Algae under stress: Characterisation and comparison of bioenergetics and
intracellular signalling during stress in two model algal systems.
Freshwater algae are important primary producers with very cosmopolitan distribution[1].
They are eukaryotic organisms that share many cellular characteristics with higher
organisms. Developing an understanding of these organisms is therefore important both
because they may serve as good model systems for eukaryotic cell stress responses and
because they have significant environmental and economic importance. Algae are
subjected to a wide range of environmental stresses in the natural environment[2]. The
current proposal will examine the relationship between two such stressors; the response
to high light stress and the response to phosphate contamination of the aqueous
environment.
High light stress is currently of relevance because there is mounting evidence that climate
change is resulting in higher light levels and particularly increased UV light
components[3] while watercourses are also experiencing increased phosphate
contamination as a result of agricultural ‘run off’ and industrial effluent discharge[4]. The
combination of these stressors will thus become increasingly important in the natural
environment. There has been some work on high light stress in algae[5-9] but
understanding of the cell biology of these responses is still poor and very little work has
been undertaken on the combined effect of pollutants and high light stress. Central to
understanding these responses is elucidation of the signalling mechanism modulating the
response, which is probably a calcium signalling pathway[10], together with an
understanding of the bioenergetics of the response which is critical to the energy budget
of the organism and hence survival.
The current project aims to characterise at a cell biology level, the stress response of two
different model systems (Chlamydomonas rheinhardtii, a mobile unicellular alga and
Mougeotia scalaris, a non-mobile alga with a well known chloroplast avoidance
response[11], to light and phosphate stress alone and in combination. The project will test
the following hypotheses:

There are significant differences in the bioenergetics of stress responses to high
light and phosphate contamination between Mougeotia scalaris and
Chlamydomonas rheinhardti.

There are significant differences in the intercellular signalling during stress
responses to high light and phosphate contamination between Mougeotia scalaris
and Chlamydomonas rheinhardti.

The stress responses in Mougeotia scalaris and Chlamydomonas rheinhardti to
high light and phosphate contamination are modulated by a dynamic intracellular
calcium signal.

The stress response in Mougeotia scalaris and Chlamydomonas rheinhardti to
high light and phosphate contamination stress in combination, differs to the stress
response to either stressor alone.
The project will be based on pure cultures of each organism kept under controlled
conditions of nutrition and light. Light stress will be delivered using a custom built
apparatus able to deliver controlled levels of high light illumination over a variable stress
period while phosphate stress will be delivered by means of addition of this contaminant
to culture media. The stress responses will be characterised by means of a combination of
Clark oxygen electrode analysis of the photosynthetic response of the organisms, by
HPLC determination of pigment changes, especially of the carotenoids together with vital
and fixed cell microscopy as outlined below.
The stress responses at an intracellular level will be assessed in terms of changes in
bioenergetics of the cells and in terms of the presence and pattern of dynamic calcium
signals using vital cell microscopy[12]. An outline of the parameters to be measured for
each of these is shown in Table 1.
Cellular bioenergetics during and following
the stress response
Distribution and movement of mitotracker
orange labelled mitochondria
Changes in the mitochondrial potential assessed
using TRME, JC-1and rhodamine -123
Changes in superoxide and peroxide production
within the mitochondria
Calcium signalling
Degree and propagation of cytoplasmic calcium
signals during the stress response and recovery
period assessed using FURA ratiometric
calcium signals
Degree of modulation of calcium signal by
mitochondrial calcium uptake / release assessed
using RHOD-AM mitochondrial targeted
calcium sensitive dye
Pattern of movement and potential changes
within calcium containing vesicles assessed
using lysosensor blue / yellow
Changes in ATP production and usage during
the response measured using a combination of
ATPase blockers and uv uncaging of caged
ATP to deliver a known ATP dose to specific
areas within the cell
Table 1: Key live cell imaging techniques proposed.
The live cell imaging will be based on an optical fluorescence microscope which has
optical sectioning capabilities based on controlled z movements and then optical
deconvolution[13] (Olympus BX61 with Hyugens and Image Pro plus software).
Data collected will be analysed by use of SPSS and Prism to characterise the stress
responses of the model organisms and to compare them to each other. Logistic regression
will be used to explore the interaction of the stressors with each other in each model
system.
It is expected that the PhD will be completed in a 5 year 3 day per week time frame. The
initial 6 months will be spent optimising culture conditions, organising the equipment
needs and developing the initial imaging set up (for example environmental chamber
optimisation and light levels etc). The next 12 months will be spent characterising the
baseline responses of the model organisms (for example the non stressed light curves in
the oxygen electrode), optimising the loading conditions for dye loading and developing
the deconvolution parameters for deconvolution of the live cell data. The 24 months
following this will be utilised collecting the experimental data on each model system
under the range of single and combined stressors using the techniques outlined above.
The final 18 months will be spent revising such experimental data as required and
completing the thesis write up. The literature review and write up will be concentrated at
the beginning and end of the project but will also be undertaken throughout the project.
References
1.
Lee, R.E., Phycology. 3 ed. 1999, Cambridge: cambridge university Press.
2.
Mendez-Alvarez, S., U. Leisinger, and R.I. Eggen, Adaptive responses in
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3.
Hader, D.P., Effects of solar UV-B radiation on aquatic ecosystems. Adv Space
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Imaging. Science, 2003. 300: p. 82-86.
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deconvolution. Cell structure and function, 2002. 27: p. 335-341.