Introduction
Increasing atmospheric levels of CO2 and the associated dissolution of
CO2 into the oceans has resulted in ocean acidification (OA), with
increased levels of pCO2, HCO3- and H+ and decreased
CO32- concentration. The acidity of surface oceans has increased
by 30 % (lowering pH by 0.1) since the Industrial Revolution and is
expected to increase by 100–150 % (0.3–0.4 pH) by the year 2100 (Orr
et al., 2005). At the same time, increased sea surface temperatures are
predicted to cause a shoaling of the surface mixed layer, which in turn will
lead to enhanced exposure to sunlight (both as photosynthetically active
radiation (PAR) and as UV radiation (UVR)). This enhanced stratification will also decrease
upward transport of nutrients from deeper, nutrient-rich layers, leading to
more frequent/marked nutrient limitation (Cermeño et al., 2008). Global
change is thus likely to cause changes in a multiplicity of factors that
influence phytoplankton growth and it is thus critical to examine OA in the
context of interactive effects with these other environmental drivers (Boyd,
2011).
Increased availability of CO2 in seawater appears in some cases to
bring a low level of benefit to growth and photosynthesis of natural
phytoplankton populations (Riebesell and Tortell, 2011, and references
therein), though in most cases laboratory experiments have shown little
effect of OA alone (Doney et al., 2009). However, the effects can differ
according to changes in solar radiation and/or other physical or chemical
factors (Gao et al., 2012a). Increased acidity of seawater may lead to
physiological stress (Pörtner and Farrell, 2008) and affect
phytoplankton nutrient uptake (Beman et al., 2011; Shi et al., 2012).
Therefore, OA could most likely result in differential effects on different
photosynthetic organisms or under different environmental conditions (Gao,
2011).
Diatoms account for about 20 % of total global primary production and
about 40 % of that in the oceans (Granum et al., 2005). Early reports
suggested that growth of diatom species could be limited by the availability
of CO2 (Riebesell et al., 1993). However, the growth rate of
diatom-dominated natural phytoplankton populations was not affected by
CO2 enrichment to 800 µatm (Tortell, 2000), and not all diatom
species were sensitive to seawater pCO2 rise under nutrient-replete
conditions in a mesocosm study (Kim et al., 2006). In laboratory
experiments, growth of Skeletonema costatum was not stimulated by elevated CO2 (800 µatm; Chen and Gao, 2011). Phaeodactylum tricornutum grown under nitrate-limited conditions also
showed no enhancement of growth under high CO2 (1000 µatm; Li et
al., 2012a). Nevertheless, in other work, the diatoms Phaeodactylum tricornutum (1000 µatm; Wu
et al., 2010) and Attheya sp. (670 µatm; King et al., 2011) showed enhanced
growth rate in nutrient-replete conditions under elevated CO2 levels.
These variable findings reflect physiologically differential responses among
different species or under different experimental or environmental
conditions. Changes in light intensity can lead to enhanced, unaffected or
inhibited growth rates under OA conditions, even for the same diatom species
(Gao et al., 2012b). Recently, microcosm studies have shown that the species
abundance and physiological responses (e.g., Chl a, DNA damage, reactive oxygen species (ROS),
photosynthetic efficiency) could be regulated by nutrients and light
availability under high CO2 conditions (Neale et al., 2014; Sobrino et
al., 2014). Therefore, the effects of OA should be considered in the context
of the influence of multiple factors, such as temperature, nutrient status,
light and UVR (Boyd, 2011; IPCC, 2011; Gao et al., 2012a).
Solar UVB radiation (280–315 nm), which is increasing due to interactions of
global change and ozone depletion (Häder et al., 2011), is known to
damage DNA (Buma et al., 2003; Gao et al., 2008), lower photosynthetic rates
(Helbling et al., 2003), perturb the uptake of nutrients (Hessen et al.,
2008) and alter morphological development (Wu et al., 2005) of
phytoplankton. In contrast, under moderate levels of solar radiation, solar
UVA radiation (315–400 nm) is known to stimulate photosynthesis (Gao et al.,
2007), signaling (Cashmore, 1998) and photo-repair of UVB-induced damage
(Buma et al., 2003) in phytoplankton. Previously, it was shown that
UV-induced inhibition of dinoflagellates was lower under nutrient-replete
conditions but higher under nutrient limitation due to less efficient
repair resulting from lowered nutrient availability (Litchman et al., 2002).
Similar enhancement of UVB impacts under nutrient (N, P) limitation were
shown for a green microalga, Dunaliella tertiolecta (Shelly et al., 2002; Heraud et al., 2005).
Recently, OA was found to enhance UVB-induced damage to a red tide alga,
Phaeocystis globosa, leading to a greater decrease in growth rate and photochemical yield under
1000 µatm CO2 (Chen and Gao, 2011).
Marine phytoplankton often experience nutrient limitation in offshore
waters; with progressive ocean warming, such limitation will be intensified
due to the decreased depth of the surface mixed layer (enhanced stratification)
(Cermeño et al., 2008). Combined effects of nutrient levels and CO2
have been reported in many studies. For example, photosynthetic carbon
fixation of the coccolithophorid Emiliania huxleyi was enhanced under high light and low
nitrogen conditions when the seawater CO2 concentration was raised to
2000 µatm (Leonardos and Geider, 2005). However, increased seawater
CO2 concentration also showed antagonistic effects with iron in
modulating (down- or up-regulating) primary production of marine
phytoplankton in the Gulf of Alaska (a nutrient-replete but low-chlorophyll
area) (Hopkinson et al., 2010). In some toxin producing species, for example
the dinoflagellate Karlodinium veneficum, toxicity was enhanced under high CO2 and low
phosphate conditions (Fu et al., 2010). However, to the best of our
knowledge, there is little information concerning the combined effects of OA
and NO3- limitation on diatoms and their susceptibility to
damage from solar UVR (280–400 nm).
Nutrient availability can influence phytoplankton responses to UV and to
CO2-induced seawater acidification. Theoretically, increased seawater
acidity can perturb the intracellular acid–base balance and thus lead to
differential interactions between nutrients and solar UVR. In this study, we
hypothesize that reduced availability of NO3- under OA would
affect the photosynthetic performance under solar radiation with or without
UVR. We used the diatom Phaeodactylum tricornutum to test this hypothesis.
Materials and methods
Growth conditions
The diatom Phaeodactylum tricornutum Bohlin (strain CCMA 106),
isolated from the South China Sea (SCS) and maintained in the Center for
Collections of Marine Bacteria and Phytoplankton (CCMBP) of the State Key
Laboratory of Marine Environmental Sciences (Xiamen University), was grown
mono-specifically in artificial seawater enriched with Aquil medium (Morel et
al., 1979). Cells were cultured in 500 mL vessels containing 250 mL of medium
under two levels of NO3- (110 µmol L-1, high nitrate –
HN and 10 µmol L-1, low nitrate – LN) and aerated with ambient (outdoor) air (low
CO2 – LC; 390 µatm) or elevated (1000µatm; high CO2 – HC) CO2 levels
within a plant CO2 chamber (HP1000G-D, Ruihua Instrument and Equipment
Co. Ltd, China). Gas flow rate was 300 mL min-1, and the CO2
concentrations varied by less than 3 % of the target value. The low
NO3- level of 10 µmol L-1 was based on its
concentration range (ca. 0–20 µmol L-1) in the oligotrophic
SCS, from where the diatom strain was isolated. Dilutions were made every
24 h, so that the seawater carbonate system was kept stable under each
CO2 level within the cell density range of 6×104 to 3×105 cells mL-1 (exponential growth phase). According to the
pre-experiment, the initial nitrate concentration of
10 µmol L-1 could be totally consumed
(0–10 µmol L-1) ; and the initial nitrate concentration of
110 µmol L-1 treatment ranged from ca. 85–110 µmol L-1 during the culture. The cells were grown at
70 µmol photons m-2 s-1 (cool white fluorescent tubes)
under a 12L : 12D photoperiod for at least 10 generations before being used
for the solar radiation treatments described below. Three independent
cultures were grown at each set of conditions.
Determination of seawater carbonate system parameters
The pH in the cultures was determined daily during the light period with a
pH potentiometric titrator (DL15, Mettler-Toledo, Schwerzenbach,
Switzerland), which was calibrated with NBS (National Bureau of Standards)
buffer solutions (Hanna). DIC (dissolved inorganic carbon) was estimated
with an automatic system (AS-C3, Apollo Scitech) linked to an infrared gas
detector (Li-Cor 7000, Li-Cor). DIC, pH, nutrient concentrations (phosphate,
10 µmol L-1; silicate, 100 µmol L-1), salinity (35)
and temperature (20 ∘C) were used to calculate the parameters of the
seawater carbonate system (HCO3-, CO32-, CO2 and
TA) using the CO2 system analyzing software CO2SYS (Lewis and
Wallace, 1998) as described previously (Li et al., 2012a). The carbonic acid
dissociation constants (K1 and K2) used were those of Roy et al. (1993), and that for boric acid (KB) was from Dickson (1990).
Radiation treatments under the solar simulator
To determine the effects of growth conditions on the sensitivity of carbon
fixation and chlorophyll fluorescence to short-term exposure to UVR, P. tricornutum cells,
grown under LC–LN, HC–LN, LC–HN and HC–HN conditions, were exposed for 1 h to different
radiation treatments with or without UVR, as follows: (1) PAR treatment, tubes
wrapped with Ultraphan film 395 (UV Opak, Digefra), exposed to PAR
alone; (2) PA treatment, tubes wrapped with Folex 320 (Montagefolie, Folex,
Dreieich, Germany), receiving wavelengths above 320 nm (PAR + UVA); (3) PAB
treatment, tubes wrapped with Ultraphan Film 295 (Digefra, Munich, Germany)
so that the cells received wavelengths above 295 nm (PAR + UVA + UVB). The
transmission spectra of the cut-off filters are available elsewhere (Zheng
and Gao, 2009). Samples were placed at a distance of 1.2 m from a solar
simulator (Sol 1200W, Dr. Hönle, Martinsried, Germany), so that the
actual PAR light intensity to which the cells were exposed within the
tubes (calculated taking into account the transmission properties of the
quartz tubes and the filters) was 44.11 Wm-2 (ca. 190.11 µmol photons m-2 s-1) which is close to the daytime mean photon flux in
the middle of the photic zone (22–36 m depth in South China Sea, SEATS
station). The corresponding UVA and UVB irradiances were 14.19Wm-2 (ca. 41.99 µmol photons m-2 s-1) and 0.75
Wm-2 (ca. 1.89 µmol photons m-2 s-1). Irradiances were
measured with a broadband filter radiometer (ELDONET, Real Time Computer,
Möhrendorf, Germany). After the radiation treatments, the cells were
replaced under their growth light level (70 µmol photons m-2 s-1) to examine the recovery of photosynthetic performance. During the
incubations, the tubes were maintained in a water bath at 20 ∘C
using a circulating cooler (Eyela, CAP-3000, Tokyo Rikakikai Co. Ltd., Tokyo,
Japan).
Measurement of carbon fixation
The 14C method was applied to measurements of marine photosynthetic
carbon fixation (Nielsen, 1952), and has been detailed with modified
protocols in many publications (Holm-Hansen and Helbling, 1995; Gao et al.,
2007). Cells were harvested in the middle of the light phase, diluted with
freshly made medium equilibrated with the designated concentrations of
CO2 to a cell concentration of 2–3×104 cells mL-1
and transferred to 35 mL quartz tubes. Each tube was injected with 100 µL–5 µCi (0.185 MBq) NaH14CO3 solution (ICN Radiochemicals).
Triplicate incubations were carried out for each treatment as mentioned
above and, additionally, three tubes were wrapped in aluminum foil and incubated
as a dark control. The cells were collected on Whatman GF/F glass filters
either immediately after 1 h exposure to the solar simulator or after a
period of recovery under their growth light for another hour. The filters were
put into 20 mL scintillation vials, fumed with HCl for 12 h and then dried
for 6 h at 45 ∘C to expel the non-fixed inorganic carbon as
CO2. Scintillation cocktail (3 mL of Tri-Carb 2800TR, Perkin
Elmer®) was added to the vials, and radioactivity in the
vials counted with a liquid scintillation counter (LS 6500, Beckman Coulter,
USA). Carbon fixation rates were calculated from these values and are
presented on a per cell or per Chl a basis.
Measurement of chlorophyll fluorescence
For chlorophyll fluorescence measurements, cell collection and radiation
treatments were carried out as described above. The effective quantum yield
was measured every 20 min either during the solar simulator exposure
or during recovery under the growth light level.
The effective quantum yield and non-photochemical quenching (NPQ)
parameters were calculated according to Genty et al. (1990) as yield =(Fm′-Ft)/Fm′ and NPQ =(Fm-Fm′)/Fm′, respectively, where Fm is the
maximum fluorescence yield after 15 min dark adaptation, Fm′ is
the light-adapted maximal chlorophyll fluorescence yield measured during the
exposures and Ft is the steady fluorescence level during the exposures.
The actinic light was set at the growth light level, and the saturating pulse
(5000 µmol photons m-2 s-1) lasted for 0.8 s.
Repair (r) and damage (k) rates during the 60 min exposure period in the
presence of UV were calculated using the Kok model (Heraud and Beardall,
2000): P/Pinitial=r/(k+r)+[k/(k+r)]e-(k+r)t, where
Pinitial and P were the yield values at the beginning and at
exposure time t.
During the recovery period, the exponential rate constant for recovery (R)
was calculated from the following equation: y=yo+b×[1-exp(-R×t)], where y represents the yield value at time t,
yo is the starting value before recovery and b is a constant.
The relative inhibitions of carbon fixation or yield caused by UVA or UVB
were calculated as follows:
InhUVR=(PPAR-PPAB)/PPAR×100%,InhUVA=(PPAR-PPA)/PPAR×100%,InhUVB=InhUVR-InhUVA;
where PPAR, PPA and PPAB represent carbon fixation or yield
values under PAR, PAR + UVA, PAR + UVA + UVB treatments, respectively.
Cells counts and chlorophyll <italic>a</italic> measurements
The cells were counted using a Z2™ Coulter Counter (Beckman, USA).
Where needed, we used the values for chlorophyll a (Chl a) contents of the
cells grown under the same CO2 and nitrate levels reported previously (Li et al., 2012a).
Total protein content, superoxide dismutase (SOD) and catalase
(CAT) measurements
To determine the total protein content and activities of superoxide dismutase (SOD) and catalase
(CAT), cells
were collected, in the middle of the light phase, onto a polycarbonate
membrane (0.22 µm, Whatman) under vacuum at a pressure of less than 0.01 MPa and washed into a 1 mL centrifuge tube with phosphate buffer (pH 7.6).
The enzyme extractions were carried out in 0.6 mL phosphate buffer (pH 7.6)
that contained 50 mM KH2PO4, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 % Triton X-100 and 1 % (w/v) polyvinylpolypyrrolidone.
The cells were broken by sonication in an ice-water bath (4 ∘C),
and the homogenized extract was centrifuged at 12 000 g (4 ∘C) for
10 min before the activities of SOD and CAT were tested with SOD and CAT
Assay Kits (Nanjing Jiancheng Biological Engineering Company, China). One
unit of SOD was defined as the amount causing a 50 % inhibition of
nitroblue tetrazolium (NBT) reduction (Wang and Wang, 2010). One unit of CAT
activity was defined as the amount required to decompose 1 µmol
H2O2 per second. The SOD and CAT activities were expressed as U
mg-1 protein and per 106 cells (Fig. S1 in the Supplement). The total protein
content was determined according to Bradford (1976) using bovine serum
albumin as the standard.
Statistical analyses and calculations
One-way analysis of variance (ANOVA) was used, followed by a multiple
comparison using a Tukey test to establish differences among the treatments.
Interactive effects among CO2, NO3- and UVR on carbon
fixation and yield were determined using a two- or three-way ANOVA to
establish significant differences among the variables.
Results
Carbon fixation
Carbon fixation was significantly inhibited by UVR in both HN and LN-grown
cells on either a per cell or per Chl a basis (Fig. 1). Under the HN conditions, the
carbon fixation rates of LC and HC cultures, compared to that of the PAR alone
treatment, were inhibited by 29.4 % (P=0.0002) and 36.7 % (P<0.0001) in the presence of UVA (PA treatment: PAR + UVA), and by 47.7 %
(P<0.0001) and 46.1 % (P=0.0029) with both UVA and UVB (PAB,
PAR + UVA + UVB) (Fig. 1a and c). However, the carbon fixation per cell in the
LC-grown cells was 10.0 % (P=0.0058) higher in those exposed to PA, and
fixation based on Chl a was higher under the PAR alone or PA treatments, by about
8.4 (P=0.0253) and 17.9 % (P=0.005) compared to that of the
HC-grown cells. For PAB treatments, there were no significant differences
between the HC- and LC-grown cells (Fig. 1a and c).
Photosynthetic carbon fixation rates of P. tricornutum under different
treatments. Photosynthetic carbon fixation rates of P. tricornutum cells represented
as rates (a, b) per cell and (c, d) per Chl a grown at ambient (390 µatm, LC)
or elevated CO2 (1000 µatm, HC) under NO3--replete (110 µmol L-1, HN)
(a, c) or NO3--limited conditions (10 µmol L-1, LN); (b, d) when exposed to PAR, PAR + UVA (PA) and
PAR + UVA + UVB (PAB) for 60 min, respectively. Vertical bars indicate ± SD; the means and standard deviations were based on three replicates. The
different lowercase letters indicate significant differences between
different treatments at P<0.05 level.
![]()
Under LN conditions, carbon fixation rates of LC- and HC-grown cells were
decreased by 14.7 (P=0.0039) and 1.1 % (P=0.8658) in the
presence of UVA (PA) and by 23.3 (P=0.0019) and 27.3 % (P=0.0123) with UVA and UVB (PAB) treatments, respectively (Fig. 1b and d),
compared with that of PAR alone treatment. This indicates that both UVA and UVB
resulted in significant impacts on the LN-grown cells under LC, but only UVB
brought about a significant reduction of the rate under HC. In the PA
treatment, the HC–LN cells fixed carbon at a rate 21.7 % (P=0.0071)
higher than in the LC–LN cells (Fig. 1b), however, there were no significant
differences between HC and LC cells in the PAR and the PAB treatments under
N limitation. Under the LN level, the carbon fixation rate per Chl a was
about 30.8 (P=0.01), 51.6 (P=0.0013) and 24.0 % (P=0.03)
higher in HC-grown than in LC-grown cells (Fig. 1d).
Photochemical quantum yield
When exposed to different irradiation treatments, photochemical quantum
yields in the cells grown under either HC or LN conditions showed
similar patterns to those grown at LC and HN conditions (Fig. 2),
decreasing rapidly during the initial 20 min and leveling off after 40 to 60 min. Under HN conditions, the yield in the HC-grown cells decreased
to a similar level among the treatments (PAR, P=0.1568; PA, P=0.0879;
PAB, P=0.1341) as that in the LC treatments (Fig. 2a and b). Under the LN
condition, the yield decreased to much lower levels compared to those under
HN treatments (Fig. 2c and d). Cells exposed to all treatments showed
recovery of the yield, under their growth light (70 µmol photons m-2 s-1), to approximately their initial levels in about 80 min (Fig. 2).
UVA- and UVB-induced inhibition of photosynthetic performance
While UVA induced significantly higher (P=0.0114) inhibition of
photosynthetic carbon fixation in the HC–HN-grown cells, but lower (P=0.0038) in the
HC–LN-grown cells (Fig. 3a and b), it did not cause significant changes in
the yield between the HC- and LC-grown cells (HN, P=0.1375; LN, P=0.0500) (Fig. 3c and d). While the contribution of UVB did not induce significant
inhibition of either carbon fixation (P=0.2308) or yield (P=0.5319) in
the HN-grown cells, under both the HC and LC conditions (Fig. 3a and c), it
caused significantly higher inhibition of the photosynthetic rate (by
203.3 %, P=0.0006) and the yield (by 76.8 %, P=0.0451) in
the HC-grown than the LC-grown cells under NO3--limited conditions
(Fig. 3b and d). Interactive effects among CO2, NO3- and
radiation treatments on yield were significant (Table 1).
The effective quantum yield of P. tricornutum under different treatments.
Changes of effective quantum yield in P. tricornutum cells at ambient (390 µatm,
LC) or elevated CO2 (1000 µatm, HC) under (a, b) NO3--replete (110 µmol L-1, HN) and (c, d) NO3--limited (10 µmol
L-1, LN) conditions when exposed to PAR, PAR + UVA (PA) and PAR + UVA + UVB
(PAB) for 60 min and another 80 min under the growth light level (the time
of the switch to growth light levels is indicated by the dashed line),
respectively. The irradiance intensities under solar simulator or growth
light were the same as mentioned above. Vertical bars show mean ± SD,
n=3.
![]()
Interactive effects among NO3- concentrations, CO2
levels and radiation treatments. Two or three way ANOVA analysis of
individual and interactive effects among NO3- concentrations,
CO2 levels and radiation treatments. The asterisks indicate significance at P<0.05. Where “Ni” indicates nitrate, “OA” CO2/ pH,
“Rad-Treat” radiation treatments, “Inh-C” inhibition of carbon fixation
and “Inh-yield” inhibition of yield.
Ni &
Ni &
OA &
Ni, OA &
Parameter
Ni
OA
Rad-Treat
OA
Rad-Treat
Rad-Treat
Rad-Treat
Carbon fixation
*
*
*
*
*
*
Inh-C
*
*
*
*
*
Yield
*
*
*
*
Inh-yield
*
*
*
*
NPQ
*
*
*
The PSII damage (k) and repair (r) rate constants (min-1)
in Phaeodactylum tricornutum cells grown in LC–HN, LC–LN, HC–HN and
HC–LN during the 60 min exposures to PAR + UVA + UVB (44.11+14.19+0.75 Wm-2). Parameters of repair and damage rates were calculated
based on Fig. 2 according to Heraud and Beardall (2000). SD was for
triplicate cultures. Treatments with the same lowercase superscript letters mean the difference is not significant. In contrast, treatments with
different lowercase superscript letters indicate the difference is
significant (P<0.05 level).
R2 for fit
Repair rate (r)
Damage rate (k)
r/k
LC–HN
> 0.99
0.044 ± 0.007a
0.068 ± 0.007a
0.666 ± 0.216ab
HC–HN
> 0.99
0.064 ± 0.019ab
0.079 ± 0.010ab
0.806 ± 0.145ab
LC–LN
> 0.99
0.054 ± 0.012ab
0.062 ± 0.008a
0.854 ± 0.138a
HC–LN
> 0.99
0.059 ± 0.005b
0.095 ± 0.010b
0.588 ± 0.073b
UV-induced inhibition of carbon fixation and PSII
activity. UVA- and UVB-induced inhibition of (a, b) photosynthetic
carbon fixation and (c, d) PSII of P. tricornutum cells grown at ambient (390 µatm,
LC) or elevated CO2 (1000 µatm, HC) under (a, c) NO3--replete (110 µmol L-1, HN) and (b, d) NO3--limited
(10 µmol L-1, LN) conditions when exposed to PAR, PAR + UVA
(PA) and PAR + UVA + UVB (PAB) for 60 min, respectively. The irradiance
intensity under solar simulator was the same as mentioned above. Vertical
bars are means ± SD, n=3, the different letters indicate significant
differences between different treatments at P<0.05 level.
![]()
Non-photochemical quenching (NPQ) of P. tricornutum under different
treatments. NPQ of P. tricornutum grown at ambient (390 µatm, LC) or elevated CO2
(1000 µatm, HC) under (a, b) NO3--replete (110 µmol L-1, HN) and (c, d) NO3--limited (10 µmol L-1, LN) conditions when
exposed to PAR, PAR + UVA (PA) and PAR + UVA + UVB (PAB) for 60 min and
another 80 min under the growth light level, respectively. The irradiance
intensities under solar simulator or growth light were the same as mentioned
above. Vertical bars means ± SD, n=3.
![]()
The exponential rate constant for recovery (R, min-1) under
growth light after 60 min exposure to solar radiation with or without UV.
Different letters of superscripts indicate significant differences between
the CO2 and NO3- treatments at P<0.05.
LC–HN
LC–LN
HC–HN
HC–LN
PAR
0.038 ± 0.006ab
0.029 ± 0.011b
0.043 ± 0.009a
0.038 ± 0.002ab
PA
0.028 ± 0.002a
0.023 ± 0.007a
0.037 ± 0.002b
0.027 ± 0.008ab
PAB
0.019 ± 0.002a
0.024 ± 0.001b
0.029 ± 0.003c
0.021 ± 0.003d
The recovery time to half maximal yield values under growth light
after 60 min exposure to solar radiation with or without UV. Different
letters of superscripts indicate significant differences between the
radiation treatments at P<0.05.
LC–HN
LC–LN
HC–HN
HC–LN
(min)
(min)
(min)
(min)
PAR
16.78 ± 2.94a
20.81 ± 5.93a
15.41 ± 2.57ab
16.79 ± 0.64a
PA
20.38 ± 1.28a
23.36 ± 4.47a
16.83 ± 0.67a
21.66 ± 4.52ab
PAB
25.82 ± 1.51b
22.73 ± 1.25a
20.05 ± 1.78b
24.64 ± 1.57b
Repair, damage rates and constant for recovery rate
The HC-grown cells had higher rates of damage, k, than the LC-grown cells
under nitrogen limitation but not under N-replete conditions (HN, P=0.2109; LN, P=0.0092; Table 2). No effect was observed for repair rates r (HN, P=0.1655; LN, P=0.5276; Table 2). The repair : damage ratios (r/k) in the HC-grown
cells showed a 21.0 % (but statistically insignificant) increase under HN
(P=0.3450) but decreased significantly by 31.1 % under LN (P=0.0320)
conditions, compared to the LC-grown cells, respectively (Table 2). Under
the low PAR, the exponential rate constant for recovery (R) showed
dependency on previous light treatments with a lowered rate in the cells
exposed to UVR, while HC stimulated the rate under the HN but not LN
conditions (Table 3). Obviously, the cells exposed to the radiation
treatments with UVB took longer (P<0.05) to recover their
photochemical yield, and pre-exposure to UVA had little (P>0.05)
effect on the recovery; HC–HN-grown cells had faster (P<0.05)
photochemical recovery (Table 4).
Non-photochemical quenching (NPQ)
Non-photochemical quenching (NPQ) showed the opposite pattern of change to
yield during both the exposure and recovery periods (Fig. 4). Under HN
conditions, HC treatments triggered the highest NPQ within 20 min,
while NPQ reached its maximal values at 40 min under the ambient (LC)
CO2 level (Fig. 4a and b). Similar trends were found in both the LN- and HN-grown cells regardless of the radiation treatments (Fig. 4). Both UVA and
UVB caused additional (P<0.05) rises in NPQ in HN-grown cells
regardless of the CO2 levels (Fig. 4a and b). However, neither UVA
nor UVB induced significant (P>0.05) change in NPQ in LN-grown
cells, regardless of the CO2 levels (Fig. 4c and d). Lower NPQ values
were found in HN-grown cells compared with LN, under either PAR alone or
PAR + UVA treatments. The addition of UVB, however, resulted in an approximately
17.0 % higher, but statistically insignificant (LC, P=0.1150; HC, P=0.1660), increase of NPQ in HN-grown compared to LN-grown cells. Transfer to the
growth light level without UV, to allow for recovery, led to a rapid decline of
NPQ with time. For the cells that were pre-exposed to the PAR + UVA + UVB
treatment, relaxation of NPQ during the recovery period showed no difference
(P>0.05) between HC- and LC-grown cells except that NPQ in the
HC–HN-grown cells declined faster (P=0.0242) than in LC–HN cells. Two-way
ANOVA showed that both nitrogen levels and radiation treatments
individually, and also interactively, affected the NPQ
(Table 1).
Protein content, SOD and CAT activities
Protein contents were enhanced in HN cultures under both LC (3.21 ± 0.98 pg cell-1) and HC (3.38 ± 1.35 pg cell-1) conditions,
compared with LN-grown cells (LC, 2.58 ± 0.46 pg cell-1; HC,
2.28 ± 0.68 pg cell-1), though statistically there were no
significant differences among the treatments (P=0.4296) (Fig. 5a). There
was no significant difference in protein content between LC and HC
treatments at a given NO3- concentration. However,
NO3- limitation enhanced SOD (LC, by 62.5 %, P=0.0004; HC, by
72.5 %, P=0.0007) and CAT (LC, by 67.5 %, P= 0.0759; HC, by
67.1 %, P=0.0747) activities in both LC- and HC-grown cells, when based
on protein content (Fig. 5b and c), though such enhancement was
insignificant (P>0.1) when normalized to per cell (Fig. S1).
Protein contents, SOD and CAT activities of P. tricornutum under different
treatments. (a) Protein contents, (b) SOD and (c) CAT activities
(represented as per milligram protein) of P. tricornutum grown at ambient (390 µatm,
LC) or elevated CO2 (1000 µatm, HC) under NO3--replete
(110 µmol L-1, HN) or NO3--limited (10 µmol L-1, LN) conditions. The
different letters above each column indicate significant differences between
different treatments at P<0.05 level. Vertical bars show mean ± SD, except the CAT value in HC–LN for which there were only two replicates,
other treatments used at least three replicates (n=3–7).
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Discussion
This study shows that nitrate limitation interacts with OA to affect the
overall impacts of solar UVR on the diatom P. tricornutum. OA and UVB caused significantly
higher inhibition of the photosynthetic rate and the quantum yield under LN
than under HN conditions. Interactive effects of reduced nitrate
availability and OA increased protein-based activity of superoxide dismutase
(SOD) and catalase (CAT) but decreased the rate of repair of PSII from
UV-induced damage. OA appeared to counteract UVB-induced damage under
NO3--replete conditions, but when combined with decreased
availability of nitrate, it increased the diatom's sensitivity to UVR.
Most diatoms have evolved CO2 concentrating mechanisms (CCMs) as a
response to low availability of CO2 in present-day oceans (Raven et
al., 2011). Increasing pCO2 may, to some extent, benefit marine
phytoplankton due to increased availability of CO2 (Burkhardt et al.,
2001; Rost et al., 2003). CCMs are known to be down-regulated under a
CO2 level doubling that of the current ambient concentration, saving
about 20 % of the energy cost for active inorganic carbon acquisition in
some diatoms (including P. tricornutum; Hopkinson et al., 2011). Such a down-regulation of
CCMs was equally obvious in P. tricornutum grown under nitrate-limited or nitrate-replete
conditions (Wu et al., 2010; Li et al., 2012a). However, this down-regulated
CCM and its effects may be mediated by many other factors. A recent study
found that different acclimation times (short term, 15–16 generations and
longer term, 33–57 generations) to increased CO2 and nitrate limitation
may have different effects on the DIC and DIN uptake rate in the diatom
Thalassiosira pseudonana, with short-term acclimated cells showing a linear correlation with changes
in fCO2, although this was not the case in long-term acclimated cells
(Hennon et al., 2014). On the other hand, the down-regulation of CCM
operation was recently shown to decrease the growth of three diatoms
(Phaeodactylum tricornutum, Thalassiosira pseudonana and Skeletonema costatum) under high levels of sunlight but to enhance it under low light
(Gao et al., 2012b). The growth rate of P. tricornutum under high CO2 (1000 µatm) decreased at light levels higher than 180 µmol m-2 s-1
to reach growth rate values lower than that of the low CO2-grown cells (Gao et al., 2012b).
In the present study, under the near-saturation light level (ca. 190 µmol photons m-2 s-1 of PAR), photosynthetic carbon fixation rate
per Chl a under nitrate-limited conditions were higher in the HC-grown
cells. Obviously, the nutrient limitation influenced the effects of OA.
UVR is known to damage photosynthetic pigments and proteins (for example D1
and rubisco proteins; Zacher et al., 2007) and therefore can reduce the
photosynthetic capacity of algae (Häder et al., 2011). UVA induced
significantly higher inhibition of carbon fixation in HC–HN- than in LC–HN-grown cells, reflecting a synergistic effect of UVA and OA; however, for the
same cells, UVB induced no greater inhibition of the photosynthetic carbon
fixation in HC compared to LC cells, which is in contrast to the findings
reported in another study (Li et al., 2012b). Many studies have shown that
the sensitivity of cells to high levels of PAR and UV under OA conditions
could be stimulated and then induce higher inhibition rate of photosynthesis
(Sobrino et al., 2008; Gao et al., 2012b; Xu and Gao, 2012). However, this
phenomenon is not always found in all species especially when the intensity
of PAR or UV is not that high. For example, a recent study reported that the
unicellular chlorophyte (Dunaliella tertiolecta) acclimated with high CO2 under nutrient-replete conditions could alleviate the stress induced by high PAR and UV
(García-Gómez et al., 2014). This could be due to energy saving
as a result of down-regulation of CCM activity. However, in the present
study, we did not find that the synergistic effects of OA and UVR induced a
higher inhibition at the light intensity of PAR + UVA + UVB (44.11+14.19+0.75 Wm-2) used, than found under LC. This may be due to the
light intensity of PAR or UVR not being high enough to exceed the energy
dissipating capacity of the cells. Furthermore, under high N the nutrient
supply would be sufficient to support the repair processes of UV- or high-PAR-induced damage. In the LN-grown cells, UVB induced greater inhibition of
both carbon fixation and yield, probably due to a decreased
repair / damage ratio (Table 2) and decreased levels of both Chl a and other
light-harvesting pigments (Li et al., 2012a) since the (re)synthesis of
both proteins and UV-screening compounds depends on nitrogen availability
(Beardall et al., 2009, 2014). Such an inhibition by UVB in
LN-grown cells was more pronounced under OA conditions (Fig. 3b and d),
though UVB appeared to counteract the OA effect under the HN condition. When
the cells are exposed to lower external pH, they need additional
energy to cope with the acid–base perturbation (Kanazawa and Kramer, 2002).
By impairing photosynthesis, nitrogen limitation could decrease the supply
of energy, especially in the presence of UVB (Döhler, 1998). Though SOD
and CAT normalized per cell showed no change in all treatments (Fig. S1),
the fact that nitrogen limitation led to decreased protein contents per cell
and with higher activity of SOD and CAT (based on protein content) implies
that these enzymes are preferentially retained in the face of decreasing
protein per cell and thus reflects an enhanced defense strategy (Fig. 5), so
that ROS that were formed under N limitation could
be scavenged. The differential impacts of UVB on HN and LN-grown cells under
the OA treatment could be due to differences in the repair and damage rates
(Table 2) and differential stimulation of periplasmic proteins (Wu and Gao,
2009), which are important transporters of ions and play important roles in
maintaining intracellular acid–base stability. On the other hand,
NO3- scarcity usually leads to an impaired PSII reaction center
activity due to decreased synthesis of key proteins, therefore, leading to
decreased quantum yields of PSII (Geider et al., 1993). In this study, P. tricornutum
showed much lower yield (Fig. 2c and d), as well as NPQ, in the nitrogen-limited cells (Fig. 4c and d), indicating smaller functional PSII reaction
centers and a lower heat dissipating capability, when combined with the OA
treatment, consistent with these cells having the highest damage and the
lowest repair (Table 2). In the HN-grown cells, better recovery of both
photosynthetic carbon fixation (data not shown) and photochemical
performance (Tables 3 and 4) under OA conditions could be attributed to
faster repair rate of PSII and related metabolic up-regulations.
The results from the present work suggest that nutrient limitation can alter
the effects of OA or UVR and their interactions. In the oligotrophic oceans,
such as the surface mixed layers of the South China Sea (SCS), where
averaged total inorganic nitrogen concentrations range from 0–20 µmol, UVB and OA can act synergistically to bring about a higher inhibition
of photosynthetic carbon fixation. Higher UVB-induced inhibition of
photosynthesis was found in pelagic low-nutrient waters than in coastal
waters in the SCS (Li et al., 2011). With enhanced stratification and
reduced thickness of the upper mixed layer due to ocean warming, fewer
nutrients will be transported from deeper layers to the photic zones, and
interactions of enhanced nutrient limitation, OA and increased solar
exposures will become the main drivers influencing marine primary production
(Gao et al., 2012a). For diatoms, such as P. tricornutum, OA and other ocean changes
may result in transitions in their vertical and horizontal distributions and
changes in phytoplankton community structure.