Task 2.1. Molecular markers
The objective of this work was to assess the use specific biomarkers
(flavodoxin, Rubisco, superoxide dismutase) in response to iron stress in
different species of phytoplankton. Most attention was given to the use of flavodoxin,
a functional homologue of ferredoxin, as a molecular marker. The protein
flavodoxin has been identified as a putative marker of iron limitation in
marine diatoms, (McKay et al. 1997, La Roche et al., 1993, 1995, Doucette et
al., 1996). The available probe for flavodoxin, a polyclonal antiserum raised
against flavodoxin isolated from the diatom Phaeodactylum tricornutum,
is primarily immunoreactive against diatoms, (La Roche et al. 1993,1996, McKay
et al. 1999).
Several Chaetoceros species were studied to assess the impact that
iron limitation had upon their growth, physiology and flavodoxin expression.
Three model species were studied, C. calcitrans, C. muelleri and C.
brevis, coastal, oceanic and Antarctic isolates with high, medium and
low iron requirements respectively. Western blot analysis demonstrated that all
three species had a measurable response to iron limitation with respect to the
abundance of flavodoxin in extracted cell protein. Flavodoxin abundance was
normalized to Rubisco abundance (also measured by western blot analysis).
Rubisco was not seen to vary significantly in iron starved or nutrient replete
samples when similar concentrations of cell protein were probed by western blot
analysis. The Rubisco signal was therefore used as a measure of biomass to
which other immuno-reaction signals could be normalized. Hence flavodoxin
abundance was expressed as the flavodoxin signal relative to the Rubisco signal
of the same sample when challenged with anti-serum raised against flavodoxin and
against Rubisco. Although flavodoxin abundance varied in each species, our
results indicated that flavodoxin expression alone could not indicate the
extent of iron limitation. In conjunction with other markers, such as
ferredoxin abundance, and physiological measurements the present anti-serum for
flavodoxin proved a useful tool to assess iron-stress in laboratory diatom
cultures. Relationships between flavodoxin expression, growth rate and various
photosynthetic parameters were established for C. muelleri and describe
the cell response to iron stress (Davey and Geider, submitted).
The use of flavodoxin in single-cell immunofluorescence assay in conjunction
with flow cytometric analysis was attempted using laboratory diatom cultures.
Although some success was achieved, poor antibody specificity proved to be
problematic. The available ferredoxin anti-serum was used in western blot
analysis and single cell assay in an attempt to quantify ferredoxin expression
in the model diatoms. The antibody concentration and specificity did not appear
to be sufficient to be used to quantify ferredoxin in whole cell assay or
non-purified cell extract of these diatoms. Attempts were made to improve the
available ferredoxin antibody and to isolate new molecular markers such as
ferritin an iron storage protein (Laulhere et al. 1991). Additional large-scale
culture work was carried out to isolate ferredoxin protein from Chaetoceros
species to be used in the production of antiserum with greater specificity.
The synthesis of the different forms of SOD (superoxide dismutase) was
studied in cultures of C. brevis which were grown in natural Antarctic
seawater. Four different treatments were applied, one with an addition of 10 nM
Fe, one with an addition of 10 nM Mn, one with an addition of 40 nM Cu and Zn
and one control. When these cultures reached the stationary growth phase they
were harvested. The proteins were isolated and separated using electrophoresis,
after which the gel was horizontally sliced. These slices were analysed using
atomic absorption spectrometry for Fe, Mn, Cu and Zn. This experiment was
repeated, however the results were not consistent. There was no clear signal of
the Mn and Fe SOD’s (~ 31 kD) or the Cu/Zn SOD (~85 kD) after staining of the
gel or in the profiles constructed after the element analysis. SOD’s were found
in all four treatments using the reaction catalysed by these enzymes. No
differences were found in any of the four treatments.
References
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