Investigating Diel Regulation in Cellular Carbon
Fixation and Rubisco Abundance in Thalassiosira
weissflogii
- Princeton University, Princeton, NJ
- Dr. Jodi Young, Department of Geosciences
- Professor Francois Morel, Department of Geosciences
Objective
- investigate diel cycles in physiological regulation of carbon fixation
and Rubisco abundance in marine diatoms
Diatoms – why should we care?
- large and ecologically significant group of organisms
- 20% of carbon fixation via photosynthesis (Falkowski and Raven)
- physiology of photosynthesis is poorly understood
- Thalassiosira weissflogii (model organism, marine diatom)
Rubisco
- ribulose-1,5-bisphosphate carboxylase/oxygenase
- often the rate-determining step of photosynthesis
- catalyzes fixation of carbon dioxide in first step of Calvin Cycle
Diel Cycles
- has been observed that diatom physiology obeys day:night cycle
- general trend indicates that activity peaks around middle of day
- lack of storage capabilities for large and abundant proteins
Methods
-
acclimated to 12:12 day:night cycle for 17 days (~20-25 generations)
lag, exponential, and stationary phase
harvesting of cells occurred after 17 days
Coulter counter (cell count)
radioactive C-14 (carbon fixation)
BCA assay (protein abundance)
Western blot (specific protein abundance)
FIRe (photosynthetic efficiency)
flow cytometry (cell division)
Cell Counts Throughout Day
Cell Counts Throughout Day
30000
25000
20000
20000
Cell Count in Cells/mL
Cell Count in Cells/mL
25000
15000
15000
10000
10000
5000
5000
0
0
5
7
9
11
13
Time of Day
15
17
19
21
5
7
9
11
13
17
19
Time of Day
1B
1A
15
2B
2A
Growth rates measured long-term ranged from 1.08 day-1 to 1.31 day-1.
Growth rates from 6 a.m. until 2 p.m. ranged from 0.37 day-1 to 0.81 day-1.
Growth rates from 2 p.m. until 7 p.m. ranged from 2.15 day-1 to 2.36 day-1.
21
Protein Levels Per Cell Throughout Day
160
0.18
140
0.16
120
0.14
Protein levels in nanograms per cell
Protein in micrograms per mL
Protein Levels Throughout Day
100
80
60
40
0.12
0.1
0.08
0.06
0.04
20
0.02
0
0
5
10
Time of Day
15
20
0
0
5
10
Time of Day
1B
2B
1B
Protein levels declined in the morning and then rose throughout the day.
Protein levels per cell followed a diel cycle that peaked at around 2 p.m.
2B
15
20
Rubisco Levels Per Cell
(2B)
Rubisco Levels Per Cell
(1B)
Relative Rubisco Levels
Relative Rubisco Levels
0.25
0.2
0.15
0.1
0.05
0
0
5
10
15
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
20
5
Rubisco Levels
Rubisco Levels
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
10
Time of Day
15
20
Rubisco Level, per unit
Total Protein (2B)
Rubisco Level, per unit
Total Protein (1B)
5
15
Time of Day
Time of Day
0
10
20
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
Time of Day
15
20
Carbon Fixation Throughout Day
Picomoles of Carbon Fixation per cell per hour
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
10
Time of Day
1A
2A
12
14
16
18
20
Fv/Fm
0.74
0.73
0.72
Arbitrary units
0.71
0.7
0.69
0.68
0.67
0.66
0
2
4
6
8
10
Time of Day
12
14
16
18
20
G1 and G2 Proportions Throughout Day (1A)
80.00%
70.00%
Proportion of G1 or G2
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
10
11
12
13
14
15
Time of Day
G1
G2
16
17
18
19
20
Results
- Observed diel cycles in
-
total protein abundance
relative Rubisco abundance
relative CdCA abundance
carbon fixation
cell division rates
photosynthetic efficiency
Next Step
- more trials may be necessary to more precisely quantify trend
- examination of RNA data using RT-qPCR to determine gene activity