A Global Regulatory Mechanism Integrates

Carbon and Energy Metabolism in

Thalassiosira pseudonana Sarah R. Smith

C. Glé, R.M. Abbriano, J.C. Traller,E. Trentacoste, A. K. Davis, O. Cook, S.

Federowicz, T. Norden-Krichmar,

A. E. Allen, and M. Hildebrand

Scripps Institution of Oceanography

University of California, San Diego

J. Craig Venter Institute

Algae Biomass Summit Orlando, Florida

September 30 – October 3, 2013

Metabolite Exudation

Respiration (CO2)

Cellular

Carbon

CO2 H

ea

t

Flu

ore

sce

nce

Light Absorption

Metabolite Exudation

Respiration (CO2)

Cellular

Carbon

CO2 H

ea

t

Flu

ore

sce

nce

Light Absorption

Silicon Starvation

What is the molecular

mechanism that integrates

these processes?

Short-term silicon starvation in T. pseudonana

0

5

10

15

20

25

30

35

40

5

10

15

20

25

30

35

40

0 4 8 12 16 20 24

Si- #1

Si- #2

Si- #3

Si- #4

Si- #5

Si- #6

Si- #7

24h 18h 4h 0h 12h 8h

Nile

Red

Flu

ore

scen

ce

(a

.u. ce

ll-1

)

Bo

dip

y F

luo

resce

nce (a

.u. c

ell -1)

• Lipid droplet formation

induced between 8-

12hr

• Chlorophyll increases

• Growth arrests

immediately

Pe

rce

nta

ge

of P

op

ula

tio

n w

ith

(n

) ch

loro

. Duration of Silicon Starvation (hours)

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 4 8 12 18 24

Duration of Silicon Starvation (hours)

Short-term silicon starvation in T. pseudonana

0

5

10

15

20

25

30

35

40

5

10

15

20

25

30

35

40

0 4 8 12 16 20 24

Si- #1

Si- #2

Si- #3

Si- #4

Si- #5

Si- #6

Si- #7

Nile

Red

Flu

ore

scen

ce

(a

.u. ce

ll-1

)

Bo

dip

y F

luo

resce

nce (a

.u. c

ell -1)

• Cell count

• Cell cycle

• Microscopy

• GC-MS FAME

• HPLC Pigments

• P-I curves

• FRRf

• mRNA

Pe

rce

nta

ge

of P

op

ula

tio

n w

ith

(n

) ch

loro

. Duration of Silicon Starvation (hours)

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 4 8 12 18 24

Duration of Silicon Starvation (hours)

http://employees.csbsju.edu/hjakubowski/classes/ch331/bind/olbindtransciption.html

Physiology and

Metabolism 1

Pre-existing suite

of regulatory

elements

2

3

New

regulatory

elements

Despite this

complexity,

do organizing

principles

emerge?

mRNA levels

Striking degree of genome-wide coordinate

regulation indicates global regulatory

mechanism

Pe

rce

nta

ge

of P

op

ula

tio

n w

ith

(n

) ch

loro

.

20%

30%

40%

50%

60%

70%

80%

90%

100%

0 4 8 12 18 24

Duration of Silicon Starvation (hours)

Carbon Fixation

Transcript levels changes

directly reflect chloroplast

replication, not changes in

C-B carbon fixation

-1

0

1

2

3

4

5

6

0 4 8 12 16 20 24

Duration of Silicon Starvation (hours)

Fo

ld C

ha

ng

e G

en

e E

xp

ressio

n

(lo

g_

2)

Calvin-Benson Cycle Genes

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 4 8 12 18 24

Duration of Silicon Starvation (hours)

μg

C f

ixed

1x10

6 c

ell

s-1

hr-1

Carbon Fixation Rate

Fatty Acid Biosynthesis

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 4 8 12 16 20 24

ACCase

MCT1

KASI

KAS3

HAD1

EAR1

KAR

Duration of Silicon Starvation (hours)

Fo

ld C

han

ge G

en

e E

xp

ressio

n (lo

g_2)

0

20

40

60

80

100

120

140

160

180

0 4 8 12 18 24

C22:6

C20:5

C18:4

C18:0

C16:4

C16:3

C16:2

C16:2

C16:1

C16:1

C16:0

C15:0

C14:0

FA

ME

(μ

g m

gD

W-1

)

Duration of Silicon Starvation (hours)

• Coordinated regulation of FAB genes with plastid

replication suggests that expression is cell cycle

progression dependent

• Metabolite flux into these pathways is what regulates lipid

accumulation

Metabolite Exudation

Respiration CO2

Cellular

Carbon

CO2

fixation Hea

t

Flu

ore

sce

nce

Light Absorption

Calvin-Benson

FA Biosynthesis

FCPs PSII & PSI

Chrysolaminarin Synthase

Several energy and carbon sinks

are coordinately regulated

4 8 12 18 24

-1

-2

-3

1

0

2

Duration of Silicon Starvation (hours)

Fo

ld C

ha

ng

e G

en

e E

xp

ressio

n (

log

_2

)

Several energy and carbon sinks

are coordinately regulated

Respiration – TPI/GAPDH

Protein Biosynthesis

Aromatic Amino Acid

Biosynthesis

Complex Lipid Biosynthesis -5

-4

-3

-2

-1

0

1

2

0 4 8 12 16 20 24

TPI/GAPDH

tRNA Synth.

EF P

EPSP Synthase

ELO

SQDG Synthase

PsbW

CDC20, activates the anaphase

promoting complex

PSII Biogenesis

Fo

ld C

ha

ng

e G

en

e E

xp

ressio

n (

log

_2

)

-5

-4

-3

-2

-1

0

1

2

0 4 8 12 16 20 24

CDC20-like

Duration of Silicon Starvation (hours)

Fo

ld C

ha

ng

e G

en

e E

xp

ressio

n (

log

_2

)

Duration of Silicon Starvation (hours)

Metabolite Exudation

Respiration CO2

Cellular

Carbon

CO2

fixation Hea

t

Flu

ore

sce

nce

Light Absorption FCPs PSII & PSI

Calvin-Benson

FA Biosynthesis Chrysolaminarin Synthase

TPI-GAPDH

tRNA synthetase EF P EPSP Synthase

Cell Cycle

Progression

There is global regulation of most genes

that can be attributed to cell cycle

progression

• This mechanism integrates expression of genes

involved in metabolite flux and cellular

energetics

• Informs metabolic engineering approaches (transcription factor and single-gene manipulations)

• How to regulate productivity of a unicellular

crop?

Fo

ld-c

ha

ng

e G

en

e E

xp

res

sio

n (

log

_2

)

Duration of Silicon Starvation (hours)

Take-Home Message

Hildebrand Lab Eric Allen Vic Vacquier Maria Vernet Al Darzins ABO Summit

Funding:

AFOSR DOE NSF

CEC LMSF DoD NDSEG