Eur. J. Phycol. (2015), 50: 469–480
Flashing light enhancement of photosynthesis and growth
occurs when photochemistry and photoprotection are balanced
in Dunaliella salina
SAID ABU-GHOSH1,3, DROR FIXLER2,3, ZVY DUBINSKY1, ALEXEI SOLOVCHENKO4,
MIRIAM ZIGMAN1, YARON YEHOSHUA1 AND DAVID ILUZ1
1
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel
Faculty of Engineering, Bar-Ilan University, Ramat-Gan 5290002, Israel
3
The Institute of Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat-Gan 5290002, Israel
4
Department of Bioengineering, Faculty of Biology, Moscow State University, 119234, GSP-1 Moscow, Russia
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2
(Received 12 January 2015; revised 26 April 2015; accepted 30 April 2015)
Photosynthesis and growth in Dunaliella salina were significantly (P ≤ 0.01) enhanced under flashing light with a light–dark cycle
tailored for the minimal activation of the violaxanthin cycle (VC), in comparison with cultures grown under constant illumination
of the same time-integrated photon dose. The experiments were performed without using inhibitors or any other experimental
conditions other than light. The flashing light (FL) was optimally balanced for the growth and photosynthesis of D. salina, which
was characterized by a decreased VC de-epoxidation rate. The high-light induction of non-photochemical quenching (NPQ) was
significantly slower in the FL-acclimated cells compared with those acclimated to continuous illumination. The comparative
investigation of the cell ultrastructure revealed distinct effects of high-light stress in cultures grown under continuous light; this
was not the case under FL. We conclude that a slower activation of the VC takes place under balanced flashing light, when each
flash is followed by a dark period long enough for utilization of the photons absorbed during the flash at a given irradiance. The
activation kinetics of the VC and, hence, the induction of NPQ seem to play an important role in the enhancement of the
photosynthesis observed in D. salina grown under flashing light. Potential use of these parameters for optimization of FL for
microalgal cultivation is discussed.
Key words: flashing light, microalgae, non-photochemical quenching, photosynthesis, ultrastructure, violaxanthin cycle
INTRODUCTION
Light energy is necessary to drive photosynthesis,
although excessively absorbed light leads to photoinhibition (Raven, 2011). Photoautotrophs, including
microalgae, are capable of growth at a wide range of
light intensities by means of different acclimation
mechanisms (Sharma et al., 2012), including xanthophyll cycles. Currently, six types of xanthophyll
cycles are known (Latowski et al., 2011), four of
which are described in algae (Lunch et al., 2013). In
chlorophytes, the violaxanthin cycle (VC) operates
based on the reversible irradiance-dependent interconversion of violaxanthin (Vio) to antheraxanthin (Ant),
and then to zeaxanthin (Zea), by Vio de-epoxidase.
The VC exerts its photoprotective effect via the accumulation of Zea-facilitating thermal dissipation of
excessive light energy, thus giving rise to non-photochemical quenching (NPQ) (Sirikhachornkit &
Niyogi, 2010; Demmig-Adams et al., 2012).
Correspondence to: Said Abu-Ghosh. (e-mail:
[email protected])
The scattering and attenuation of light penetrating
the water surface, as well as the lens-like light-focusing properties of surface waves, form (in natural water
bodies as well as in artificial cultivation systems)
fluctuating illumination domains, with irradiances
ranging from supersaturating to complete darkness
(Iluz et al., 2012). Since the VC operates on a timescale of seconds to seasons (Demmig-Adams et al.,
2012), it is crucial to microalgae dwelling in habitats
with abrupt fluctuations of irradiance for the establishment of an optimal balance of light harvesting and
photoprotection (Lohr & Wilhelm, 1999; DemmigAdams & Adams, 2006; Jahns & Holzwarth, 2012;
Lunch et al., 2013).
Fluctuating light environment, referred to as intermittent or flashing light (FL), has been found to have a
beneficial effect on microbial photosysnthesis in some
instances, especially in cultures illuminated by flashes
of supersaturating intensity (Nedbal et al., 1996; Park
& Lee, 2001; Vejrazka et al., 2013). As reviewed by
Zarmi et al. (2013), this effect is explained by the
optimal balance of flash duration and intensity vs.
ISSN 0967-0262 (print)/ISSN 1469-4433 (online)/15/040469-480 © 2015 British Phycological Society
http://dx.doi.org/10.1080/09670262.2015.1069404
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S. Abu-Ghosh et al.
470
the following dark period, allowing efficient photochemical utilization of the absorbed light energy. Although
the regulatory mechanisms, including those related to
VC operation and NPQ induction, were thought to play
an important role in the FL effect in real systems, this
question remains largely open (Zarmi et al., 2013). The
role of the VC in enhancing the photosynthesis of
microalgae acclimated to flashing light (FL) is insufficiently studied. It was reported that Zea accumulates in
the cells of the chlorophyte Chlorella pyrenoidosa
under single-turnover flashes fired at a low (non-actinic) frequency, although no relationship was found
between the kinetics of Zea formation and the acclimation to FL (Schubert et al., 1994).
To examine the role of the VC in acclimation to and
regulation of photosynthesis under FL, we chose the
chlorophyte Dunaliella salina, a model species for
studying carotenoid (Car) metabolism (Smith et al.,
2010). This microalga is capable of growth in different
light zones due to the versatility of its photosynthetic
apparatus (Vasilikiotis & Melis, 1994; Kleinegris et al.,
2010; Fu et al., 2013) and the ability to accumulate high
amounts of β-carotene (Jin et al., 2003; Ye et al., 2008)
in response to high light (Lamers et al., 2010).
Besides its importance as a model system, D.
salina is a biotechnologically important species
used for the industrial production of β-carotene
(Lamers et al., 2012) by means of the cultivation of
shallow, open ponds at high cell densities (BenAmotz, 2004; Borowitzka, 2013). Such cultivation
conditions lead to the formation of a steep light
gradient, ranging from supersaturating irradiances
at the surface (when the absorbed light energy is
lost due to NPQ build-up) to light-limiting conditions at the bottom of the pond (leading to net energy
loss due to respiration).
In this context, diverse parameters affected by
light fluctuations, such as changes in chlorophyll
(Chl) fluorescence (Vincent, 1980), the composition
of photosynthetic pigments (Perry et al., 1981;
Sukenik et al., 1987) and ultrastructural features of
the cell, as well as growth, oxygen evolution and
dark respiration rates, and VC activation kinetics,
have been investigated. In our study, D. salina was
grown in a flat-panel photobioreactor at various constant-light (CL) and FL settings, mimicking, to a
certain extent, the irradiance fluctuations in natural
water bodies and microalgal cultivation facilities. We
report here that FL conditions, combining high incident photon flux with dark intervals that were sufficiently long for the utilization of the light absorbed
during the flash, enhance the growth of and photosynthesis in D. salina, thus reducing the manifestations of high light stress. It was also demonstrated
that a slowing down of VC activation was characteristic of the FL enhancement of microalgal photosynthesis and, hence, enhancement of culture
growth under FL of supersaturating intensity.
MATERIALS AND METHODS
Microalgal strain and growth conditions
Dunaliella salina was obtained from the Interuniversity
Institute for Marine Sciences, Eilat, Israel, and grown at
23°C in a sterile, filtered F/2 medium (Guillard, 1975) with
27 g l−1 sea salts obtained from Instant Ocean (Blacksburg,
Virginia, USA). The F/2 medium was modified by increasing
the nitrogen concentration to 1.5 g l−1 NaNO3, and adjusted
to a pH of 7.6. Precultures were grown at 85 μmol photons
m−2 s−1 under weekly dilution.
Special care was taken to achieve uniform irradiance in the
photobioreactor in order to minimize interference by mutual
shading of the cells for the reliable examination of FL effects.
The experiments were conducted in a 70 ml flat-panel custom-designed photobioreactor of ~1 cm light-path (Figs S1–
S3). Illumination was provided with a home-made panel of
six EdiStar 120-W point-source light-emitting diodes (LED)
(Edison, Karlsbad, Germany) generating, under software
control (LabView 8.6, National Instruments, Austin, Texas,
USA), square-wave flashes of programmable intensity, frequency, and time. To efficiently acclimate the cells, cultures
were exposed to the different light regimes for 24 h a day
without dark periods. Temperature was maintained at 23°C.
The parameters of the FL, such as flash time (tf), dark time
(td), duty cycle [φ = tf/(tf + td) (Vejrazka et al., 2011)] and
incident intensities, are summarized in Table 1. Light intensity was adjusted daily, based on readings made along the
whole optical path of the photobioreactor with a built-in
spherical PAR sensor (Fig. S2). To avoid interference from
ambient light, all experiments were performed in a dark
room. For inorganic carbon supply and mixing, the suspension was constantly bubbled with a CO2 (5% v/v):air mixture
(Sforza et al., 2012) at a rate of 70 ml min−1. The pH of the
medium was maintained at ~8 with 40 mM Tris HCl buffer.
Algal growth was monitored via optical density at 750 nm
(OD750) using a NanoDrop 2000c spectrophotometer
Table 1. Irradiation parameters used in the cultivation of Dunaliella salina.
Designation
CL500
FL75067%
10 Hz
FL100050%
50 Hz
FL200025%
50 Hz
Incident irradiance (μmol
photons m−2 s−1)
500
Flash frequency Flash time
(Hz)
(ms)
Continuous
∞
Dark time
(ms)
Duty cycle, φ
(%)
Time-integrated irradiance (μmol
photons m−2 s−1)
—
100
500
750
10
67
33
67
500
1000
50
10
10
50
500
2000
50
5
15
25
500
Flash-photoacclimated microalgae
(Thermo Scientific, Wilmington, DE); cell count with a BD
FACSCalibur flow cytometer (Breda, the Netherlands) and
dry mass accumulation (Abu-Ghosh et al., 2015b). Two
independent experimental repetitions were carried out with
four samples analysed from each. Average values ± SD are
presented unless stated otherwise.
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Monitoring of photosynthetic activity
To enable variable Chl fluorescence monitoring, the photobioreactor was equipped with a novel PAM system,
AlgaeGrowth-PAM, designed in our laboratory and manufactured by Heinz Walz (Effeltrich, Germany) (Figs S1–S2),
controlled by custom WinControl-3 software (Fig. S4). This
software uses two empirical functions to estimate the photosynthesis cardinal data: the functions REG1 and REG2 have
been introduced by Platt et al. (1980) and Jassby & Platt
(1976), respectively, to describe classical light response
curves of photosynthesis. Photosynthetic O2 production and
respiration rates were recorded in the microalgal suspensions
in the late exponential phase (day 8) at actinic light of 110,
150, 230, 330, 480 and 1000 μmol photons m−2 s−1, with a
Clark-type oxygen electrode (Hansatech Instruments Ltd.,
Norfolk, UK) at 23°C in a sterile, closed chamber under
vigorous stirring. Exponential fitting was performed to calculate the alpha value and to extract Pmax values.
The D. salina cultures acclimated to different CLs and FLs
(Table 1), were dark-adapted for 60 min at 23°C prior to Fv/
Fm measurements. The minimum fluorescence level (Fo) in
dark-adapted samples was measured using a weak (< 10 μmol
photons m−2 s−1) measuring beam, which did not, under our
experimental conditions, trigger the Chl fluorescence transient. The algal cultures were subjected to a 0.6 s saturating
pulse (10 000 μmol photons m−2 s−1) to measure maximum
photosystem II (PSII) fluorescence in the dark-adapted state
(Fm). Then the cultures were illuminated with stepwise
increasing actinic light (0, 110, 150, 230, 330, 480 and
1000 μmol photons m−2 s−1) for a total period of 16 min,
followed by 30 min of dark recovery. During the actinic
illumination and subsequent dark recovery, maximum PSII
fluorescence in the light-adapted state (Fm’) was measured
using the same saturating pulses. During Chl fluorescence
measurements, the cell suspensions were not stirred to avoid
interference from ‘unquenched’ cells moving in and out of
the measuring light path. The potential maximum efficiency
of PSII was calculated as Fv/Fm = (Fm−Fo)/Fm, and the
Stern–Volmer coefficient of non-photochemical quenching
(NPQ) was calculated as (Fm−Fm′)/Fm′ (Demmig-Adams
et al., 1996).
Monitoring of the VC operation in Dunaliella
suspensions
Absorbance in the blue-green region of the spectrum (sensitive to carotenoid (Car) and chlorophyll (Chl) concentrations), corrected by subtraction of that in the green region
(sensitive mainly to Chl), was reported to reflect the interconversion of the VC xanthophylls (Peguero-Pina et al.,
2008; Garbulsky et al., 2011). The absorbance band used
for monitoring of the VC operation (505 nm) was shifted
aside from the xanthophylls’ main absorption maxima to
avoid strong, overlapping interference from Chl b (Johnson
471
et al., 2009). The reference band (565 nm) was selected in
the region which is predominantly governed by Chl absorption. As a result, the kinetics of ΔA505 calculated as ΔA505(t)
= A505(t) − A565(t) was used as a proxy for Zea buildup in
the course of VC upregulation (Johnson et al., 2009).
Spectra in the 400–700 nm range were recorded using
a Cary 300 UV-visible spectrophotometer (Agilent
Technologies, California, USA).
Fresh precultures of D. salina grown, as described above,
in CL or FL were dark-acclimated and illuminated with
actinic light of μmol photons m−2 s−1 for 10 min, and then
dark-relaxed for the same period at 23°C. According to the
results of our pilot experiments, this actinic irradiance readily
induces the de-epoxidation of Vio but does not induce the
slow, reversible photoinhibitory component (qI) of NPQ in
D. salina. The 3 ml samples were taken from the lightstressed and dark-relaxed cultures at one-minute intervals.
The absorption spectra of the samples were measured in a
1 cm glass cuvette (NanoColor 919 33, Düren, Germany). At
least three measurements were carried out for each light
treatment.
Pigment analysis
About 10 ml of each culture was taken during the late exponential growth phase of photoacclimation and then darkacclimated for 60 min (Bonente et al., 2012). Three millilitres
of the dark-adapted sample was passed through a glass fibre
filter (Whatman GF/C, Schleicher and Schuell Co., Keene,
New Hampshire, USA), and the filter with the cells was flashfrozen in liquid nitrogen. The same volume of the darkadapted cell suspension was illuminated with actinic light
of 700 µmol photons m−2 s−1 for 10 min and processed as the
dark-acclimated sample. The frozen discs were extracted
with 5 ml of 90% (v/v) acetone by homogenizing for 1 min.
The extract was filtered, and the filtrate was retained. The
pigments were re-extracted from the filter debris by adding 2
ml of 90% (v/v) acetone and mixing vigorously. The extract
was filtered again and pooled with the filtrate from the previous extraction. A 15 µl aliquot of the pooled extract was
injected into an Elite LaChrom HPLC (Hitachi, Tokyo,
Japan) equipped with an autosampler (L-2200) and a diode
array detector (L-2455). Carotenoids and chlorophylls were
separated on a Varian Microsorb-MV 100-S C18 (250 mm
long, 4.6 mm i.d.) cartridge column (Waters, Milford,
Massachusetts, USA) at 30°C, as previously described
(Müller-Moulé et al., 2002). Chlorophyll in photoacclimated
algal cells was extracted with acetone, as previously
described. The amounts of Chl and Car in the extracts were
determined according to Solovchenko et al. (2010). Two
independently grown cultures were analysed for each light
treatment, each in triplicate.
Transmission electron microscopy (TEM)
Samples were fixed in a 0.1 M cacodylate buffer containing
3% glutaraldehyde (v/v) overnight at 4°C, and post-fixed in
1% OsO4 in the same buffer for 2 h. The samples were then
dehydrated through a graded ethanol series [50, 70, 90 and
100% (v/v in double-distilled H2O) for 10 min, twice at each
concentration] at room temperature and embedded in araldite
resin (Sigma-Aldrich, St. Louis, Missouri, USA). Sections of
S. Abu-Ghosh et al.
472
0.05 μm thickness were obtained with an ultramicrotome,
stained in uranyl acetate in ethanol for 30 min at room
temperature, and viewed using a Tecnai T12 transmission
electron microscope (FEI, Hillsboro, Oregon, USA).
RESULTS
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Effect of flashing light on growth and pigment
composition of the culture
All cultures were exposed to FL, with the same timeintegrated photon flux equivalent to 500 µmol photons
m−l s−s of CL (designated as CL500). This particular
PAR intensity was chosen, according to the results of
pilot experiments, as a trade-off between the photon
flux suitable for culture growth and the necessity to
impose a light stress sufficient for VC activation in D.
salina (Figs S5–S8). The substantial differences in the
cell number and biomass accumulation of the cultures
grown at different light regimes were recorded at an
early stationary phase (3rd day of cultivation;
Figs S5–S8). The cultures grown under FL with different light/dark-period ratios, generally displayed
higher growth rates in comparison with the CL cultures (Figs 1 & 2); the highest increase was recorded
under 1000 μmol photons m−2 s−1/50 Hz (designated
as FL100050%
50 Hz ).
The volumetric contents of Chl and Car (mg l−1)
and their ratios in D. salina grown under different CL
and FL regimes for 8 d are presented in Table 2. Total
Chl contents in all FL cultures changed synchronously
with that of Car, resulting in similar Car/Cl ratios
< 0.25. In contrast, a pronounced accumulation of
Car was found in the CL cultures (~4 times higher
than in the FL cultures), resulting in 2–4 times higher
Car/Chl ratio, indicative of a higher degree of highlight stress in the CL cultures.
Photosynthesis and respiration rates
The first estimation of photosynthetic activity of the
cultures acclimated to different light regimes was
done via Fv/Fm measurements. Remarkably, the
potential maximum quantum yield of PS II of the D.
salina cultures grown under FL was higher than in
those grown under CL (Fig. 3).
The photosynthesis of D. salina acclimated to CL
and FL was further investigated via comparison of
oxygen evolution-based light (P-I) curves. Figure 4
shows the photosynthetic capacity of the cultures, as
manifested by the maximum photosynthesis rate,
Pmax, and the slope of the initial linear increase in
O2, indicative of the photon-use efficiency (Neale
et al., 1993).
In the actinic irradiance range used in this work (up
to 1000 μmol photons m−2 s−1), we did not observe
light saturation of photosynthetic oxygen evolution.
To estimate the potential Pmax values, we extrapolated
Figs 1-2. Growth kinetics of Dunaliella salina grown under a
constant illumination of 500 μmol photons m−2 s−1 (solid
symbols) and different combinations of flashing-light parameters (open symbols) equivalent to the same time-integrating
irradiance. Fig. 1. Cell number. Fig. 2. Dry mass. Error bars
indicate for SD (n = 8).
Table 2. Pigment composition in Dunaliella salina acclimated
to different light regimes.
Growth conditions
CL500
FL75067%
10 Hz
FL100050%
50 Hz
FL200025%
50 Hz
Total Chl
mg L−1
Total Car
mg L−1
1.54 ± 0.04
0.83 ± 0.01
Car:Chl (wt/wt)
0.54 ± 0.04
0.27 ± 0.05
a
0.19 ± 0.01a
1.30 ± 0.07
0.20 ± 0.05
a
0.15 ± 0.01a
1.65 ± 0.07
0.40 ± 0a
1.45 ± 0.04
0.24 ± 0.02a
Chl and Car represent chlorophyll and carotenoids.
a
Significantly different from the constant-light-grown cells (Student’s
t-test; P ≤ 0.01).
Flash-photoacclimated microalgae
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FL100050%
50 Hz cultures displayed the highest photosynthetic activity, with approximately a 2.3-fold increase
over the CL-acclimated cultures. The slopes of the P-I
curves of the FL-acclimated cells were steeper than
those of the CL-grown cells (e.g. 0.17 vs. 0.076 for
FL100050%
50 Hz and CL, respectively), suggesting higher
photosynthetic efficiency under the FL.
Since the productivity of microalgae depends, aside
from photosynthesis, on the respiration rate (Quinn
et al., 2011), we compared the dark oxygen consumption-rate in the CL- and FL-acclimated cells (Fig. 5).
The results show that in CL-grown cells, the respiration rate was higher than in the FL-grown cells, indicative of lower energy consumption for maintenance
and, hence, increased net productivity of photosynthesis under FL.
Flashing-light-acclimated D. salina displays slower
Zea formation rate and kinetics of NPQ induction
under high light
Fig. 3. Potential maximum quantum yield of photosystem II
(Fv/Fm) of Dunaliella salina acclimated to flashing light of
different regimes compared with constant high light, of the
same photon dose. **Significant difference (Student’s t-test, P
< 0.01); *Significant difference (Student’s t-test, P < 0.05).
Error bars indicate for SD (n = 2).
the best-fit exponentials, describing the P-I curves to
saturating light intensities. The CL-acclimated cultures had an estimated light-saturated rate of photosynthesis (PEmax ) of 58.7 µmol O2 (mmol Chl)−1s−1,
while PEmax of FL-acclimated cells ranged between
57.6 and 133.7 µmol O2 (mmol Chl)−1s−1. The
Fig. 4. P-I curves of photosynthesis recorded in Dunaliella
salina cultures after acclimation to constant (closed squares)
and different combinations of flashing-light parameters, as
measured after 60 min of dark adaptation (see the Materials
and Methods section).
The rate of Zea formation estimated via ΔOD505
kinetics under the actinic light of 700 μmol photons
m−2 s−1 was significantly higher in the cultures acclimated to CL compared with those acclimated to FL
(see the τ½ values in Fig. 6), with no significant
differences in the starting Zea levels after 60 min of
dark relaxation (Fig. S9).
To characterize further the relevance of the ΔOD505
signal to the upregulation of the VC, the induction
kinetics of NPQ were also recorded and compared
with the actual rates of VC activation in the D. salina
cultures acclimated to CL or FL.
Fig. 5. Respiration rates of Dunaliella salina cultures acclimated to constant (closed squares) and different combinations
of flashing-light parameters (open symbols), as measured after
60 min of dark adaptation (for designations, see Fig. 4).
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rates, specifically in the first minute of induction
(0.76 ± 0.07, 0.79 ± 0.07 and 0.91 ± 0.08, in
25%
67%
FL100050%
50 Hz , FL200050 Hz and FL75010 Hz , respectively), than those of CL500-acclimated cells (1.01 ±
0.005). The observed differences in the NPQ induction rates correlated well with the changes in de-epoxidation (DEP) (Fig. 7). In the CL500-acclimated cells,
Vio was de-epoxidized almost completely during 10
min of irradiation with actinic light (DEP = 91.6 ±
0.3%). At the same time, the DEP values obtained
under the same conditions in the FL-acclimated cells
were significantly lower. Notably, the DEP values
after 60 min of dark-acclimation in the FL-grown
cells were also lower in comparison with the darkrelaxed CL cultures, indicating a lower sustained DEP
level of the VC under FL.
Fig. 6. Kinetics of zeaxanthin formation in the cells of
Dunaliella salina acclimated to constant light of 500 μmol
photons m−2 s−1 (closed symbols) or different flashing-light
regimes of the same time-integrated photon dose (open symbols) recorded as ΔA505. Characteristic half-times of zeaxanthin formation are indicated. The kinetics were recorded in
the samples dark-adapted for 60 min and irradiated with 700
μmol photons m−2 s−1 for 10 min. Averaged kinetics are presented (SD < 10% of the corresponding averages, n = 8).
Fig. 7. Correlation of the violaxanthin-cycle activation (bars)
and the buildup of NPQ (symbols, right scale) in the cells of
Dunaliella salina acclimated to a constant light of 500 μmol
photons m−2 s−1 or different flashing-light regimes of the same
time-integrated photon dose (see Table 1). De-epoxidation
state (DEPs) % [(zeaxanthin + 0.5 antheraxanthin)/(violaxanthin +antheraxanthin + zeaxanthin)] a, bBoth of the NPQ
and DEPs are significantly different (Student’s t-test, P <
0.01). SD ≤ 10%.
Collectively, Fig. 7 shows that the FL-acclimated
cultures displayed a slower kinetics of Zea
formation in comparison with those recorded in
the CL-acclimated cultures. In addition, the
FL-acclimated cells showed lower NPQ induction
Changes in the cell ultrastructure under different
illumination regimes
To obtain a deeper insight into the CL and FL
responses of the photosynthetic apparatus of D. salina
described above, we studied the ultrastructure of the
cells acclimated to CL or FL, paying special attention
to the rearrangements of the plastid ultrastructure as
well as to the plastidial and cytoplasmic inclusions
(Figs 8–13). The D. salina cells grown at a low irradiance of 20 μmol photons m−2 s−1 were characterized
by a well-developed grana-lamellar system with a
large number of thylakoid stacks (Fig. 8). The cells
grown at a high constant irradiance (CL500) were
larger and contained a high number of starch grains
and significantly reduced chloroplasts with obvious
signs of light stress, such as swelling of the thylakoid
lumen (Fig. 9).
Two FL treatments were chosen for the comparative study of FL effects on cell ultrastructure:
25%
FL100050%
50 Hz andFL200050 Hz . Generally, the FLacclimated cells also displayed the common features of high-light acclimation, such as reduction
of the chloroplasts (Fig. 9). At the same time, their
thylakoids were more structurally intact, displaying
a lower degree of swelling (Figs 10–13).
The CL- and FL-acclimated D. salina cells were
significantly different in their plastidial and cytoplasmic inclusions, harbouring reserve compounds.
Thus, the CL-acclimated cells contained predominantly starch grains, large in size and number
(Fig. 9). In contrast, as shown in the EM graphs,
FL-acclimated cells accumulated mainly cytoplasmic lipid droplets, and only a limited accumulation
of starch grains was observed in this case (Figs 10,
12). Both CL- and FL-acclimated cells contained
plastoglobuli in their chloroplasts.
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Flash-photoacclimated microalgae
Figs 8-13. Representative transmission electron micrographs of Dunaliella salina cells acclimated to: 8. constant low light (20 μmol
photons m−2 s−1); Fig. 9. constant high light (500 μmol photons m−2 s−1); Figs 10, 11. flashing light of 1000 μmol photons m−2 s−1/50
Hz of 50% duty cycle; or Figs 12, 13. 2000 μmol photons m−2 s−1/50 Hz of 50% duty cycle. LD, lipid droplets; Pg, plastoglobuli; SG,
starch grains; Th, thylakoids. Arrows in (9) point to the swollen thylakoids.
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DISCUSSION
When continuously illuminated with high fluxes of
PAR, the cells of photoautotrophic organisms are normally able to utilize only a small fraction of the
absorbed light energy. This phenomenon is mainly
due to limitations of the dark photosynthesis reactions,
non-photochemical quenching and photoinhibition
(Lunch et al., 2013; Zarmi et al., 2013). In particular,
we observed a decline in Fv/Fm in the cultures grown
under high constant light (Fig. S8), which most likely
manifest photoinhibition (Jin et al., 2003). On the
contrary, intermittent or flashing light, even of supersaturating intensity, could be efficiently utilized for
photochemistry provided that the FL timescale (flashto-dark ratio) is synchronized with the physiological
requirement of the cell, as determined by its photosynthetic capacity (Zarmi et al., 2013). If this condition is not fulfilled, the efficiency of photochemistry
under FL is reduced due to the engagement of different photoprotective mechanisms, with a large contribution, as shown in this work, of the upregulation of
the VC and the corresponding increase of NPQ.
We have tested the hypothesis that FL with appropriately balanced intensity, frequency, and flash-todark ratio, induces only a limited de-epoxidation of
Vio, so that upregulation of the VC and, hence, NPQ
induction occurs at a slower rate and to a lower extent
in comparison with CL of the same time-integrated
PAR dose. In addition, our data confirmed that the VC
is fully functional under FL of high-incident intensity
(Iluz et al., 2012) in the chlorophyte D. salina.
Balanced flashing light enhances photosynthesis and
increases growth rate
Under CL, D. salina growth rate increased as a function of irradiance but not at supersaturating light intensities (Figs S5–S7), when the cultures apparently
suffered from photoinhibition.
In contrast to CL, FL with appropriately balanced
parameters is expected to efficiently drive photosynthesis, even at very high PAR intensities (Kok, 1953;
Dubinsky et al., 1986; Abu-Ghosh et al., 2015a). It is
known that the effect of FL depends on the duty-cycle,
i.e. on the ratio of the light energy delivered by a
single flash and the dark time available for its utilization (Park & Lee, 2001; Yoshimoto et al., 2005;
Kliphuis et al., 2010; Iluz et al., 2012). Indeed, judging from the oxygen evolution-based light curves,
this was the case under our experimental conditions,
e.g. in the FL100050%
100 Hz (Figs 1, 2 & 4). This particular
combination of the FL parameters turned optimal for
D. salina, probably because its duty cycle (10 ms:10
ms, on:off) is close to the average turnover time of a
single photosynthetic unit in microalgae (Dubinsky
et al., 1986). In contrast, the effect of FL with
higher flash frequencies (e.g. FL200025%
50 Hz under our
476
conditions, Figs 1, 2 & 6) is very similar to that of CL
since the photosynthetic apparatus effectively ‘integrates’ the portions of the absorbed light that come at
too fast a rate (Park & Lee, 2001).
An additional factor facilitating the increase in net
productivity and, hence, higher growth rate under a
balanced FL could be the lower expenditure of photosynthates for cell maintenance. Indeed, the respiration
rate was lower in the FL-acclimated cells in comparison to those acclimated to the CL (Fig. 5).
Acclimation of the pigment apparatus to flashing light
It was found that D. salina cultures acclimated to CL or
FL exhibited distinct changes in total Chl and Car
contents (Table 2). Thus, under CL, the Car/Chl ratio
was considerably (2–4 times) higher than under FL.
Such an increase in relative Car content might result in
a greater degree of high-light stress under CL
(Solovchenko, 2013) and, in particular, it might alter
light energy transfer efficiency of PS II, as was reported
for the green microalga Chlorella pyrenoidosa, when
treated with brief flashes (Schubert et al., 1994).
Probably, the acclimation of D. salina to FL conditions involves the adjustment of its photosynthetic
pigment composition. On one hand, the flashing
light did not impair the general ability of D. salina
cells to acclimate to high irradiances. On the other
hand, the lower Car to Chl ratio suggests that these
cultures are less stressed under FL in comparison with
those grown under CL conditions (Solovchenko,
2013). At the same time, this response does not preclude the possible increase in capability of NPQ
induction under high light (Belgio et al., 2014).
Response of the violaxanthin cycle to flashing light
and its relationships with NPQ buildup
Operational VC is important for photoprotection.
Indeed, its suppression, e.g. by a mutation (such as
npq1 in Arabidopsis) results in increased damage to
PSII under high light (Kalituho et al., 2007).
However, permanent upregulation of the VC, like
that in npq2 mutants of Arabidopsis or in zea1 mutants
of Chlamydomonas, leads to a high NPQ level even at
low light. These organisms are characterized by a
significant sustained level of the absorbed light energy
loss (Niyogi, 1999; Thaipratum et al., 2009), resulting
in reduced growth rates under limiting light conditions
when compared with the wild type (Dall’osto et al.,
2005).
The patterns of VC activation under FL and its
significance for the enhancement of photosynthesis
in microalgae under FL are still unclear. To elucidate this, we studied the VC dynamics in D. salina
cultures grown under different combinations of
supersaturating flashes and dark periods (Table 1).
In spite of a high intensity of a single flash,
Downloaded by [Moskow State Univ Bibliote] at 02:54 24 November 2015
Flash-photoacclimated microalgae
D. salina efficiently photosynthesized when acclimated to FL conditions (Fig. 4), but not to a high
or supersaturating CL (Figs 4, S5–S8, respectively).
The dark periods following the flashes also facilitated the photochemical utilization of the photons
delivered by the flash. As a result, a relatively
slower activation of the VC was observed under
FL in comparison with CL. In the latter case, the
high-frequency flashes were separated by dark periods that were too short, resulting in an increased
high-light stress in comparison to the cultures
grown at FL of lower frequency (FL100050%
50 Hz ), or
even at higher photon flux (FL200025%
).
50 Hz
Although the lowest rate of Zea formation, as evidenced by HPLC and ΔA505 kinetics (Fig. 6), was
recorded in FL200025%
50 Hz cultures, this combination of
FL parameters did not result in the highest growth
rate, implying the possible induction of a photoinhibitory mechanism under such a high irradiance in spite
of a shorter flash time. The highest growth rate was
displayed by the culture grown under FL100050%
50 Hz,
suggesting that this combination of FL parameters is
optimally balanced for D. salina under our experimental conditions.
To better understand the possible significance of the
VC operation in the FL-acclimated microalgal cells,
we followed the dynamics of NPQ induction in their
cultures. As follows from Fig. 7, the magnitude of
NPQ increase was lower in the FL-grown cells than
in the CL-grown cells, which is in accord with the
kinetics of Zea formation recorded as ΔA505.
We suggest that the decreased thermal dissipation
of the light energy not stored as products of photosynthesis, namely the NPQ, in flashing-light acclimated cultures and their faster relaxation time might
provide a major contribution to the phenomenon of
flashing-light enhancement of photosynthesis. The
reduction of NPQ losses under flashing-light regimes
leaves a larger fraction of harvested light energy available for photochemical storage.
The flashing light is less stressful to the algal cell than
an equal dose of constant light
Conspicuous responses of microalgal cells to highlight stress were apparent at the ultrastructural level.
The reduction of the chloroplasts and their thylakoids,
and the development of storage structures (usually
starch grains and lipid droplets) harbouring the
excessively fixed carbon that could not be utilized
for cell growth and maintenance under the stressful
conditions, were the main ultrastructural changes
(Solovchenko, 2010).
To obtain an additional qualitative estimation of the
degree of high-light stress imposed by the different
illumination regimes tested in this work, we investigated the ultrastructure of D. salina cells by TEM
477
during the late-exponential phase of photoacclimation
(Figs 8–13). It was found that high CL irradiance
brought about profound changes consistent with the
high-light response outlined above. In particular,
high-CL acclimated cells possessed significantly
reduced chloroplasts with a lower number of grana,
decreased stacking, and numerous large starch grains.
Notably, a thylakoid swelling was also observed in
these cells (Fig. 9). These ultrastructural features
could be putatively related to a more profound NPQ
increase, as in higher plants (Johnson et al., 2011).
However, a certain degree of photoinhibition in the
CL-acclimated cells cannot be ruled out, since similar
alterations in the thylakoid structure were previously
associated with photoinhibition rather than NPQ
(Majeran et al., 2001).
In spite of the considerable reduction of the chloroplast, the thylakoids of the FL-acclimated D. salina
cells were more ultrastructurally intact, regardless of
the incident irradiance level (Figs 11, 13). At the same
time, the fine structure of thylakoids was slightly
different in the different FL treatments studied. Thus,
in the FL100050%
50 Hz treatment (resulting in the highest
enhancement of the culture growth), the thylakoid
membranes were less expanded than in the cells
grown under the less optimal FL conditions, leading
to an increase in DEPs and NPQ (e.g. FL200025%
50 Hz ;
Figs 12, 13). The minor alteration of the thylakoid
ultrastructure is probably a manifestation of the
NPQ-related response of the photosynthetic apparatus
to different FL conditions observed against the background of the major ultrastructural changes triggered
by high-light acclimation.
CONCLUSIONS
We demonstrated that the balanced FL parameters
enhancing photosynthesis and the growth of D. salina
are correlated with a slow and limited activation of the
VC. However, less optimal FL-parameter combinations lead to a more rapid buildup of the VC and to a
correspondingly higher induction of NPQ. There are
grounds to conclude that the degree of VC engagement under FL reflects the degree of the synchronization between the timescale of the FL and that of light
absorption and utilization by the microalgal cell. In
view of these findings, the dynamics of the NPQ level
could be suggested as a reliable criterion for the selection and fine-tuning of optimal FL parameters for the
cultivation of microalgae, at least in the case of
D. salina. Another promising direction for the
improvement of microalgal growth under FL conditions is replacing the dark interval between the flashes
with intermittent decreases of irradiance. This is
expected to facilitate the relaxation of photoprotective
mechanisms and increase the overall photosynthetic
energy budget.
S. Abu-Ghosh et al.
ACKNOWLEDGEMENTS
We thank Professor Moshe Ben-Tzion for the LabView
programming; Eli Zimmerman for help with the construction of the light panel; Dr Erhard Pfündel (Waltz)
for providing the program used in chlorophyll fluorescence inductions, Dr Tatiana Babushkin for assistance
with the electron microscopy; Professor Alexander V.
Ruban from Queen Mary University of London and Dr
Ir. Packo Lamers of Wageningen University for scientific
advice; and Sharon Victor for critically reading the
manuscript. A.S. acknowledges the financial support of
Russian Scientific Fund (grant number 14-50-00029).
The author Said Abu-Ghosh dedicates this work to his
parents.
Downloaded by [Moskow State Univ Bibliote] at 02:54 24 November 2015
CONFLICT OF INTEREST
478
In some cases, the standard deviation (SD) is smaller
than the marker size.
Supplementary Fig. S9. HPLC analysis of the zeaxanthin levels in Dunaliella salina after accumulation to
different light regimes followed by a dark adaptation
period of 60 min. Data are normalized to 100 chlorophyll
molecules and are means ± SD from at least three replicates. No significant differences between the samples
(Student’s t-test, P < 0.05).
AUTHOR CONTRIBUTIONS
S. Abu-Ghosh: designing and conducting experiments and
writing manuscript; Z. Dubinsky and D. Iluz: original concept;
D. Fixler; results discussion and partial financing; A.
Solovchenko: drafting and editing manuscript; M. Zigman:
assistance with culture experiments; and Y. Yehoshua: providing culture rooms.
The authors declare that there are no conflicts of interest
associated with this article.
REFERENCES
SUPPLEMENTARY INFORMATION
The following supplementary material is available via
the Supplementary Content tab on the article’s online
page at http://dx.doi.org/10.1080/09670262.2015.
1069404
Supplementary Figs S1, S2. A novel OXY-PAM fluorometer operating under control of the WinControl-3
program. S1. General view of the OXY-PAM fluorometer. S2. Dimensions of the working chamber of the
novel OXY-PAM fluorometer.
Supplementary Fig. S3. Detailed structure of the novel
Oxy-PAM system: 1. AlgaeGrowth-PAM (AGPA000x);
2. USB-cable; 3. Power Supply (24V/90W, APSA000x).
Sensors included to this set are: 4. Algae PAR/Temp.
(APTA000x) with 5. Temperature-Sensor; 6; Spherical
PAR-Sensor (SQUSxxx) and 7. Custom-built PAM
sensor; 8. Oxygen-Sensor (AOSA000x). The cuvette:
9. Algae Cuvette (ACSA000x); 10. Stirr-Box
(ASBA000x) and 11. LED-Panel (ALPA000x). The
arrow indicates the location of the fluorescence sensor.
Supplementary Fig. S4. Sample of chlorophyll fluorescence data of Dunaliella salina, monitored by the novel
home-designed Oxy-PAM [manufactured by WALZ
(Effeltrich, Germany)] using WinControl-3 program.
Supplementary Figs S5-S8. Growth kinetics and photosynthetic efficiency of Dunaliella salina under different
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