Elemental Stoichiometry and Photophysiology Regulation of Synechococcus sp. PCC7002 Under Increasing Severity of Chronic Iron Limitation

Elemental Stoichiometry and Photophysiology Regulation of Synechococcus sp. PCC7002 Under... Abstract Iron (Fe) is an essential cofactor 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, the total cellular content of pigments decreased. Additionally, with increasing severity of Fe limitation, intertwined responses of PSII 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 with 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. 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 et al. 1999). However, in the oxidative conditions of the modern ocean, the low solubility of Fe leads to low Fe availability. Indeed, >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-Chl content (Martin and Fitzwater 1988, Tsuda 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 with Fe stress with other principal metabolic pathways (Scherer et al. 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 et al. 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 a dearth of Fe on cyanobacteria are focused on responses under Fe starvation (no Fe addition in the culture) vs. Fe-replete conditions (Sandström et al. 2002, Ludwig and Bryant 2012), studies concentrating on 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 when 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 homeostasis mechanisms dramatically different from Fe-starved cells. For instance, chronic Fe-limited Synechoccocus sp. PCC7002 down-regulated enzymes involved in sugar catabolism, through down-regulation of enzymes participating in the oxidative pentose phosphate pathway (Osanai et al. 2005, Ludwig and Bryant 2012, Blanco-Ameijeiras et al. 2017), while almost no changes in enzyme expression of carbon metabolism were reported for Fe-starved Synechoccocus sp. PCC7002 (Ludwig and Bryant 2012). In cyanobacteria, the metabolic strategies to cope with a dearth of Fe include a decrease in cellular growth rates (Wilhelm et al. 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 et al. 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, the 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 one 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 Chl a fluorescence methods, different parameters of the single turnover fluorescence induction (FRR-ST) curves were determined and the photophysiological parameters for each treatment were derived. These parameters reflect different aspects of cellular status and photophysiological responses of PSII. The acclimation mechanisms regulating photosynthetic electron transport and energy dissipation were investigated in dark-acclimated cells and cells exposed to increasing photosynthetic active radiation (PAR) for a short time. These results support previous responses of this strain to increasing Fe limitation at the transcriptomic level (Blanco-Ameijeiras et al. 2017). We thus provide 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 The growth rate (μ) 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 and strong Fe limitation, but then increased again 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 (0.17 fmol cell–1; Fig. 1). The POC, PON and POP production rates (POCprod, PONprod and POPprod) decreased linearly with increasing Fe 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). Fig. 1 View largeDownload slide Cellular volume, growth, production rates and stoichiometry of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). The values in the pie charts represent the cellular content (fmol cell–1) of the two major elements in the particulate organic matter [carbon (POCcq) and nitrogen (PONcq)]. The values of particulate organic phosphate (POPcq) represent <1% and are not visible in the pie charts. The size of the pies is a proportional representation of the cell volume. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). Error bars indicate the SD between three biological replicates. Fig. 1 View largeDownload slide Cellular volume, growth, production rates and stoichiometry of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). The values in the pie charts represent the cellular content (fmol cell–1) of the two major elements in the particulate organic matter [carbon (POCcq) and nitrogen (PONcq)]. The values of particulate organic phosphate (POPcq) represent <1% and are not visible in the pie charts. The size of the pies is a proportional representation of the cell volume. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). Error bars indicate the SD between three biological replicates. The cellular content of Chl a decreased by 90% with increasing Fe limitation, between Fe-replete conditions and severe Fe limitation (Fig. 2). Similarly, the cellular content of the accessory pigments β-carotene and zeaxanthin also decreased linearly with increasing severity of Fe limitation (Fig. 2). Fig. 2 View largeDownload slide Cellular pigment content of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). Represented values were calculated as the average of three biological replicates. Fig. 2 View largeDownload slide Cellular pigment content of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). Represented values were calculated as the average of three biological replicates. Biophysical properties of PSII under increasing chronic Fe limitation The PSII functional absorption cross-section determined in dark-acclimated samples (σPSII) was significantly higher under Fe-replete and severe Fe limitation treatments than in mild and strong Fe-limited treatments (Fig. 3A). Despite their large σPSII, severely Fe-limited cells showed a significantly lower degree of connectivity between PSII 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, The re-oxidation time of the 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 (PBSs) and 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 with 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 indicates 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/FmFb), 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/FmFb 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/FmFfsw), which showed a gradual decrease from 0.38 to 0.24 with increasing severity of Fe limitation (Fig. 3E). Fig. 3 View largeDownload slide Photophysiological parameters determined in Synechococcus sp. PCC7002 acclimated to different levels of Fe availability. (A) PSII effective absorption cross-section (σPSII); (B) excitonic connectivity among PSII reaction centers (ρ); (C) re-oxidation time of the primary quinone-type acceptor QA (τ); (D) non-photochemical quenching (NSV) and (E) Fv/Fm, commonly used as a measurement of the PSII photochemical efficiency (which needs to be carefully considered in cyanobacteria) corrected by Fb and by Ffsw. Filled symbols show the results from correction by Fb and open symbols show the results from correction by Ffsw only. Error bars indicate the SD of three biological replicates. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). The differences between the data sets corrected by Fb and by Ffsw were significantly different (P <0.001) only in (D) and (E). Fig. 3 View largeDownload slide Photophysiological parameters determined in Synechococcus sp. PCC7002 acclimated to different levels of Fe availability. (A) PSII effective absorption cross-section (σPSII); (B) excitonic connectivity among PSII reaction centers (ρ); (C) re-oxidation time of the primary quinone-type acceptor QA (τ); (D) non-photochemical quenching (NSV) and (E) Fv/Fm, commonly used as a measurement of the PSII photochemical efficiency (which needs to be carefully considered in cyanobacteria) corrected by Fb and by Ffsw. Filled symbols show the results from correction by Fb and open symbols show the results from correction by Ffsw only. Error bars indicate the SD of three biological replicates. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). The differences between the data sets corrected by Fb and by Ffsw were significantly different (P <0.001) only in (D) and (E). 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 1). Minimum (F0) and maximum fluorescence (Fm) increased with increasing Fe limitation (Fig. 4A, B), while increasing irradiance levels led to a decrease in these fluorescence levels for all the treatments. Under severe Fe limitation, the maximum electron transport rate (ETRmax Fb) and light saturation threshold (EK Fb) were significantly higher than in the other treatments (Table 1; Fig. 4C), whereas maximum light use efficiency (αFb) was higher under Fe-replete conditions than in the Fe-limited treatments (Table 1). 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 1). 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 (8–53 μmol photons m–2 s–1) and decreased with increasing irradiance. However, in the Fe-limited treatments, the maximum σPSII' was achieved at a 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 μmol photons m–2 s–1), the re-oxidation time of the 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). Table 1 Photophysiological parameters under different concentrations of dissolved inorganic iron (Fe') Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Results are given as means ± SD between three biological replicates. Superscript letters indicate statistical differences (P < 0.001) amongst the iron treatments. The absolute electron transport rate under growth PAR (ETR53; e– PSII–1 s–1), maximum absolute electron transport rate (ETRmax; e– PSII s–1), maximum light use efficiency (α; dimensionless) and light saturation threshold (EK; µmol photons m–2 s–1) were derived from light curve (LC) analysis. Table 1 Photophysiological parameters under different concentrations of dissolved inorganic iron (Fe') Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Results are given as means ± SD between three biological replicates. Superscript letters indicate statistical differences (P < 0.001) amongst the iron treatments. The absolute electron transport rate under growth PAR (ETR53; e– PSII–1 s–1), maximum absolute electron transport rate (ETRmax; e– PSII s–1), maximum light use efficiency (α; dimensionless) and light saturation threshold (EK; µmol photons m–2 s–1) were derived from light curve (LC) analysis. Fig. 4 View largeDownload slide Photophysiological parameters determined in relation to increasing irradiances from 0 to 856 µmol photons m–2 s–1 in Synechococcus sp. PCC7002 acclimated to different Fe availability. (A) Minimum fluorescence (F0Fb, dark acclimated; and F'Fb, light acclimated); (B) maximum fluorescence [Fm(')Fb]; (C) absolute electron transport rate (ETR); (D) PSII effective absorption cross-section [σPSII(')Fb]; (E) non-photochemical quenching (NSVFb); and (F) reoxidation time of the primary quinone-type acceptor QA [τ(')Fb]. Continuous lines (C) represent the curve fitted for each Fe concentration according to Webb et al. (1974) using the beta phase fit. Dotted lines (A, B, D, E, F) are spline lines connecting data points from a given growth condition, for clarity. The gray bar indicates the growth light level for the cultures. Error bars indicate the SD of three biological replicates. Fig. 4 View largeDownload slide Photophysiological parameters determined in relation to increasing irradiances from 0 to 856 µmol photons m–2 s–1 in Synechococcus sp. PCC7002 acclimated to different Fe availability. (A) Minimum fluorescence (F0Fb, dark acclimated; and F'Fb, light acclimated); (B) maximum fluorescence [Fm(')Fb]; (C) absolute electron transport rate (ETR); (D) PSII effective absorption cross-section [σPSII(')Fb]; (E) non-photochemical quenching (NSVFb); and (F) reoxidation time of the primary quinone-type acceptor QA [τ(')Fb]. Continuous lines (C) represent the curve fitted for each Fe concentration according to Webb et al. (1974) using the beta phase fit. Dotted lines (A, B, D, E, F) are spline lines connecting data points from a given growth condition, for clarity. The gray bar indicates the growth light level for the cultures. Error bars indicate the SD of three biological replicates. Under Fe-replete conditions, the Fv/FmFb determined following the LC and 10 min of dark acclimation was 30% lower than the initial Fv/FmFb 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. Fig. 5 View largeDownload slide Comparison of photosynthetic yield (Fv/FmFb) in dark-acclimated cells before and after the light curve (LC). Empty bars represent measurements performed in dark-acclimated samples (60 min) before the LC and filled bars represent measurements performed in dark-acclimated samples (10 min) after the LC. Error bars indicate the SD of three biological replicates. Italic letters indicate statistically significant differences amongst treatments (P < 0.001). Gray values on the top indicate the percentage change in Fv/Fm following 10 min of dark acclimation after the LC with respect to Fv/Fm initially determined in 60 min dark-acclimated samples, before the LC. Fig. 5 View largeDownload slide Comparison of photosynthetic yield (Fv/FmFb) in dark-acclimated cells before and after the light curve (LC). Empty bars represent measurements performed in dark-acclimated samples (60 min) before the LC and filled bars represent measurements performed in dark-acclimated samples (10 min) after the LC. Error bars indicate the SD of three biological replicates. Italic letters indicate statistically significant differences amongst treatments (P < 0.001). Gray values on the top indicate the percentage change in Fv/Fm following 10 min of dark acclimation after the LC with respect to Fv/Fm initially determined in 60 min dark-acclimated samples, before the LC. Relationship between photophysiological parameters and state transitions Under Fe-replete conditions, plots of NSVFb (our metric of non-photochemical excitation dissipation), vs. τ' (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 light onset (8 μmol photons m–2 s–1). With further increases in light up to 128 μmol photons m–2 s–1, NSVFb and τ' remained almost unchanged. However, after exposure to yet higher irradiances from 239 up to 856 μmol photons m–2 s–1, the increase in NSVFb was accompanied by a decrease of τ' as downstream electron transport accelerated. Under mild and strong Fe limitation, the overall relationship between NSVFb and τ' presented three main sections (Fig. 6B, C). Fig. 6 View largeDownload slide Relationship between non-photochemical quenching (NSVFb) and the re-oxidation time of the primary acceptor QA (τ') in Synechococcus sp. PCC7002 acclimated to different Fe availability (A–D) and under different irradiances from 0 up to 856 µmol photons m–2 s–1. Numbers in parentheses indicate the PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). Error bars indicate the SD of three biological replicates. Fig. 6 View largeDownload slide Relationship between non-photochemical quenching (NSVFb) and the re-oxidation time of the primary acceptor QA (τ') in Synechococcus sp. PCC7002 acclimated to different Fe availability (A–D) and under different irradiances from 0 up to 856 µmol photons m–2 s–1. Numbers in parentheses indicate the PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). Error bars indicate the SD of three biological replicates. In the first section, between darkness and 8 μmol photons m–2 s–1, NSVFb remained similar, while τ' increased. In the second section, between 8 and 128 μmol photons m–2 s–1, NSVFb and τ' declined. Over the third section, at irradiances >128 μmol photons m–2 s–1, NSVFb increased again while τ' 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 μmol 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. Fig. 7 View largeDownload slide Correlation between PSII effective absorption cross-section (σPSII') and the product of the non-photochemical quenching by the re-oxidation time of the primary acceptor QA (NSVFb×τ'). σPSII' is negatively correlated with NSVFb×τ' in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1 (P < 0.05, represented by continuous lines). Pearson product moment correlation coefficients of –0.83, –0.82, –0.98 and –0.92 were calculated for increasing concentrations of Fe'. Numbers in parentheses indicate the instantaneous PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). Fig. 7 View largeDownload slide Correlation between PSII effective absorption cross-section (σPSII') and the product of the non-photochemical quenching by the re-oxidation time of the primary acceptor QA (NSVFb×τ'). σPSII' is negatively correlated with NSVFb×τ' in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1 (P < 0.05, represented by continuous lines). Pearson product moment correlation coefficients of –0.83, –0.82, –0.98 and –0.92 were calculated for increasing concentrations of Fe'. Numbers in parentheses indicate the instantaneous PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). 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 μmol photons m–2 s–1, while with higher irradiances the ST size decreased rapidly (Fig. 8). Under Fe limitation, the ST 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 μmol photons m–2 s–1. Under severe Fe limitation, the calculated ST size was <1, suggesting that no ST was operating. Fig. 8 View largeDownload slide Size of state transitions in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1. The gray horizontal line indicates a state transition equal to 1, which typically corresponds to dark acclimation, i.e. no state transition. The gray vertical bar indicates the growth light level. Error bars indicate the SD of three biological replicates. Fig. 8 View largeDownload slide Size of state transitions in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1. The gray horizontal line indicates a state transition equal to 1, which typically corresponds to dark acclimation, i.e. no state transition. The gray vertical bar indicates the growth light level. Error bars indicate the SD of three biological replicates. Discussion Macromolecular allocation under Fe limitation In line with results previously reported (Wilhelm et al. 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 to 0 .037 d–1; Fitzwater et al. 1996), one of the major HNLC regions with the Southern Ocean. Under nutrient starvation (nitrogen and phosphorus), different phytoplanktonic groups increase lipid and carbohydrate storage, while cellular protein contents either remain steady or decrease (Bertilsson et al. 2003, Wagner et al. 2006, Jakob et al. 2007, Halsey et al. 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 coding 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 et al. 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 accompany 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 et al. 2010). This decrease in C:N with increasing severity of 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 the severity of Fe limitation. The first response was observed under mild and strong Fe limitation, where a decrease in σPSII 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 τ observed under mild and strong Fe limitation relative to Fe-replete conditions indicated that re-oxidation of the QA pool was slower, showing downstream limitations on the electron transport. Mild Fe limitation resulted in up-regulation of the gene isiA and down-regulation of psaA and psaB, encoding PSI proteins, which then remained unchanged with further increasing severity of Fe limitation (Blanco-Ameijeiras et al. 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 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 σPSII observed under mild and strong Fe limitation could be associated with the increase of IsiA competing with the PSII for excitation energy from the PBSs, as previously reported (Park et al. 1999, Sandström et al. 2001). In fact, work by Wilson et al. (2007) using Fe-limited IsiA mutants, in combination with PBS mutants and orange carotenoid protein (OCP) mutants, suggest that PBSs 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 in Fe-replete conditions, suggesting that the equilibration of excitons with 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 σPSII 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 σPSII could then be explained by the remaining PBS antennae serving a small population of active PSII reaction centers organized in a puddle rather than a lake antenna bed. The lower expression of genes coding for PSII reaction centers reported by the transcriptomic analysis supports this hypothesis (Blanco-Ameijeiras et al. 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 et al. 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, coding for a putative PBS degradation protein. As in this study σPSII was determined with three blocks of light-emitting diodes (LEDs), the spectral shifts due to different pigment complements might contribute to some of the changes observed. However, the concentration of PBS pigments relative to other pigments of the photosynthetic apparatus is not expected to change dramatically in Fe-limited cyanobacteria, as demonstrated by 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 components of the electron transport chain, such as PSII, Cyt b6f and ATPase, whereas the respiratory terminal oxidase Cyt 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 expected 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 τ' 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 downstream electron transport, which is accompanied by an increase in non-photochemical quenching. This quenching under high light could presumably be attributed to the OCP, reported as a key cyanobacterial 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 et al. 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 μmol photons m–2 s–1, rather than at growth irradiance (53 μmol photons m–2 s–1), suggesting that Fe limitation hampers the state transition. This hypothesis of inefficient STs 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 an NADH2 dehydrogenase subunit (Blanco-Ameijeiras et al. 2017), which is required for the transition to state 2 in the dark by mediating electron flow into the intersystem electron carriers in the dark (Huang et al. 2003). Under severe Fe limitation, the light to dark STs were absent. The decrease in τ' 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 of NSVFb vs. σPSII and τ' vs. 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 that under Fe-replete conditions. However, the comparison of Fv/FmFb 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 et al. 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 of mild to strong Fe limitation compared with severe limitation. Under mild and strong Fe limitation, there was a decrease in LET accompanied by progressive loss of state transitions. Under severe Fe limitation, STs 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. MaterialS 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 2), and finally filter-sterilized (0.2 μm polycarbonate membrane, Whatman). Culture medium was amended with different concentrations of dissolved Fe (Table 2), 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 et al. 2001, Buck et al. 2015) and oceanic low Fe (Fitzsimmons et al. 2013, Buck et al. 2015) regions, respectively. Manipulations were conducted in a trace metal-clean AirClean Systems (AC600 Series PCR Workstation, STAR LAB). All labware and material used were cleaned to remove any trace metal background contamination by soaking them for 1 week in 0.01% Citranox (ALCONOX) and Milli-Q rinsing, followed by 1 week soaking in 1.2 M HCl prior to extensive rinsing with ultrapure water (18.2 MΩ) obtained from a Milli-Q Direct System (Merk Millipore). Unless otherwise specified, all solutions used in this study were prepared using analytical grade chemicals (Sigma-Aldrich) 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), 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 d for at least 22 generations prior to the experiments. To this end, experimental bottles were inoculated with the respective acclimated cultures into 2,000 ml of medium with an initial concentration of 10.7×104 ± 2.3×104cell ml–1, in each polycarbonate bottle. The cells were grown in three biological replicates of each experimental treatment and harvested after 4 d of incubation. Table 2 Concentrations of macronutrients, trace metals and vitamins added to the synthetic oceanic seawater Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 All the elements except vitamins were prepared from ICP-MS standard solutions (Fluka). Bioavailable concentrations were estimated from dissolved inorganic iron (Fe'), calculated with MINEQL+ 4.6 (Schecher and McAvoy 1994). Table 2 Concentrations of macronutrients, trace metals and vitamins added to the synthetic oceanic seawater Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 All the elements except vitamins were prepared from ICP-MS standard solutions (Fluka). Bioavailable concentrations were estimated from dissolved inorganic iron (Fe'), calculated with MINEQL+ 4.6 (Schecher and McAvoy 1994). 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) with a 45 μm capillary. Based on the cell density, growth rate (µ) was calculated according to Equation 1. µ=(Lnc1−Lnc0)/Δt (1) where c0 and c1 are the cell counts in millilitres 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% HCl for 24 h to remove particulate inorganic carbon (Verardo et al. 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). POC and PON content were corrected for blank measurements and normalized to total cell counts to calculate cellular quotas (POCcq and PONcq). POCprod and PONprod were calculated multiplying the cellular quota by µ. Results of production rates were expressed in moles of carbon or nitrogen per cell per day, accordingly. Particulate organic phosphate (POP) content Organic phosphorus compounds were digested in the presence of the oxidizing decomposition reagent Oxisolv (Merck Millipore) 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) and 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). The 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® HPLC system equipped with a temperature-controlled auto-sampler L-2200, a DAD detector L-2450 (Hitachi High Technologies Inc.) and a Spherisorb ODS-2 column (25 cm×4.6 mm, 5 μm particle size; Waters Corp.). Pigment separation was achieved using a LiChrospher® 100 RP-18 guard cartridge (Merck KGaA). Peaks detected at 440 nm were identified and quantified via co-chromatography of pigment standards obtained from DHI Lab Products 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.) that circulated Milli-Q water at 22ºC 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 1 h 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 Chl), 530 and 624 nm (preferentially absorbed by the PBSs) 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 the 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 fell within the optimum range of 0.04–0.05 and the curve of fluorescence increase reached 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 six times with an interval of 3 s. Using FastPro8 GUI, the acquisitions were fitted to the KPF biophysical model (Kolber et al. 1998) to determine dark-adapted F0, Fm, σPSII, τ and 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 PBSs 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 an energetically detached photosynthetic antenna complex in the cyanobacterium Synechocystis sp. PCC6803 (Ihalainen et al. 2005, Schrader et al. 2011). This phenomenon has been estimated to represent almost 50% of the pigment content in the phytoplankton cells present in high-nutrient low-Chl 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 (Cadoret 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−repleteFv/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 data set of dark-acclimated measurements was independently corrected for the two types of fluorescence baseline corrections (Ffsw and Fb), and the resulting estimates were compared. The use of the sub index Ffsw or Fb with the photophysiological parameters indicates the correction applied. The Fv/Fm was calculated following Equation 3. Fv/Fm=(Fm-F0)/Fm (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 LC were conducted by exposing each treatment to a set of increasing irradiance levels (8, 23, 53, 128, 239, 465 and 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 (σPSII'), re-oxidation time of the 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 performed before the LC to determine the recovery capacity after exposure to high light intensities. The recovery yield was calculated as the percentage of variation with respect to the dark-acclimated measurement before the LC. Absolute PSII ETRs at each PAR were calculated according to Equation 6 (Suggett et al. 2006, Suggett et al. 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-1s-1)=σPSII×[(Fq'/Fm')/(Fv/Fm)]×PAR (6) The relationship between NSVFb and τ(')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 STs 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) However, 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 analysis of variance (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.). Supplementary Data Supplementary data are available at PCP online. Funding This work was funded by the Swiss National Science Foundation [FNS PP00P2-138955 to C.S.H.]; the Schmidheiny Foundation [to S.B.A.]; the Helmholtz Association [Young Investigators Group EcoTrace, VH-NG-901 to S.T.)]; and the Canada Research Chairs program [to D.A.C.]. Acknowledgments The authors are grateful to Marc C. Moore for his comments on 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. Disclosures The authors have no conflicts of interest to declare. References Behrenfeld M.J. , Worthington K. , Sherrell R.M. , Chavez F.P. , Strutton P. , McPhaden M. ( 2006 ) Controls on tropical Pacific Ocean productivity revealed through nutrient stress diagnostics . Nature 442 : 1025 – 1028 . Google Scholar CrossRef Search ADS PubMed Behrenfeld M.J. , Milligan A.J. ( 2013 ) Photophysiological expressions of iron stress in phytoplankton . Annu. Rev. Mar. Sci. 5 : 217 – 246 . Google Scholar CrossRef Search ADS Bertilsson S. , Berglund O. , Karl D.M. , Chisholm S.W. ( 2003 ) Elemental composition of marine Prochlorococcus and Synechococcus: implications for the ecological stoichiometry of the sea . Limnol. Oceanogr. 48 : 1721 – 1731 . Google Scholar CrossRef Search ADS Bibby T.S. , Nield J. , Partensky F. , Barber J. ( 2001 ) Oxyphotobacteria: antenna ring around photosystem I . Nature 413 : 590 – 590 . Google Scholar CrossRef Search ADS PubMed Blanco-Ameijeiras S. , Cosio C. , Hassler C.S. ( 2017 ) Long-term acclimation to iron limitation reveal new insights in metabolism regulation of Synechococcus sp . Front. Mar. Sci. 4 . Boyd P.W. , Ellwood M.J. ( 2010 ) The biogeochemical cycle of iron in the ocean . Nat. Geosci. 3 : 675 – 682 . Google Scholar CrossRef Search ADS Bruland K.W. , Rue E.L. , Smith G.J. ( 2001 ) Iron and macronutrients in California coastal upwelling regimes: implications for diatom blooms . Limnol. Oceanogr. 46 : 1661 – 1674 . Google Scholar CrossRef Search ADS Buck K.N. , Sohst B. , Sedwick P.N. ( 2015 ) The organic complexation of dissolved iron along the U.S. GEOTRACES (GA03) North Atlantic Section . Deep Sea Res. II 116 : 152 – 165 . Google Scholar CrossRef Search ADS Cadoret J.-C. , Demoulière R. , Lavaud J. , van Gorkom H.J. , Houmard J. , Etienne A.-L. , et al. ( 2004 ) Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA) . Biochim. Biophys. Acta 1659 : 100 – 104 . Google Scholar CrossRef Search ADS PubMed Campbell D. , Hurry V. , Clarke A.K. , Gustafsson P. , Oquist G. ( 1998 ) Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation . Microbiol. Mol. Biol. Rev . 62 : 667 – 683 . Google Scholar PubMed Campbell D. , Oquist G. ( 1996 ) Predicting light acclimation in cyanobacteria from non-photochemical quenching of photosystem II fluorescence, which reflects state transitions in these organisms . Plant Physiol. 111 : 1293 – 1298 . Google Scholar CrossRef Search ADS PubMed Ermakova M. , Huokko T. , Richaud P. , Bersanini L. , Howe C.J. , et al. ( 2016 ) Distinguishing the roles of thylakoid respiratory terminal oxidases in the cyanobacterium Synechocystis sp. PCC 6803 . Plant Physiol . 171 : 1307 – 1319 . Google Scholar PubMed Ferreira F. , Straus N.A. ( 1994 ) Iron deprivation in cyanobacteria . J. Appl. Phycol. 6 : 199 – 210 . Google Scholar CrossRef Search ADS Finkel Z.V. , Follows M.J. , Liefer J.D. , Brown C.M. , Benner I. , Irwin A.J. , et al. ( 2016 ) Phylogenetic diversity in the macromolecular composition of microalgae . PLoS One 11 : e0155977 . Google Scholar CrossRef Search ADS PubMed Fitzsimmons J.N. , Zhang R. , Boyle E.A. ( 2013 ) Dissolved iron in the tropical North Atlantic Ocean . Mar. Chem . 154 : 87 – 99 . Google Scholar CrossRef Search ADS Fitzwater S.E. , Coale K.H. , Gordon R.M. , Johnson K.S. , Ondrusek M.E. ( 1996 ) Iron deficiency and phytoplankton growth in the Equatorial Pacific . Deep Sea Res. Part II 43 : 995 – 1015 . Google Scholar CrossRef Search ADS Flombaum P. , Gallegos J.L. , Gordillo R.A. , Rincon J. , Zabala L.L. , Jiao N. , et al. ( 2013 ) Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus . Proc. Natl. Acad. Sci. USA 110 : 9824 – 9829 . Google Scholar CrossRef Search ADS Fraser J.M. , Tulk S.E. , Jeans J.A. , Campbell D.A. , Bibby T.S. , Cockshutt A.M. , et al. ( 2013 ) Photophysiological and photosynthetic complex changes during iron starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC . PLoS One 8 : e59861 . Google Scholar CrossRef Search ADS PubMed Geider R. , Roche J.L. ( 2002 ) Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis . Eur. J. Phycol. 37 : 1 – 17 . Google Scholar CrossRef Search ADS Giovagnetti V. , Ruban A.V. ( 2017 ) Detachment of the fucoxanthin chlorophyll a/c binding protein (FCP) antenna is not involved in the acclimative regulation of photoprotection in the pennate diatom Phaeodactylum tricornutum . Biochim. Biophys. Acta 1858 : 218 – 230 . Google Scholar CrossRef Search ADS PubMed Greene R.M. , Geider R.J. , Falkowski P.G. ( 1991 ) Effect of iron limitation on photosynthesis in a marine diatom . Limnol. Oceanogr. 36 : 1772 – 1782 . Google Scholar CrossRef Search ADS Greene R.M. , Geider R.J. , Kolber Z. , Falkowski P.G. ( 1992 ) Iron-induced changes in light harvesting and photochemical energy conversion processes in eukaryotic marine algae . Plant Physiol . 100 : 565 – 575 . Google Scholar CrossRef Search ADS PubMed Halsey K.H. , Jones B.M. ( 2015 ) Phytoplankton strategies for photosynthetic energy allocation . Annu. Rev. Mar. Sci. 7 : 265 – 297 . Google Scholar CrossRef Search ADS Halsey K.H. , Milligan A.J. , Behrenfeld M.J. ( 2010 ) Physiological optimization underlies growth rate-independent chlorophyll-specific gross and net primary production . Photosynth. Res. 103 : 125 – 137 . Google Scholar CrossRef Search ADS PubMed Hansen H.P. , Korolef F. ( 1983 ) Determination of nutrients. In Methods of Seawater Analysis . Edited by Grasshoff K. , Kremling K. , Ehrhardt M. pp. 150 – 228 . Weinheim, Germany : Wiley Verlag Chemie GmbH . Hassler C.S. , Alasonati E. , Mancuso Nichols C.A. , Slaveykova V.I. ( 2011 ) Exopolysaccharides produced by bacteria isolated from the pelagic Southern Ocean—role in Fe binding, chemical reactivity, and bioavailability . Mar. Chem . 123 : 88 – 98 . Google Scholar CrossRef Search ADS Hilton J. , Lishman H. , Mackness S. , Heaney S.I. ( 1986 ) An automated method for the analysis of ‘particulate’ carbon and nitrogen in natural waters . Hydrobiologia 141 : 269 – 271 . Google Scholar CrossRef Search ADS Ho T.-Y. , Quigg A. , Finkel Z.V. , Milligan A.J. , Wyman K. , Falkowski P.G. , et al. ( 2003 ) The elemental composition of some marine phytoplankton . J. Phycol. 39 : 1145 – 1159 . Google Scholar CrossRef Search ADS Huang C. , Yuan X. , Zhao J. , Bryant D.A. ( 2003 ) Kinetic analyses of state transitions of the cyanobacterium Synechococcus sp. PCC 7002 and its mutant strains impaired in electron transport . Biochim. Biophys. Acta 1607 : 121 – 130 . Google Scholar CrossRef Search ADS PubMed Ihalainen J.A. , D'Haene S. , Yeremenko N. , van Roon H. , Arteni A.A. , Boekema E.J. , et al. ( 2005 ) Aggregates of the chlorophyll-binding protein isia (cp43') dissipate energy in cyanobacteria . Biochemistry 44 : 10846 – 10853 . Google Scholar CrossRef Search ADS PubMed Ivanov A.G. , Krol M. , Sveshnikov D. , Selstam E. , Sandstrom S. , Koochek M. , et al. ( 2006 ) Iron deficiency in cyanobacteria causes monomerization of photosystem I trimers and reduces the capacity for state transitions and the effective absorption cross section of photosystem I in vivo . Plant Physiol . 141 : 1436 – 1445 . Google Scholar CrossRef Search ADS PubMed Jakob T. , Wagner H. , Stehfest K. , Wilhelm C. ( 2007 ) A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation . J. Exp. Bot. 58 : 2101 – 2112 . Google Scholar CrossRef Search ADS PubMed Jiang H.-B. , Lou W.-J. , Ke W.-T. , Song W.-Y. , Price N.M. , Qiu B.-S. , et al. ( 2015 ) New insights into iron acquisition by cyanobacteria: an essential role for ExbB–ExbD complex in inorganic iron uptake . ISME J. 9 : 297 – 309 . Google Scholar CrossRef Search ADS PubMed Kirilovsky D. ( 2007 ) Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism . Photosynth. Res. 93 : 7 – 16 . Google Scholar CrossRef Search ADS PubMed Kolber Z.S. , Prášil O. , Falkowski P.G. ( 1998 ) Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols . Biochim. Biophys. Acta 1367 : 88 – 106 . Google Scholar CrossRef Search ADS PubMed Kretz C.B. , Bell D.W. , Lomas D.A. , Lomas M.W. , Martiny A.C. ( 2015 ) Influence of growth rate on the physiological response of marine Synechococcus to phosphate limitation . Front. Microbiol. 6 : 85 . Google Scholar CrossRef Search ADS PubMed Kroon B.M.A. , Thoms S. ( 2006 ) From electron to biomass: a mechanistic model to describe phytoplankton photosynthesis and steady-state growth rates . J. Phycol. 42 : 593 – 609 . Google Scholar CrossRef Search ADS Liu S.-W. , Qiu B.-S. ( 2012 ) Different responses of photosynthesis and flow cytometric signals to iron limitation and nitrogen source in coastal and oceanic Synechococcus strains (Cyanophyceae) . Mar. Biol. 159 : 519 – 532 . Google Scholar CrossRef Search ADS Ludwig M. , Bryant D.A. ( 2012 ) Acclimation of the global transcriptome of the cyanobacterium Synechococcus sp. strain PCC 7002 to nutrient limitations and different nitrogen sources . Front. Microbiol. 3 : 145 . Google Scholar PubMed Ma W. , Ogawa T. , Shen Y. , Mi H. ( 2007 ) Changes in cyclic and respiratory electron transport by the movement of phycobilisomes in the cyanobacterium Synechocystis sp. strain PCC 6803 . Biochim. Biophys. Acta 1767 : 742 – 749 . Google Scholar CrossRef Search ADS PubMed Macey A.I. , Ryan-Keogh T. , Richier S. , Moore C.M. , Bibby T.S. ( 2014 ) Photosynthetic protein stoichiometry and photophysiology in the high latitude North Atlantic . Limnol. Oceanogr. 59 : 1853 – 1864 . Google Scholar CrossRef Search ADS Mackey K.R.M. , Paytan A. , Grossman A.R. , Bailey S. ( 2008 ) A photosynthetic strategy for coping in a high-light, low-nutrient environment . Limnol. Oceanogr. 53 : 900 – 913 . Google Scholar CrossRef Search ADS Mackey K.R.M. , Post A.F. , McIlvin M.R. , Cutter G.A. , John S.G. , Saito M.A. , et al. ( 2015 ) Divergent responses of Atlantic coastal and oceanic Synechococcus to iron limitation . Proc. Natl. Acad. Sci. USA 112 : 9944 – 9949 . Google Scholar CrossRef Search ADS Mahmood A. , Abualhaija M.M. , van den Berg C.M.G. , Sander S.G. ( 2015 ) Organic speciation of dissolved iron in estuarine and coastal waters at multiple analytical windows . Mar. Chem . 177 : 706 – 719 . Google Scholar CrossRef Search ADS Martin J.H. , Fitzwater S.E. ( 1988 ) Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic . Nature 331 : 341 – 343 . Google Scholar CrossRef Search ADS McKew B.A. , Davey P. , Finch S.J. , Hopkins J. , Lefebvre S.C. , Metodiev M.V. , et al. ( 2013 ) The trade-off between the light-harvesting and photoprotective functions of fucoxanthin-chlorophyll proteins dominates light acclimation in Emiliania huxleyi (clone CCMP 1516) . New Phytol. 200 : 74 – 85 . Google Scholar CrossRef Search ADS PubMed Misumi M. , Kato H. , Tomo T. , Sonoike K. ( 2015 ) Relationship between photochemical quenching and non-photochemical quenching in six species of cyanobacteria reveals species difference in redox state and species commonality in energy dissipation . Plant Cell Physiol . 57 : 1510 – 1517 . Google Scholar PubMed Moore C.M. , Mills M.M. , Arrigo K.R. , Berman-Frank I. , Bopp L. , Boyd P.W. , et al. ( 2013 ) Processes and patterns of oceanic nutrient limitation . Nat. Geosci. 6 : 701 – 710 . Google Scholar CrossRef Search ADS Mullineaux C.W. , Allen J.F. ( 1986 ) The state 2 transition in the cyanobacterium Synechococcus 6301 can be driven by respiratory electron flow into the plastoquinone pool . FEBS Lett . 205 : 155 – 160 . Google Scholar CrossRef Search ADS Mullineaux C.W. , Allen J.F. ( 1990 ) State 1–State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II . Photosynth. Res. 23 : 297 – 311 . Google Scholar CrossRef Search ADS PubMed Murphy C.D. , Ni G. , Li G. , Barnett A. , Xu K. , Grant-Burt J. , et al. ( 2017 ) Quantitating active photosystem II reaction center content from fluorescence induction transients . Limnol. Oceanogr. Methods 15 : 54 – 69 . Google Scholar CrossRef Search ADS Noffke N. , Christian D. , Wacey D. , Hazen R.M. ( 2013 ) Microbially induced sedimentary structures recording an ancient ecosystem in the ca. 3.48 billion-year-old dresser formation, Pilbara, Western Australia . Astrobiology 13 : 1103 – 1124 . Google Scholar CrossRef Search ADS PubMed Öquist G. ( 1974 ) Iron deficiency in the blue-green alga Anacystis nidulans: changes in pigmentation and photosynthesis . Physiol. Plant. 30 : 30 – 37 . Google Scholar CrossRef Search ADS Osanai T. , Kanesaki Y. , Nakano T. , Takahashi H. , Asayama M. , Shirai M. , et al. ( 2005 ) Positive regulation of sugar catabolic pathways in the cyanobacterium Synechocystis sp. PCC 6803 by the group 2 σ factor SigE . J. Biol. Chem. 280 : 30653 – 30659 . Google Scholar CrossRef Search ADS PubMed Oxborough K. ( 2014 ) FAstPro8 GUI and FRRf3 Systems Documentation . 2230-801-HB edn. Chelsea Technologies Group , West Molesey, UK . Oxborough K. , Baker N.R. ( 1997 ) Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components—calculation of qP and Fv/Fm without measuring Fo . Photosynth. Res . 54 : 135 – 142 . Google Scholar CrossRef Search ADS Park Y.-I. , Sandstrom S. , Gustafsson P. , Oquist G. ( 1999 ) Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation . Mol. Microbiol. 32 : 123 – 129 . Google Scholar CrossRef Search ADS PubMed Pitchford J.W. , Brindley J. ( 1999 ) Iron limitation, grazing pressure and oceanic high nutrient–low chlorophyll (HNLC) regions . J. Plankton Res . 21 : 525 – 547 . Google Scholar CrossRef Search ADS Price N.M. , Harrison G.I. , Hering J.G. , Hudson R.J. , Nirel P.M.V. , Palenik B. , et al. ( 1989 ) Preparation and chemistry of the artificial algal culture medium . Aquil. Biol. Oceanogr . 6 : 443 – 461 . Ralph P.J. , Gademann R. ( 2005 ) Rapid light curves: a powerful tool to assess photosynthetic activity . Aquat. Bot . 82 : 222 – 237 . Google Scholar CrossRef Search ADS Raven J.A. , Evans M.C.W. , Korb R.E. ( 1999 ) The role of trace metals in photosynthetic electron transport in O2-evolving organisms . Photosynth. Res . 60 : 111 – 150 . Google Scholar CrossRef Search ADS Riethman H.C. , Sherman L.A. ( 1988 ) Immunological characterization of iron-regulated membrane proteins in the cyanobacterium Anacystis nidulans R2 . Plant Physiol . 88 : 497 – 505 . Google Scholar CrossRef Search ADS PubMed Ryan-Keogh T.J. , Macey A.I. , Cockshutt A.M. , Moore C.M. , Bibby T.S. ( 2012 ) The cyanobacterial chlorophyll-binding-protein IsiA acts to increase the in vivo effective absorption cross-section of PSI under iron limitation . J. Phycol . 48 : 145 – 154 . Google Scholar CrossRef Search ADS PubMed Sandström S. , Park Y.-I. , Öquist G. , Gustafsson P. ( 2001 ) CP43′, the isiA gene product, functions as an excitation energy dissipator in the cyanobacterium Synechococcus sp. PCC 7942 . Photochem. Photobiol . 74 : 431 – 437 . Google Scholar CrossRef Search ADS PubMed Sandström S. , Ivanov A.G. , Park Y.-I. , Oquist G. , Gustafsson P. ( 2002 ) Iron stress responses in the cyanobacterium Synechococcus sp . Physiol. Plant. 116 : 255 – 263 . Google Scholar CrossRef Search ADS PubMed Schecher W.D. , McAvoy C.D. ( 1994 ) MINEQL+: A Chemical Equilibrium Program for Personal Computers. Environmental Research Software , Hallowell, ME . Scherer S. , Stürzl E. , Büger P. ( 1982 ) Interaction of respiratory and photosynthetic electron transport in Anabaena variabilis Kütz . Arch. Microbiol. 132 : 333 – 337 . Google Scholar CrossRef Search ADS Schrader P.S. , Milligan A.J. , Behrenfeld M.J. ( 2011 ) Surplus photosynthetic antennae complexes underlie diagnostics of iron limitation in a cyanobacterium . PLoS One 6 : e18753 . Google Scholar CrossRef Search ADS PubMed Schuback N. , Schallenberg C. , Duckham C. , Maldonado M.T. , Tortell P.D. ( 2015 ) Interacting effects of light and iron availability on the coupling of photosynthetic electron transport and CO2-assimilation in marine phytoplankton . PLoS One 10 : e0133235 . Google Scholar CrossRef Search ADS PubMed Sedoud A , López-Igual R. , Ur Rehman A. , Wilson A. , Perreau F. , Boulay C. , et al. ( 2014 ) The cyanobacterial photoactive orange carotenoid protein is an excellent singlet oxygen quencher . Plant Cell 26 : 1781 – 1791 . Google Scholar CrossRef Search ADS PubMed Simis S.G.H. , Huot Y. , Babin M. , Seppälä J. , Metsamaa L. ( 2012 ) Optimization of variable fluorescence measurements of phytoplankton communities with cyanobacteria . Photosynth. Res. 112 : 13 – 30 . Google Scholar CrossRef Search ADS PubMed Suggett D.J. , Moore C.M. , Marañón E. , Omachi C. , Varela R.A. , Aiken J. , et al. ( 2006 ) Photosynthetic electron turnover in the tropical and subtropical Atlantic Ocean . Deep Sea Res. II 53 : 1573 – 1592 . Google Scholar CrossRef Search ADS Suggett D.J. , Moore C.M. , Hickman A.E. , Geider R.J. ( 2009 ) Interpretation of fast repetition rate (FRR) fluorescence: signatures of phytoplankton community structure versus physiological state . Mar. Ecol. Prog. Ser. 376 : 1 – 19 . Google Scholar CrossRef Search ADS Sun J. , Golbeck J.H. ( 2015 ) The presence of the IsiA–PSI supercomplex leads to enhanced Photosystem I electron throughput in iron-starved cells of Synechococcus sp. PCC 7002 . J. Phys. Chem. B 119 : 13549 – 13559 . Google Scholar CrossRef Search ADS PubMed Sunda W.G. ( 1989 ) Trace metal interactions with marine phytoplankton . Biol. Oceanogr . 6 : 411 – 442 . Sunda W.G. , Huntsman S.A. ( 1995 ) Iron uptake and growth limitation in oceanic and coastal phytoplankton . Mar. Chem . 50 : 189 – 206 . Google Scholar CrossRef Search ADS Thompson A.W. , Huang K. , Saito M.A. , Chisholm S.W. ( 2011 ) Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability . ISME J. 5 : 1580 – 1594 . Google Scholar CrossRef Search ADS PubMed Thurotte A. , Lopez-Igual R. , Wilson A. , Comolet L. , Bourcier de Carbon C. , Xiao F. , et al. ( 2015 ) Regulation of orange carotenoid protein activity in cyanobacterial photoprotection . Plant Physiol. 169 : 737 – 747 . Google Scholar CrossRef Search ADS PubMed Tsuda A. ( 2003 ) A mesoscale iron enrichment in the Western Subarctic Pacific induces a large centric diatom bloom . Science 300 : 958 – 961 . Google Scholar CrossRef Search ADS PubMed Vassiliev I.R. , Kolber Z. , Wyman K.D. , Mauzerall D. , Shukla V.K. , Falkowski P.G. , et al. ( 1995 ) Effects of iron limitation on Photosystem II composition and light utilization in Dunaliella tertiolecta . Plant Physiol. 109 : 963 – 972 . Google Scholar CrossRef Search ADS PubMed Verardo D.J. , Froelich P.N. , McIntyre A. ( 1990 ) Determination of organic carbon and nitrogen in marine sediments using the Carlo Erba NA-1500 analyzer . Deep Sea Res. A . 37 : 157 – 165 . Google Scholar CrossRef Search ADS Vrede T. , Dobberfuhl D.R. , Kooijman S.A.L.M. , Elser J.J. ( 2004 ) Fundamental connections among organism C:N:P stoichiometry, macromolecular composition, and growth . Ecology 85 : 1217 – 1229 . Google Scholar CrossRef Search ADS Wagner H. , Jakob T. , Wilhelm C. ( 2006 ) Balancing the energy flow from captured light to biomass under fluctuating light conditions . New Phytol. 169 : 95 – 108 . Google Scholar CrossRef Search ADS PubMed Webb W.L. , Newton M. , Starr D. ( 1974 ) Carbon dioxide exchange of Alnus rubra . Oecologia 17 : 281 – 291 . Google Scholar CrossRef Search ADS PubMed Wilhelm S.W. , Maxwell D.P. , Trick C.G. ( 1996 ) Growth, iron requirements, and siderophore production in iron-limited Synechococcus PCC 72 . Limnol. Oceanogr. 41 : 89 – 97 . Google Scholar CrossRef Search ADS Wilhelm S.W. , MacAuley K. , Trick C.G. ( 1998 ) Evidence for the importance of catechol-type siderophores in the iron-limited growth of a cyanobacterium . Limnol. Oceanogr. 43 : 992 – 997 . Google Scholar CrossRef Search ADS Wilhelm S.W. , Trick C.G. ( 1995 ) Physiological profiles of Synechococcus (cyanophyceae) in iron-limiting continuous cultures . J. Phycol. 31 : 79 – 85 . Google Scholar CrossRef Search ADS Wilson A. , Boulay C. , Wilde A. , Kerfeld C.A. , Kirilovsky D. ( 2007 ) Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins . Plant Cell 19 : 656 – 672 . Google Scholar CrossRef Search ADS PubMed Wright S.W. , Jeffrey S.W. , Mantoura R.F.C. , Llewellyn C.A. , Bjornland T. , Repeta D. , et al. ( 1991 ) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton . Mar. Ecol. Prog. Ser. 77 : 183 – 196 . Google Scholar CrossRef Search ADS Xu K. , Grant-Burt J.L. , Donaher N. , Campbell D.A. ( 2017 ) Connectivity among photosystem II centers in phytoplankters: patterns and responses . Biochim. Biophys. Acta 1858 : 459 – 474 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations 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 PSII fluorescence determined with continuous background illumination Fe iron Fe' dissolved inorganic Fe Fb cellular baseline fluorescence correction by fluorescence from phycobilisomes and PSI Ffsw baseline fluorescence correction by fluorescence from filtered growth medium FRR-ST single turnover fluorescence induction curves Fm dark-adapted PSII maximum fluorescence Fm' maximum PSII fluorescence determined with continuous background illumination Fq'/Fm' effective PSII quantum yield determined with continuing background illumination Fv/Fm the PSII photochemical efficiency in dark-acclimated cells F0 dark-adapted PSII minimum fluorescence LC measurements performed in cells exposed to increasing light for a short time LED light-emitting diode LET linear electron transport from PSII to PSI NSV normalized Stern–Volmer photochemical quenching OCP orange carotenoid protein PAR photosynthetic active radiation PBS phycobilisome 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 ST state transition α maximum light use efficiency Δt time of incubation in days μ growth rate ρ degree of connectivity between PSII reaction centers determined in dark acclimated samples ρ' degree of connectivity between PSII reaction centers determined with continuous background illumination σPSII PSII functional absorption cross-section determined in dark-acclimated samples σPSII' PSII functional absorption cross-section determined in illuminated cells τ re-oxidation time of the plastoquinone primary acceptor QA determined in the dark τ' re-oxidation time of primary quinone-type acceptor QA determined in illuminated cells © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

Elemental Stoichiometry and Photophysiology Regulation of Synechococcus sp. PCC7002 Under Increasing Severity of Chronic Iron Limitation

Loading next page...
 
/lp/ou_press/elemental-stoichiometry-and-photophysiology-regulation-of-C0MfHncBeI
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
ISSN
0032-0781
eISSN
1471-9053
D.O.I.
10.1093/pcp/pcy097
Publisher site
See Article on Publisher Site

Abstract

Abstract Iron (Fe) is an essential cofactor 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, the total cellular content of pigments decreased. Additionally, with increasing severity of Fe limitation, intertwined responses of PSII 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 with 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. 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 et al. 1999). However, in the oxidative conditions of the modern ocean, the low solubility of Fe leads to low Fe availability. Indeed, >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-Chl content (Martin and Fitzwater 1988, Tsuda 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 with Fe stress with other principal metabolic pathways (Scherer et al. 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 et al. 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 a dearth of Fe on cyanobacteria are focused on responses under Fe starvation (no Fe addition in the culture) vs. Fe-replete conditions (Sandström et al. 2002, Ludwig and Bryant 2012), studies concentrating on 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 when 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 homeostasis mechanisms dramatically different from Fe-starved cells. For instance, chronic Fe-limited Synechoccocus sp. PCC7002 down-regulated enzymes involved in sugar catabolism, through down-regulation of enzymes participating in the oxidative pentose phosphate pathway (Osanai et al. 2005, Ludwig and Bryant 2012, Blanco-Ameijeiras et al. 2017), while almost no changes in enzyme expression of carbon metabolism were reported for Fe-starved Synechoccocus sp. PCC7002 (Ludwig and Bryant 2012). In cyanobacteria, the metabolic strategies to cope with a dearth of Fe include a decrease in cellular growth rates (Wilhelm et al. 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 et al. 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, the 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 one 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 Chl a fluorescence methods, different parameters of the single turnover fluorescence induction (FRR-ST) curves were determined and the photophysiological parameters for each treatment were derived. These parameters reflect different aspects of cellular status and photophysiological responses of PSII. The acclimation mechanisms regulating photosynthetic electron transport and energy dissipation were investigated in dark-acclimated cells and cells exposed to increasing photosynthetic active radiation (PAR) for a short time. These results support previous responses of this strain to increasing Fe limitation at the transcriptomic level (Blanco-Ameijeiras et al. 2017). We thus provide 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 The growth rate (μ) 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 and strong Fe limitation, but then increased again 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 (0.17 fmol cell–1; Fig. 1). The POC, PON and POP production rates (POCprod, PONprod and POPprod) decreased linearly with increasing Fe 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). Fig. 1 View largeDownload slide Cellular volume, growth, production rates and stoichiometry of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). The values in the pie charts represent the cellular content (fmol cell–1) of the two major elements in the particulate organic matter [carbon (POCcq) and nitrogen (PONcq)]. The values of particulate organic phosphate (POPcq) represent <1% and are not visible in the pie charts. The size of the pies is a proportional representation of the cell volume. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). Error bars indicate the SD between three biological replicates. Fig. 1 View largeDownload slide Cellular volume, growth, production rates and stoichiometry of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). The values in the pie charts represent the cellular content (fmol cell–1) of the two major elements in the particulate organic matter [carbon (POCcq) and nitrogen (PONcq)]. The values of particulate organic phosphate (POPcq) represent <1% and are not visible in the pie charts. The size of the pies is a proportional representation of the cell volume. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). Error bars indicate the SD between three biological replicates. The cellular content of Chl a decreased by 90% with increasing Fe limitation, between Fe-replete conditions and severe Fe limitation (Fig. 2). Similarly, the cellular content of the accessory pigments β-carotene and zeaxanthin also decreased linearly with increasing severity of Fe limitation (Fig. 2). Fig. 2 View largeDownload slide Cellular pigment content of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). Represented values were calculated as the average of three biological replicates. Fig. 2 View largeDownload slide Cellular pigment content of the cyanobacterium Synechococcus sp. PCC7002 under different levels of dissolved inorganic iron (Fe'). Represented values were calculated as the average of three biological replicates. Biophysical properties of PSII under increasing chronic Fe limitation The PSII functional absorption cross-section determined in dark-acclimated samples (σPSII) was significantly higher under Fe-replete and severe Fe limitation treatments than in mild and strong Fe-limited treatments (Fig. 3A). Despite their large σPSII, severely Fe-limited cells showed a significantly lower degree of connectivity between PSII 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, The re-oxidation time of the 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 (PBSs) and 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 with 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 indicates 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/FmFb), 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/FmFb 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/FmFfsw), which showed a gradual decrease from 0.38 to 0.24 with increasing severity of Fe limitation (Fig. 3E). Fig. 3 View largeDownload slide Photophysiological parameters determined in Synechococcus sp. PCC7002 acclimated to different levels of Fe availability. (A) PSII effective absorption cross-section (σPSII); (B) excitonic connectivity among PSII reaction centers (ρ); (C) re-oxidation time of the primary quinone-type acceptor QA (τ); (D) non-photochemical quenching (NSV) and (E) Fv/Fm, commonly used as a measurement of the PSII photochemical efficiency (which needs to be carefully considered in cyanobacteria) corrected by Fb and by Ffsw. Filled symbols show the results from correction by Fb and open symbols show the results from correction by Ffsw only. Error bars indicate the SD of three biological replicates. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). The differences between the data sets corrected by Fb and by Ffsw were significantly different (P <0.001) only in (D) and (E). Fig. 3 View largeDownload slide Photophysiological parameters determined in Synechococcus sp. PCC7002 acclimated to different levels of Fe availability. (A) PSII effective absorption cross-section (σPSII); (B) excitonic connectivity among PSII reaction centers (ρ); (C) re-oxidation time of the primary quinone-type acceptor QA (τ); (D) non-photochemical quenching (NSV) and (E) Fv/Fm, commonly used as a measurement of the PSII photochemical efficiency (which needs to be carefully considered in cyanobacteria) corrected by Fb and by Ffsw. Filled symbols show the results from correction by Fb and open symbols show the results from correction by Ffsw only. Error bars indicate the SD of three biological replicates. Different superscript italic letters indicate that differences among the Fe treatments are statistically significant (P < 0.001). The differences between the data sets corrected by Fb and by Ffsw were significantly different (P <0.001) only in (D) and (E). 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 1). Minimum (F0) and maximum fluorescence (Fm) increased with increasing Fe limitation (Fig. 4A, B), while increasing irradiance levels led to a decrease in these fluorescence levels for all the treatments. Under severe Fe limitation, the maximum electron transport rate (ETRmax Fb) and light saturation threshold (EK Fb) were significantly higher than in the other treatments (Table 1; Fig. 4C), whereas maximum light use efficiency (αFb) was higher under Fe-replete conditions than in the Fe-limited treatments (Table 1). 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 1). 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 (8–53 μmol photons m–2 s–1) and decreased with increasing irradiance. However, in the Fe-limited treatments, the maximum σPSII' was achieved at a 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 μmol photons m–2 s–1), the re-oxidation time of the 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). Table 1 Photophysiological parameters under different concentrations of dissolved inorganic iron (Fe') Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Results are given as means ± SD between three biological replicates. Superscript letters indicate statistical differences (P < 0.001) amongst the iron treatments. The absolute electron transport rate under growth PAR (ETR53; e– PSII–1 s–1), maximum absolute electron transport rate (ETRmax; e– PSII s–1), maximum light use efficiency (α; dimensionless) and light saturation threshold (EK; µmol photons m–2 s–1) were derived from light curve (LC) analysis. Table 1 Photophysiological parameters under different concentrations of dissolved inorganic iron (Fe') Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Fe' (pM) 36.7 3.83 0.47 0.047 ETR53Fb 13.48 ± 0.14b 10.87 ± 0.57a 11.44 ± 0.66a 11.50 ± 0.39a ETRmaxFb 76.37 ± 3.14b 64.42 ± 2.58b 66.89 ± 9.54b 114.94 ± 22.13a αFb 0.29 ± 0.01b 0.24 ± 0.01a 0.26 ± 0.02a 0.24 ± 0.01a EkFb 264.97 ± 8.01b 266.24 ± 3.68b 260.23 ± 21.85b 472.29 ± 93.14a Results are given as means ± SD between three biological replicates. Superscript letters indicate statistical differences (P < 0.001) amongst the iron treatments. The absolute electron transport rate under growth PAR (ETR53; e– PSII–1 s–1), maximum absolute electron transport rate (ETRmax; e– PSII s–1), maximum light use efficiency (α; dimensionless) and light saturation threshold (EK; µmol photons m–2 s–1) were derived from light curve (LC) analysis. Fig. 4 View largeDownload slide Photophysiological parameters determined in relation to increasing irradiances from 0 to 856 µmol photons m–2 s–1 in Synechococcus sp. PCC7002 acclimated to different Fe availability. (A) Minimum fluorescence (F0Fb, dark acclimated; and F'Fb, light acclimated); (B) maximum fluorescence [Fm(')Fb]; (C) absolute electron transport rate (ETR); (D) PSII effective absorption cross-section [σPSII(')Fb]; (E) non-photochemical quenching (NSVFb); and (F) reoxidation time of the primary quinone-type acceptor QA [τ(')Fb]. Continuous lines (C) represent the curve fitted for each Fe concentration according to Webb et al. (1974) using the beta phase fit. Dotted lines (A, B, D, E, F) are spline lines connecting data points from a given growth condition, for clarity. The gray bar indicates the growth light level for the cultures. Error bars indicate the SD of three biological replicates. Fig. 4 View largeDownload slide Photophysiological parameters determined in relation to increasing irradiances from 0 to 856 µmol photons m–2 s–1 in Synechococcus sp. PCC7002 acclimated to different Fe availability. (A) Minimum fluorescence (F0Fb, dark acclimated; and F'Fb, light acclimated); (B) maximum fluorescence [Fm(')Fb]; (C) absolute electron transport rate (ETR); (D) PSII effective absorption cross-section [σPSII(')Fb]; (E) non-photochemical quenching (NSVFb); and (F) reoxidation time of the primary quinone-type acceptor QA [τ(')Fb]. Continuous lines (C) represent the curve fitted for each Fe concentration according to Webb et al. (1974) using the beta phase fit. Dotted lines (A, B, D, E, F) are spline lines connecting data points from a given growth condition, for clarity. The gray bar indicates the growth light level for the cultures. Error bars indicate the SD of three biological replicates. Under Fe-replete conditions, the Fv/FmFb determined following the LC and 10 min of dark acclimation was 30% lower than the initial Fv/FmFb 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. Fig. 5 View largeDownload slide Comparison of photosynthetic yield (Fv/FmFb) in dark-acclimated cells before and after the light curve (LC). Empty bars represent measurements performed in dark-acclimated samples (60 min) before the LC and filled bars represent measurements performed in dark-acclimated samples (10 min) after the LC. Error bars indicate the SD of three biological replicates. Italic letters indicate statistically significant differences amongst treatments (P < 0.001). Gray values on the top indicate the percentage change in Fv/Fm following 10 min of dark acclimation after the LC with respect to Fv/Fm initially determined in 60 min dark-acclimated samples, before the LC. Fig. 5 View largeDownload slide Comparison of photosynthetic yield (Fv/FmFb) in dark-acclimated cells before and after the light curve (LC). Empty bars represent measurements performed in dark-acclimated samples (60 min) before the LC and filled bars represent measurements performed in dark-acclimated samples (10 min) after the LC. Error bars indicate the SD of three biological replicates. Italic letters indicate statistically significant differences amongst treatments (P < 0.001). Gray values on the top indicate the percentage change in Fv/Fm following 10 min of dark acclimation after the LC with respect to Fv/Fm initially determined in 60 min dark-acclimated samples, before the LC. Relationship between photophysiological parameters and state transitions Under Fe-replete conditions, plots of NSVFb (our metric of non-photochemical excitation dissipation), vs. τ' (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 light onset (8 μmol photons m–2 s–1). With further increases in light up to 128 μmol photons m–2 s–1, NSVFb and τ' remained almost unchanged. However, after exposure to yet higher irradiances from 239 up to 856 μmol photons m–2 s–1, the increase in NSVFb was accompanied by a decrease of τ' as downstream electron transport accelerated. Under mild and strong Fe limitation, the overall relationship between NSVFb and τ' presented three main sections (Fig. 6B, C). Fig. 6 View largeDownload slide Relationship between non-photochemical quenching (NSVFb) and the re-oxidation time of the primary acceptor QA (τ') in Synechococcus sp. PCC7002 acclimated to different Fe availability (A–D) and under different irradiances from 0 up to 856 µmol photons m–2 s–1. Numbers in parentheses indicate the PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). Error bars indicate the SD of three biological replicates. Fig. 6 View largeDownload slide Relationship between non-photochemical quenching (NSVFb) and the re-oxidation time of the primary acceptor QA (τ') in Synechococcus sp. PCC7002 acclimated to different Fe availability (A–D) and under different irradiances from 0 up to 856 µmol photons m–2 s–1. Numbers in parentheses indicate the PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). Error bars indicate the SD of three biological replicates. In the first section, between darkness and 8 μmol photons m–2 s–1, NSVFb remained similar, while τ' increased. In the second section, between 8 and 128 μmol photons m–2 s–1, NSVFb and τ' declined. Over the third section, at irradiances >128 μmol photons m–2 s–1, NSVFb increased again while τ' 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 μmol 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. Fig. 7 View largeDownload slide Correlation between PSII effective absorption cross-section (σPSII') and the product of the non-photochemical quenching by the re-oxidation time of the primary acceptor QA (NSVFb×τ'). σPSII' is negatively correlated with NSVFb×τ' in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1 (P < 0.05, represented by continuous lines). Pearson product moment correlation coefficients of –0.83, –0.82, –0.98 and –0.92 were calculated for increasing concentrations of Fe'. Numbers in parentheses indicate the instantaneous PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). Fig. 7 View largeDownload slide Correlation between PSII effective absorption cross-section (σPSII') and the product of the non-photochemical quenching by the re-oxidation time of the primary acceptor QA (NSVFb×τ'). σPSII' is negatively correlated with NSVFb×τ' in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1 (P < 0.05, represented by continuous lines). Pearson product moment correlation coefficients of –0.83, –0.82, –0.98 and –0.92 were calculated for increasing concentrations of Fe'. Numbers in parentheses indicate the instantaneous PAR (µmol photons m–2 s–1) to which the cells were exposed for 5 min during the light curve (LC). 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 μmol photons m–2 s–1, while with higher irradiances the ST size decreased rapidly (Fig. 8). Under Fe limitation, the ST 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 μmol photons m–2 s–1. Under severe Fe limitation, the calculated ST size was <1, suggesting that no ST was operating. Fig. 8 View largeDownload slide Size of state transitions in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1. The gray horizontal line indicates a state transition equal to 1, which typically corresponds to dark acclimation, i.e. no state transition. The gray vertical bar indicates the growth light level. Error bars indicate the SD of three biological replicates. Fig. 8 View largeDownload slide Size of state transitions in Synechococcus sp. PCC7002 acclimated to different Fe availability under different irradiances from 8 to 856 µmol photons m–2 s–1. The gray horizontal line indicates a state transition equal to 1, which typically corresponds to dark acclimation, i.e. no state transition. The gray vertical bar indicates the growth light level. Error bars indicate the SD of three biological replicates. Discussion Macromolecular allocation under Fe limitation In line with results previously reported (Wilhelm et al. 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 to 0 .037 d–1; Fitzwater et al. 1996), one of the major HNLC regions with the Southern Ocean. Under nutrient starvation (nitrogen and phosphorus), different phytoplanktonic groups increase lipid and carbohydrate storage, while cellular protein contents either remain steady or decrease (Bertilsson et al. 2003, Wagner et al. 2006, Jakob et al. 2007, Halsey et al. 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 coding 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 et al. 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 accompany 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 et al. 2010). This decrease in C:N with increasing severity of 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 the severity of Fe limitation. The first response was observed under mild and strong Fe limitation, where a decrease in σPSII 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 τ observed under mild and strong Fe limitation relative to Fe-replete conditions indicated that re-oxidation of the QA pool was slower, showing downstream limitations on the electron transport. Mild Fe limitation resulted in up-regulation of the gene isiA and down-regulation of psaA and psaB, encoding PSI proteins, which then remained unchanged with further increasing severity of Fe limitation (Blanco-Ameijeiras et al. 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 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 σPSII observed under mild and strong Fe limitation could be associated with the increase of IsiA competing with the PSII for excitation energy from the PBSs, as previously reported (Park et al. 1999, Sandström et al. 2001). In fact, work by Wilson et al. (2007) using Fe-limited IsiA mutants, in combination with PBS mutants and orange carotenoid protein (OCP) mutants, suggest that PBSs 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 in Fe-replete conditions, suggesting that the equilibration of excitons with 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 σPSII 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 σPSII could then be explained by the remaining PBS antennae serving a small population of active PSII reaction centers organized in a puddle rather than a lake antenna bed. The lower expression of genes coding for PSII reaction centers reported by the transcriptomic analysis supports this hypothesis (Blanco-Ameijeiras et al. 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 et al. 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, coding for a putative PBS degradation protein. As in this study σPSII was determined with three blocks of light-emitting diodes (LEDs), the spectral shifts due to different pigment complements might contribute to some of the changes observed. However, the concentration of PBS pigments relative to other pigments of the photosynthetic apparatus is not expected to change dramatically in Fe-limited cyanobacteria, as demonstrated by 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 components of the electron transport chain, such as PSII, Cyt b6f and ATPase, whereas the respiratory terminal oxidase Cyt 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 expected 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 τ' 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 downstream electron transport, which is accompanied by an increase in non-photochemical quenching. This quenching under high light could presumably be attributed to the OCP, reported as a key cyanobacterial 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 et al. 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 μmol photons m–2 s–1, rather than at growth irradiance (53 μmol photons m–2 s–1), suggesting that Fe limitation hampers the state transition. This hypothesis of inefficient STs 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 an NADH2 dehydrogenase subunit (Blanco-Ameijeiras et al. 2017), which is required for the transition to state 2 in the dark by mediating electron flow into the intersystem electron carriers in the dark (Huang et al. 2003). Under severe Fe limitation, the light to dark STs were absent. The decrease in τ' 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 of NSVFb vs. σPSII and τ' vs. 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 that under Fe-replete conditions. However, the comparison of Fv/FmFb 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 et al. 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 of mild to strong Fe limitation compared with severe limitation. Under mild and strong Fe limitation, there was a decrease in LET accompanied by progressive loss of state transitions. Under severe Fe limitation, STs 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. MaterialS 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 2), and finally filter-sterilized (0.2 μm polycarbonate membrane, Whatman). Culture medium was amended with different concentrations of dissolved Fe (Table 2), 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 et al. 2001, Buck et al. 2015) and oceanic low Fe (Fitzsimmons et al. 2013, Buck et al. 2015) regions, respectively. Manipulations were conducted in a trace metal-clean AirClean Systems (AC600 Series PCR Workstation, STAR LAB). All labware and material used were cleaned to remove any trace metal background contamination by soaking them for 1 week in 0.01% Citranox (ALCONOX) and Milli-Q rinsing, followed by 1 week soaking in 1.2 M HCl prior to extensive rinsing with ultrapure water (18.2 MΩ) obtained from a Milli-Q Direct System (Merk Millipore). Unless otherwise specified, all solutions used in this study were prepared using analytical grade chemicals (Sigma-Aldrich) 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), 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 d for at least 22 generations prior to the experiments. To this end, experimental bottles were inoculated with the respective acclimated cultures into 2,000 ml of medium with an initial concentration of 10.7×104 ± 2.3×104cell ml–1, in each polycarbonate bottle. The cells were grown in three biological replicates of each experimental treatment and harvested after 4 d of incubation. Table 2 Concentrations of macronutrients, trace metals and vitamins added to the synthetic oceanic seawater Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 All the elements except vitamins were prepared from ICP-MS standard solutions (Fluka). Bioavailable concentrations were estimated from dissolved inorganic iron (Fe'), calculated with MINEQL+ 4.6 (Schecher and McAvoy 1994). Table 2 Concentrations of macronutrients, trace metals and vitamins added to the synthetic oceanic seawater Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 Total concentration (M) Bioavailable Fe concentration (M) Macronutrients NaNO3 3.00E-04 NaH2PO4·2H2O 1.00E-05 Na2SiO3·5H2O 1.00E-04 Trace metals ZnCl2 6.00E-07 CoCl2 1.00E-07 MnCl2 1.35E-07 Na2MoO4 1.00E-08 NiCl2 6.00E-08 Na2EDTA 6.00E-05 CuCl2 1.20E-08 Na2SeO3 1.00E-09 Vitamins Thiamine HCl 2.96E-07 Biotin 2.05E-09 Vitamin B12 3.69E-10 Fe treatments used FeCl3 (Fe-replete treatment) 2.00E-07 33.67E-12 FeCl3 (mild Fe limitation treatment) 2.00E-08 3.83E-12 FeCl3 (strong Fe limitation treatment) 2.00E-09 0.47E-12 FeCl3 (severe Fe limitation treatment) 0.00E+00 0.047E-12 All the elements except vitamins were prepared from ICP-MS standard solutions (Fluka). Bioavailable concentrations were estimated from dissolved inorganic iron (Fe'), calculated with MINEQL+ 4.6 (Schecher and McAvoy 1994). 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) with a 45 μm capillary. Based on the cell density, growth rate (µ) was calculated according to Equation 1. µ=(Lnc1−Lnc0)/Δt (1) where c0 and c1 are the cell counts in millilitres 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% HCl for 24 h to remove particulate inorganic carbon (Verardo et al. 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). POC and PON content were corrected for blank measurements and normalized to total cell counts to calculate cellular quotas (POCcq and PONcq). POCprod and PONprod were calculated multiplying the cellular quota by µ. Results of production rates were expressed in moles of carbon or nitrogen per cell per day, accordingly. Particulate organic phosphate (POP) content Organic phosphorus compounds were digested in the presence of the oxidizing decomposition reagent Oxisolv (Merck Millipore) 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) and 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). The 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® HPLC system equipped with a temperature-controlled auto-sampler L-2200, a DAD detector L-2450 (Hitachi High Technologies Inc.) and a Spherisorb ODS-2 column (25 cm×4.6 mm, 5 μm particle size; Waters Corp.). Pigment separation was achieved using a LiChrospher® 100 RP-18 guard cartridge (Merck KGaA). Peaks detected at 440 nm were identified and quantified via co-chromatography of pigment standards obtained from DHI Lab Products 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.) that circulated Milli-Q water at 22ºC 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 1 h 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 Chl), 530 and 624 nm (preferentially absorbed by the PBSs) 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 the 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 fell within the optimum range of 0.04–0.05 and the curve of fluorescence increase reached 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 six times with an interval of 3 s. Using FastPro8 GUI, the acquisitions were fitted to the KPF biophysical model (Kolber et al. 1998) to determine dark-adapted F0, Fm, σPSII, τ and 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 PBSs 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 an energetically detached photosynthetic antenna complex in the cyanobacterium Synechocystis sp. PCC6803 (Ihalainen et al. 2005, Schrader et al. 2011). This phenomenon has been estimated to represent almost 50% of the pigment content in the phytoplankton cells present in high-nutrient low-Chl 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 (Cadoret 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−repleteFv/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 data set of dark-acclimated measurements was independently corrected for the two types of fluorescence baseline corrections (Ffsw and Fb), and the resulting estimates were compared. The use of the sub index Ffsw or Fb with the photophysiological parameters indicates the correction applied. The Fv/Fm was calculated following Equation 3. Fv/Fm=(Fm-F0)/Fm (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 LC were conducted by exposing each treatment to a set of increasing irradiance levels (8, 23, 53, 128, 239, 465 and 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 (σPSII'), re-oxidation time of the 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 performed before the LC to determine the recovery capacity after exposure to high light intensities. The recovery yield was calculated as the percentage of variation with respect to the dark-acclimated measurement before the LC. Absolute PSII ETRs at each PAR were calculated according to Equation 6 (Suggett et al. 2006, Suggett et al. 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-1s-1)=σPSII×[(Fq'/Fm')/(Fv/Fm)]×PAR (6) The relationship between NSVFb and τ(')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 STs 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) However, 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 analysis of variance (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.). Supplementary Data Supplementary data are available at PCP online. Funding This work was funded by the Swiss National Science Foundation [FNS PP00P2-138955 to C.S.H.]; the Schmidheiny Foundation [to S.B.A.]; the Helmholtz Association [Young Investigators Group EcoTrace, VH-NG-901 to S.T.)]; and the Canada Research Chairs program [to D.A.C.]. Acknowledgments The authors are grateful to Marc C. Moore for his comments on 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. Disclosures The authors have no conflicts of interest to declare. References Behrenfeld M.J. , Worthington K. , Sherrell R.M. , Chavez F.P. , Strutton P. , McPhaden M. ( 2006 ) Controls on tropical Pacific Ocean productivity revealed through nutrient stress diagnostics . Nature 442 : 1025 – 1028 . Google Scholar CrossRef Search ADS PubMed Behrenfeld M.J. , Milligan A.J. ( 2013 ) Photophysiological expressions of iron stress in phytoplankton . Annu. Rev. Mar. Sci. 5 : 217 – 246 . Google Scholar CrossRef Search ADS Bertilsson S. , Berglund O. , Karl D.M. , Chisholm S.W. ( 2003 ) Elemental composition of marine Prochlorococcus and Synechococcus: implications for the ecological stoichiometry of the sea . Limnol. Oceanogr. 48 : 1721 – 1731 . Google Scholar CrossRef Search ADS Bibby T.S. , Nield J. , Partensky F. , Barber J. ( 2001 ) Oxyphotobacteria: antenna ring around photosystem I . Nature 413 : 590 – 590 . Google Scholar CrossRef Search ADS PubMed Blanco-Ameijeiras S. , Cosio C. , Hassler C.S. ( 2017 ) Long-term acclimation to iron limitation reveal new insights in metabolism regulation of Synechococcus sp . Front. Mar. Sci. 4 . Boyd P.W. , Ellwood M.J. ( 2010 ) The biogeochemical cycle of iron in the ocean . Nat. Geosci. 3 : 675 – 682 . Google Scholar CrossRef Search ADS Bruland K.W. , Rue E.L. , Smith G.J. ( 2001 ) Iron and macronutrients in California coastal upwelling regimes: implications for diatom blooms . Limnol. Oceanogr. 46 : 1661 – 1674 . Google Scholar CrossRef Search ADS Buck K.N. , Sohst B. , Sedwick P.N. ( 2015 ) The organic complexation of dissolved iron along the U.S. GEOTRACES (GA03) North Atlantic Section . Deep Sea Res. II 116 : 152 – 165 . Google Scholar CrossRef Search ADS Cadoret J.-C. , Demoulière R. , Lavaud J. , van Gorkom H.J. , Houmard J. , Etienne A.-L. , et al. ( 2004 ) Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA) . Biochim. Biophys. Acta 1659 : 100 – 104 . Google Scholar CrossRef Search ADS PubMed Campbell D. , Hurry V. , Clarke A.K. , Gustafsson P. , Oquist G. ( 1998 ) Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation . Microbiol. Mol. Biol. Rev . 62 : 667 – 683 . Google Scholar PubMed Campbell D. , Oquist G. ( 1996 ) Predicting light acclimation in cyanobacteria from non-photochemical quenching of photosystem II fluorescence, which reflects state transitions in these organisms . Plant Physiol. 111 : 1293 – 1298 . Google Scholar CrossRef Search ADS PubMed Ermakova M. , Huokko T. , Richaud P. , Bersanini L. , Howe C.J. , et al. ( 2016 ) Distinguishing the roles of thylakoid respiratory terminal oxidases in the cyanobacterium Synechocystis sp. PCC 6803 . Plant Physiol . 171 : 1307 – 1319 . Google Scholar PubMed Ferreira F. , Straus N.A. ( 1994 ) Iron deprivation in cyanobacteria . J. Appl. Phycol. 6 : 199 – 210 . Google Scholar CrossRef Search ADS Finkel Z.V. , Follows M.J. , Liefer J.D. , Brown C.M. , Benner I. , Irwin A.J. , et al. ( 2016 ) Phylogenetic diversity in the macromolecular composition of microalgae . PLoS One 11 : e0155977 . Google Scholar CrossRef Search ADS PubMed Fitzsimmons J.N. , Zhang R. , Boyle E.A. ( 2013 ) Dissolved iron in the tropical North Atlantic Ocean . Mar. Chem . 154 : 87 – 99 . Google Scholar CrossRef Search ADS Fitzwater S.E. , Coale K.H. , Gordon R.M. , Johnson K.S. , Ondrusek M.E. ( 1996 ) Iron deficiency and phytoplankton growth in the Equatorial Pacific . Deep Sea Res. Part II 43 : 995 – 1015 . Google Scholar CrossRef Search ADS Flombaum P. , Gallegos J.L. , Gordillo R.A. , Rincon J. , Zabala L.L. , Jiao N. , et al. ( 2013 ) Present and future global distributions of the marine Cyanobacteria Prochlorococcus and Synechococcus . Proc. Natl. Acad. Sci. USA 110 : 9824 – 9829 . Google Scholar CrossRef Search ADS Fraser J.M. , Tulk S.E. , Jeans J.A. , Campbell D.A. , Bibby T.S. , Cockshutt A.M. , et al. ( 2013 ) Photophysiological and photosynthetic complex changes during iron starvation in Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC . PLoS One 8 : e59861 . Google Scholar CrossRef Search ADS PubMed Geider R. , Roche J.L. ( 2002 ) Redfield revisited: variability of C:N:P in marine microalgae and its biochemical basis . Eur. J. Phycol. 37 : 1 – 17 . Google Scholar CrossRef Search ADS Giovagnetti V. , Ruban A.V. ( 2017 ) Detachment of the fucoxanthin chlorophyll a/c binding protein (FCP) antenna is not involved in the acclimative regulation of photoprotection in the pennate diatom Phaeodactylum tricornutum . Biochim. Biophys. Acta 1858 : 218 – 230 . Google Scholar CrossRef Search ADS PubMed Greene R.M. , Geider R.J. , Falkowski P.G. ( 1991 ) Effect of iron limitation on photosynthesis in a marine diatom . Limnol. Oceanogr. 36 : 1772 – 1782 . Google Scholar CrossRef Search ADS Greene R.M. , Geider R.J. , Kolber Z. , Falkowski P.G. ( 1992 ) Iron-induced changes in light harvesting and photochemical energy conversion processes in eukaryotic marine algae . Plant Physiol . 100 : 565 – 575 . Google Scholar CrossRef Search ADS PubMed Halsey K.H. , Jones B.M. ( 2015 ) Phytoplankton strategies for photosynthetic energy allocation . Annu. Rev. Mar. Sci. 7 : 265 – 297 . Google Scholar CrossRef Search ADS Halsey K.H. , Milligan A.J. , Behrenfeld M.J. ( 2010 ) Physiological optimization underlies growth rate-independent chlorophyll-specific gross and net primary production . Photosynth. Res. 103 : 125 – 137 . Google Scholar CrossRef Search ADS PubMed Hansen H.P. , Korolef F. ( 1983 ) Determination of nutrients. In Methods of Seawater Analysis . Edited by Grasshoff K. , Kremling K. , Ehrhardt M. pp. 150 – 228 . Weinheim, Germany : Wiley Verlag Chemie GmbH . Hassler C.S. , Alasonati E. , Mancuso Nichols C.A. , Slaveykova V.I. ( 2011 ) Exopolysaccharides produced by bacteria isolated from the pelagic Southern Ocean—role in Fe binding, chemical reactivity, and bioavailability . Mar. Chem . 123 : 88 – 98 . Google Scholar CrossRef Search ADS Hilton J. , Lishman H. , Mackness S. , Heaney S.I. ( 1986 ) An automated method for the analysis of ‘particulate’ carbon and nitrogen in natural waters . Hydrobiologia 141 : 269 – 271 . Google Scholar CrossRef Search ADS Ho T.-Y. , Quigg A. , Finkel Z.V. , Milligan A.J. , Wyman K. , Falkowski P.G. , et al. ( 2003 ) The elemental composition of some marine phytoplankton . J. Phycol. 39 : 1145 – 1159 . Google Scholar CrossRef Search ADS Huang C. , Yuan X. , Zhao J. , Bryant D.A. ( 2003 ) Kinetic analyses of state transitions of the cyanobacterium Synechococcus sp. PCC 7002 and its mutant strains impaired in electron transport . Biochim. Biophys. Acta 1607 : 121 – 130 . Google Scholar CrossRef Search ADS PubMed Ihalainen J.A. , D'Haene S. , Yeremenko N. , van Roon H. , Arteni A.A. , Boekema E.J. , et al. ( 2005 ) Aggregates of the chlorophyll-binding protein isia (cp43') dissipate energy in cyanobacteria . Biochemistry 44 : 10846 – 10853 . Google Scholar CrossRef Search ADS PubMed Ivanov A.G. , Krol M. , Sveshnikov D. , Selstam E. , Sandstrom S. , Koochek M. , et al. ( 2006 ) Iron deficiency in cyanobacteria causes monomerization of photosystem I trimers and reduces the capacity for state transitions and the effective absorption cross section of photosystem I in vivo . Plant Physiol . 141 : 1436 – 1445 . Google Scholar CrossRef Search ADS PubMed Jakob T. , Wagner H. , Stehfest K. , Wilhelm C. ( 2007 ) A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation . J. Exp. Bot. 58 : 2101 – 2112 . Google Scholar CrossRef Search ADS PubMed Jiang H.-B. , Lou W.-J. , Ke W.-T. , Song W.-Y. , Price N.M. , Qiu B.-S. , et al. ( 2015 ) New insights into iron acquisition by cyanobacteria: an essential role for ExbB–ExbD complex in inorganic iron uptake . ISME J. 9 : 297 – 309 . Google Scholar CrossRef Search ADS PubMed Kirilovsky D. ( 2007 ) Photoprotection in cyanobacteria: the orange carotenoid protein (OCP)-related non-photochemical-quenching mechanism . Photosynth. Res. 93 : 7 – 16 . Google Scholar CrossRef Search ADS PubMed Kolber Z.S. , Prášil O. , Falkowski P.G. ( 1998 ) Measurements of variable chlorophyll fluorescence using fast repetition rate techniques: defining methodology and experimental protocols . Biochim. Biophys. Acta 1367 : 88 – 106 . Google Scholar CrossRef Search ADS PubMed Kretz C.B. , Bell D.W. , Lomas D.A. , Lomas M.W. , Martiny A.C. ( 2015 ) Influence of growth rate on the physiological response of marine Synechococcus to phosphate limitation . Front. Microbiol. 6 : 85 . Google Scholar CrossRef Search ADS PubMed Kroon B.M.A. , Thoms S. ( 2006 ) From electron to biomass: a mechanistic model to describe phytoplankton photosynthesis and steady-state growth rates . J. Phycol. 42 : 593 – 609 . Google Scholar CrossRef Search ADS Liu S.-W. , Qiu B.-S. ( 2012 ) Different responses of photosynthesis and flow cytometric signals to iron limitation and nitrogen source in coastal and oceanic Synechococcus strains (Cyanophyceae) . Mar. Biol. 159 : 519 – 532 . Google Scholar CrossRef Search ADS Ludwig M. , Bryant D.A. ( 2012 ) Acclimation of the global transcriptome of the cyanobacterium Synechococcus sp. strain PCC 7002 to nutrient limitations and different nitrogen sources . Front. Microbiol. 3 : 145 . Google Scholar PubMed Ma W. , Ogawa T. , Shen Y. , Mi H. ( 2007 ) Changes in cyclic and respiratory electron transport by the movement of phycobilisomes in the cyanobacterium Synechocystis sp. strain PCC 6803 . Biochim. Biophys. Acta 1767 : 742 – 749 . Google Scholar CrossRef Search ADS PubMed Macey A.I. , Ryan-Keogh T. , Richier S. , Moore C.M. , Bibby T.S. ( 2014 ) Photosynthetic protein stoichiometry and photophysiology in the high latitude North Atlantic . Limnol. Oceanogr. 59 : 1853 – 1864 . Google Scholar CrossRef Search ADS Mackey K.R.M. , Paytan A. , Grossman A.R. , Bailey S. ( 2008 ) A photosynthetic strategy for coping in a high-light, low-nutrient environment . Limnol. Oceanogr. 53 : 900 – 913 . Google Scholar CrossRef Search ADS Mackey K.R.M. , Post A.F. , McIlvin M.R. , Cutter G.A. , John S.G. , Saito M.A. , et al. ( 2015 ) Divergent responses of Atlantic coastal and oceanic Synechococcus to iron limitation . Proc. Natl. Acad. Sci. USA 112 : 9944 – 9949 . Google Scholar CrossRef Search ADS Mahmood A. , Abualhaija M.M. , van den Berg C.M.G. , Sander S.G. ( 2015 ) Organic speciation of dissolved iron in estuarine and coastal waters at multiple analytical windows . Mar. Chem . 177 : 706 – 719 . Google Scholar CrossRef Search ADS Martin J.H. , Fitzwater S.E. ( 1988 ) Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic . Nature 331 : 341 – 343 . Google Scholar CrossRef Search ADS McKew B.A. , Davey P. , Finch S.J. , Hopkins J. , Lefebvre S.C. , Metodiev M.V. , et al. ( 2013 ) The trade-off between the light-harvesting and photoprotective functions of fucoxanthin-chlorophyll proteins dominates light acclimation in Emiliania huxleyi (clone CCMP 1516) . New Phytol. 200 : 74 – 85 . Google Scholar CrossRef Search ADS PubMed Misumi M. , Kato H. , Tomo T. , Sonoike K. ( 2015 ) Relationship between photochemical quenching and non-photochemical quenching in six species of cyanobacteria reveals species difference in redox state and species commonality in energy dissipation . Plant Cell Physiol . 57 : 1510 – 1517 . Google Scholar PubMed Moore C.M. , Mills M.M. , Arrigo K.R. , Berman-Frank I. , Bopp L. , Boyd P.W. , et al. ( 2013 ) Processes and patterns of oceanic nutrient limitation . Nat. Geosci. 6 : 701 – 710 . Google Scholar CrossRef Search ADS Mullineaux C.W. , Allen J.F. ( 1986 ) The state 2 transition in the cyanobacterium Synechococcus 6301 can be driven by respiratory electron flow into the plastoquinone pool . FEBS Lett . 205 : 155 – 160 . Google Scholar CrossRef Search ADS Mullineaux C.W. , Allen J.F. ( 1990 ) State 1–State 2 transitions in the cyanobacterium Synechococcus 6301 are controlled by the redox state of electron carriers between Photosystems I and II . Photosynth. Res. 23 : 297 – 311 . Google Scholar CrossRef Search ADS PubMed Murphy C.D. , Ni G. , Li G. , Barnett A. , Xu K. , Grant-Burt J. , et al. ( 2017 ) Quantitating active photosystem II reaction center content from fluorescence induction transients . Limnol. Oceanogr. Methods 15 : 54 – 69 . Google Scholar CrossRef Search ADS Noffke N. , Christian D. , Wacey D. , Hazen R.M. ( 2013 ) Microbially induced sedimentary structures recording an ancient ecosystem in the ca. 3.48 billion-year-old dresser formation, Pilbara, Western Australia . Astrobiology 13 : 1103 – 1124 . Google Scholar CrossRef Search ADS PubMed Öquist G. ( 1974 ) Iron deficiency in the blue-green alga Anacystis nidulans: changes in pigmentation and photosynthesis . Physiol. Plant. 30 : 30 – 37 . Google Scholar CrossRef Search ADS Osanai T. , Kanesaki Y. , Nakano T. , Takahashi H. , Asayama M. , Shirai M. , et al. ( 2005 ) Positive regulation of sugar catabolic pathways in the cyanobacterium Synechocystis sp. PCC 6803 by the group 2 σ factor SigE . J. Biol. Chem. 280 : 30653 – 30659 . Google Scholar CrossRef Search ADS PubMed Oxborough K. ( 2014 ) FAstPro8 GUI and FRRf3 Systems Documentation . 2230-801-HB edn. Chelsea Technologies Group , West Molesey, UK . Oxborough K. , Baker N.R. ( 1997 ) Resolving chlorophyll a fluorescence images of photosynthetic efficiency into photochemical and non-photochemical components—calculation of qP and Fv/Fm without measuring Fo . Photosynth. Res . 54 : 135 – 142 . Google Scholar CrossRef Search ADS Park Y.-I. , Sandstrom S. , Gustafsson P. , Oquist G. ( 1999 ) Expression of the isiA gene is essential for the survival of the cyanobacterium Synechococcus sp. PCC 7942 by protecting photosystem II from excess light under iron limitation . Mol. Microbiol. 32 : 123 – 129 . Google Scholar CrossRef Search ADS PubMed Pitchford J.W. , Brindley J. ( 1999 ) Iron limitation, grazing pressure and oceanic high nutrient–low chlorophyll (HNLC) regions . J. Plankton Res . 21 : 525 – 547 . Google Scholar CrossRef Search ADS Price N.M. , Harrison G.I. , Hering J.G. , Hudson R.J. , Nirel P.M.V. , Palenik B. , et al. ( 1989 ) Preparation and chemistry of the artificial algal culture medium . Aquil. Biol. Oceanogr . 6 : 443 – 461 . Ralph P.J. , Gademann R. ( 2005 ) Rapid light curves: a powerful tool to assess photosynthetic activity . Aquat. Bot . 82 : 222 – 237 . Google Scholar CrossRef Search ADS Raven J.A. , Evans M.C.W. , Korb R.E. ( 1999 ) The role of trace metals in photosynthetic electron transport in O2-evolving organisms . Photosynth. Res . 60 : 111 – 150 . Google Scholar CrossRef Search ADS Riethman H.C. , Sherman L.A. ( 1988 ) Immunological characterization of iron-regulated membrane proteins in the cyanobacterium Anacystis nidulans R2 . Plant Physiol . 88 : 497 – 505 . Google Scholar CrossRef Search ADS PubMed Ryan-Keogh T.J. , Macey A.I. , Cockshutt A.M. , Moore C.M. , Bibby T.S. ( 2012 ) The cyanobacterial chlorophyll-binding-protein IsiA acts to increase the in vivo effective absorption cross-section of PSI under iron limitation . J. Phycol . 48 : 145 – 154 . Google Scholar CrossRef Search ADS PubMed Sandström S. , Park Y.-I. , Öquist G. , Gustafsson P. ( 2001 ) CP43′, the isiA gene product, functions as an excitation energy dissipator in the cyanobacterium Synechococcus sp. PCC 7942 . Photochem. Photobiol . 74 : 431 – 437 . Google Scholar CrossRef Search ADS PubMed Sandström S. , Ivanov A.G. , Park Y.-I. , Oquist G. , Gustafsson P. ( 2002 ) Iron stress responses in the cyanobacterium Synechococcus sp . Physiol. Plant. 116 : 255 – 263 . Google Scholar CrossRef Search ADS PubMed Schecher W.D. , McAvoy C.D. ( 1994 ) MINEQL+: A Chemical Equilibrium Program for Personal Computers. Environmental Research Software , Hallowell, ME . Scherer S. , Stürzl E. , Büger P. ( 1982 ) Interaction of respiratory and photosynthetic electron transport in Anabaena variabilis Kütz . Arch. Microbiol. 132 : 333 – 337 . Google Scholar CrossRef Search ADS Schrader P.S. , Milligan A.J. , Behrenfeld M.J. ( 2011 ) Surplus photosynthetic antennae complexes underlie diagnostics of iron limitation in a cyanobacterium . PLoS One 6 : e18753 . Google Scholar CrossRef Search ADS PubMed Schuback N. , Schallenberg C. , Duckham C. , Maldonado M.T. , Tortell P.D. ( 2015 ) Interacting effects of light and iron availability on the coupling of photosynthetic electron transport and CO2-assimilation in marine phytoplankton . PLoS One 10 : e0133235 . Google Scholar CrossRef Search ADS PubMed Sedoud A , López-Igual R. , Ur Rehman A. , Wilson A. , Perreau F. , Boulay C. , et al. ( 2014 ) The cyanobacterial photoactive orange carotenoid protein is an excellent singlet oxygen quencher . Plant Cell 26 : 1781 – 1791 . Google Scholar CrossRef Search ADS PubMed Simis S.G.H. , Huot Y. , Babin M. , Seppälä J. , Metsamaa L. ( 2012 ) Optimization of variable fluorescence measurements of phytoplankton communities with cyanobacteria . Photosynth. Res. 112 : 13 – 30 . Google Scholar CrossRef Search ADS PubMed Suggett D.J. , Moore C.M. , Marañón E. , Omachi C. , Varela R.A. , Aiken J. , et al. ( 2006 ) Photosynthetic electron turnover in the tropical and subtropical Atlantic Ocean . Deep Sea Res. II 53 : 1573 – 1592 . Google Scholar CrossRef Search ADS Suggett D.J. , Moore C.M. , Hickman A.E. , Geider R.J. ( 2009 ) Interpretation of fast repetition rate (FRR) fluorescence: signatures of phytoplankton community structure versus physiological state . Mar. Ecol. Prog. Ser. 376 : 1 – 19 . Google Scholar CrossRef Search ADS Sun J. , Golbeck J.H. ( 2015 ) The presence of the IsiA–PSI supercomplex leads to enhanced Photosystem I electron throughput in iron-starved cells of Synechococcus sp. PCC 7002 . J. Phys. Chem. B 119 : 13549 – 13559 . Google Scholar CrossRef Search ADS PubMed Sunda W.G. ( 1989 ) Trace metal interactions with marine phytoplankton . Biol. Oceanogr . 6 : 411 – 442 . Sunda W.G. , Huntsman S.A. ( 1995 ) Iron uptake and growth limitation in oceanic and coastal phytoplankton . Mar. Chem . 50 : 189 – 206 . Google Scholar CrossRef Search ADS Thompson A.W. , Huang K. , Saito M.A. , Chisholm S.W. ( 2011 ) Transcriptome response of high- and low-light-adapted Prochlorococcus strains to changing iron availability . ISME J. 5 : 1580 – 1594 . Google Scholar CrossRef Search ADS PubMed Thurotte A. , Lopez-Igual R. , Wilson A. , Comolet L. , Bourcier de Carbon C. , Xiao F. , et al. ( 2015 ) Regulation of orange carotenoid protein activity in cyanobacterial photoprotection . Plant Physiol. 169 : 737 – 747 . Google Scholar CrossRef Search ADS PubMed Tsuda A. ( 2003 ) A mesoscale iron enrichment in the Western Subarctic Pacific induces a large centric diatom bloom . Science 300 : 958 – 961 . Google Scholar CrossRef Search ADS PubMed Vassiliev I.R. , Kolber Z. , Wyman K.D. , Mauzerall D. , Shukla V.K. , Falkowski P.G. , et al. ( 1995 ) Effects of iron limitation on Photosystem II composition and light utilization in Dunaliella tertiolecta . Plant Physiol. 109 : 963 – 972 . Google Scholar CrossRef Search ADS PubMed Verardo D.J. , Froelich P.N. , McIntyre A. ( 1990 ) Determination of organic carbon and nitrogen in marine sediments using the Carlo Erba NA-1500 analyzer . Deep Sea Res. A . 37 : 157 – 165 . Google Scholar CrossRef Search ADS Vrede T. , Dobberfuhl D.R. , Kooijman S.A.L.M. , Elser J.J. ( 2004 ) Fundamental connections among organism C:N:P stoichiometry, macromolecular composition, and growth . Ecology 85 : 1217 – 1229 . Google Scholar CrossRef Search ADS Wagner H. , Jakob T. , Wilhelm C. ( 2006 ) Balancing the energy flow from captured light to biomass under fluctuating light conditions . New Phytol. 169 : 95 – 108 . Google Scholar CrossRef Search ADS PubMed Webb W.L. , Newton M. , Starr D. ( 1974 ) Carbon dioxide exchange of Alnus rubra . Oecologia 17 : 281 – 291 . Google Scholar CrossRef Search ADS PubMed Wilhelm S.W. , Maxwell D.P. , Trick C.G. ( 1996 ) Growth, iron requirements, and siderophore production in iron-limited Synechococcus PCC 72 . Limnol. Oceanogr. 41 : 89 – 97 . Google Scholar CrossRef Search ADS Wilhelm S.W. , MacAuley K. , Trick C.G. ( 1998 ) Evidence for the importance of catechol-type siderophores in the iron-limited growth of a cyanobacterium . Limnol. Oceanogr. 43 : 992 – 997 . Google Scholar CrossRef Search ADS Wilhelm S.W. , Trick C.G. ( 1995 ) Physiological profiles of Synechococcus (cyanophyceae) in iron-limiting continuous cultures . J. Phycol. 31 : 79 – 85 . Google Scholar CrossRef Search ADS Wilson A. , Boulay C. , Wilde A. , Kerfeld C.A. , Kirilovsky D. ( 2007 ) Light-induced energy dissipation in iron-starved cyanobacteria: roles of OCP and IsiA proteins . Plant Cell 19 : 656 – 672 . Google Scholar CrossRef Search ADS PubMed Wright S.W. , Jeffrey S.W. , Mantoura R.F.C. , Llewellyn C.A. , Bjornland T. , Repeta D. , et al. ( 1991 ) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton . Mar. Ecol. Prog. Ser. 77 : 183 – 196 . Google Scholar CrossRef Search ADS Xu K. , Grant-Burt J.L. , Donaher N. , Campbell D.A. ( 2017 ) Connectivity among photosystem II centers in phytoplankters: patterns and responses . Biochim. Biophys. Acta 1858 : 459 – 474 . Google Scholar CrossRef Search ADS PubMed Abbreviations Abbreviations 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 PSII fluorescence determined with continuous background illumination Fe iron Fe' dissolved inorganic Fe Fb cellular baseline fluorescence correction by fluorescence from phycobilisomes and PSI Ffsw baseline fluorescence correction by fluorescence from filtered growth medium FRR-ST single turnover fluorescence induction curves Fm dark-adapted PSII maximum fluorescence Fm' maximum PSII fluorescence determined with continuous background illumination Fq'/Fm' effective PSII quantum yield determined with continuing background illumination Fv/Fm the PSII photochemical efficiency in dark-acclimated cells F0 dark-adapted PSII minimum fluorescence LC measurements performed in cells exposed to increasing light for a short time LED light-emitting diode LET linear electron transport from PSII to PSI NSV normalized Stern–Volmer photochemical quenching OCP orange carotenoid protein PAR photosynthetic active radiation PBS phycobilisome 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 ST state transition α maximum light use efficiency Δt time of incubation in days μ growth rate ρ degree of connectivity between PSII reaction centers determined in dark acclimated samples ρ' degree of connectivity between PSII reaction centers determined with continuous background illumination σPSII PSII functional absorption cross-section determined in dark-acclimated samples σPSII' PSII functional absorption cross-section determined in illuminated cells τ re-oxidation time of the plastoquinone primary acceptor QA determined in the dark τ' re-oxidation time of primary quinone-type acceptor QA determined in illuminated cells © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Plant and Cell PhysiologyOxford University Press

Published: May 30, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off