Investigating the protection mechanisms of the
photosynthetic apparatus of Nannochloropsis oceanica
Gunvor Røkke, Thor Bernt Melø, Martin Frank Hohmann-Marriott
Abstract
Introduction
Methods
Results
Conclusion
Abstract
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Nannochloropsis is a heterokont alga emerging as a model organism. It is currently being
investigated both for its abilities to produce lipids (Singh & Gu, 2010) and commercially
interesting pigments (Lubián et al., 2000).
We investigated how the photosynthetic machinery of Nannochloropsis oceanica responds to
high light and anaerobic conditions. High light could potentially damage PSII, and therefore
many algae species have developed protection mechanisms, such as the xanthophyll cycle (Jahns
& Holzwarth, 2012) or state transitions (Haldrup et al., 2001).
We gained valuable insight into the interpretation of PAM fluorometry saturating light pulse
analysis in Nannochloropsis, that is incompatible to conventional interpretation in other model
organisms. This revised interpretation of PAM fluorometry in Nannochloropsis helps us to reevaluate experimental data. Our results shows that Nannochloropsis carries out state transitions,
while the xanthophyll cycle only plays a minor role in photoacclimation.
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Introduction
Nannochloropsis
The xanthophyll cycle
State transitions
The PQ pool
Alage and lipids
Nannochloropsis
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Introduction
•
Heterokont microalga
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Diameter: ~ 3 µm
•
Haploid genome of about 28,7 Mb
(Vieler et al., 2012)
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Performs oxygenic photosynthesis
•
Enthusiastic lipid producer
–
ω3-fatty acids
–
Saturated fatty acids relevant for
biodiesel production
The xanthophyll cycle
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Introduction
The xanthophyll cycle is one of the most important light protection
mechanisms known to date. It is initialized by decreased lumenal pH
in the thylakoids, which normally occurs when the photosynthetic activity is high, and the alga has an
increased rate of water splitting and plastoquinone transport. The resulting low lumenal pH activates
violaxanthin de-epoxidase, which converts violaxanthin (light harvesting) to zeaxanthin
(photoprotective) (Cao, 2013). Zeaxanthin disposes of excess photons by emission of heat, and thus
protects PSII from damage. The xanthophyll cycle has previously shown to be active in Nannochloropsis.
State transitions
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Introduction
State transitions, alongside with the xanthophyll cycle, is one of the
most important light photoprotection mechanisms in algae.
State transitions are initiated in conditions reducing the plastoquinone pool. Reduced
plastoquinones activates a thylakoid-bound kinase, which enables light harvesting complexes from
LHCII associated with PSII to travel to the far more efficient PSI (Haldrup, 2011). This process
balances out the ratio of light distributed to the two photosystems, and make the algae able to
function more efficiently in high-light conditions. It also preventins photo damage on PSII.
State transitions has so far not been detected in Nannochloropsis.
The plastoquinone pool
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Introduction
The plastoquinone (PQ) pool, and especially its degree of reduction,
plays a role in the onset of photoprotection mechanisms. The PQ pool
is mainly regulated by the outer environment of the algae. The most obvious environmental
factor impacting the PQ pool is high light. During high light, LHCII will harvest more light, and
the rate of water splitting and plastoquinone transport will increase. Although less evident, this
will also happen in anaerobic conditions.
In anaerobic conditions, the algae’s mitochondria are deprived of their usual electron acceptor,
and excess electrons from mitochondrial electron transport will instead be used to reduce NAD+.
Through a line of electron transfer reactions, these exess electrons can also find their way to the
chloroplast, and cause the plastoquinone pool to be even more reduced than in high light.
Algae as lipid producers
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Introduction
With rising concentrations of climate gasses in the atmosphere, and an
increased will to find alternative carbon sources, algae could be the solution!
Microalgae produces relatively large amounts of lipids (Xiao, 2013; Radakovits, 2012), and these
lipids can be extracted and converted to biodiesel. Another interesting group of lipids produced
by microalgae is polyunsaturated fatty acid (Vieler, 2012). In addition, as photosynthetic
organisms, they produce commercially interesting antioxidants (Lubián, 2000). The production of
both lipids and antioxidants can be regulated by external conditions, and both the lipid synthesis
pathways and the antioxidant production pathways are tightly connected with photosynthesis.
Materials and methods
Cultivation
Experiment setup
77K spectroscopy
PAM fluorometry
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Cultivation
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Strain:
–
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Growth temperature:
–
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18 °C
Light intensity:
–
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Nannochloropsis oceanica CCMP1779
200 µmol photons/m2s
Chlorophyll a concentration used in
measurements:
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40 µg chl a/ml
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Methods
Experiment setup
•
Investigation of the redox state of the PQ pool in Nannochloropsis
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Investigated in high light (with and withouth DBMIB) and in anaerobic
conditions
•
–
Investigated by PAM fluorometry and analysis of fluorescence induction curves
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Contionous measurement performed over time
Investigation of photoprotection mechanisms in Nannochloropsis
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Investigated in anaerobic conditions and high light
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Investigated by utilizing CCCP, DTT and nigericin
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Investigated by PAM fluorometry and 77K spectroscopy
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PAM fluorometry performed as continous measurement over time
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Samples for 77K taken at t = 0, t = 5 m, t = 10 m, t = 30 m, t = 60 m, t = 90 m
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Methods
77K spectroscopy
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Methods
77K spectroscopy was performed using a
custom made setup coupled to a Jaz
spectrophotometer (Ocean Optics). Spectra
were recorded by the SpectraSuite software
(Ocean Optics).
The recorded spectra were base line
corrected and normalized.
PAM fluorometry
PAM fluorometry was performed using a multicolour
PAM fluorometer (Walz) coupled to an oxygen measuring
device.
Four different measuring lights were utilized in a 4
minute sequence (see below).
Once every minute, a saturating light pulse of 440 nm
was given.
Subsequent to measurements, the raw data was analysed
by a custom made MATLAB program.
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Methods
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Results
Redox status of the PQ pool
Photoaclimation mechanisms
Redox status of the Nannochloropsis PQ pool
The difference in maximum fluorescence (Fm)
between darkness (A’) and high light (A’’),
aerobic conditions (B’) and anaerobic
conditions (B’’), and darkness without DBMIB
(C’), darkness with DBMIB (C’’), and high light
with DBMIB (C’’’) was investigated by PAM
fluorometry.
The results show that the maximum
fluorescence was highest when high light and
DBMIB was utilized simultaneously. The Fm
values being different in the three
experiments also indicate that the redox state
of the PQ pool is not the same in the
investigated conditions.
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Results
Further
Redox status of the Nannochloropsis PQ pool
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Results
The fluorescence induction curves recorded by PAM
fluorometry (upper figure) were analyzed further,
and the rate of fluorescence decay was found for all
phases of all experiments (lower figure). QA is the
main fluorescence modulator in Nannochloropsis, and
fluorescence decreases when QA transfers an electron
to the PQ pool. A low rate of decay indicates a high
degree of reduction in the PQ pool. The analysis
shows that high light by itself is not sufficient to
obtain a reduced PQ pool in Nannochloropsis.
Further
Redox status of the Nannochloropsis PQ pool
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Results
Conclusions:
•
The underlying assumptions for SAT pulse analysis (saturating light pulses leading to
a reduced plastoquinone pool) are not valid in Nannochloropsis
•
Our results show that the plastoquinone pool has a higher degree of reduction in
anaerobic condition compared to in high light.
Conclusions with relevance to photoaclimation:
•
Conventional interpretation of SAT pulse analysis in Nannochloropsis could lead to an
under-reporting of photoprotection mechanisms. This might be the reason why state
transitions have never been observed in this alga.
Photoacclimation in Nannochloropsis
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Results
In order to investigate photoacclimation mechanisms in Nannochloropsis, we subjected
the alga to high light and anaerobic conditions. To find the contribution of the
xanthophyll cycle to the overall non-photochemical quenching (NPQ), three different
inhibitors were utilized. Two of these, CCCP and nigericin, are known to remove any pH
gradients over membranes, while DTT is a direct inhibitor of the enzyme violaxanthinde-epoxidase. Our results show that despite what has earlier been assumed,
Nannochloropsis does indeed seem to carry out state transitions. Our results also show
that the xanthophyll cycle plays a smaller role in the overall photoacclimation than
previously thought.
Further
Photoacclimation in Nannochloropsis
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Results
Nannochloropsis cells were subjected to
anaerobic conditions, sampled over time and
investigated by 77K spectroscopy. The results
show a shift of pigments from photosystem II
(emitting fluorescence at 687 nm) to
photosystem I (emitting fluorescence at 720
nm) over time. This indicates that
Nannochloropsis does indeed carry out state
transitions as part of its photoacclimation.
Further
Photoacclimation in Nannochloropsis
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Results
Nannochloropsis cells were subjected
to high light, sampled over time and
investigated by 77K spectroscopy.
The results obtained in high light
does indeed show a slight shift of
fluorescence intensity towards PSI
over time, but this is less evident than
in anaerobic conditions, indicating
that the plastoquinone pool is less
reduced in high light, than in
anaerobic conditions.
Further
Photoacclimation in Nannochloropsis
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Results
Maximum fluorescence over time found in Nannochloropsis cells subjected to anaerobic conditions (left figure)
and high light (right figure). Fluorescence emission is in an inverse relationship with NPQ, so a decrease in
fluorescence indicates an increase in NPQ. If the xanthophyll cycle was an essential part of the photoacclimation
in Nannochloropsis, a difference should have been observed between the fluorescence kinetics in the experiment
where no inhibitor was utilized, and the other experiments. This is not the case here. Instead, NPQ is increasing in
all anaerobic experiments, while staying relatively stable in all high light experiment. This indicates state
transition activity rather than an active xanthophyll cycle.
Conclusion
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Our results indicate that Nannochloropsis might be a high light-acclimated organism. High light does not
lead to significant changes in the redox state of the plastoquinone pool. Anaerobic conditions significantly
increases the reduction degree, but we still observed that the PQ pool is not completely reduced until an
inhibitor is added.
Keeping this in mind, we investigated photoacclimation mechanisms in Nannochloropsis, and despite what
has previously been assumed, we found Nannochloropsis to perform state transitions, while our results
indicate that the xanthophyll cycle contributes to non-photochemical quenching to a minor extent.
References
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