14 kDa subunit 32 0 g11697.t1 F-type H+-transporting ATPase subunit beta 3 22 1 g11946.t2 FAD/NAD(P)-binding oxidoreductase 62 2 g12869.t1 Cytochrome c 82 3 g18128.t4 F-type H+-transporting ATPase subunit alpha 3 22 4 g2416.t1 Ubiquinol:Cytochrome c oxidoreductase Cytochrome c1 32 5 g2977.t1 Cytochrome c oxidase subunit II 82 6 g9598.t1 Inorganic pyrophosphatase 4

(((((((((((((((((

Changes in oxidative phosphorylation

CS

IDH MDH

Pyruvate

Acetyl-CoA

citrate

isocitrate

2-ketoglutarate succinate

Fumarate

oxaloacetate

SDH

TCA Cycle

Ala$Ser$Gly$Thr$Trp$

acetoacetate

Leu$Lys$Phe$Tyr$

Ile$Leu$Thr$Trp$

Arg$Gln$Glu$Pro$

Ile$Met$Val$

Asp$Arg$

Asp$Phe$Tyr$

PDH

Alternate(routes(of((acetyl/CoA(genera2on(via(amino(acid(

catabolism(

Red: Increased Green: Decreased Gray: no change * : not found

*

*

*

Schematic representation of lipid accumulation based on current data

PEP

pyruvate

pyruvate

PDH

starch

glucose

G-6P

Fru-6P

Fru-1,6P2

G-1P

PGM

FBP

TPI

PGI

6-PGL

6-PG

G6-PDH

6PGHD

PFK

HK

AMY

ALD FBA

ACL

LIGHT

PDH CS

IDH MDH

acetyl-CoA

malonyl-CoA

FFA

ACC

MCAT FAS

acetate ACS

G3P

3-PGA

RuBP

Ru5P

RuBisCO

PRK

TK

PGK

CO2

G3P DHAP

chlororespiration

C5 and C6 sugars

glucose PGI GAPDH PMG ENO

pyruvate Acetyl-CoA

citrate

isocitrate

2-ketoglutarate succinate

malate

oxaloacetate

citrate oxaloacetate malate

SDH

CS

MDH

MS ICL

malate

glyoxylate

citrate

isocitrate

succinate

oxaloacetate

acetyl-CoA

ACO

ACS

LPA

GPAT PA

LPAAT DAG

PL DGAT FFA

ACSL Acyl-CoA

TAG

Calvin Cycle

TCA Cycle

Glyoxylate Cycle

C16:0; C16:1 (9); C16:3 (7,10,13); C18:0; C18:1 (9); C18:1 (11); C18:2 (9,12); C18:3 (5,9,12)

G3P

plastid mitochondria

ER

acetyl-CoA

oxaloacetate

Summary: u  Under N2 stress, growth is stopped, leads to massive reprogramming in the

metabolism.

u  Decreased photosynthetic capacity, while accumulating starch, citrate, succinate, methionine trehelose, and lipids .

u  For lipid synthesis acetyl-CoA substrate is generated from the degradation of amino acids and accumulation of citrate.

u  Down regulation of IDH leads to build up of isocitrate within mitochondria, suppress citrate to isocitrate turnover and excess citrate then trafficked out in cytosol to fuel lipid biosynthesis

u  Citrate accumulated via the TCA and glyoxylate cycles is converted to acetyl CoA and oxaloacetate by the activity of ACL.

u  High ACL activity, builds up citrate, decreases levels of isocitrate, are typical features found in most oil producing organisms, such as Candida curvata, Lipomyces starkeyii, Rhodosporidium toruloides, Mucor circinelloides and Mortierella alpina.

Acknowledgements

FATTTLab ²  Prof. Concetta DiRusso ²  Prof. Paul Black ²  Dr. James Allen ² Mark Behrens ²  Tu Boqiang

UNL Collaborators

²  Dr. Ron Cerney ²  Dr. Tom Elthon ²  Dr. Wayne Reikhoff ²  Dr. Girish Rasineni ²  Dr. Jiri Admac ²  Prof. Donald Weeks

Penn State University

²  Prof. Bruce A Stanley

This work was supported by Nebraska EPSCoR

Thank you !!! Questions …………plz