Experiments with Thalassiosira weissflogii (TW) in Relation to Changing Oceanic Carbon

Dioxide Concentrations

Raheel AnwarMorel Lab

CO2 Concentrations and Why They’re ImportantThe changing environment has led to higher

dissolved cabron dioxide concentrations in the world’s oceans due to global warming and the greenhouse gas effect

Higher CO2 concentrations,which can easily be converted to bicarobonate, cause changes in the pH or acidity of the world’s oceans

http://switchboard.nrdc.org/blogs/lsuatoni/why_scientists_agree_ocean_aci.html

The CDCA EnzymeCadmium carbonic anhydrase

Catalyzes the conversion of CO2 into bicarbonate and viceversa

Cadmium is a normally toxic metal, but here, it can act as a coenzyme for the reaction

Why TW and CDCA?Knowing where and in what quanitities this

enzyme is located in the algae is importantTells us how this species and other algae like it

can cope with the changing conditions of the oceans

Allow us to anticipate the effects of future changes

Overview of WorkStudied the expression of the CDCA enzyme

in TW under different pH conditionsIsolated chloroplasts to determine CDCA

activity and localization

CDCA AnalysisLooked at TW cells growing under:

High (8.6)Medium (8.2) and Low (7.8) pH conditions.

Western Blot

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000

CDCA Expression under Different pH Conditions

LowMediumHigh

Intensity of CDCA band in units

Isolation of ChloroplastsDeveloped a procedure for the isolation of chloroplasts First, sonication was used to break open cells without

entirely destroying all the chloroplasts Cells are resuspended in a buffer designed to protect

the chloroplasts that are now floating free in the solutionSolution is filtered on a 10 micron filter, allowing TW

cells, most of which are 10 micron or bigger to be caught on the filter, while chloroplasts, which necessarily are smaller, pass through.

This reduces whole cell contaminants remarkablyChloroplasts are then isolated on a gradient for further

analysis

MIMS Assay of CDCA activity for Chloroplast ExtractResults showed that CDCA activity was not

present in the chloroplastsReasons:

CDCA enzyme could be located outside of the chloroplasts

Whole cell activity is ruled out since the pellet of chloroplasts is washed many times

Chloroplasts could get inactivated, but fluroescence microscopy shows only slight decrease in output

Signals from the nucleus may be necessary for CA activity

ConclusionsOverall, higher concentrations of CDCA are

found when the pH is higher, giving us clues on how algae in the oceans are effected

Isolation of chloroplasts showed no signs of CDCA activity

Thanks toPEI/Grand ChallengesDr. Yan XuProfessor Morel