Increased light‐enhanced dark respiration under warming suggests intensified metabolic coupling in an Arctic diatom

Temperature affects functional traits such as growth rates, biomass production or cellular quotas in Arctic phytoplankton to different degrees, likely resulting from diverging temperature sensitivities of underlying physiological key processes. We used membrane-inlet mass spectrometry to measure 16O2, 18O2 and carbon dioxide (CO2) fluxes to quantify photosynthetic O2 production ( PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ ), photosynthetic C-fixation ( PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ ), respiratory CO2 release ( R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ ), respiratory O2 consumption in the dark ( R O 2 $$ {\mathrm{R}}_{O_2} $$ ) and light-enhanced dark respiration (LEDR) as well as fast repetition rate fluorometry to quantify quantum yields and electron transport in the Arctic diatom Thalassiosira hyalina under different warming scenarios. At higher temperatures, cells upregulate light harvesting abilities to compensate for a lower maximum photosystem II (PSII) quantum yield, thereby maintaining O2 production and likely also thylakoid proton motive force (PMF). Furthermore, substantial LEDR under warming suggests that plastidial reductant is increasingly rerouted towards mitochondrial O2 consumption to support a sufficient ATP : NADPH ratio inside chloroplasts. In line with the indicated reductant import, the mitochondrial citric acid cycle is suppressed, minimizing respiratory CO2 loss despite stimulated LEDR. This suggests that the optimization of the plastidial ATP : NADPH ratio is critical for higher net biomass retention under warming. Our findings underline the necessity to interpret subprocesses of photosynthesis and respiration independently and explain the often-observed enhanced biomass accumulation in a warmer Arctic Ocean. The Arctic Ocean is most prone to climate change, with warming rates being higher than the global average, especially in the Fram Strait area (Carvalho & Wang, 2020; Constable et al., 2022; Rantanen et al., 2022). In addition to the general trend of rising mean sea surface temperatures, marine heatwaves have notably increased in frequency, duration and intensity (Hobday et al., 2016; Oliver et al., 2019). Hence, the Arctic is already exposed to exceptional degrees of ocean warming, and model simulations project further temperature increases, especially in the ever extending sea-ice-free regions. Since phytoplankton represent the base of the marine food web and contribute significantly to marine biogeochemical cycling (Falkowski et al., 1998; Field et al., 1998), their responses to warming have the potential to impact the entire Arctic ecosystem. Temperature as a universal driver affects molecular movement and consequently all physical and (bio)chemical reactions (Brown et al., 2004; Pearle et al., 2010), including also those key processes involved in primary production. As single-celled phototrophic organisms, phytoplankton perform oxygenic photosynthesis to convert light energy into (bio)chemical energy (Falkowski & Raven, 2013; Falciatore et al., 2022). First, the excitation energy harvested by pigment molecules is used to successively extract electrons from water, which are subsequently passed on to the photosynthetic electron transport chain (ETC) and ultimately lead to the generation of reductants, that is, electron carriers like NADPH. With the flow of electrons along the ETC, a PMF is established, which is later exploited for the generation of ATP. Both ATP and NADPH subsequently fuel the Calvin cycle, where the enzyme Ribulose-1,5-bisphosphate-carboxylase/-oxygenase (RubisCO) catalyzes the fixation of CO2 into organic biomass. The ATP : NADPH ratio created in the photosynthetic light reactions, however, is typically too low to satisfy the stoichiometric demands of the Calvin cycle (Allen et al., 2005; Lepetit et al., 2022), so that cells experience an overreduction of the chloroplasts. Therefore, alternative electron flows (AEFs) are necessary to either increase the relative amount of ATP or to divert excess NADPH (Allen et al., 2005; Curien et al., 2016). AEFs comprise cyclic electron flow, which is known to be prominent in polar diatoms (Goldman et al., 2015), oxygen (O2) consuming water-to-water cycles, such as the Mehler reaction and the plastid terminal oxidase (PTOX). The metabolic coupling of the chloroplasts with other cellular compartments represents another mechanism, in which excess reductant from the plastid is dissipated to, for example, the cytoplasm and the mitochondria. All these processes either support the PMF, and thus ATP production, or dissipate reductants in order to increase the ATP : NADPH ratio (e.g. Asada, 2000; Bailleul et al., 2015; Curien et al., 2016; Nawrocki et al., 2019; Lepetit et al., 2022). Regarding temperature sensitivity, light harvesting is typically less sensitive than the use of ATP and NADPH in metabolic processes (Baker et al., 1988; Raven & Geider, 1988). Consequently, any temperature-dependent change in the biochemical consumption of ATP and NADPH will require physiological adjustments on multiple levels. Therefore, AEFs can function as regulatory valves to maintain metabolic homeostasis inside the chloroplast and across cellular compartments, especially under stressful environmental conditions, as induced by increasing light intensities or temperatures, for example. Ecophysiological processes, including cell division, photosynthesis and respiration, are typically stimulated by elevated temperature as a result of accelerated diffusion rates, membrane fluidity and enzyme activity (Los et al., 2013; Young et al., 2015; Padfield et al., 2016; Schuback et al., 2017). However, temperature-induced changes are process-specific (e.g. Baker et al., 2016; Barton et al., 2020; Rehder et al., 2024), and cells require physiological regulation, such as the above-described AEFs, to overcome metabolic imbalances and to avoid detrimental temperature effects (Rehder et al., 2023). Consequently, continuous adjustment of physiological processes is critical for phytoplankton to maintain metabolic homeostasis over the widest possible range of experienced temperatures. Arctic phytoplankton has evolved to be surprisingly plastic toward ocean warming (Hoppe et al., 2018; Rehder et al., 2024; Wolf et al., 2024). Previous studies, for instance, found that many prominent Arctic phytoplankton species currently live substantially below their optimal temperatures and thus, it is expected that these organisms will benefit from moderate warming. In this study, we hypothesized that Arctic phytoplankton modulate photophysiology as well as AEF operation to adjust photosynthetic and respiratory processes to warmer temperatures. To resolve these regulatory efforts, we acclimated the Arctic diatom T. hyalina to different temperatures (2°C, 6°C and 10°C) and measured photophysiological parameters as well as rates of 16O2 evolution, 18O2 uptake and CO2 fluxes by means of fast repetition rate fluorometry (FRRf) and membrane-inlet mass spectrometry (MIMS), respectively. We cultured the Arctic diatom T. hyalina (KB3 SS5, isolated 2021 in Svalbard, Norway) at 2°C, 6°C and 10°C in 0.2-μm sterile-filtered Arctic seawater (Salinity 31), enriched with vitamins and trace metals according to f/2 media (Guillard & Ryther, 1962). Nitrate, silicate and phosphate were added in concentrations of 100, 100 and 6 μmol l−1, respectively. The irradiance was set to continuous light at 30 μmol photons m−2 s−1 to mimic a polar day light climate. Cells were acclimated to experimental conditions for at least seven generations as semi-continuous dilute batch cultures in aerated 2 l glass bottles (Schott Instruments, Mainz, Germany) under continuous supply of humidified air (pCO2 of 400 μatm) generated in a gas mixing system (CGM 2000; MCZ Umwelttechnik, Bad Nauheim, Germany). To ensure temperature stability, culture bottles were submersed in temperature-controlled aquaria. Cell concentrations during cultivation never exceeded 8000 cells ml−1 to ensure nutrient replete conditions and stable carbonate chemistry. Particulate organic carbon (POC) samples were filtered onto precombusted (12 h, 500°C) glass fiber filters (GF/F, 0.7 μm nominal pore size; Whatman, Maidstone, UK). After drying for at least 24 h at 60°C, filters were submitted to elemental analysis (EuroVector EA 3000, Pavia, Italy) using the flash combustion technique (Knap et al., 1996). Chl a samples were filtered onto precombusted (12 h, 500°C) GF/F (0.7 μm; Whatman), shock frozen in liquid nitrogen and stored at −80°C until extraction. Chl a was extracted overnight in 90% acetone (Sigma-Aldrich) with an additional cell disruption using a cell-mill (Precellys 24; Bertin, Montigny-le-Bretonneux, France). Extracts were centrifuged (13 000 g for 5 min; Sigma 4K10), and Chl a concentration in the supernatant was determined using the fluorometric ‘acidification method’ (Turner Trilogy Fluorometer; Turner Designs, San Jose, CA, USA; Knap et al. (1996)). Photophysiological characteristics of PSII were assessed by means of Chl a variable fluorescence using an FRRf (FastOcean; Chelsea Technologies, Yateley, UK) combined with the FastAct2 Laboratory system (Chelsea Technologies). Light-emitting diodes were set to 450 nm emission wavelength to fully saturate all PSII reaction centers on short timescales. We used the FRRf in single turnover mode, in which the saturation phase comprised 100 flashlets on a 2 μs pitch, and the relaxation phase comprised 40 flashlets on a 60 μs pitch. All measurements were conducted in a temperature-controlled cuvette at the respective acclimation temperature after dark acclimation for 45 min. Light acclimation indices (Ik) were calculated from absETRs following Trimborn et al. (2017). Optical antenna sizes (σLHCII) were obtained by dividing σPSII; dark by (Fv/Fm). A MIMS (Isoprime, GV Instruments, Manchester, UK) was used to measure (1) photosynthetic O2 production during light ( PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ ), (2) respiratory O2 consumption during dark ( R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ ) and light (so-called LEDR) and (3) photosynthetic net C-fixation in the light ( PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ ) and respiratory CO2 release in the dark ( R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ ). These rates were determined by measuring changes in gas concentrations of 16O2, 18O2 and CO2 within each biological replicate. Calibrations followed the procedures described in Rokitta & Rost (2012) for 16O2 and CO2. To calibrate for 18O2, a normal 16O2 calibration was performed and the acceleration voltage of the mass spectrometer was adjusted to redirect the 16O2 beam to the 18O2 detector. All fluxes were corrected for instrumental consumption of 16O2 and 18O2 under each temperature treatment. For 18O2 consumption measurements, the assay medium was purged with N2 gas overnight to remove all 16O2. Subsequently, the medium was transferred into the MIMS cuvette and equilibrated with 18O2 gas. The bioassays were performed using concentrated cell suspensions. To this end, cells were concentrated by gentle filtration over polycarbonate filters (Isopore TSTP, 3 μm pore size, Merck, Darmstadt, Germany) and resuspended in O2- and CO2-free culture medium buffered to a pHNBS of 7.9 (50 mM HEPES). Subsequently, cells were transferred to the temperature- and light-controlled MIMS cuvette and spiked with 18O2 reaching a final O2 concentration of c. 21%. Dissolved inorganic carbon (DIC) was added to the cuvette to yield typical concentration of seawater (c. 2200 μmol l−1). Carbonic anhydrase was added (500 μg l−1 final concentration) to ensure instantaneous equilibration of the carbonate system (Rokitta & Rost, 2012). Using the CO2 : DIC ratios obtained in the calibrations, CO2 traces were converted to total carbon fluxes. Flux measurements were performed in of 3 at light intensities and μmol photons m−2 for net and rates of PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ as well as LEDR ( R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ in the followed & we mean R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ rates from the R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ rates in the light to LEDR light and C-fixation rates were obtained from CO2 fluxes during the light rates of R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ were obtained from CO2 fluxes during the dark rates of C-fixation ( PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ ) were calculated by mean R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ rates from net C-fixation rates during After the Chl a samples were from the cuvette to the obtained rates on Chl and respiratory and were calculated from rates as PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ and R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ according to Rehder et al. All acclimation and photophysiological parameters are as the mean of biological obtained from gas measurements are in biological for the For acclimation and photophysiological normal was using the and were using the We performed followed by to for using et al., 2019). The of was set to rates of T. hyalina were c. at and 6°C and significantly to at 10°C The was at and significantly to at 6°C and 10°C The Chl a was at 6°C and increased from cell to cell at 10°C The Chl a : ratio was at at and 6°C increased significantly to at 10°C Thalassiosira hyalina a PSII quantum yield of at and which significantly to at 10°C The yield of energy of PSII under experimental light intensity increased from at to at 10°C The electron transport rate under experimental light intensity was at 100 PSII s−1 across all temperatures, and the light acclimation (Ik) increased from c. 100 μmol photons m−2 s−1 at to c. μmol photons m−2 s−1 at 10°C The of PSII (σLHCII) increased from PSII at to PSII at and the of PSII significantly from μs at to μs at 10°C photosynthetic O2 production rates ( PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ ) rates under experimental light intensity ( PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ at 60 μmol O2 Chl of the acclimation temperature μmol photons m−2 PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ was higher than at experimental light and increased with acclimation temperatures from μmol O2 Chl at to μmol O2 Chl at 10°C experimental light photosynthetic C-fixation rates ( PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ ) at μmol Chl at and 6°C to μmol Chl at 10°C PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ was also higher than under experimental light the general with of μmol Chl at and 6°C and a to μmol Chl at 10°C were c. at and increased to at 10°C under experimental light intensity O2 consumption rates were higher in the dark of acclimation temperature and light to light-enhanced dark respiration experimental light LEDR at and 6°C (c. μmol O2 Chl increased to μmol O2 Chl at 10°C LEDR increased substantially from μmol O2 Chl at to μmol O2 Chl at 10°C R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ at and 6°C (c. 30 μmol O2 Chl and increased to μmol O2 Chl at 10°C CO2 release rates ( R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ ) in the dark were at μmol Chl and to μmol Chl at 6°C and 10°C As a from 0.7 to and 10°C cellular as by growth rates, benefit from the degrees of warming. growth rates at under the lower temperatures and and at the temperature of 10°C In a on the T. hyalina using a higher light intensity μmol photons m−2 the conditions, a temperature with growth rates of was at (Rehder et al., 2024). In the study, a of cell was likely to the light μmol photons m−2 This is by light acclimation indices being higher than the experimental light intensity increased further at higher temperatures, that the with warming. This is in with on and polar phytoplankton species as well as & 2005; & Rehder et al., 2024; Wolf et al., and that light is a general photophysiological in Hence, especially Arctic phytoplankton will likely benefit from a combined increase in temperature and light as it is for the Arctic Ocean (e.g. Constable et al., 2022; Rehder et al., 2024). Photophysiological parameters a lower of the light harvesting under increasing temperatures the in PSII quantum yield the increase in energy of PSII as well as the relative electron transport rates under experimental light conditions a in PSII of gas measurements PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ under all acclimation temperatures which has also in (e.g. & 2005; Rehder et al., 2023). To cells increase their biomass a : and the antenna of indicated by the increased As a absETRs PSII were on a of the acclimation With this mechanism, cells increase light harvesting to compensate for a less thereby maintaining PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ on a PSII under higher temperatures a of reductant into However, cells were to fully this higher in electron transport under the conditions, the of photosynthesis was reductant in the the This that the to adjust the light harvesting under acclimation temperatures is a to maintain physiological homeostasis in photosynthetic light reactions, especially the light The rates of PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ under all temperatures that the production of NADPH and of the PMF during the light reactions This also that the Calvin cycle activity and thus photosynthetic C-fixation ( PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ ) by the temperatures. to a moderate warming cells were to maintain the physiological rates of PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ as well as R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ in light and LEDR and R O 2 $$ {\mathrm{R}}_{{\mathrm{O}}_2} $$ ) at process-specific This is in with a on the diatom (Rehder et al., and that also the Arctic diatom T. hyalina the to physiological homeostasis under moderate warming scenarios. Since the electron flow of the photosynthetic light reactions typically a relative excess of NADPH over ATP with to the of the Calvin cycle (Allen et al., 2005; Curien et al., 2016; Lepetit et al., 2022), cells require AEFs to modulate the plastidial ATP : NADPH Our gas measurements the of at least O2 consuming AEF to dissipate as by a higher O2 consumption rate in the light to the dark which is in line with (Falkowski et al., et al., et al., Lepetit et al., 2022). a O2 consumption can from different reductant consuming with the photosynthetic electron such as 2004; et al., et al., the Mehler reaction Asada, 2000; Curien et al., as well as a of reductant from the chloroplasts to the et al., 2015; Lepetit et al., 2022; et al., 2024). The of such a metabolic coupling is well in and et al., & et al., 2004; Rehder et al., 2023). In this mechanism, excess reductant is from chloroplasts in the for example, and to the cytoplasm and the & et al., After the reductant has transferred to in the it the respiratory ETC, where it is ultimately transferred to O2 at or the alternative oxidase & The of reductant a in the mitochondrial and as a the citric acid cycle that during is under elevated temperature has for the diatom (Rehder et al., and is also by the from 0.7 to with warming as well as the LEDR. are to to an of in a mode, in order to reductant for respiratory ATP production & The low in T. hyalina can be is that is from the citric acid cycle, a for the metabolic coupling being the to dissipate especially under warming. warming, increased of carbon has & 2023). A of into the thylakoid under these conditions generated during the photosynthetic light the PMF (Rokitta et al., 2022). cells to maintain PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ and PMF generation in the a enhanced activity result in a lower ATP : NADPH and thus a higher excess of NADPH. Therefore, it can be that under warming NADPH has to be rerouted to the respiratory ETC, the of the citric acid cycle under 6°C and as by the R CO 2 $$ {\mathrm{R}}_{{\mathrm{CO}}_2} $$ and These findings are that reductant and of mitochondrial activity are key in the physiological responses of to temperatures. the ratio of PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ to PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ stable under moderate warming, by a stable of c. under warming T. hyalina experienced a PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ despite PS O 2 $$ {\mathrm{PS}}_{{\mathrm{O}}_2} $$ resulting in an of photosynthetic processes, by a of In the of cell and photosynthetic at which is in line with Rehder et al. The of PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ either from detrimental temperature effects on of the Calvin cycle et al., or from further increased consumption of the PMF by a under these conditions, as by & Young these temperature responses from enzyme or increased PMF consumption be in of the for the PS CO 2 $$ {\mathrm{PS}}_{{\mathrm{CO}}_2} $$ it is in line with the general that processes involved in the photosynthetic light reactions are less temperature sensitive than those in the Calvin cycle & Geider, and has for different in to warming (Baker et al., 2016; Rehder et al., 2024). a of the Calvin cycle and also PMF consumption by the to the of the photosynthetic under warming, which was also in increased and LEDR the this of LEDR was likely to a overreduction of the chloroplast This that the metabolic coupling of chloroplasts and under that it is also an regulatory under This physiological under increasing temperatures, and the by the Arctic diatom T. Cells increased light harvesting to compensate for the photosynthetic of PSII and the light under elevated temperatures. to maintain O2 production and thus likely PMF, of the temperature treatment. The increased LEDR under warming that cells increased the photosynthetic ATP : NADPH ratio by plastidial reductant into the mitochondria. This to fuel mitochondrial ATP from the chloroplast the citric acid cycle that is the respiratory CO2 As a the net retention can be over a range of experienced temperatures, despite The responses of O2 and CO2 fluxes can thus result in diverging and temperature This the to O2 and CO2 fluxes To further the metabolic coupling and involved transport measurements of as well as and Furthermore, this metabolic coupling also in phytoplankton the described for the surprisingly of Arctic phytoplankton to with higher temperatures and explain the of biomass accumulation under moderate warming. We for support with MIMS measurements and for support with FRRf measurements as well as the We also for support in measurements of was by for and was and by All the study, performed the the all and the with of and The that support the findings are in the and respiratory at in light intensity of 30 μmol photons m−2 s−1 at different acclimation temperatures (2°C, 6°C and 10°C) in Thalassiosira Photophysiological parameters obtained from a fluorescence measurements at different acclimation temperatures (2°C, 6°C and 10°C) in Thalassiosira is for the or of any by the than be to the The is for the or of any by the than be to the for the The with to in and in any