Stress Acclimation and Death-related Genes in the diatom Thalassiosira pseudonana Amelia...
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Stress Acclimation and Death-related Genes in the diatom Thalassiosira pseudonana Amelia Min-Venditti 1 , Kimberlee Thamatrakoln 2 , Kay Bidle 2 1 Department of Ecology and Evolutionary Biology, Rice University 2 Advisors, Department of Marine and Coastal Sciences, Rutgers University Abstract Introduction Materials and Methods Results Results References Acknowledgements QuickTime™ and a decompressor are needed to see this picture. Diatoms are the dominant class of unicellular marine phytoplankton living in the ocean’s euphotic zone. Thalassiosira pseudonana is a centric diatom whose whole genome has been sequenced. A suite of death-related genes were recently found to be over-expressed in growing cells starved for iron (Fe), and, as such, are hypothesized to have cell functions other than death. If these genes confer an ecological advantage to diatoms under nutrient stress, it may help us understand their retention in unicellular genomes. This project tested whether the over-expression of two putative death- related genes (DSPs) increases fitness under Fe stress. We compared the growth and photophysiology of wild-type cells to that of clones over-expressing distinct DSP- green fluorescent proteins. DSP expression was verified during the time course using both epifluorescence microscopy and Western blot analysis. Finally, we correlated the expression of DSPs with known subcellular markers of stress and death. Although they account for less than 1% of the biomass on Earth, phytoplankton make up about 50% of carbon-based primary production, and diatoms alone account for almost 40% (1). A largely unknown part of diatom life cycles are the cellular mechanisms behind the diatom transition from bloom to post-bloom life stages as a function of nutrient availability (2). Fe often limits diatom growth in the ocean and has been shown to induce programmed cell death (PCD), a genetically controlled form of cell suicide (3). Intuitively, genes related to PCD have an evolutionary fitness of zero in unicellular organisms, so it is of interest why and how PCD genes evolved in phytoplankton. Previous work in the Bidle lab discovered a suite of genes strongly induced in Fe-stressed, yet growing cells. These included putative so-called “death-specific proteins”, DSP1 and DSP2. These findings suggest that genes previously thought to be death-specific may also play important roles in diatom acclimation to environmental stresses (3). The purpose of this study was to characterize the physiology of two clones that had been previously generated in the Bidle lab. T. pseudonana clones 18-58 and 37-22 were engineered to over-express TpDSP1 (protein ID 11118) and TpDSP2 (protein ID 11117), respectively, using a a light-driven fcp promoter (4). They also contain a fusion with green fluorescent protein (GFP), which serves as a reporter of expression and is detectable by fluorescence screening and immunoblotting. (1) Bidle, K.D. and P.G. Falkowski. 2004. Cell death in planktonic photosynthetic microorganisms. Nature Reviews Microbiology. 2:643–655. (2) Falkowski, P.G., et al. 2004. The Evolution of Modern Eukaryotic Phytoplankton. Science. 305: 354–360. (3) Bidle, K.D. and S.J. Bender. 2008. Iron Starvation and Culture Age Activate Metacaspases and Programmed Cell Death in the Marine Diatom Thalassiosira pseudonana. Eukaryotic Cell. 7: 223-236 (4) Poulsen, N. and P.M. Chesley. 2006. Molecular Genetic Manipulation of the Diatom Thalassiosira pseudonana (Bacillariophyceae). Journal of Phycology. 42: 1059-1065. Microscope results confirmed that clones 18-58 and 37-22 display GFP fluorescence, which suggests that the DSP- GFP fusion protein is being expressed. In the Western blot, however, some faint immunohybridzation was seen for wild-type at the right size for the DSP-GFP protein. Though at a much lower level than the clones, this indicates possible contamination from one of the clones. The cell abundance for wild-type, 18-58, and 37-22 was similar, rising through exponential phase, until around day 4, when it plateaued in response to physiological stress. The Fv/Fm values stayed relatively high for the first four days, then declined noticeably, as expected in Fe- staved cells. The caspase specific activity assay showed an inherently different subcellular response; caspase activity in all cells, which is part of the stress/death machinery, was over two orders of magnitude higher after the cells reached stationary phase than during exponential phase. The activity between cultures was also very different. During exponential phase, the wild-type activity was over an order of magnitude higher than the clones, then during stationary phase the values were more similar. Finally, when cell health was declining rapidly, caspase activity among the samples was within the same order of magnitude. Wild-type and clones 18-58 and 37-22 were grown in NEPC artificial seawater media, without Fe. Cultures were exposed to constant light at 18°C and constant bubbling. Photosystem II efficiency (Fv/Fm) was measured using fluorescence induction and relaxation (FIRe). Cell abundance was determined daily using a Coulter Multisizer II. Cell biomass was harvested daily, and pellets were stored at -80°C for analysis by Western blot, microscopy and a caspase activity assay. Figure 2a. Average cell concentration (lines) and caspase specific activity (bars) for wild-type, 18-58 and 37-22 clones during exponential, stationary, and death phases. The fold change in caspase activity compared to wild-type is shown in parenthesis for the two clones. Figure 2b. Average photosynthetic efficiency (Fv/Fm) for wild type and clones over the time course. A lower Fv/Fm indicates decreasing health. Figure 4. Western blot of wild-type, 18-58 and 37-22 cell extracts probed with anti-GFP antibody. Strong immunohybridization to the expected ~52 kD DSP-GFP fusion protein was observed in both clones. Some hybridization to the wild-type proteins also occurred. Figure 3. Differential interference contrast (DIC) and epifluorescence micrographs to visualize GFP, chlorophyll, and nuclear DNA (via Hoechst staining). L-R: various diatoms with characteristic silica shells; a satellite image of a phytoplankton bloom; T. pseudonana cells, the first lying on its side and the circular one in valve view. Discussion This research was possible due to the RIOS NSF REU program. Thanks to Liti Haramaty, Chris Brown, Frank Natale, and the rest of the Bidle lab for tactical and practical help. Thanks to Kay Bidle and Kim Thamatrakoln for intellectual support and mentoring. Protein was extracted using 2% SDS then was run by SDS- PAGE. Proteins were transferred to a PVDF membrane, which was probed with with GFP antiserum to measure GFP expression. Protein was extracted using sonication, then caspase specific activity was found by measurement of IETD-AFC substrate cleavage normalized to protein concentration. Fv/Fm of Fe- time course 0.200 0.250 0.300 0.350 0.400 0.450 0.500 0.550 0.600 0.650 0.700 1 2 3 4 5 6 7 Day wt 18-58 37-22 Activities and Growth Curve 1 (1.09) (1.5) (3.3) (1.8) (10) (23) 0 1000 2000 3000 4000 5000 6000 7000 8000 1 2 3 4 5 6 7 Day 1.00E+04 1.00E+05 1.00E+06 1.00E+07 cell concentration (cells/mL) wt activity 18-58 activi 37-22 activi wt 18-58 37-22 DSP1 or DSP2 fcp GFP Figure 1. DSP-GFP fusion vector Wild-type 18-58 37-22 Hoechst GFP Chlorophyll DIC
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Transcript of Stress Acclimation and Death-related Genes in the diatom Thalassiosira pseudonana Amelia...
Stress Acclimation and Death-related Genes in the diatom Thalassiosira pseudonana
Amelia Min-Venditti1, Kimberlee Thamatrakoln2 , Kay Bidle2
1 Department of Ecology and Evolutionary Biology, Rice University 2 Advisors, Department of Marine and Coastal Sciences, Rutgers University
Abstract
Introduction
Materials and Methods
Results
Results
References
Acknowledgements
QuickTime™ and a decompressor
are needed to see this picture.
Diatoms are the dominant class of unicellular marine phytoplankton living in the ocean’s euphotic zone. Thalassiosira pseudonana is a centric diatom whose whole genome has been sequenced. A suite of death-related genes were recently found to be over-expressed in growing cells starved for iron (Fe), and, as such, are hypothesized to have cell functions other than death. If these genes confer an ecological advantage to diatoms under nutrient stress, it may help us understand their retention in unicellular genomes. This project tested whether the over-expression of two putative death-related genes (DSPs) increases fitness under Fe stress. We compared the growth and photophysiology of wild-type cells to that of clones over-expressing distinct DSP-green fluorescent proteins. DSP expression was verified during the time course using both epifluorescence microscopy and Western blot analysis. Finally, we correlated the expression of DSPs with known subcellular markers of stress and death.
Although they account for less than 1% of the biomass on Earth, phytoplankton make up about 50% of carbon-based primary production, and diatoms alone account for almost 40% (1). A largely unknown part of diatom life cycles are the cellular mechanisms behind the diatom transition from bloom to post-bloom life stages as a function of nutrient availability (2). Fe often limits diatom growth in the ocean and has been shown to induce programmed cell death (PCD), a genetically controlled form of cell suicide (3).
Intuitively, genes related to PCD have an evolutionary fitness of zero in unicellular organisms, so it is of interest why and how PCD genes evolved in phytoplankton. Previous work in the Bidle lab discovered a suite of genes strongly induced in Fe-stressed, yet growing cells. These included putative so-called “death-specific proteins”, DSP1 and DSP2. These findings suggest that genes previously thought to be death-specific may also play important roles in diatom acclimation to environmental stresses (3).
The purpose of this study was to characterize the physiology of two clones that had been previously generated in the Bidle lab. T. pseudonana clones 18-58 and 37-22 were engineered to over-express TpDSP1 (protein ID 11118) and TpDSP2 (protein ID 11117), respectively, using a a light-driven fcp promoter (4). They also contain a fusion with green fluorescent protein (GFP), which serves as a reporter of expression and is detectable by fluorescence screening and immunoblotting.
(1) Bidle, K.D. and P.G. Falkowski. 2004. Cell death in planktonic photosynthetic microorganisms. Nature Reviews Microbiology. 2:643–655. (2) Falkowski, P.G., et al. 2004. The Evolution of Modern Eukaryotic Phytoplankton. Science. 305: 354–360.(3) Bidle, K.D. and S.J. Bender. 2008. Iron Starvation and Culture Age Activate Metacaspases and Programmed Cell Death in the Marine Diatom Thalassiosira pseudonana. Eukaryotic Cell. 7: 223-236 (4) Poulsen, N. and P.M. Chesley. 2006. Molecular Genetic Manipulation of the Diatom Thalassiosira pseudonana (Bacillariophyceae). Journal of Phycology. 42: 1059-1065.
Microscope results confirmed that clones 18-58 and 37-22 display GFP fluorescence, which suggests that the DSP-GFP fusion protein is being expressed. In the Western blot, however, some faint immunohybridzation was seen for wild-type at the right size for the DSP-GFP protein. Though at a much lower level than the clones, this indicates possible contamination from one of the clones.The cell abundance for wild-type, 18-58, and 37-22 was similar, rising through exponential phase, until around day 4, when it plateaued in response to physiological stress.The Fv/Fm values stayed relatively high for the first four days, then declined noticeably, as expected in Fe-staved cells. The caspase specific activity assay showed an inherently different subcellular response; caspase activity in all cells, which is part of the stress/death machinery, was over two orders of magnitude higher after the cells reached stationary phase than during exponential phase. The activity between cultures was also very different. During exponential phase, the wild-type activity was over an order of magnitude higher than the clones, then during stationary phase the values were more similar. Finally, when cell health was declining rapidly, caspase activity among the samples was within the same order of magnitude.
Wild-type and clones 18-58 and 37-22 were grown in NEPC artificial seawater media, without Fe. Cultures were exposed to constant light at 18°C and constant bubbling.
Photosystem II efficiency (Fv/Fm) was measured using fluorescence induction and relaxation (FIRe).
Cell abundance was determined daily using a Coulter Multisizer II.
Cell biomass was harvested daily, and pellets were stored at -80°C for analysis by Western blot, microscopy and a caspase activity assay.
Figure 2a. Average cell concentration (lines) and caspase specific activity (bars) for wild-type, 18-58 and 37-22 clones during exponential, stationary, and death phases. The fold change in caspase activity compared to wild-type is shown in parenthesis for the two clones.
Figure 2b. Average photosynthetic efficiency (Fv/Fm) for wild type and clones over the time course. A lower Fv/Fm indicates decreasing health.
Figure 4. Western blot of wild-type, 18-58 and 37-22 cell extracts probed with anti-GFP antibody. Strong immunohybridization to the expected ~52 kD DSP-GFP fusion protein was observed in both clones. Some hybridization to the wild-type proteins also occurred.
Figure 3. Differential interference contrast (DIC) and epifluorescence micrographs to visualize GFP, chlorophyll, and nuclear DNA (via Hoechst staining).
L-R: various diatoms with characteristic silica shells; a satellite image of a phytoplankton bloom; T. pseudonana cells, the first lying on its side and the circular one in valve view.
Discussion
This research was possible due to the RIOS NSF REU program. Thanks to Liti Haramaty, Chris Brown, Frank Natale, and the rest of the Bidle lab for tactical and practical help. Thanks to Kay Bidle and Kim Thamatrakoln for intellectual support and mentoring.
Protein was extracted using 2% SDS then was run by SDS-PAGE. Proteins were transferred to a PVDF membrane, which was probed with with GFP antiserum to measure GFP expression.
Protein was extracted using sonication, then caspase specific activity was found by measurement of IETD-AFC substrate cleavage normalized to protein concentration.
Fv/Fm of Fe- time course
0.200
0.250
0.300
0.350
0.400
0.450
0.500
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1 2 3 4 5 6 7
Day
Fv/F
m
wt
18-58
37-22
Activities and Growth Curve
1(1.09)
(1.5)(3.3)(1.8)(10)
(23)0
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1 2 3 4 5 6 7Day
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1 m
g-1
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1.00E+06
1.00E+07
cell c
oncentr
ati
on (
cells/m
L)
wt activity
18-58 activity
37-22 activity
wt
18-58
37-22
DSP1 or DSP2fcp GFP Figure 1. DSP-GFP fusion vector
Wild-type 18-58 37-22
Ho
ech
st
G
FP
Ch
loro
ph
yll
DIC
