Interacting Effects of Light and Iron Availability on the Coupling of Photosynthetic Electron Transport and CO2-Assimilation in Marine Phytoplankton

Abstract

Iron availability directly affects photosynthesis and limits phytoplankton growth over vast oceanic regions. For this reason, the availability of iron is a crucial variable to consider in the development of active chlorophyll a fluorescence based estimates of phytoplankton primary productivity. These bio-optical approaches require a conversion factor to derive ecologically-relevant rates of CO2-assimilation from estimates of electron transport in photosystem II. The required conversion factor varies significantly across phytoplankton taxa and environmental conditions, but little information is available on its response to iron limitation. In this study, we examine the role of iron limitation, and the interacting effects of iron and light availability, on the coupling of photosynthetic electron transport and CO2-assimilation in marine phytoplankton. Our results show that excess irradiance causes increased decoupling of carbon fixation and electron transport, particularly under iron limiting conditions. We observed that reaction center II specific rates of electron transport (ETR(RCII), mol e- mol RCII(-1) s(-1)) increased under iron limitation, and we propose a simple conceptual model for this observation. We also observed a strong correlation between the derived conversion factor and the expression of non-photochemical quenching. Utilizing a dataset from in situ phytoplankton assemblages across a coastal--oceanic transect in the Northeast subarctic Pacific, this relationship was used to predict ETR(RCII): CO2-assimilation conversion factors and carbon-based primary productivity from FRRF data, without the need for any additional measurements.

Fig 1. Map of sampling stations along the Line-P transect in the NE subarctic Pacific.

The iron addition experiment was initiated at station P20, located in iron-limited high nutrient low chlorophyll (HNLC) waters. Sampling depths at other stations along the transect were: 30 m at P4; 5 m, 25 m and 40 m at P12, P16, P20 and P26.

Fig 2. Response of chl a biomass and photophysiology during the on-board iron addition experiment.

Shown are changes in (a) [chl a], (b) F_v/F_m, and (c) σ_PSII. Error bars represent standard errors from three biological replicates and are sometimes smaller than the symbol.

Fig 3. Response of rates of CO₂-assimilation (mol C mol chl a ^-1 s^-1) and ETR_RCII (mol e^- mol RCII ^-1 s^-1) during the iron addition experiment.

Both rates were measured as a function of irradiance, and PvsE curves were fit with the exponential model of Webb et al. [74]. Shown are mean values from three biological replicates where error bars represent standard error of mean and are sometimes smaller than symbols.

Fig 4. Time-course of α (a-c) and P_max (d-f) of CO₂-assimilation, ETR_RCII and the derived conversion factor Φ_e:C/n_PSII during the iron addition experiment.

The conversion factor Φ_e:C/n_PSII under light limiting conditions is derived from values in (a) and (b). Similarly, the conversion factor Φ_e:C/n_PSII at light saturation is derived from the values in (d) and (e). The error in (a), (b), (c), and (d) is the 95% confidence interval of the parameter derived from the fit to data from three biological replicates, and the error in (c) and (f) is the propagated error from (a)/(b) and (d)/(e), respectively.

Fig 5. Light dependency of ChlF-derived parameters from FRRF measurements on day three after iron addition and in the iron-limited control treatment.

The parameter F_q′/F_v′ (a) represents the efficiency of charge separation in functional RCII and is an estimate of the fraction of open RCII (i.e. Q_A oxidized) at any given light level. The parameter F_v′/F_m′ (b) represents the efficiency of excitation energy capture by the fraction of open RCII and can be used to quantify the extent to which non-photochemical quenching in the PSII antenna competes with photochemistry for excitation energy. The parameter F_q′/F_m′ (c) represents the overall quantum efficiency of photochemical energy conversion in PSII (Φ′_PSII). See text for a full description of these parameters and their interpretation. Error bars represent standard errors from three biological replicates and are often smaller than symbols.

Fig 6. Changes in the light dependency of the conversion factor Φ_e:C/n_PSII (a-e) and NPQ_NSV (f-j) over the course of the iron addition experiment.

Units of in Φ_e:C/n_PSII are (mol e- mol C) / (mol chl a mol RCII^-1). The curves were derived by dividing corresponding values of ETR_RCII and CO₂-assimilation from the PvsE curves presented in Fig 3. NPQ was estimated as the normalized Stern-Volmer quenching coefficient NPQ_NSV = F_o′/F_v′ and is unitless [65]. Error bars are the standard error from three biological replicates and often smaller than symbols.

Fig 7. Relationship between the conversion factor Φ_e:C/n_PSII and NPQ_NSV values during the iron addition experiment.

Values of Φ_e:C/n_PSII were derived from PvsE curves of CO₂-assimilation and ETR_RCII at irradiances corresponding to each ETR_RCII-PvsE curve light level. Units of Φ_e:C/n_PSII are (mol e^- mol C^-1) / (mol chl a mol RCII^-1). NPQ_NSV values were derived as F_o′/F_v′ for each light level of the SSLC. Data points represent means and standard errors for parameters derived from three biological replicates. A quadratic fit through all data points (Φ_e:C/n_PSII = -733.21 NPQ²+8792.4 NPQ– 1477.1) is statistically significant (R² = 0.70, p-value < 0.0001).

Fig 8. Conceptual diagram visualizing the concept of excess excitation pressure and its dissipation before and after charge separation in RCII.

(A) Absorption of light energy by pigments in the light harvesting antenna of PSII cannot be controlled biologically, and rises linearly with incident light intensity. However, rates of linear electron transport (LET) and CO₂-assimilation saturate at a light intensity determined by the physiological state of the phytoplankton, resulting in a typical PvsE curve. Under optimal growth conditions, it is the resupply of NADP^-(predominantly from CO₂-assimilation) which limits LET, while under short-term exposure to excess light and under iron limitation, the ‘bottleneck’ of LET will be located before PSI. Whenever exitonic influx exceeds the chemical outflux at the level of RCII, excess excitation pressure needs to be safely dissipated to prevent photodamage. (B) Under optimal growth conditions and sub-saturating light, all absorbed photons are used for charge separation in RCII, and the majority of electrons will be used for LET and CO₂-assimilation, resulting in minimum Φ_e:C. (C) Conditions of high excitation pressure can be caused by short-term exposure to high light, but also by iron limitation, which comprises the functioning of the ETC and has been shown to create a ‘bottle neck’ for LET before PSI. Under these conditions, PTOX-mediated pseudo-cyclic electron flow (e.g. [–58,62,105,113]), cyclic electron transport around PSII (e.g. [107,108,114]), and charge recombination in RCII (e.g. [109,110,115]), have been suggested to safely dissipate excess excitation energy after RCII (but before PSI). Up-regulation of these alternative electron flow pathways could explain the high ETR_RCII (and Φ_e:C/n_PSII) observed in our iron-limited samples. Excess excitation energy can also be dissipated in the light harvesting antenna, before charge separation in RCII. Collectively, a number of different molecular processes dissipating excess excitation energy in the PSII antenna can be quantified as NPQ_NSV.

Fig 9. Rates of CO₂-assimilation (mol C mol chl a ^-1 hr^-1) derived from FRRF measurements plotted against rates measured by ¹⁴C-assimilation experiments.

Samples were taken at one to three depths at five stations along Line-P in the NE subarctic Pacific (see Fig 1). FRRF based PvsE curves were used to derive ETR_RCII and NPQ_NSV at 8 light levels for each sample, and Φ_e:C/n_PSII values were then derived from the relationship presented in Fig 7. Φ_e:C/n_PSII and ETR_RCII for each light level were used to calculate CO₂-assimilation rates. Error bars for predicted CO₂-assimilation rates represent the propagated error from the ChlF yields of the last three ST acquisitions of each light level during the FRRF PvsE curve used to derive NPQ_NSV and ETR_RCII. Error bars for measured CO₂-assimilation rates represent the mean coefficient of variance derived from all duplicate measurements (n = 46). The correlation between all predicted and measured data points (n = 95) was statistically significant (Spearman’s r = 0.90, two-tailed p-value < 0.0001). All statistics are for non log-transformed data.