Global climate change leads to simultaneous changes in multiple
environmental drivers in the marine realm. Although physiological
characterization of coccolithophores has been studied under climate change, there is limited knowledge on the biochemical responses of this
biogeochemically important phytoplankton group to changing multiple
environmental drivers. Here, we investigate the interactive effects of
reduced phosphorus availability (4 to 0.4 µmol L-1), elevated pCO2 concentrations (426 to 946 µatm), and increasing light intensity (40 to 300 µmol photons m-2 s-1) on elemental content and macromolecules of the cosmopolitan coccolithophore Emiliania huxleyi. Reduced phosphorus availability reduces particulate organic nitrogen (PON) and protein contents per cell under 40 µmol photons m-2 s-1 but not under 300 µmol photons m-2 s-1. Reduced phosphorus
availability and elevated pCO2 concentrations act synergistically to
increase particulate organic carbon (POC) and carbohydrate contents per cell under 300 µmol photons m-2 s-1 but not under 40 µmol photons m-2 s-1. Reduced phosphorus availability, elevated pCO2 concentrations, and increasing light intensity act synergistically to increase the allocation of POC to carbohydrates. Under elevated pCO2 concentrations and increasing light intensity, enhanced carbon fixation could increase carbon storage in the phosphorus-limited regions of the oceans where E. huxleyi dominates the phytoplankton assemblages. In each type of light intensity, elemental-carbon-to-phosphorus (C:P) and nitrogen-to-phosphorus (N:P) ratios decrease with increasing growth rate. These results suggest that coccolithophores could reallocate chemical elements and energy to synthesize macromolecules efficiently, which allows them to regulate their
elemental content and growth rate to acclimate to changing environmental
conditions.
National Natural Science Foundation of China4180612932001180Introduction
The continuous increase in atmospheric CO2 levels, as a consequence of
anthropogenic activities, leads to global and ocean warming, which in turn
shoals the ocean upper mixed layer (UML), hinders upward transport of
nutrients from deeper oceans to the UML, and increases light exposure to
phytoplankton cells dwelling therein (Steinacher et al., 2010; Wang et al.,
2015). The dissolution of CO2 in the oceans is causing a significant
chemical shift toward higher CO2 and proton ([H+]) concentrations,
a process defined as ocean acidification (OA; Caldeira and Wickett, 2003).
Environmental changes in the UML will expose phytoplankton cells to
physiological stress, and understanding the effects of changing multiple
environmental drivers on the physiology and biochemistry of marine
phytoplankton is important for projections of changes in the biogeochemical
roles of phytoplankton in the future ocean (Gao et al., 2019).
Coccolithophores take up carbon dioxide (CO2) to produce particulate
organic carbon (POC) via photosynthesis and use bicarbonate
(HCO3-) and calcium (Ca2+) to synthesize calcium carbonate
plates (coccoliths, particulate inorganic carbon (PIC)) and release CO2 via calcification; they also play a critical role in the marine carbon cycle (Rost and Riebesell, 2004). The cosmopolitan coccolithophore Emiliania huxleyi typically forms extensive blooms that are
easily detected by satellite remote sensing due to high light (HL) scattering
caused by coccoliths (Terrats et al., 2020; He et al., 2022). Within E. huxleyi blooms in polar and subpolar oceans, dissolved nitrate and phosphate concentrations in surface seawater could be as low as 0.95 and 0.16 µmol L-1, respectively (Townsend et al., 1994); light intensity is higher than 300 µmol photons m-2 s-1 (Tyrrell and Merico, 2004); and the mean concentrations of seawater CO2 increased by 21.0 %–43.3 % which weakens the oceanic CO2 uptake from the atmosphere (Kondrik et al., 2018). Emiliania huxleyi is also the dominant phytoplankton species in the lower photic zone in the north-eastern Caribbean Sea (western Atlantic Ocean) (Jordan and Winter, 2000) and in the South Pacific Gyre where dissolved nitrate and phosphate concentrations are about 1.0 and 0.2 µmol L-1, respectively, and light intensity is lower than 20 µmol photons m-2 s-1 (Beaufort et al., 2008;
Perrin et al., 2016). In the future ocean, numerous environmental factors
will simultaneously change, and the extent of these changes may increase (Gao
et al., 2019). To explore how E. huxleyi acclimates to simultaneous changes in macronutrient concentration, light intensity, and CO2 level, it is interesting to investigate their physiological and biochemical processes, which can also help to project the effect of coccolithophores on the ocean carbon cycle and ecological systems.
For more than a decade, research has shown that E. huxleyi cells developed several strategies to acclimate to reduced phosphorus availability, increasing light intensity, and ocean acidification (Leonardos and Geider, 2005; McKew et al., 2015; Wang et al., 2022). Interactive effects of phosphorus availability and light intensity have shown that under phosphorus-limited conditions, cells increased expression and the activity of alkaline phosphatase, and took up and used phosphorus efficiently under high light (HL) intensity, whereas they lowered the phosphorus uptake rate under low light (LL) intensity (Riegman et al., 2000; Perrin et al., 2016). In addition, the positive effect of reduced phosphorus availability on cellular POC and PIC contents of E. huxleyi was further enhanced by increasing light intensity due to high light-induced increases in CO2 and HCO3- uptake rates under low phosphate availability (Leonardos and Geider, 2005). The negative effect of reduced phosphorus availability on cellular particulate organic phosphorus (POP) content was partly compensated by the increased PO43- uptake rate under increasing light intensity (Perrin et al., 2016). On the other hand, several studies report that ocean acidification and reduced phosphorus availability acted synergistically to increase the cellular POC content, especially at high light intensity, and acted antagonistically to affect the cellular PIC content of E. huxleyi (Leonardos and Geider, 2005; Matthiessen et al.,
2012; Zhang et al., 2020). In addition, ocean acidification normally
amplified the positive effect of increasing light intensity on cellular POC
content (Rokitta and Rost, 2012; Heidenreich et al., 2019). Due to the high
proton-concentration-induced reduction in the HCO3- uptake rate, ocean acidification could weaken or counteract the positive effect of increasing light intensity on cellular PIC content (Rokitta and Rost, 2012; Kottmeier et al., 2016). Overall, while recent studies have focused on the physiological performance of E. huxleyi and their effects on marine biogeochemical cycling of carbon, little information is available about the biochemical response of E. huxleyi to reduced phosphorus availability, increasing light intensity, and ocean acidification.
The objective of this study is to investigate the combined effects of
reduced phosphorus availability, increasing light intensity, and ocean
acidification on cellular elemental contents, the carbon (C) : nitrogen (N) : phosphorus (P) ratio, and macromolecules of E. huxleyi and to analyse the effects of macromolecules on elemental contents. Under reduced phosphorus availability, increasing light intensity, and ocean acidification, we hypothesize that increased cellular POC content is more likely to be caused by increased carbohydrate content. In addition, we discuss the potential mechanisms for changing cellular PIC content in response to changed levels of phosphate, light, and CO2, which is important for projections of changes in coccolithophore biogeochemistry and ecology in the future ocean.
Materials and methodsExperimental setup
The Emiliania huxleyi strain RCC1266 (morphotype A) was originally isolated from shelf waters around Ireland (49∘30′ N, 10∘30′ W) in 2007 and obtained from the Roscoff algal culture collection (Fig. S1 in the Supplement). Emiliania huxleyi was cultured under a 14 h : 10 h light : dark cycle (light period: 06:00 to 20:00 CST, China standard time) in a thermo-controlled
incubator (MGC-400H, Shanghai Yiheng Scientific Instrument) at 20 ∘C in
semicontinuous cultures. The artificial seawater (ASW) medium was prepared
according to Berges et al. (2001) with a salinity of 33 psu, a boron
concentration of 372 µmol L-1, the addition of 2350 µmol L-1 bicarbonate to achieve the total alkalinity (TA) of 2350 µmol L-1, and enrichment with 64 µmol L-1 NO3-, f/8
concentrations for trace metals and vitamins (Guillard and Ryther, 1962).
The experiment was conducted in two parts (Fig. S2). The first part (Part 1)
was performed at 40 µmol photons m-2 s-1 (low light
intensity, LL), and the second one (Part 2) was at 300 µmol photons m-2 s-1 (high light intensity, HL). The LL intensity used here
corresponds to the lower end of the irradiance range of the UML, and the HL
intensity represents the irradiance in the surface ocean (Jin et al.,
2016; Perrin et al., 2016). For each part of the experiment, dissolved
inorganic phosphorus (DIP) concentration and ocean acidification were
combined in a fully factorial design: high DIP concentration (4 µmol L-1)+ low CO2 level (426 µatm, current CO2 level)
(HP + LC, treatment 1 in LL and treatment 5 in HL), high DIP concentration
(4 µmol L-1)+ high CO2 level (946 µatm, future CO2
level) (HP + HC, treatment 2 in LL and treatment 6 in HL), low DIP
concentration (0.43 µmol L-1)+ low CO2 level (426 µatm)
(LP + LC, treatment 3 in LL and treatment 7 in HL), and low DIP
concentration (0.43 µmol L-1)+ high CO2 level (946 µatm)
(LP + HC, treatment 4 in LL and treatment 8 in HL). High DIP concentration
is replete for the physical process of E. huxleyi, and at the end of the incubation,
low DIP concentration limits the growth of E. huxleyi (see below). There were eight
treatments in total and four biological replicates for each treatment (Fig. S2). In all cases, cell densities were lower than 78 000 cells mL-1, and
the cells were acclimated to each treatment for at least eight generations
before physiological and biochemical parameters were measured.
At LL intensity (Part 1), for the treatments of HP + LC and HP + HC, the ASW
media were enriched with 4 µmol L-1 PO43- and aerated
for 24 h at 20 ∘C with filter-sterilized (PTFE filter, 0.22 µm pore
size, Nantong) air pumped from the room. The pHTotal (total scale)
values of the media under both HP + LC and HP + HC treatments were about
8.04. The dry air was humidified with Milli-Q water prior to the aeration to
minimize evaporation. Under the HP + HC treatment, the pHTotal values of the media were adjusted to 7.74 by stepwise additions of
CO2-saturated seawater. For the treatments of LP + LC and LP + HC, the ASW media were enriched with 0.4 µmol L-1 PO43- and aerated for 24 h at 20 ∘C with filtered room air. Under the LP + HC treatment, the pHTotal values of the media were also adjusted to 7.74 as described above. The HP + LC, HP + HC, LP + LC and LP + HC seawater was
then filtered (0.22 µm pore size, Polycap 75 AS, Whatman) and carefully
pumped into autoclaved 50 mL (for TA measurements), 600 mL (for
pre-experimental cultures), and 2350 mL (for experimental cultures)
polycarbonate bottles (Nalgene) with no headspace to minimize gas exchange.
The cells were inoculated to achieve an initial density of 5000 cells
mL-1 in the HP + LC and HP + HC conditions, respectively, and cultured for 2 d then diluted to the initial density again. These processes were
performed three times in 600 mL bottles for pre-experimental cultures at 40 µmol photons m-2 s-1 (LL) of photosynthetically active radiation (PAR; measured using a LI-190SA quantum sensor, Beijing Ligaotai
Technology Co. Ltd.). In the main experimental cultures, the cells were
transferred from 600 to 2350 mL bottles at the same time and cultured
for another 2 d (Fig. S2b). Culture bottles were rotated 10 times until
cells were mixed at 09:00, 13:00, and 19:00 CST. On the second day of the
main experimental cultures, subsamples were taken for measurements of cell
densities; pHTotal; TA; cellular contents of total particulate carbon
(TPC); particulate organic carbon (POC), nitrogen (PON), and phosphorus
(POP); carbohydrate; and protein. At the end of the cultures under the
previous conditions, cell samples with an initial density of 5000 cells mL-1 were transferred from the HP + LC condition (treatment 1) to the LP + LC
condition (treatment 3) and from the HP + HC condition (treatment 2) to the LP + HC
condition (treatment 4) at LL intensity. The cells were acclimated to
the LP + LC and LP + HC conditions for eight generations before subsamples were taken for measurements, which allows cells to have enough time periods to
change growth rate against the low DIP concentration.
At HL intensity (Part 2), samples grown under the HP + LC and HP + HC
conditions were transferred from 40 (LL) to 300 µmol photons m-2 s-1 (HL) of PAR with an initial cell density of 5000 cells mL-1.
The cells were cultured under the HP + LC and HP + HC conditions for 2 d,
respectively, and then diluted back to the initial cell density. These
processes were performed three times in 600 mL bottles at HL intensity, and
then the main experimental cultures were conducted in 2350 mL bottles. On
the second day of the incubation, subsamples were taken for measurements of
the parameters. After that, cell samples with an initial density of 5000 cells mL-1 were transferred from the HP + LC condition (treatment 5) to the
LP + LC condition (treatment 7), and from the HP + HC condition (treatment 6) to the
LP + HC condition (treatment 8). At HL intensity, cell samples were
acclimated for at least eight generations in LP + LC and LP + HC conditions,
respectively, before subsamples were taken for measurements.
Phosphate concentration and carbonate chemistry measurements
In the beginning and on the second day of the incubations, samples for
determinations of phosphate concentration (20 mL), pHTotal value (20
mL), and total alkalinity (TA) (50 mL) were respectively filtered (PTFE
filter, 0.22 µm pore size, Nantong) 7 h after the onset of the light period (at 13:00 CST). The dissolved inorganic phosphorus (DIP) concentration was
measured using a spectrophotometer (SP-722, Shanghai Spectrum Instruments)
following the phosphomolybdate method (Hansen and Koroleff, 1999). The
bottle for pH measurement was filled from bottom to top with overflow and
closed without a headspace. The pHTotal value was measured immediately at 20 ∘C using a pH meter which was corrected with a standard buffer of
defined pH in seawater (Dickson, 1993). The TA samples were treated with a 10 µL saturated HgCl2 solution and stored in the dark at 4.0 ∘C, and
TA was measured at 20 ∘C by potentiometric titration (AS-ALK1+, Apollo SciTech) according to Dickson et al. (2007). Carbonate chemistry parameters
were estimated from TA and pHTotal using the CO2SYS program of Pierrot
et al. (2006) with carbonic acid constants, K1 and K2, taken from
Roy et al. (1993).
Cell density and elemental content measurements
Cell densities were monitored daily at 13:30 with 20 mL samples, and fresh media with the same DIP concentration and carbonate
chemistry as in the initial treatment conditions were added as top-up. Cell
densities were determined using a Multisizer™ 3 Coulter Counter (Beckman Coulter). Growth rates were calculated for each replicate according
to the equation: μ=(lnN1-lnN0)/d, where N1 and
N0 refer to the cell densities on the second day and in the beginning of
the main experiment, respectively, and d is the growth period in days.
After mixing, samples for determinations of TPC (300 mL), POC and PON (300 mL), and POP (300 mL) were obtained by filtering them onto the GF/F filters
(precombusted at 450 ∘C for 6 h) at the same time (14:00 CST) in each
treatment. For POC and PON measurements, samples were fumed with HCl for 12 h to remove inorganic carbon. Samples of TPC, POC, and PON were dried at
60 ∘C for 12 h and analysed using an Elementar CHNS analyser (Vario EL
cube, GmbH, Germany). Cellular particulate inorganic carbon (PIC) content
was calculated as the difference between cellular TPC and POC contents
(Fabry and Balch, 2010). To remove dissolved inorganic phosphorus from the
GF/F filters, POP samples were rinsed three times with 0.17 mol L-1
Na2SO4. After that, 2 mL 0.017 mol L-1 MgSO4 solution
was added onto filters, and POP samples were dried at 90 ∘C for 12 h
and then combusted at 500 ∘C for 6 h to remove POC and digested by 0.2 mol L-1 HCl (Solórzano and Sharp, 1980). Phosphorus concentration
was measured using a microplate reader (Thermo Fisher) following the
ammonium molybdate method (Chen et al., 1956) using adenosine-5'-triphosphate
disodium trihydrate as a standard.
Protein and carbohydrate measurements
Samples for determinations of protein (600 mL) and carbohydrate (600 mL)
were respectively filtered onto polycarbonate filters (0.6 µm pore
size, Nuclepore, Whatman) and onto precombusted GF/F filters at 14:30.
Protein samples were extracted by bead milling (FastPrep Lysing Matrix D) in a 0.5 mL protein extraction buffer (lithium dodecyl sulfate,
ethylene diamine tetraacetic acid, Tris, glycerol, and 4-(2-aminoethyl)
benzenesulfonyl fluoride hydrochloride). Bead milling was performed four
times for 1 min at 6.5 m s-1, and samples were placed on ice for 2 min
between each round of bead milling to prevent degradation. The samples were
then centrifuged at 10 000×g for 5 min (Centrifuge 5418 R,
Eppendorf, Germany), and extracted protein in the supernatant was quantified
using the BCA assay with bovine gamma globulin as a standard using a
microplate reader (Ni et al., 2016). Carbohydrate samples were hydrolyzed
with 12.00 mol L-1 H2SO4 in the dark for 1 h and diluted by
Milli-Q water to a final H2SO4 concentration of 1.20 mol L-1.
Then, samples were sonicated for 5 min, vortexed for 30 s, and boiled at
90 ∘C for 3 h (Pakulski and Benner, 1992). The extracted carbohydrate
was determined by a phenol sulfuric reaction with D-glucose as standard
(Masuko et al., 2005).
Data analysis
The percentages of carbon in carbohydrate and protein are 40 % and 53 %, respectively, and the percentage of nitrogen in protein is 16 % (Geider and LaRoche, 2002). A three-way analysis of variance (ANOVA) was used to
determine the main effects of the DIP concentration, light intensity, and
CO2 level, as well as their interactions on each variable. A Tukey post hoc test was performed to identify significant differences between two DIP
concentrations, two light intensities, and two CO2 levels. A
Shapiro–Wilk test was conducted to analyse the normality of residuals, and a
Levene test was conducted graphically to test for homogeneity of variances.
The significant difference between treatments was set at p<0.05.
All data analyses were conducted using the statistical software R with the
packages carData, lattice, and nlme (R version 3.5.0).
Carbonate chemistry parameters and dissolved inorganic phosphorus
(DIP) concentration at the end of the incubation. The values are means
± standard deviation (SD) of four replicates. Respectively, LL and HL
represent 40 and 300 µmol photons m-2 s-1 of
photosynthetically active radiation (PAR), and HP and LP represent 4 and
0.43 µmol L-1 PO43- at the beginning of the incubations.
During the incubations, organismal activity significantly reduces dissolved
inorganic phosphorus (DIP) concentrations (Table 1). Under high phosphorus
(HP) treatment, DIP concentrations decrease by 20.32 % in low light (LL)
and low CO2 (LC), by 22.32 % in LL and high CO2 (HC), by
27.66 % in high light (HL) and LC, and by 31.58 % in HL and HC. Under
low phosphorus (LP) treatment, DIP concentrations decrease from 0.43 µmol L-1 at the beginning of the experiment to be lower than 0.04 µmol L-1 (the detection limit) at the end of the incubation in LL
and LC conditions, in LL and HC conditions, in HL and LC conditions, and in
HL and HC conditions.
During the incubations, at LL intensity, pHT values increase, on
average, by 0.02 in HP + LC, by 0.03 in HP + HC, by 0.09 in LP + LC, and by
0.10 in LP + HC conditions (Table 1). At HL intensity, pHT values
increase by 0.05 in HP + LC, by 0.06 in HP + HC, by 0.12 in LP + LC, and by
0.09 in LP + HC conditions. Correspondingly, at LL intensity, seawater
CO2 concentrations decrease by 5.53 % in HP + LC, by 6.89 % in
HP + HC, by 22.76 % in LP + LC, and by 22.77 % in LP + HC. At HL
intensity, seawater CO2 concentrations decrease by 16.18 % in
HP + LC, by 16.41 % in HP + HC, by 28.92 % in LP + LC, and by 22.30 %
in LP + HC. Overall, organismal activity has larger effects on carbonate
chemistry under the LP treatment than under the HP treatment.
Growth rate (a), cellular contents of particulate organic carbon
(POC, b), nitrogen (PON, c), phosphorus (POP, d), and particulate
inorganic carbon (PIC, e) of Emiliania huxleyi RCC1266 in the treatments of high phosphorus
availability and low CO2 level (HP + LC), high phosphorus availability
and high CO2 level (HP + HC), low phosphorus availability and low
CO2 level (LP + LC), and low phosphorus availability and high CO2
level (LP + HC) under low light (empty, 40 µmol photons m-2 s-1) and high light (filled, 300 µmol photons m-2 s-1)
intensities. Different letters represent significant differences in each
parameter between treatments (p<0.05). The data represent the
means and standard deviation of four independent cultures.
Growth rate (d-1); cellular contents of POC, PON, POP, PIC,
carbohydrate (CHO), and protein (Pro) (pg cell-1); the ratios of POC : PON, POC : POP, PON : POP, and PIC : POC; the percentages of POC allocated to carbohydrate (CHO–C : POC) and protein (Pro–C : POC); and the
percentage of PON allocated to protein (Pro–N : PON) (%). LC and HC
represent low CO2 (426 µatm) and high CO2 (946 µatm)
levels, respectively. Please see Table 1 for more detailed information.
The effect of increasing light intensity on growth rate is positive, which
can be seen by comparing growth rate in the HL regimes with their paired LL
regimes (Fig. 1a; Table 2), though the extent of the increase in growth rate
depends on CO2 levels and phosphate availability. Compared to the LL
intensity, growth rates at HL intensity increased by 48.48 % in HP + LC,
by 50.87 % in HP + HC, by 60.86 % in LP + LC, and by 60.80 % in
LP + HC (Tukey post hoc test, all values of p<0.01). The effect of
increasing CO2 levels on the growth rate depends on light intensity and
phosphate availability (Fig. 1a). Compared to the LC level, growth rates in the HC level decreased by 3.08 % in the LL and HP condition (p=0.48), by 16.13 %
in the LL and LP condition (p<0.01), by 1.50 % in the HL and HP condition
(p=0.68), and by 16.27 % in the HL and LP condition (p<0.01). The
effect of reduced phosphorus availability on the growth rate is negative, and the extent of the reduction in growth rate depends on light intensity and CO2 levels (Fig. 1a). Compared to the HP availability, growth rates in LP
availability decreased by 8.46 % in the LL and LC condition (p<0.01),
by 20.81 % in the LL and HC condition (p<0.01), and by 15.63 % in
HL and HC condition (p<0.01); they did not change significantly in the
HL and LC condition (p=0.99). These results show that high CO2 levels and low phosphorus availability acted synergistically to reduce the
growth rate of E. huxleyi, and increasing light intensity could partly counteract this response (Table S1 in the Supplement).
Cellular POC, PON, POP, and PIC contents
The effect of increasing light intensity on cellular POC content is
positive, which was observed by comparing POC content in all the HL regimes
with their paired LL regimes (Fig. 1b). The extent of the increase in POC
content depends on CO2 levels and phosphate availability. Compared to the
LL intensity, cellular POC contents at HL intensity increased by 27.15 %
in HP + LC, by 26.51 % in HP + HC, by 43.24 % in LP + LC, and by
58.13 % in LP + HC conditions (Tukey post hoc test, all values of p<0.01). The effect of increasing CO2 levels on POC content is
light- and phosphate-dependent; it can be seen by comparing POC content in
the HC regimes with their paired LC regimes (Fig. 1b). At LL intensity,
cellular POC contents are not significantly different between HP + LC,
HP + HC, LP + LC, and LP + HC conditions (all values of p>0.1).
At HL intensity, compared to LC level, cellular POC contents in the HC level
increased by 5.12 % in the HP condition (p=0.74) and by 8.28 % in the LP
condition (p=0.07). The effect of phosphate reduction on POC content is
light- and CO2-dependent, which can be seen by comparing POC content in
the LP regimes with that in their paired HP regimes (Fig. 1b). At LL
intensity, cellular POC contents did not significantly differ between LP and
HP availability. At HL intensity, compared to HP availability, cellular POC
contents in LP availability increased by 11.80 % in the LC condition (p=0.02) and by 15.28 % in the HC condition (p<0.01). These results
show that ocean acidification and reduced phosphorus availability acted
synergistically to increase POC contents in the HL condition but not in the LL condition (Table S1).
The effect of increasing light intensity on cellular PON content depends on
CO2 levels and phosphate availability (Fig. 1c). Compared to the LL
intensity, cellular PON contents at HL intensity increased by 12.03 % in the
HP + LC condition (p=0.27), by 19.54 % in the HP + HC condition (p<0.01), by 22.68 % in the LP + LC condition (p<0.01), and by 30.90 %
in the LP + HC condition (p<0.01). The effect of increasing CO2
levels on PON content is light- and phosphate-dependent, which can be seen by
comparing POC content in the HC regimes with their paired LC regimes (Fig. 1c). Compared to the LC level, cellular PON contents in the HC level did not change
significantly in the LL + HP, LL + LP, and HL + LP
conditions but increased by 14.68 % in the HL + HP condition (p=0.02).
The effect of phosphate reduction on PON content is CO2- and light-dependent, which can be seen by comparing PON content in the LP regimes with
that in their paired HP regimes (Fig. 1c). Compared to the HP availability,
cellular PON contents in LP availability decreased by 16.59 % in the LL and LC
condition (p=0.01), by 24.03 % in the LL + HC condition (p<0.01), by 8.35 % in the HL + LC condition (p=0.43), and by 17.32 % in the
HL + HC condition (p<0.01). These results show that increasing
light intensity compensated for the negative effect of phosphate reduction
on PON content (Table S1).
The effect of increasing light intensity on POP content is positive and can
be seen by comparing POP content in the HL regimes with their paired LL
regimes, though the extent of the increase in POP content depends on CO2
levels and phosphate availability (Fig. 1d). Compared to the LL intensity,
cellular POP contents at HL intensity increased by 35.79 % in HP + LC, by
41.70 % in HP + HC, by 57.22 % in LP + LC, and by 56.44 % in LP + HC
conditions (Tukey post hoc test, all values of p<0.01). Ocean
acidification did not change the POP contents significantly under all
treatments used here (all values of p>0.53) (Fig. 1d). Reduced
phosphorus availability significantly decreased the POP contents, which can
be seen by comparing POP content in the LP regimes with their paired HP
regimes, though the extent of the reduction in POP content depends on light
intensity and CO2 levels (Fig. 1d). Compared to the HP availability,
cellular POP contents in LP availability decreased by 52.96 % in the LL and LC
condition, by 54.03 % in the LL + HC condition, by 46.11 % in the HL + LC
condition, and by 49.51 % in the HL + HC condition (all values of p<0.01). These results show that reduced phosphorus availability had a larger
effect on POP content than that of ocean acidification and increasing light
intensity (Table S1).
The effect of increasing light intensity on PIC content is positive, which
can be seen by comparing PIC content in the HL regimes with their paired LL
regimes, though the extent of increase in PIC content depends on CO2
levels and phosphorus availability (Fig. 1e). Compared to the LL intensity,
cellular PIC contents at HL intensity increased by 77.87 % in HP + LC, by
70.28 % in HP + HC, by 98.31 % in LP + LC, and by 90.68 % in LP + HC
conditions (Tukey post hoc test, all values of p<0.01). The effect
of increasing CO2 levels on PIC content is negative and can be seen by
comparing PIC content in the HC regimes with those in their paired LC
regimes (Fig. 1e). The extent of the reduction in PIC content depends on light
intensity and phosphorus availability. Compared to the LC level, cellular PIC
contents under ocean acidification decreased by 31.43 % in the LL and HP
condition (p=0.09), by 16.00 % in the LL and LP condition (p=0.67), by
35.02 % in the HL and HP condition (p<0.01), and by 21.12 % in the HL
and LP condition (p<0.01). The effect of phosphate reduction on PIC
content is positive, which can be seen by comparing PIC content in the LP
regimes with their paired HP regimes, though the extent of the increase in PIC
content depends on light intensity and CO2 levels (Fig. 1e). Compared
to the HP availability, cellular PIC contents in LP availability increased by
16.00 % in the LL and LC condition (p=0.83), by 41.26 % in the LL and HC
condition (p=0.16), by 29.98 % in the HL and LC condition (p<0.01), and by 60.44 % in the HL and HC condition (p<0.01). These
results show that high light intensity and low phosphorus availability acted
synergistically to increase the PIC content, which counteracts the negative
effect of ocean acidification on PIC content (Table S1).
Cellular contents of carbohydrate (a) and protein (b); the
percentages of POC allocated to carbohydrate (c) and protein (d); and the
percentage of PON allocated to protein (e) of E. huxleyi RCC1266 in the treatments of
high phosphorus availability and low CO2 level (HP + LC), high
phosphorus availability and high CO2 level (HP + HC), low phosphorus
availability and low CO2 level (LP + LC), and low phosphorus
availability and high CO2 level (LP + HC) under low light (empty) and
high light (filled) intensities. Different letters represent significant
differences in each parameter between treatments (p<0.05). The
data represent the means and standard deviation of four independent
cultures.
Carbohydrate and protein contents
The effect of increasing light intensity on carbohydrate content is positive
and can be seen by comparing carbohydrate content in the HL regimes with
their paired LL regimes, though the extent of the increase in carbohydrate
depends on CO2 levels and phosphorus availability (Fig. 2a). Compared
to the LL intensity, cellular carbohydrate contents at HL intensity increased by
148.81 % in the HP + LC condition, by 139.42 % in the HP + HC condition, by
179.12 % in the LP + LC condition, and by 204.42 % in the LP + HC condition (all
values of p<0.01). The effect of increasing CO2 levels on
carbohydrate content is light- and phosphate-dependent, which can be seen by
comparing carbohydrate content in the HC regimes with their paired LC
regimes (Fig. 2a). Compared to the LC level, cellular carbohydrate contents
under ocean acidification did not change significantly under the LL condition
and increased by 21.32 % in the HL and HP condition (p=0.02) and by
18.45 % in the HL and LP condition (p<0.01). The effect of phosphate
reduction on carbohydrate content is light- and CO2-dependent and can be
seen by comparing carbohydrate content in the LP regimes with their paired
HP regimes (Fig. 2a). Compared to the HP availability, cellular carbohydrate
contents in the LP availability did not change significantly in the LL and LC
and LL and HC conditions (both p>0.65) but increased by
40.13 % in the HL and LC condition (p<0.01) and by 36.00 % in the HL
and HC condition (p<0.01). These results show that increasing light
intensity dominantly increased carbohydrate content, and ocean acidification
and reduced phosphorus availability acted synergistically to increase
carbohydrate contents under high light intensity (Table S1).
The effect of increasing light intensity on cellular protein content is
positive, which can be seen by comparing protein content in the HL regimes
with their paired LL regimes, though the extent of the increase in protein
content depends on CO2 level and phosphorus availability (Fig. 2b).
Compared to the LL intensity, cellular protein contents at the HL intensity
increased by 24.76 % in the HP + LC condition, by 30.43 % in the HP + HC
condition, by 68.09 % in the LP + LC condition, and by 65.39 % in the LP + HC
condition (all values of p<0.01). The effect of increasing CO2
levels on protein content can be seen by comparing protein content in the HC
regimes with their paired LC regimes (Fig. 2b). Compared to the LC level,
cellular protein contents under ocean acidification did not change
significantly in the LL and HP condition, in the LL and LP condition, in the HL and HP
condition, and in the HL and LP condition (all values of p>0.09).
The effect of phosphate reduction on protein content is light- and CO2-dependent, which can be seen by comparing protein content in the LP regimes
with their paired HP regimes (Fig. 2b). Compared to the HP availability,
cellular protein content in the LP availability decreased by 27.88 % in the LL and
LC condition and by 28.80 % in the LL and HC condition (both p<0.01) but
did not change significantly in the HL and LC condition and in the HL and HC
condition (both p>0.11). These results show that high light
intensity counteracted the negative effect of low phosphorus availability on
protein content, and ocean acidification had less of an effect on protein content (Table S1).
Percentage of POC allocated to carbohydrate (carbohydrate–C <inline-formula><mml:math id="M564" display="inline"><mml:mo>:</mml:mo></mml:math></inline-formula> POC) and
protein (protein–C <inline-formula><mml:math id="M565" display="inline"><mml:mo>:</mml:mo></mml:math></inline-formula> POC)
Increasing light intensity increased the percentage of POC allocated to
carbohydrate (carbohydrate–C : POC), which can be seen by comparing
carbohydrate–C : POC in the HL regimes with their paired LL regimes, though
the extent of increase in carbohydrate–C : POC depends on CO2 levels
and phosphorus availability (Fig. 2c). Compared to the LL intensity,
carbohydrate–C : POC at the HL intensity increased by 95.60 % in the HP + LC
condition, by 97.69 % in the HP + HC condition, by 95.05 % in the LP + LC
condition, and by 83.37 % in the LP + HC condition (all values of p<0.01). The effect of increasing CO2 levels on carbohydrate–C : POC is
light- and phosphate-dependent and can be seen by comparing carbohydrate–C : POC in the HC regimes with their paired LC regimes (Fig. 2c). Compared to the
LC level, carbohydrate–C : POC under ocean acidification increased by
20.12 % in the LL and HP condition, by 11.42 % in the LL and LP condition, by
20.36 % in the HL and HP condition, and by 4.40 % in the HL and LP condition
(all values of p>0.08). The effect of phosphate reduction on
carbohydrate–C : POC is light- and CO2-dependent, which can be seen by
comparing carbohydrate–C : POC in the LP regimes with that in their paired
HP regimes (Fig. 2c). Compared to the HP availability, carbohydrate–C : POC in
LP availability increased by 25.61 % in the LL and LC condition (p=0.16),
by 17.37 % in the LL and HC condition (p=0.47), by 25.81 % in the HL and LC
condition (p<0.01), and by 8.11 % in the HL and HC condition (p<0.05). These results show that increasing light intensity, ocean
acidification, and reduced phosphorus availability acted synergistically to
increase the percentage of POC allocated to carbohydrate (Table S1).
Increasing the light intensity did not significantly change the percentage of the POC allocated to protein (protein–C : POC) under the phosphorus
availability and CO2 levels used here (Fig. 2d). Ocean acidification
did not significantly affect the protein–C : POC in the LL and HP, LL and
LP, HL and HP, and HL and LP conditions. The effect of phosphate
reduction on protein–C : POC is light- and CO2-dependent, which can be
seen by comparing the protein–C : POC in the LP regimes with their paired
HP regimes (Fig. 2d). Compared to the HP availability, protein–C : POC in LP
availability decreased by 27.39 % in the LL and LC condition (p<0.01), by 23.05 % in the LL and HC condition (p<0.01), by 12.81 %
in the HL and LC condition (p=0.09), and by 21.77 % in the HL and HC condition
(p<0.01). These results show that reduced phosphorus availability
dominantly reduced the protein–C : POC, and the increasing light intensity and
ocean acidification had less of an effect on protein–C : POC (Table S1). On the other hand, increasing light intensity, reduced phosphorus availability, and
ocean acidification did not significantly change the percentage of PON
allocated to protein (protein–N : PON) (Fig. 2e).
Cellular POC : POP ratio (a), PON : POP ratio (b), and protein
content (c) of E. huxleyi RCC1266 as a function of growth rate, and cellular POC
content as a function of carbohydrate (d) in the treatments of high
phosphorus availability and low CO2 level (HP + LC, □), high
phosphorus availability and high CO2 level (HP + HC, ○), low
phosphorus availability and low CO2 level (LP + LC, △), and low
phosphorus availability and high CO2 level (LP + HC, ◊) under low light
(LL, empty) and high light (HL, filled) intensities. Each point indicates an
individual replicate under each treatment.
Elemental stoichiometry and protein content as a function of growth rate
Reduced phosphorus availability increased the POC : PON ratio, and the
extent of the increase was larger under the HL intensity than the LL intensity (Fig. S5a). At
LL and HL intensities, both the POC : POP ratio and PON : POP ratio were
linearly and negatively correlated with growth rates (Fig. 3a, b). In LL and
HL conditions, the POC : POP ratio decreased linearly with increasing growth
rate (R2=0.71, F=32.08, p<0.01 in LL condition; R2=0.53, F=14.63, p<0.01 in HL condition). Similarly, in LL and
HL conditions, the PON : POP ratio decreased linearly with increasing growth
rate (R2=0.69, F=29.23, p<0.01 in LL condition; R2=0.50, F=13.31, p<0.01 in HL condition). In all treatments,
the protein content increased linearly with increasing growth rate (R2=0.76, F=151.14, p<0.01) (Fig. 3c), and the POC content increased
linearly with increasing carbohydrate content (R2=0.94, F=435.10, p<0.01) (Fig. 3d).
Discussion
Coccolithophores play a complex role in the marine carbon cycle through
production and export of organic carbon to depth but also through the
carbonate counter pump, and their responses to global climate change could
have significant consequences for marine carbon cycling (Riebesell et al.,
2017). The bloom-forming coccolithophore E. huxleyi, dominating the assemblages in
seawater under limited phosphorus condition, is likely to be exposed to
increasing light intensity and ocean acidification in the future ocean
(Kubryakova et al., 2021). In this study, we observed that increasing light
intensity compensates for the negative effects of low phosphorus
availability on cellular protein and nitrogen contents (Figs. 1 and 2).
Reduced phosphorus availability, increasing light intensity, and ocean
acidification act synergistically to increase cellular contents of
carbohydrate and POC and the allocation of POC to carbohydrate. These
changes in E. huxleyi could provide vital information for evaluating the carbon cycle in marine ecosystems under global change.
Ribonucleic acid (RNA) is the main phosphorus-containing macromolecule
within the cell (Geider and La Roche, 2002). Therefore, the reduced
phosphorus availability dominantly reduces the RNA content (Fig. S6), which
contributes to low POP contents (McKew et al., 2015) (Fig. 1d). In
eukaryotic cells, ribosomal RNA (rRNA) constitutes about 80 % of the total RNA and is mainly used to create ribosome (Dyhrman, 2016). Thus, reduced RNA contents decrease the numbers of ribosome, which has the potential to decrease protein synthesis (Dyhrman, 2016; Rokitta et al., 2016). On the other hand, the separation of the regression line between the POC : POP ratio (or PON : POP ratio) and growth rate under low and high light intensities suggests different POP storage contents in E. huxleyi among different light intensities (Perrin
et al., 2016). Low light intensity down-regulates the expression of genes
related to nitrate reductase and nitrite reductase; it then reduces the
nitrate uptake and assimilation efficiency of E. huxleyi and other phytoplankton
species (Perrin et al., 2016; Lu et al., 2018), which exacerbates the
negative effect of low phosphorus availability on protein synthesis and PON
contents (Figs. 1c and 2b). Besides that, low light intensity significantly
reduces the rates of RNA synthesis, carbohydrate synthesis, and cell division
(Zhang et al., 2021), adding to the negative effect of low phosphorus
availability on the growth rate of E. huxleyi (Fig. 1a). Under high light intensity and low CO2 level, reduced phosphorus availability did not change growth rate
and protein content (Figs. 1a and 2b), which suggests that E. huxleyi might compensate
for low phosphate-induced decreases in ribosome content by increasing
protein synthesis efficiency under increasing light intensity (Reith and
Cattolico, 1985). Under high light intensity and ocean acidification,
reduced phosphorus availability did not significantly change cellular
protein content while reducing the growth rate, which might indicate the lowered
protein synthesis efficiency (McKew et al., 2015).
Several studies report that reduced phosphorus availability (0.4–0.5 µmol L-1) did not change growth rates significantly during the
short-time (2 or 3 d) incubations under low CO2 level and high light
intensity (Rokitta et al., 2016; Zhang et al., 2020; Wang et al., 2022). The
reasons could be that E. huxleyi cells developed high affinity for phosphate, increased the uptake rate of phosphate (Wang et al., 2022), and could replace the phospholipid membrane with a non-phosphorus membrane during the short-time
incubation of phosphorus limitation (Shemi et al., 2016). Our data showed
that reduced phosphorus availability and ocean acidification acted
synergistically to reduce the growth rate under both low and high light
intensities (Fig. 1a). One of the reasons could be that low pH value under
ocean acidification up-regulates the expressions of a series of genes
involved in ribosome metabolism, such as genes of large subunit ribosomal
protein L3, L38E, and L30E (RP-L3, RP-L38E, RP-L30E) and small subunit ribosomal protein S3E, S5E, and
SAE (PR-S3E, PR-S5E, RP-SAE) in E. huxleyi (Wilson and Doudna Cate, 2012) (Fig. S7). Under ocean
acidification, up-regulation of the expression of these genes has the potential
to drive cells to allocate more phosphorus to synthesize ribosome and to
reduce the allocation of phosphorus to DNA replication (Rokitta et al.,
2011), which exacerbates the limitation of reduced phosphorus availability
on the rate of cell division in E. huxleyi (Rouco et al., 2013). Under
phosphorus-replete conditions, more phosphorus is reallocated to ribosome
metabolism under ocean acidification which could facilitate nitrogen
assimilation (Fig. 2b). Overall, under high light intensity, ocean
acidification is likely to facilitate E. huxleyi cells to increase nitrogen content in
phosphorus-replete conditions and to reduce the growth rate in
phosphorus-limited conditions.
In this study, we found that low light intensity dominantly limits carbon
assimilation of E. huxleyi and reduces the effects of phosphate availability and ocean acidification on cellular carbohydrate and POC contents (Figs. 1b and 2a).
However, under high light intensity, E. huxleyi had high carbohydrate and POC contents
while having low growth rates under reduced phosphorus availability and ocean
acidification (Figs. 1a, b and 2a), which suggests that the carbon assimilation
rate did not change significantly, while the cell division rate decreased
(Matthiessen et al., 2012; Perrin et al., 2016). Furthermore, carbohydrate
is a carbon- and energy-storing macromolecule, and protein is related to
growth rate (Geider and La Roche, 2002). Under high light intensity, reduced
phosphorus availability, and ocean acidification, E. huxleyi cells could synthesize
more carbohydrate to store carbon and energy but did not increase protein
content, which contributes to the large percentage of POC allocated to
carbohydrate (Fig. 2c).
The physiological reasons for the reduced calcification rate under ocean
acidification could be due to the high proton-concentration-induced reduction in the HCO3- uptake rate (Meyer and Riebesell, 2015; Kottmeier et al.,
2016). The molecular mechanisms for low PIC content under ocean
acidification may be due to down-regulation of a series of genes potentially
involved in ion transport and pH regulation, such as genes of calcium and proton
exchanger (CAX3), sodium and proton exchanger (NhaA2), and membrane-associated proton pump
(PATP) (Mackinder et al., 2011; Lohbeck et al., 2014). On the other hand,
increasing light intensity up-regulates a series of genes related to ion
transport, such as the gene of CAX3, the gene of the Cl- and HCO3- exchanger, and genes of various subunits of a vacuolar H+–ATPase (V–ATPase) (Rokitta et al., 2011). Up-regulation of these genes in high light intensity
has the potential to facilitate cells to take up HCO3- and
Ca2+ and to pump proton outside the cells; this then leads to large PIC
content of E. huxleyi (Kottmeier et al., 2016). Our data suggest that increasing light
intensity counteracts the negative effect of ocean acidification on the cellular
PIC content of E. huxleyi (Fig. 1e). These results are consistent with the findings of
Feng et al. (2020), who reported that combinations of increasing light
intensity and ocean acidification increase the expression of genes involved
in calcium-binding proteins (CAM and GPA), which has the potential to increase
the calcium influx into cells and then compensate for the effect of reduced
HCO3- uptake rate on calcification. It is also suggested that
increasing light intensity could cause cells to remove H+ faster, which
neutralizes the effect of high proton concentration on calcification (Jin et
al., 2017). On the other hand, reduced phosphorus availability extends the
G1 phase of the cell cycle where calcification occurs, which prolongs the
calcification time and then increases cellular PIC content (Müller et
al., 2008). In addition, reduced phosphorus availability up-regulates
expressions of genes of Ca2+ uptake, proton removal, and carbonic
anhydrase and then increases coccolith production (Wang et al., 2022),
which contribute to a larger PIC content and counteract the negative effect
of ocean acidification on PIC contents (Borchard et al., 2011) (Fig. 1e).
Furthermore, one of the reasons for larger PIC contents under reduced
phosphorus availability and increasing light intensity conditions is likely
due to larger and more numerous coccoliths (Gibbs et al., 2013; Perrin et
al., 2016). Overall, responses of calcification of E. huxleyi to ocean climate change
are more complex than previously thought (Meyer and Riebesell, 2015), and it is
worth exploring the underlying mechanisms of calcification under changing
multiple environmental drivers (Mackinder et al., 2011; Feng et al., 2020).
Cellular POP content of E. huxleyi generally decreased, and the POC : POP ratio and PON : POP ratio increased with reducing phosphorus availability (Leonardo and
Geider, 2005; McKew et al., 2015). The negative correlations between the growth
rate and POC : POP ratio or PON : POP ratio under each type of light intensity are
consistent with the growth rate hypothesis (Fig. 3), which proposes that the
growth rate increases with increasing RNA : protein ratio. Phosphorus in RNA
accounts for a high percentage of total POP, whereas nitrogen in protein is
the main form of PON (Zhang et al., 2021), and the growth rate hypothesis
suggests that the growth rate could increase with decreasing POC : POP ratio or
PON : POP ratio (Sterner and Elser, 2002). Our results suggest that E. huxleyi could
reallocate chemical elements and energy to synthesize carbohydrate, protein,
and RNA efficiently and then regulate its elemental stoichiometry and
growth rate to acclimate to reduced phosphorus availability, ocean
acidification, and increasing light intensity (Moreno and Martiny, 2018).
In the future ocean, large carbohydrate and POC contents, POC : PON ratio,
and POC : POP ratio of coccolithophores indicate increases in carbon export to the deep ocean that may affect the efficiency of the biological carbon pump and the marine biogeochemical cycle of carbon (Meyer and Riebesell,
2015). In addition, increased cellular PIC content under phosphorus-limited conditions may have the potential to weaken CO2 uptake of the
oceans in phosphorus-limited marine environments. In summary, responses of
coccolithophores to climate change are likely to affect the marine carbon
cycle in the future (Riebesell et al., 2017).
Data availability
The data are available upon request to the corresponding author
(yongzhang@fjnu.edu.cn).
The supplement related to this article is available online at: https://doi.org/10.5194/bg-20-1299-2023-supplement.
Author contributions
YZ (yongzhang@fjnu.edu.cn), ZL, and KX contributed to the design of the experiment. YZ (yongzhang@fjnu.edu.cn), YZ
(qsx20211022@student.fjnu.edu.cn), SM, HC, and RH performed this experiment
and biochemical analyses. YZ (yongzhang@fjnu.edu.cn) wrote the first draft of the paper. All authors contributed to the data analyses and editing
of the paper.
Competing interests
The contact author has declared that none of the authors has any competing interests.
Disclaimer
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Acknowledgements
We would like to thank Zoe V. Finkel for providing the
Emilianiahuxleyi RCC1266 strain, Vinitha Ebenezer for her helpful revision of the paper,
and Peng Jin and two anonymous reviewers for their helpful suggestions
which have helped us to improve the paper.
Financial support
This research has been supported by the National Natural Science Foundation of China (grant nos. 41806129 and 32001180).
Review statement
This paper was edited by Koji Suzuki and reviewed by three anonymous referees.
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