Article
Reference
Elemental stoichiometry and photophysiology regulation of Synechococcus sp. PCC7002 under increasing severity of chronic
iron limitation
BLANCO AMEIJEIRAS, Sonia, et al .
Abstract
Iron (Fe) is an essential co-factor for many metabolic enzymes of photoautotrophs. Although Fe limits phytoplankton productivity in broad areas of the ocean, phytoplankton have adapted their metabolism and growth to survive in these conditions. Using the euryhaline cyanobacterium Synechococcus sp. PCC7002 we investigated the physiological responses to long-term acclimation to four levels of Fe availability representative of the contemporary ocean (36.7, 3.83, 0.47 and 0.047 pM Fe'). With increasing severity of Fe limitation, Synechococcus sp. cells gradually decreased their volume and growth while increasing their energy allocation into organic carbon and nitrogen cellular pools. Furthermore, total cellular content of pigments decreased. Additionally, with increasing severity of Fe limitation, intertwined responses of photosystem II functional cross-section (σPSII), re-oxidation time of the plastoquinone primary acceptor QA (τ), and non-photochemical quenching revealed a shift in the photophysiological response between mild to strong Fe limitation compared to severe limitation. Under mild and strong Fe limitation there [...]
BLANCO AMEIJEIRAS, Sonia, et al . Elemental stoichiometry and photophysiology regulation of Synechococcus sp. PCC7002 under increasing severity of chronic iron limitation. Plant and Cell Physiology , 2018
DOI : 10.1093/pcp/pcy097
Available at:
Title
Elemental stoichiometry and photophysiology regulation of Synechococcus sp. PCC7002 under increasing severity of chronic iron limitation
Short title
Stoichiometry & photophysiology of Synechococcus
Corresponding author
S. Blanco-Ameijeiras
Department F.-A. Forel for Environmental and Aquatic Sciences/Faculty of Science//University of Geneva/Boulevard Carl-Vogt 66/Geneva 4/1211/Switzerland/[email protected]
Subject areas
(2) environmental and stress responses & (5) photosynthesis, respiration and bioenergetics
8 color figures, 2 tables and type, 1 supplementary figure and 1 supplementary table.
Elemental stoichiometry and photophysiology regulation of Synechococcus sp. PCC7002 under increasing severity of chronic iron limitation
Stoichiometry & photophysiology of Synechococcus
Sonia Blanco-Ameijeiras1 Sophie A. M. Moisset1
Scarlett Trimborn2,3 Douglas A. Campbell4
Jasmin P. Heiden2,3 Christel S. Hassler1
1 Department F.-A. Forel for Environmental and Aquatic Sciences, Faculty of Science, University of Geneva, Boulevard Carl-Vogt 66, 1211 Geneva 4, Switzerland
2 Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany
3 Marine Botany, University of Bremen, Leobener Strasse NW2-A, 28359 Bremen, Germany
4 Biology, Faculty of Science, Mount Allison University, E4L1G7, Sackville NB, Canada
Abbreviations
c0 cell counts in ml at the beginning of the experiment incubation
c1 cell counts in ml at the end of the experiment incubation
C:N carbon to nitrogen ratio
C:N:P carbon:nitrogen:phosphorus stoichiometry EK light saturation threshold
ETRmax maximum electron transport rate
ETR53 ETR under growth PAR
F’ minimum photosystem II fluorescence determined with continuous background illumination
Fe iron
Fe’ dissolved inorganic Fe
Fb cellular baseline fluorescence correction by fluorescence from phycobilisomes and photosystem I
Ffsw baseline fluorescence correction by fluorescence from filtered growth medium
FRRf fast repetition rate fluorometer
FRR-ST single turnover fluorescence induction curves
Fm dark-adapted photosystem II maximum fluorescence
Fm’ maximum photosystem II fluorescence determined with continuous background illumination
Fq’/Fm’ typically represents effective photosystem II quantum yield determined with continuing background illumination
Fv/Fm commonly used as a measurement of the photosystem II photochemical efficiency in dark acclimated cells
F0 dark-adapted photosystem II minimum fluorescence
LC measurements performed in short-term exposed cells to increasing light
LEDs light-emitting diodes
LET linear electron transport from photosystem II to photosystem I
NSV normalized Stern Volmer photochemical quenching
OCP orange carotenoid protein
PAR photosynthetic active radiation
PBS phycobilisomes
POCcq particulate organic carbon cell quota
POCprod particulate organic carbon production rate
PONcq particulate organic nitrogen cell quota
PONprod particulate organic nitrogen production rate
POPcq particulate organic phosphate
POPprod particulate organic phosphate production rate
PSI photosystem I
PSII photosystem II
ST calculated size of state transitions
Zea zeaxanthin
α maximum light use efficiency
β-Carot β-carotene
Δt time of incubation in days
growth rate
ρ degree of connectivity between photosystem II reaction centers
ρ’ degree of connectivity between photosystem II reaction centers determined with continuous background illumination
PSII photosystem II functional absorption cross-section determined in dark acclimated samples
σPSII’ photosystem II functional absorption cross-section determined in illuminated cells
τ re-oxidation time of the plastoquinone primary acceptor QA determined in dark τ’ re-oxidation time of primary quinone-type acceptor QA determined in illuminated cells
Abstract
Iron (Fe) is an essential co-factor for many metabolic enzymes of photoautotrophs. Although Fe limits phytoplankton productivity in broad areas of the ocean, phytoplankton have adapted their metabolism and growth to survive in these conditions. Using the euryhaline cyanobacterium Synechococcus sp. PCC7002 we investigated the physiological responses to long-term acclimation to four levels of Fe availability representative of the contemporary ocean (36.7, 3.83, 0.47 and 0.047 pM Fe’). With increasing severity of Fe limitation, Synechococcus sp. cells gradually decreased their volume and growth while increasing their energy allocation into
organic carbon and nitrogen cellular pools. Furthermore, total cellular content of pigments decreased. Additionally, with increasing severity of Fe limitation, intertwined responses of photosystem II functional cross-section (σPSII), re-oxidation time of the plastoquinone primary acceptor QA (τ), and non-photochemical quenching revealed a shift in the photophysiological response between mild to strong Fe limitation compared to severe limitation. Under mild and strong Fe limitation there was a decrease in linear electron transport accompanied by progressive loss of state transitions. Under severe Fe limitation, state transitions seemed to be largely supplanted by alternative electron pathways. In addition, mechanisms to dissipate energy excess and minimize oxidative stress associated with high irradiances increased with increasing severity of Fe limitation. Overall, our results establish the sequence of physiological strategies adopted by the cells under increasing severity of chronic Fe limitation, within a range of Fe concentrations relevant to modern ocean biogeochemistry.
Keywords
Synechococcus, Photophysiology, Iron limitation, Stoichiometry
Introduction
Photoautotrophs use light to fix inorganic carbon into organic molecules through photosynthesis. Cyanobacteria were the first oxygenic photoautotrophs to appear about 3.48 billion years ago (Noffke et al., 2013). Their evolution under the reductive conditions of ancient oceans favored the ‘luxurious’ use of iron (Fe) in many redox catalysts involved in cellular processes including respiration, macronutrient assimilation and detoxification of reactive oxygen species (Sunda, 1989; Sunda and Huntsman, 1995; Raven, Evans and Korb, 1999). However, in the oxidative conditions of the modern ocean the low solubility of Fe leads to low Fe availability. Indeed, more than 30% of the world’s oceans have anomalously low phytoplankton biomass despite persistent concentrations of macronutrients due to Fe limitation (Pitchford and Brindley, 1999; Boyd and Ellwood, 2010). The role of Fe as a limiting nutrient has been well established in the Subarctic North and Equatorial Pacific as well as in the Southern Ocean, regions of high-nutrient low-chlorophyll content (Martin and Fitzwater, 1988; Tsuda et al., 2003;
Behrenfeld et al., 2006). Furthermore, Fe can co-limit primary productivity in other regions of the Pacific, Atlantic and Indian Oceans (Moore et al., 2013), where Synechococcus sp. cyanobacteria form prominent blooms (Flombaum et al., 2013).
In cyanobacteria, photosynthesis and respiration share several protein complexes connecting photosynthetic responses associated to Fe stress with other principal metabolic pathways (Scherer, Stürzl and Böger, 1982;
Campbell et al., 1998). In order to mitigate the effects of Fe depletion, cyanobacteria typically decrease their cellular Fe quotas and enhance Fe uptake (Wilhelm, MacAuley and Trick, 1998; Jiang et al., 2015), which is usually accompanied by lowered photosynthetic performance (Liu and Qiu, 2012; Fraser et al., 2013). While the vast majority of studies investigating the effects of Fe dearth on cyanobacteria are focused on responses under Fe starvation (no Fe addition in the culture) versus Fe replete conditions (Sandström et al., 2002; Ludwig and Bryant, 2012), studies concentrating in the physiological transition into Fe starvation are scarce (Ryan-Keogh et
al., 2012). Given that chronic Fe limitation is widespread in the contemporary ocean (Moore et al., 2013),
acclimation to a progressive decrease in Fe availability is essential to decipher the roles of different processes participating in homeostasis facing Fe limitation. Various studies have indeed claimed that acclimation to Fe stress provides a better mechanistic understanding of natural phytoplankton homeostasis (Thompson et al., 2011; Liu and Qiu, 2012; Mackey et al., 2015). Transcriptomic analysis demonstrated that chronically Fe-limited Synechoccocus sp. PCC7002 showed dramatically different homeostasis mechanisms than Fe starved cells. For instance, chronic
Fe-limited Synechoccocus sp. PCC7002 down-regulated enzymes involved in the sugar catabolism, through downregulation of enzymes participating in the oxidative pentose phosphate pathway (Osanai et al., 2005; Ludwig and Bryant, 2012; Blanco-Ameijeiras, Cosio and Hassler, 2017), while almost no changes in enzyme expression of the carbon metabolism were reported for Fe starved Synechoccocus sp. PCC7002 (Ludwig and Bryant, 2012).
In cyanobacteria, the metabolic strategies to cope with Fe dearth include a decrease in cellular growth rates (Wilhelm, Maxwell and Trick, 1996) and a modification of carbon:nitrogen:phosphorus (C:N:P) stoichiometry (Geider and Roche, 2002). These changes reflect the differential allocation of resources amongst different macromolecular pools (Wagner, Jakob and Wilhelm, 2006; Halsey and Jones, 2015). Even though the C:N:P stoichiometry has been investigated in cyanobacteria under nutrient replete conditions and nitrogen limitation (Geider and Roche, 2002; Ho et al., 2003; Finkel et al., 2016), information on elemental stoichiometry for Fe-limited non-diazotrophic cyanobacteria is still lacking. Typically, cellular growth rate and macromolecular composition greatly affect the degree of reduction of biomass, being directly related to the number of electrons needed to synthetize 1 mol of carbon biomass from carbon dioxide (Kroon and Thoms, 2006).
In this study we investigate how Fe limitation modulates growth and elemental stoichiometry as well as their subsequent effects on the electron transport chain in the non-diazotrophic euryhaline Synechococcus sp. PCC7002.
To this end, we grew this strain under four contrasting levels of Fe availability representative of the range found
in the modern ocean under four dissolved inorganic Fe (Fe’) concentrations (36.67, 3.83, 0.47 and 0.047 pM Fe’).
Hereafter, the different Fe treatments used will be referred to as Fe replete (36.67 pM Fe’), mild Fe limitation (3.83 pM Fe’), strong Fe limitation (0.47 pM Fe’) and severe Fe limitation (0.047 pM Fe’). Using variable chlorophyll a (Chl a) fluorescence methods different parameter of the Single turnover fluorescence induction (FRR-ST) curves were determined and the photophysiological parameters for each treatment where derived. These parameters reflect different aspects of cellular status and photophysiological responses of photosystem II (PSII).
The acclimation mechanisms regulating photosynthetic electron transport and energy dissipation were investigated in dark acclimated cells and cells short-term exposed to increasing photosynthetic active radiation (PAR). These results support previous responses of this strain in response to increasing Fe-limitation at the transcriptomic level (Blanco-Ameijeiras, Cosio and Hassler, 2017). We provide thus a global perspective on the shifts in energy balance and dissipation mechanisms in response to increasing severity of chronic Fe limitation.
Results
Cellular growth and composition under increasing chronic Fe limitation
Growth rate () of Synechococcus sp. PCC7002 decreased by 64% with increasing severity of Fe limitation,
from Fe replete conditions to severe Fe limitation (Fig. 1). Concomitantly, cellular volume decreased up to 26%
with increasing Fe limitation, showing a linear correlation (r2 = 0.845; P <0.001; Fig. 1). With increasing Fe limitation, the particulate organic carbon cell quota (POCcq) and particulate organic nitrogen cell quota (PONcq) decreased between Fe replete conditions to strong Fe limitation, but then re-increased back up to values close to replete conditions under severe Fe limitation (Fig. 1). The particulate organic phosphate cell quota (POPcq) linearly decreased between Fe replete conditions (0.84 fmol cell-1) to severe Fe limitation
limitation between Fe replete and strong Fe limitation, but remained similar between strong and severe Fe limitation (Fig. 1). With increasing Fe availability, the carbon to nitrogen (C:N) ratio showed a positive linear correlation with growth rate (r2 = 0.998; P <0.001; Fig. 1).
The cellular content of Chl a decreased by 90% with increasing Fe limitation, between Fe replete conditions to severe Fe limitation (Fig. 2). Similarly, the cellular content of the accessory pigments β-carotene (β-Carot) and Zeaxanthin (Zea), also decreased linearly with increasing severity of Fe limitation (Fig. 2).
Biophysical properties of PSII under increasing chronic Fe limitation
The PSII functional absorption cross-section determined in dark acclimated samples (PSII) was significantly
higher under Fe replete and severe Fe limitation treatments than in mild and strong Fe limited treatments (Fig. 3A).
Despite their large PSII, severely Fe limited cells showed significantly lower degree of connectivity between photosystem II reaction centers (ρ) than in the other treatments with higher Fe availability, showing a loss of excitonic connectivity among PSII centers, represented by ρ in dark acclimated samples (Fig. 3B). Meanwhile, re-oxidation time of primary quinone-type acceptor QA in dark acclimated samples (τ) showed no difference between the three Fe limited treatments but was significantly shorter under Fe replete conditions, reflecting faster re-oxidation of PSII (Fig. 3C). The values obtained for the normalized Stern Volmer photochemical quenching corrected by filtered growth medium baseline fluorescence (NSVFfsw) were significantly higher than those corrected by cellular baseline fluorescence (NSVFb) from phycobilisomes (PBS) and photosystem I (PSI) for all the Fe treatments, with the largest magnitude of difference (47%) observed under severe Fe limitation (Fig. 3D).
Despite differences in magnitude associated to the correction method, NSVFb and NSVFfsw under severe Fe limitation were both significantly higher than under Fe replete conditions. Thereafter, the use of the sub index Ffsw
or Fb with the photophysiological parameters indicate the correction applied. The Fv/Fm (commonly used as a
measurement of the PSII photochemical efficiency in dark acclimated cells), when corrected by the cellular baseline fluorescence (Fv/Fm Fb), brought the uncorrected Fv/Fm obtained for 36.7 pM Fe’ (0.43) to values close to Fe repletion (0.45). This rise in Fv/Fm Fb, was due to the decrease in minimum fluorescence (F0) achieved through the Fb correction. Fv/Fm Fb was similar among the Fe replete, mild and strong Fe limitation treatments (Fig. 3E).
However, under severe Fe limitation, Fv/Fm Fb (0.39 0.03) was significantly lower than under mild Fe-limitation (0.44 0.02; P <0.001). These results contrasted with Fv/Fm corrected by filtered growth medium baseline
fluorescence (Fv/Fm Ffsw), which showed a gradual decrease, from 0.38 to 0.24 with increasing severity of Fe limitation (Fig. 3E).
Photosynthetic performance under chronic Fe limitation
The effects of short-term exposure of the cells to increasing light (LC measurements) was investigated in all Fe treatments (Fig. 4; Table 2). Minimum (F0) and maximum fluorescence (Fm) increased with increasing Fe limitation (Fig. 4A and 4B), while increasing irradiance levels led to a decrease in these fluorescence levels for all the treatments. Under severe Fe limitation, maximum electron transport rate (ETRmax Fb) and light saturation threshold (EK Fb) were significantly higher than in the other treatments (Table 2; Fig. 4C), whereas maximum light use efficiency (αFb) was higher under Fe replete conditions than in the Fe limited treatments (Table 2). The ETR under growth PAR (ETR53 Fb) was significantly higher under Fe replete conditions, while no significant differences were observed amongst the Fe limitation treatments (Table 2). The PSII functional absorption cross-section determined in illuminated cells (σPSII’) was also greatly affected by Fe limitation with increasing irradiance levels (Fig. 4D). Under Fe replete conditions, σPSII’ reached the highest values at low to medium irradiance levels (8 to 53 mol photons m-2 s-1) and decreased with increasing irradiance. But in the
Fe limited treatments the maximum σPSII’ was achieved at higher irradiance (128 µmol photons m-2 s-1) and decreased at yet higher light levels. σPSII’ was significantly higher under severe Fe limitation than under
Fe replete conditions between 128 and 465 µmol photons m-2 s-1. After short-term exposures to increasing light, the strongest effect on NSVFb was observed under severe Fe limitation, where NSVFb increased up to 6 when cells were exposed to 856 µmol photons m-2 s-1 (Fig. 4E). In contrast, in the treatments with higher Fe availability, the NSVFb ranged between 1.19 and 1.51 at low PAR and only slightly increased at high PARs (Fig. 4E). With low irradiance levels (up to 53 mol photons m-2 s-1) the re-oxidation time of primary
quinone-type acceptor QA determined in illuminated cells (τ’) was higher under Fe limitation than under Fe replete conditions. With higher irradiance levels it decreased to values similar to Fe replete conditions (Fig. 4F).
Under Fe replete conditions, the Fv/FmFb determined following the LC and 10 min of dark acclimation was 30%
lower than the initial Fv/Fm Fb determined in 60 min dark acclimated samples, before the LC (Fig. 5).The Fv/FmFb
after 10 min in the dark after the LC measurements gradually increased with increasing Fe limitation. Under severe Fe limitation, the Fv/FmFb determined after LC was 20% higher than its initial value.
Relationship between photophysiological parameters and state transitions
Under Fe replete conditions plots of NSVFb, (our metric of non-photochemical excitation dissipation),versusτ’
(our metric of downstream electron transport), showed a V-shape with increasing irradiances (Fig. 6A). There was a sharp decrease in NSVFb reaching a minimum between darkness and the light onset (8 mol photons m-2 s-1).
With further increases in light up to 128 mol photons m-2 s-1, NSVFb and ‘ remained almost unchanged. But after
exposition to yet higher irradiances from 239 up to 856 mol photons m-2 s-1, the increase in NSVFb was accompanied by a decrease of ’ as downstream electron transport accelerated. Under mild and strong Fe limitation, the overall relationship between NSVFb and ’presented three main sections (Fig. 6B and 6C).
In the first section, between darkness and 8 mol photons m-2 s-1, NSVFb remained similar, while ’ increased. In the second section, between 8 and 128 mol photons m-2 s-1, NSVFb and ’ declined. Over the third section, at irradiances above 128 mol photons m-2 s-1, NSVFb increased again while ‘ further decreased. The main difference
between mild and strong Fe limitation was the lower slope of the second section and greater NSVFb value at 0.47 pM Fe’. Finally, for cells acclimated to severe Fe limitation, the large error bars observed between 0 and 239 mol photons m-2 s-1 prevent the extrapolation of a convincing pattern, but for higher light intensities NSVFb
increased as for the other experimental treatments (Fig. 6D). We observed that these light dependent changes in NSVFb and τ’, when merged in the product NSVFb×τ’, were negatively correlated with σPSII’ for each Fe treatment (Fig. 7). However, each Fe regime showed a different slope and intercept.
Under Fe replete conditions, the calculated size of the state transition (ST) was large in the dark and with low irradiance levels, reaching the maximum size at 128 mol photons m-2 s-1, while with higher irradiances the state
transition size decreased rapidly (Fig. 8). Under Fe limitation, the state transition remained about 1 with low irradiances, as typically occurs in dark conditions. Under strong and severe Fe limitation maximum size was attained between 53 and 128 mol photons m-2 s-1. Under severe Fe limitation, calculated state transition size was
smaller than 1, suggesting no state transition was operating.
Discussion
Macromolecular allocation under Fe limitation
In line with results previously reported (Wilhelm, Maxwell and Trick, 1996; Sandström et al., 2002), Fe limited Synechococcus sp. PCC7002 showed a decline in cell volume and growth rate. The growth rate of 0.29 d-1 reported for Synechococcus sp. PCC7002 under severe Fe limitation is in good agreement with the values reported for Fe limited natural phytoplankton communities from the Equatorial Pacific (ranging from 0.17 and .037 d-1; Fitzwater et al., 1996), one of the major HNLC regions with the Southern Ocean. Under nutrient starvation (nitrogen,
phosphorus) different phytoplanktonic groups increase lipid and carbohydrate storage, while cellular protein contents either remain steady or decrease (Bertilsson et al., 2003; Wagner, Jakob and Wilhelm, 2006; Jakob et al., 2007; Halsey, Milligan and Behrenfeld, 2010). The decrease of POCcq observed in mild and strong Fe limited Synechococcus sp. PCC7002 suggested a decrease in the allocation of organic carbon into lipids and/or
carbohydrates, whereas the decreased PONcq pointed towards a decreased accumulation of proteins and nucleotides. However, the increase of POCcq and PONcq observed from strong to severe Fe limitation suggested a change in the metabolic acclimation to severe Fe limitation. This hypothesis was also supported by enhanced expression of genes encoding for proteins involved in the translation of RNA into peptides, as well as genes specifically involved in amino acid biosynthesis pathways reported for the same strain under severe Fe limitation (Blanco-Ameijeiras, Cosio and Hassler, 2017).
The increase in POCcq and PONcq under severe Fe limitation reflected profound shifts in the cellular macromolecular composition (Geider and Roche, 2002; Finkel et al., 2016), which come along with variation in cellular energy demands (Kroon and Thoms, 2006). Thus, increasing the allocation of nitrogen into biomass under increasing Fe limitation would also increase the cellular demand for electrons, which might impair growth
(Vrede et al., 2004). Indeed, we observed significant correlations between elemental stoichiometry (C:N) and growth rate with increasing Fe limitation, showing that these parameters remained closely linked under Fe limitation. The low C:N determined in this study for Fe replete Synechococcus sp. PCC7002 (4.25 mol mol-1) compared well with the lower range of the previously reported values of C:N for cyanobacteria, ranging between 3.6 and 7.8 mol mol-1 under nutrient replete conditions (Bertilsson et al., 2003; Kretz et al., 2015; Finkel et al., 2016). The decrease in C:N ratio observed in Synechococcus sp. PCC7002 with increasing severity of Fe limitation, contrasts with the responses typically observed in cyanobacteria and other phytoplankton groups under nutrient starvation (Bertilsson et al., 2003; Jakob et al., 2007; Halsey, Milligan and Behrenfeld, 2010). This decrease in C:N with increasing severityof Fe limitation was attributed to larger increases in PONcq than in POCcq.
Photophysiological parameters in dark acclimated cells
Under increasing severity of Fe limitation the decrease in the cellular content of Chl a (Wilhelm and Trick, 1995), as well as a shift in the peak of Chl a absorbance (Öquist, 1974), previously reported in the literature reflect accumulation of IsiA complexes (Bibby et al., 2001). Our photophysiological results pointed towards a transition between two main responses according to severity of Fe limitation. The first response was observed under mild and strong Fe limitation, where a decrease in PSII with respect to Fe replete conditions in dark acclimated Synechococcus sp. PCC7002 indicated that the cells underwent important adjustments in their excitation capture
capacity at the PSII level (Fig. 3). Furthermore, the increase of observed under mild and strong Fe limitation
relative to Fe replete conditions indicated that re-oxidation of the QA pool was slower, showing down-stream limitations on the electron transport. Mild Fe limitation resulted in up-regulation of the gene isiA and down-regulation of psaA and psaB, encoding for PSI proteins, which then remained unchanged with further increasing severity of Fe limitation (Blanco-Ameijeiras, Cosio and Hassler, 2017). These results support a decrease in linear electron transport from PSII to PSI (LET). Indeed, it has been previously reported that IsiA forms a ring
complex around the PSI, increasing the effective cross-section of PSI (Ryan-Keogh et al., 2012), which leads to an increase in the electron flux through each remaining PSI under Fe limitation (Sun and Golbeck, 2015). Several studies have also shown that IsiA free complexes can also act as effective energy dissipators and be involved in photoprotective mechanisms (Park et al., 1999; Sandström et al., 2001; Ihalainen et al., 2005). In this context, the significantly lower values of PSII observed under mild and strong Fe limitation could be associated to the increase
of IsiA competing with the PSII for excitation energy from the PBS as previously reported (Park et al., 1999;
Sandström et al., 2001). In fact, work from Wilson et al. (2007) using Fe limited IsiA mutants, in combination with PBS mutants and orange carotenoid protein (OCP) mutants, suggest that phycobilisomes transfer absorbed energy to IsiA, which represents a non photochemical quenching mechanism. Finally, ρ remained similar under mild and strong Fe limitation as well as Fe replete conditions, suggesting that the equilibration of excitons to other open PSII reaction centers could act as a protective mechanism minimizing PSII reaction center over-reduction (Liu and Qiu, 2012).
The second phase of response was observed under severe Fe limitation, where ρ significantly decreased, indicating excitonic disconnection of PSII reaction centers, and PSII was significantly enhanced up to values similar to those
registered under Fe replete treatments. Xu et al. (2017) propose that ρ is negatively correlated to the average distance among active PSII reaction centers. In this context, we suspect that under severe Fe limitation a decline in active PSII reaction centers lowers connectivity. The increase in PSII could then be explained by the remaining
PBS antenna serving a small population of active PSII reaction centers organized in a puddle rather than a lake antenna bed. The lower expression of genes encoding for PSII reaction centers reported by the transcriptomic analysis supports this hypothesis (Blanco-Ameijeiras, Cosio and Hassler, 2017). Suggesting, therefore, that under severe Fe limitation the transfer of excitation energy into photochemistry is limited by the number of photochemical traps available per PSII antenna unit. A similar response has been also reported for the cyanobacterium Anacystis nidulans R2 (Riethman and Sherman, 1988) and the eukaryotes Dunaliella tertiolecta
and Phaeodactylum tricornutum under Fe limitation (Greene, Geider and Falkowski, 1991; Greene et al., 1992;
Vassiliev et al., 1995). Despite the observed decrease in pigments per cell under severe Fe limitation, transcriptomic results suggest that no significant PBS degradation took place under strong and severe Fe limitation, because of a significant down-regulation of nbla, encoding for a putative PBS degradation protein. As in this study
PSII was determined with three blocks of light-emitting diodes (LEDs), the spectral shifts due to different pigments
complements might contribute to some of the changes observed. However, the concentration of PBS pigments relative to other pigments of the photosynthetic apparatus are not expected to dramatically change in Fe limited cyanobacteria as demonstrates the work of Ferreira and Straus (1994) in the cyanobacteria Synechocystis PCC6803. Transcriptomic data have also shown that under severe Fe limitation Synechococcus sp. PCC7002 down-regulated the expression of genes encoding for components of the electron transport chain, such as PSII, cytochrome b6f and ATPase, whereas the respiratory terminal oxidase cytochrome oxidase II was up-regulated (Blanco-Ameijeiras et al., 2017). Therefore, the contribution of alternative electron pathways, where electrons flow from PSII to a terminal oxidase, utilizing O2 as the terminal electron acceptor (Ermakova et al., 2016), may be relevant under severe Fe limitation (Behrenfeld and Milligan, 2013). Thus, the pool of PSII would remain highly oxidized while the LET through PSI would be expect to decrease (Behrenfeld and Milligan, 2013; Ermakova et al., 2016).
Tolerance to short-term exposure to high PARs under chronic Fe limitation
Short-term exposure to increasing PARs also showed specific responses according to the severity of Fe limitation in Synechococcus sp. PCC7002. Under Fe replete conditions the “V” shape of the relationship between NSVFb and
‘ observed was in good agreement with observations in other cyanobacteria species grown in Fe replete conditions (Mullineaux and Allen, 1986; Misumi et al., 2015). In the dark, the respiratory electron transport typically drives Fe replete cells into state 2 with a down regulation of fluorescence emission from PSII (Mullineaux and Allen,
1990; Campbell et al., 1998; Huang et al., 2003). The decrease in NSVFb observed upon onset of illumination in Fe replete Synechococcus (Fig. 6A) was in agreement with previous studies reporting that oxidation of the plastoquinone pool upon illumination induced a transition to state 1 where the fluorescence yield of PSII increases (Campbell and Oquist, 1996; Misumi et al., 2015). The shortened τ observed with further increasing irradiance indicates an acceleration of the down-stream electron transport, which is accompanied by an increase in non-photochemical quenching. This quenching under high light could be presumably attributed to the OCP, reported as a key cyanobacteria energy dissipating mechanism under high irradiance (Kirilovsky, 2007;
Wilson et al., 2007; Sedoud et al., 2014; Thurotte et al., 2015). The transcriptomic analysis demonstrated that the orthologous gene encoding OCP was constitutively expressed under all the Fe treatments tested in Synechococcus sp. PCC7002 (Blanco-Ameijeiras, Cosio and Hassler, 2017).
Under mild and strong Fe limitation Synechococcus sp. PCC7002 showed similar responses to short-term exposure to high PARs. The transition to state 1 was only achieved between 53 and 128 mol photons m-2 s-1, rather than at
growth irradiance (53 mol photons m-2 s-1), suggesting that Fe limitation hampers the state transition. This hypothesis of inefficient state transitions under mild and strong Fe limitation is in good agreement with previous studies reporting increasing half-time of the state 2 transition in Fe starved Synechococcus sp. PCC7942 and Synechocystis sp. PCC6803 (Mullineaux and Allen, 1986; Ivanov et al., 2006). In this regard, decreasing NADH
dehydrogenase and respiration activity have been suggested as primary drivers to decrease the electron transport to the soluble electron carriers, resulting in an oxidized plastoquinone pool in the dark (Huang et al., 2003;
Ma et al., 2007). Indeed, Fe limited Synechococcus sp. PCC7002 have shown a significant down-regulation of ndhF, encoding for a NADH2 dehydrogenase subunit (Blanco-Ameijeiras, Cosio and Hassler, 2017), which is required for the transition to state 2 in the dark by mediating electron flow into the inter-system electron carriers in the dark (Huang et al., 2003).
Under severe Fe limitation the light to dark state transition were absent. The decrease in ‘ observed with increasing
irradiance, suggests an acceleration of the downstream electron transport and non-photochemical quenching increased to very high levels. In combination with the low growth rate observed under severe Fe limitation, these results suggest that the increased downstream electron rate is less efficient in terms of ATP production. Therefore, the significantly higher EkFb and ETRmaxFb reported under severe Fe limitation might be attributed to alternative electron pathways. Similar results were previously reported for Fe limited phytoplankton communities from the Pacific Ocean after short-term exposure to high irradiance (Mackey et al., 2008; Schuback et al., 2015), as well as for monoclonal cultures of the diatom Thalassiosira oceanica and the prymnesiophyte Chrysochromulina polylepis (Schuback et al., 2015).
The photophysiological response of Synechococcus sp. PCC7002 under Fe limitation with increasing irradiance showed that increases in non-photochemical quenching and slower re-oxidation time of the plastoquinone pool, led to a decrease in σPSII’, which in general is consistent with a state 2 transition. These results showed that changes in σPSII’ were positively correlated with NSVFb×τ’, although significant correlations between NSVFb versus σPSII
and τ’ versus σPSII were not observed (Supplementary Fig. S1). The mechanistic relationship remains unclear. With
increasing severity of Fe limitation the NSVFb capacity significantly increased with increasing irradiance. As NSVFb is an unbound ratio (Giovagnetti and Ruban, 2017), the high values obtained for severe Fe limited cells under high irradiance do not imply that the dissipation of non-photochemical quenching was 4-fold of that under Fe replete conditions. However, the comparison of Fv/Fm Fb before and after LC measurements, suggests an increasing efficiency of photoprotective mechanisms with increasing severity of Fe limitation in Synechococcus sp. PCC7002. In addition, up-regulation of genes involved in mitigation of oxidative stress was
also reported for this strain under severe Fe limitation (Blanco-Ameijeiras, Cosio and Hassler, 2017).
Conclusion
Our results establish a sequence in physiological strategies to respond to progressive Fe limitation in the euryhaline Synechococcus sp. PCC7002. With increasing Fe limitation, the cells gradually decreased their volume and growth
rate, while their elemental stoichiometry dramatically shifted, indicating an increasing energy allocation into the organic nitrogen and carbon pools. Photophysiological analysis, in combination with previous transcriptomic analysis of Synechococcus sp. PCC7002 under identical environmental conditions, revealed a shift in the photophysiological response between mild to strong Fe limitation compared to severe limitation. Under mild and strong Fe limitation there was a decrease in linear electron transport accompanied by progressive loss of state transitions. Under severe Fe limitation, state transitions seemed to be largely supplanted by alternative electron pathways. In addition, mechanisms to dissipate energy excess and minimize oxidative stress associated with high irradiances increased with increasing severity of Fe limitation.
Material and Methods
Culture conditions
Synechococcus sp. PCC7002 was grown in chemically characterized synthetic oceanic seawater (Hassler et al.,
2011), which was chelexed (Chelex-100, BioRad; Price et al., 1989), enriched with macronutrients (also chelexed), trace metals and vitamins (Table 1) and finally filter-sterilized (0.2 m polycarbonate membrane, Whatman).
Culture medium was amended with different concentrations of dissolved Fe (Table 1), resulting in four Fe’
concentrations (36.67, 3.83, 0.47 and 0.047 pM Fe’), calculated with MINEQL+ 4.6 (Schecher and McAvoy, 1994). Chosen Fe’ concentrations represent estuarine and coastal (Mahmood et al., 2015), oceanic upwelled Fe-rich (Bruland, Rue and Smith, 2001; Buck, Sohst and Sedwick, 2015) and oceanic low Fe (Fitzsimmons, Zhang and Boyle, 2013; Buck, Sohst and Sedwick, 2015) regions, respectively. Manipulations were conducted in a trace metal-clean AirClean Systems (AC600 Series PCR Workstation, STAR LAB, Creedmoor, NC, USA). All labware and material used were cleaned to remove any trace metal background contamination through 1 week soaking in 0.01% Citranox (ALCONOX, White Plains, NY, USA) and Milli-Q rinsing, followed by 1 week soaking in 1.2 M hydrochloric acid prior to extensive rinsing with ultrapure water (18.2 M) obtained from a
Milli-Q Direct System (Merk Millipore, Darmstadt, Germany). Unless otherwise specified, all solutions used in this study were prepared using analytical grade chemicals (Sigma-Aldrich, Buchs, Switzerland) and Milli-Q water.
Cultures of Synechococcus sp. PCC7002 were grown in semi-continuous batch cultures at 22°C in a RUMED 34001 Light thermostat equipped with Daylight fluorescent tubes (Rubarth Apperate GmbH, Laatzen, Germany), where PAR was 50 μmol photons m-2 s-1 under a 12:12 h light:dark cycle. The cultures were acclimated to the selected Fe’ concentrations in semi-continuous diluted batch cultures using a dilution/re-inoculation period of 7 days for at least 22 generations prior the experiments. To this end, experimental bottles were inoculated with the respectiveacclimated cultures into 2000 ml of media with an initial concentration of 10.7×104 ±2.3×104cell ml-1,
in each polycarbonate bottles. The cells were grown in three biological replicates of each experimental treatment and harvested after 4 days of incubation.
Cell size and growth rate
Cell size and cell counts were determined in vivo using the cell counter and analyzer system CASY Model TTC (Roche Innovartis, Reutlingen, Germany) with a 45 m capillary. Based on the cell density, growth rate (µ) was calculated according to equation 1.
µ = (Ln c1 – Ln c0) / Δt (1)
where c0 and c1 are the cell counts in ml at the beginning and at the sampling day of the experiment, respectively, and Δt is the period of incubation in days.
Particulate organic carbon (POC) and nitrogen (PON) content
Aliquots of 100-250 ml of culture were concentrated through filtration onto pre-combusted GF/F filters (0.7 µm nominal pore size, 47 mm, Whatman) and stored at -20°C. Before analysis, the filters were fumed with 37%
hydrochloric acid for 24 h to remove particulate inorganic carbon (Verardo, Froelich and McIntyre, 1990). After drying at 60°C for 24 h, the filters were packaged in pre-combusted aluminum foil (Hilton et al., 1986) and analyzed on a Perking Elmer 2400 Series II CHNS/O Elemental Analyzer using an organic analytical standard of cystine (PerkinElmer, Shelton, USA). POC and PON content were corrected for blank measurements and normalized to total cell counts to calculate cellular quotas (POCcq and PONcq). POC and PON production rates (POCprod and PONprod) were calculated multiplying the cellular quota by µ. Results of production rates were expressed in mol of carbon or nitrogen cell-1 d-1, accordingly.
Particulate organic phosphate (POP) content
Organic phosphorus compounds were digested in presence of the oxidizing decomposition reagent Oxisolv (Merck Millipore, Darmstadt, Germany) under high temperature (~121°C) and pressure (~100 kPa) to obtain dissolved orthophosphate. After addition of ascorbic acid (39.6 mM final concentration (Fisher Scientific UK, Leics, UK) and a 10% v/v of a reagent solution (3.6 M sulfuric acid, 13.8 mM ammonium heptamolybdate and 1.95 mM potassium antimonyl tartrate) the orthophosphate formed a blue heteropoly acid that was determined by spectrophotometric analysis (Hansen and Korolef, 1983). A calibration series, ranging from 0 to 500 µg l-1 was prepared with known amounts of H3PO4 in H2O Titrisol (Merck Millipore, Darmstadt, Germany). POP cell quota (POPcq) and POP production rate (POPprod) were calculated as described before for POC and PON.
Pigment content
Aliquots of 100-250 ml of culture were concentrated through filtration on 47 mm GF/F filters, snap frozen with liquid nitrogen and stored at -80°C until analysis. Pigments were extracted through manual homogenization in 90:10 acetone:water, incubated for 24 h at 4°C in the dark and filtered (4 mm nylon syringe filters, 0.45 µm pore size) prior to analysis. Analyses were performed using a Hitachi LaChromElite® high-performance-liquid- chromatography (HPLC) system equipped with a temperature controlled auto-sampler L-2200, a DAD detector L-2450 (Hitachi High Technologies Inc., Schaumburg, IL, USA) and a Spherisorb ODS-2 column (25 cm×4.6 mm, 5 μm particle size; Waters Corp., Milford, MA, USA). Pigments separation was achieved using a LiChrospher® 100 RP-18 guard cartridge (Merck KGaA, Darmstadt, Germany). Peaks detected at 440 nm were identified and quantified via co-chromatography of pigment standards obtained from DHI Lab Products (ORT, Denmark) using the software EZChrom Elite ver. 3.1.3 following Wright et al. (1991).
Variable Chl a fluorescence
Chl a fluorescence measurements were performed using the fast repetition rate fluorometer (FRRf) FastOcean PTX coupled to a FastAct base unit (Chelsea Technologies Group Ltd, West Molesey, UK) that circulated Milli-Q water at 22 ºC around the sample to keep the temperature constant during measurements. Three biological replicates were measured for each Fe treatment during the light phase of the light:dark cycle. Photophysiological measurements were performed at least 3 h after the onset of incubation light and after 1h dark acclimation immediately prior to analysis. FRR-ST curves consisted of a saturation phase comprising 100 flashlets on a 2 μs pitch and a relaxation phase comprising 40 flashlets on a 50 μs pitch. Excitation light was produced by a block of 450 (preferentially absorbed by chlorophyll), 530 and 624 nm (preferentially absorbed by the phycobilisomes) LEDs with intensities of 0.66 × 1022, 0.40 × 1022 and 1.49 × 1022 photons m-2 s-1, respectively. The relative intensities of the three different LED bands were chosen on basis of the automatic optimization performed by the system for each sample. The automatic optimization tested different LED intensities and voltage of the photomultiplier tube, until the value of RσPII generated by the FastPro8 felt within the optimum range of 0.04 to 0.05 and the curve of fluorescence increase reaches a plateau at the end of the saturation phase. This optimization gave the same optimal settings for all the samples (Supplementary Table S1). Twelve FRR-ST induction curves, spaced by 120 ms, were averaged using the software FastPro8 GUI (Chelsea Technologies Group Ltd.) into a single induction curve. This was repeated 6 times with an interval of 3 seconds. Using FastPro8 GUI, the acquisitions were fitted to the KPF biophysical model (Kolber, Prášil and Falkowski, 1998) to determine dark-adapted minimum (F0) and maximum (Fm) photosystem II (PSII) fluorescence yields, PSII functional absorption cross-section (PSII), re-oxidation time of primary quinone-type acceptor QA (τ) and degree of connectivity between PSII reaction centers (ρ). All photophysiological parameters were corrected by F0 determined for each sample on 0.2 µm filtered growth media (fsw; Ffsw; Supplementary Table S1). The corrections Ffsw were performed using the blank correction option of the FastPro8 GUI software.
In cyanobacteria, F0 does not arise exclusively from PSII alone. Cellular baseline fluorescence (Fb) from phycobilisomes (PBS) and PSI can significantly contribute to the F0 signal (Campbell et al., 1998; Simis et al., 2012; Murphy et al., 2017). Therefore, the values of fluorescence-based parameters that use F0 in their calculation, such as Fv/Fm, commonly used as a measurement of the PSII photochemical efficiency, must be interpreted with caution. In addition, under Fe limitation, an increase of the non-variable component of the fluorescence yield per unit of Chl a was associated with the expression product of isiA and accumulation of energetically detached photosynthetic antenna complex in the cyanobacterium Synechocystis sp. PCC6803 (Ihalainen et al., 2005;
Schrader, Milligan and Behrenfeld, 2011). This phenomenon has been estimated to represent almost 50% of the pigment content in the phytoplankton cells present in high-nutrient low-chlorophyll regions and waters of the Fe-limited subpolar North Atlantic (Behrenfeld et al., 2006; Macey et al., 2014). In this context, thermal dissipation by excitation of IsiA was reported to contribute up to 38% of non-photochemical quenching in Synechocystis sp.
PCC6803 under Fe starvation (Cadonet et al., 2004). In order to account for Fb associated with energetically detached photosynthetic antenna complexes, all acquisitions were also corrected for the Fb according to equation 2 (Oxborough, 2014) using the blank correction option of the FastPro8 GUI software:
Fb = Fm – Fv / (Fe replete Fv/Fm) (2)
where a value of 0.455 of Fv/Fm, determined in nutrient replete cultures, was used as ‘Fe replete Fv/Fm’. In fact, the
‘true Fv/Fm’ from PSII alone is expected to be about 0.75 (Campbell et al., 1998). Here, the dataset of dark acclimated measurements was independently corrected for the two types of fluorescence baseline corrections (Ffsw
and Fb) and the resulting estimates compared. The use of the sub index Ffsw or Fb with the photophysiological parameters indicate the correction applied.
The Fv/Fm was calculated following equation 3.
In cyanobacteria, measured non-photochemical quenching encompasses thermal energy dissipation (Kirilovsky, 2007; Thurotte et al., 2015) and fast state transitions to distribute excitation between PSII and PSI (Campbell et al., 1998). Non-photochemical quenching (NSV) was calculated using the normalized Stern-Volmer following equation 4 (McKew et al., 2013; Oxborough, 2014).
NSV = (Fm/Fv) - 1 = F0/Fv (4)
Fluorescence light curves (LC) were conducted exposing each treatment to a set of increasing irradiance levels (8, 23, 53, 128, 239, 465, 856 µmol photons m-2 s-1) each applied for 5 min. At each PAR step, the minimum (F’) and maximum (Fm’) PSII fluorescence, PSII functional absorption cross-section (PSII’), re-oxidation time of primary quinone-type acceptor QA (τ’) and degree of connectivity between PSII reaction centers (ρ’) were determined with continuing background illumination. The F0 of these measurements were Fb corrected in order to account for the distorting effect induced by energetically detached photosynthetic antenna complexes in Chl a fluorescence and thereby in the discussion of the results.
The two baseline corrections Fb and Ffsw (presented above) were applied for each measurement again. The Fq’/Fm’, typically representing effective PSII quantum yield in ambient light, was calculated following equation 5.
Fq’/Fm’ = (Fm’- F’) / Fm’ (5)
NSV for each PAR was calculated using the normalized Stern-Volmer following equation 4 (McKew et al., 2013).
After the LC, the samples were acclimated to dark for 10 min and a final measurement in the dark was performed and compared with the initial dark acclimated measurement preformed before the LC to determine the recovery
capacity after exposure to high light intensities. The recovery yield was calculated as the percentage of variation respect to the dark acclimated measured before the LC.
Absolute PSII electron transport rates (ETR) at each PAR were calculated according to equation 6 (Suggett et al., 2006, 2009). The ETR curve was fitted using the model from Ralph and Gademann (2005) with the beta phase fit.
From the fitted curve, maximum ETR (ETRmax), maximum light use efficiency (α) and the light saturation threshold (Ek) were determined.
ETR (e- PSII-1 s-1) = PSII × ((Fq’/Fm’) / (Fv/Fm)) × PAR (6)
The relationship between NSVFb and (’)Fb with increasing irradiances was used as indicative of the changes of state transition in response to increasing instantaneous light intensities in cells acclimated to different Fe availability according to Misumi et al. (2015). Thus, inflection points in the resulting curve would suggest a change in the state transition. In addition, the size of the state transitions during the LC, was calculated following equation 7 (Oxborough and Baker, 1997; Campbell et al., 1998).
ST = (Fm’-F0’) / (Fm-F0) (7)
where F0’ was calculated according to equation 8 (Oxborough and Baker, 1997).
F0’ = F0 / (Fv /Fm+ F0/Fm’) (8)
Although, a weakness of this parameter is that it can be influenced by additional non-photochemical quenching mechanisms at play (Oxborough and Baker, 1997).
Statistical analysis
All data are given as the means of the three biological replicates and its standard deviation. Significant differences between the treatments were tested using one-way ANOVA followed by post hoc (Holm-Sidak method) tests. The significance level was set to 0.05. Correlation between pairs of variables was tested using Pearson product moment correlation with the significance level set to 0.05. Statistical analyses were performed using SigmaPlot (SysStat Software Inc., San Jose, CA, USA).
Funding
This work was founded by the Swiss National Science Foundation (FNS PP00P2-138955 to CSH), the Schmidheiny Foundation to SBA, the Helmholtz association (Young Investigators Group EcoTrace, VH-NG-901 to ST) and the Canada Research Chairs program (to DAC).
Disclosures
The authors have no conflicts of interest to declare.
Acknowledgments
Authors are grateful to Marc C. Moore for his comments in a previous version of this manuscript, Damien J. E.
Cabanes for measurements of total Fe concentration in the inorganic seawater matrix used for the experiments and Fabrice Carnal for his support and stimulating discussions.
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