a global regulatory mechanism integrates carbon...
TRANSCRIPT
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