Temperature sensitivity of carbon concentrating mechanisms in the diatom Phaeodactylum tricornutum

Marine diatoms are key primary producers across diverse habitats in the global ocean. Diatoms rely on a biophysical carbon concentrating mechanism (CCM) to supply high concentrations of CO2 around their carboxylating enzyme, RuBisCO. The necessity and energetic cost of the CCM are likely to be highly sensitive to temperature, as temperature impacts CO2 concentration, diffusivity, and the kinetics of CCM components. Here, we used membrane inlet mass spectrometry (MIMS) and modeling to capture temperature regulation of the CCM in the diatom Phaeodactylum tricornutum (Pt). We found that enhanced carbon fixation rates by Pt at elevated temperatures were accompanied by increased CCM activity capable of maintaining RuBisCO close to CO2 saturation but that the mechanism varied. At 10 and 18 °C, diffusion of CO2 into the cell, driven by Pt’s ‘chloroplast pump’ was the major inorganic carbon source. However, at 18 °C, upregulation of the chloroplast pump enhanced (while retaining the proportion of) both diffusive CO2 and active HCO3− uptake into the cytosol, and significantly increased chloroplast HCO3− concentrations. In contrast, at 25 °C, compared to 18 °C, the chloroplast pump had only a slight increase in activity. While diffusive uptake of CO2 into the cell remained constant, active HCO3− uptake across the cell membrane increased resulting in Pt depending equally on both CO2 and HCO3− as inorganic carbon sources. Despite changes in the CCM, the overall rate of active carbon transport remained double that of carbon fixation across all temperatures tested. The implication of the energetic cost of the Pt CCM in response to increasing temperatures was discussed. Supplementary Information The online version contains supplementary material available at 10.1007/s11120-023-01004-2.


Introduction
Diatoms are responsible for 40% of marine carbon fixation (Field et al. 1998) and are found across all latitudes of the ocean. Increasing anthropogenic inputs of CO 2 into the atmosphere is driving both ocean warming and an increase in the ocean's absorption of anthropogenic CO 2 leading to decreasing oceanic pH (ocean acidification) and changes in oceanic carbon chemistry with increasing concentrations of dissolved CO 2 and HCO 3 − (Zeebe 2012;Cheng et al. 2022). While different marine phytoplankton species have differing responses to the elevation of CO 2 (Dutkiewicz et al. 2015), diatoms' ability to fix carbon appears largely insensitive to ocean acidification due to their highly effective carbon concentrating mechanisms (CCMs) that can utilize the much larger pool of HCO 3 − available in seawater. Most diatoms studied to date possess a biophysical CCM that uses a compartment (pyrenoid) capable of elevating CO 2 around the CO 2 fixing enzyme, RuBisCO (Roberts et al. 2007;Hopkinson et al. 2016). Some diatoms may also possess a biochemical CCM (e.g., a C4 pathway) but this is still open for debate (Reinfelder et al. 2000;Morel et al. 2002;Roberts et al. 2007;Clement et al. 2016) and is not the focus of this study. While pyrenoids are common in diatoms, the mechanism by which the CCM elevates CO 2 around RuBisCO varies among species. Broadly, there appears to be two types of CCM, (1) those where inorganic carbon (C i ) uptake is dependent on an extracellular carbonic anhydrase (eCA) and (2) those lacking an eCA and instead rely on HCO 3 − transporters on the cell membrane for C i uptake (Tsuji et al. 2017a).
While algal CCMs are highly effective, they are also energy intensive (Raven 2010;Raven et al. 2014). CCMs elevate pyrenoid CO 2 concentrations (needed to nearly saturate RuBisCO) to levels that far exceed CO 2 concentrations in seawater, creating large concentration gradients, which encourages diffusive CO 2 efflux across highly permeable membranes. Manipulation of internal CO 2 pools and active transport of HCO 3 − across membranes requires energy input. Numerous studies have shown that increased extracellular CO 2 or changes in extracellular or intracellular pH can reduce the energetic requirement of the CCM (Hopkinson 2014;Mangan et al. 2016), however, there has been mixed evidence in diatoms on whether this can result in enhanced carbon fixation rates (Gao et al. 2014). Optimal CCM function requires synergistically balancing different CCM components, which facilitate HCO 3 − and CO 2 influx into the cell, recycling of leaked CO 2 and the maintenance of high CO 2 concentrations inside the pyrenoid.
Temperature plays a large but relatively understudied role on the CCM. As temperatures increase, the solubility of CO 2 and O 2 in seawater is reduced, along with a decrease in RuBisCO substrate affinity and CO 2 /O 2 specificity (Galmés et al. 2016). However, membrane permeability, rates of diffusion and biochemical reaction rates all increase with rising temperatures, until an optimum is reached (Beardall et al. 2009;Li and Young 2022). It is likely that the temperature sensitivity of these different CCM components vary, leading to imbalanced C i fluxes with fluctuating temperature. Therefore, diatoms need to regulate their CCM components to maintain balanced C i fluxes to support high photosynthetic efficiency. There has been extensive research on the response of C4 plants (biochemical CCM) to varying temperature (Sage and Kubien 2007) but little is known about CCM response to temperature in the marine environment. While temperature fluctuations may not be as large in marine compared to terrestrial environments, marine heatwaves can be 5-6 °C above average temperatures and are devastating for marine ecosystems (Frölicher and Laufkötter 2018). It is likely these marine heatwaves are becoming more common, particularly in coastal regions (Lima and Wethey 2012;Hughes et al. 2018).
Phaeodactylum tricornutum (Pt) is a model coastal marine diatom with a well-established CCM model at 20 °C (Hopkinson et al. 2011;Hopkinson 2014). While Pt is of low abundance in the open ocean, it is widely distributed globally from sub-polar to tropical environments and able to grow between 5 and 28 °C in the lab (Kudo et al. 2000;De Martino et al. 2011;Rastogi et al. 2020). Here, we acclimated the model diatom Pt to three different temperatures over its growth range and measured CCM activity using membrane inlet mass spectrometry (MIMS). Experimental data were fit within the Pt CCM model, based on Fragilariopsis cylindrus (Fcyl) CCM (Li and Young 2022) adapted from the "chloroplast pump" model (Hopkinson 2014), to explore how the Pt CCM functions across temperatures.

Cell culture conditions and growth rate
Phaeodactylum tricornutum CCMP632 (Pt) cells were cultured in buffered (25 mM HEPES, pH 8.10) f/2 media with salinity at 35‰. The pH of the culture media was adjusted to 8.10 for each growth temperature, i.e., 10 °C, 18 °C and 25 °C. Light intensity was set at ~ 120 µmol m −2 s −1 with 16 h:8 h, light:dark cycle. Cell cultures were subcultured weekly and acclimated at each temperature for at least two weeks. Initial inoculum was 1 × 10 4 cells ml −1 (or 1 × 10 5 cells ml −1 at 10 °C) at day zero and cells were harvested during exponential growth phase, at cell densities between 5 × 10 5 and 1 × 10 6 cells ml −1 on the day of each experiment. Growth rates were calculated as described in Li and Young (2022). Beckman Coulter Z2 cell counter was used to determine cell density and size, assuming a spherical cell shape for diameter calculation. The nonspherical shape of Pt cells, which determines the cell surface area, does not impact the estimation of volume-based mass transfer coefficients.

Membrane inlet mass spectrometry
Membrane Inlet Mass Spectrometry (MIMS), including gas concentration calibrations and data analyses are as described in Li and Young (2022) for Fcyl. Photosynthesis and C i usage measurements with Pt cells were carried out in a similar fashion though at different temperatures (10, 18 and 25 °C) with the presence of the eCA inhibitor acetazolamide (AZ) as detailed in previous studies (Hopkinson et al. 2011;Hopkinson 2014;Li and Young 2022). Like our Fcyl experiments, 18 O, 13 C-labeled NaHCO 3 (final concentration ~ 2 mM) was used to determine HCO 3 − dehydration rate, CO 2 /HCO 3 − mass transfer coefficient (f c , f b ) and CA activities at different temperatures (Li and Young 2022). Pt cells were concentrated before loading into the MIMS chamber with final average densities of 3 ~ 4 × 10 6 cells ml −1 .

Photosynthetic rate and C i uptake versus DIC concentration
Additional experiments to investigate the apparent kinetic properties of Pt C i uptake were conducted by measuring rates of O 2 evolution under varying dissolved inorganic carbon concentrations ([DIC]). Similar to the previous study of Hopkinson (2014), stepwise increases in [DIC] were created by loading 13 C-NaHCO 3 into the MIMS chamber in the presence of 25 mM HEPES buffered (pH 8.1) artificial seawater. For each different [DIC], Pt cells were treated to a dark-light-dark cycle until quasi-steady state was reached (see Fig. S1, e.g.). The CO 2 and O 2 signals were used for calculating photosynthetic rate and Ci uptake according to earlier studies (Badger et al. 1994;Hopkinson et al. 2011;Li and Young 2022). The cytosolic CO 2 concentration was estimated using following equation: where [CO 2 ] cyt and [CO 2 ] bulk are the cytosolic and bulk CO 2 concentrations respectively; U CO2 is the CO 2 uptake rate and f c is the mass transfer coefficient of CO 2 . The rate of photosynthesis (O 2 evolution) as a function of [CO 2 ] was plotted and fit to the Michaelis-Menten equation.

RuBisCO kinetics and quantification
In vitro RuBisCO kinetics at different temperatures (6, 12, 18, 25 °C) were measured according to previous studies (Sharwood et al. 2008;Young et al. 2016). Briefly, RuBisCO active sites were quantified using [ 14 C] 2-CABP binding assay as described by Sharwood et al. (2008). For the ribulose-P 2 -dependent 14 CO 2 -fixation assays, crude extracts of soluble diatom protein were incubated with 15 mM NaH 14 CO 3 and 15 mM MgCl 2 at the relevant temperature for 10-15 min to activate RuBisCO. This extract was added to 7 ml septum-capped scintillation vials containing reaction buffer (0.5 ml of 100 mM EPPS-NaOH, pH 8, 15 mM MgCl 2 , 0.6 mM ribulose-P 2 , 0.1 mg ml −1 CA) equilibrated with 21% (v/v) O 2 in N 2 and five differing concentrations of 14 CO 2 (between 10 and 115 μM). Values for the half saturation constant for CO 2 in air (K Cair ) and maximal carboxylase activity in air (V Cmax ) were extrapolated from the data using the Michaelis-Menten equation as described previously (Sharwood et al. 2008;Whitney et al. 2011). The carboxylation turnover rate of RuBisCO (k catC ) was calculated by dividing V Cmax by the number of RuBisCO active sites as determined in [ 14 C] 2-CABP-binding assay.
Temperature dependence of k catC and K Cair was described by fitting following equation as described by Arcus et al. (2016): The best-fit constants (a, b, c) were used to interpolate k catC and K Cair values at different temperature (T).
Western Blots were used to quantify the relative abundance of RuBisCO in Pt cells cultured at different temperatures, with experimental details described previously (Li and Young 2022). However, Western Blots using commercial anti-RbcL antibody and standards consistently underestimated absolute RuBisCO concentrations in diatoms. Thus, for modeling purposes, average RuBisCO quantities were estimated by assuming Pt RuBisCO reaches 80% saturation rate during photosynthesis at 18 °C, based on previous modeling and field research work (Hopkinson 2014.

Modeling and statistical analyses
We previously adapted the "chloroplast pump" models (Hopkinson 2014;Li and Young 2022) to simulate the C i fluxes of Fcyl. By adjusting the HCO 3 − uptake behavior observed in our Pt data, as well as other physiochemical parameters acquired in this study, we modified the Fcyl CCM model to simulate Pt C i fluxes during steady state photosynthesis at different temperatures. The model was constrained to describe observed CO 2 concentrations, net O 2 evolution rates and C i uptake rates. The Python script with modeling parameter files can be found on GitHub (https:// github. com/ limen gwsu/ Pt_ CCM_T). All descriptive statistical analyses including mean, stand deviation, tukey HSD p values, etc. were calculated using Python packages including pandas, numpy, scipy (Virtanen et al. 2020) and statsmodels (Seabold and Perktold 2010).

Growth rates and photosynthesis with temperature
Pt net photosynthesis increased steadily across all temperatures tested, with an ~ fourfold increase between 10 and 25 °C, as measured by the rate of O 2 evolution in the light (Fig. 1a). Respiration (O 2 consumption in the dark) also increased significantly at 25 °C compared with 10 and 18 °C (p ≤ 0.001, Fig. 1b). The estimated daily net O 2 evolution (calculated as 1/3 dark respiration and 2/3 light O 2 evolution over a 16:8 daylight cycle), increased with temperature (p ≤ 0.001, Fig. 1c). However, the specific growth rate (d −1 ) of Pt only increased between 10 and 18 °C, not between 18 and 25 °C (Fig. 1c). This disparity could be attributed to changing rates of respiration and photosynthesis over a diel cycle that were not captured in our short-term MIMS experiments. In addition, cell size also increased significantly between 18 and 25 °C (p ≤ 0.001), though only by ~ 10% in volume, and may partly explain the difference observed between daily net photosynthesis and growth rates (Figs. 1c, S2).

RuBisCO kinetics and quantity
Faster growth and photosynthetic rates at higher temperatures could be attributed to the temperature-driven increase in RuBisCO catalytic activity (k catC ). From 10 to 25 °C, RuBisCO k catC increased from 0.9 to 3.3 s −1 , an almost four-fold increase between 10 and 25 °C, similar in magnitude to the increase in photosynthetic oxygen evolution (Fig. 2a). Between 10 and 25 °C, there is also a 2.5-fold increase in K Cair from 22 to 55 µM (Fig. 2a), thus at higher temperatures RuBisCO needs a higher CO 2 concentration for similar saturation levels. As K Cair was less temperature sensitive than k catC , carboxylation efficiency (k catC /K Cair ) increases with temperature.
There was little change in RuBisCO abundance per cell across temperatures in Pt (Figs. 2b, S3a). The Western Blot method tracks changes in relative abundance but appears to underestimate diatom RbcL quantity in Pt, which was also observed in our previous work on Fcyl (Li and Young 2022). Our estimation of average gross CO 2 fixation rates at RuBisCO (based on estimated gross O 2 evolution rates, Fig. S3b), increased from 0.90 to 3.0 × 10 -17 mol cell −1 s −1 with a temperature increase from 10 to 25 °C. Dividing the gross CO 2 fixation rates by k catC , the results lead to an average minimum RuBisCO quantity at 1 × 10 -17 mol per Pt cell. For modeling purposes and to estimate the relative substrate (CO 2 ) concentration at RuBisCO, 80% saturation was assumed at 18 °C, which resulted in an estimated abundance of 1.24 × 10 -17 mol RuBisCO per Pt cell across different temperatures. Combined with K Cair data, we estimated the average CO 2 concentration around RuBisCO during steady state photosynthesis to be 95, 147, and 151 µM at 10, 18, and 25 °C, respectively.

Inorganic carbon usage
An increase in cellular inorganic carbon (C i ) uptake was required to supply the faster carboxylation rates and maintain higher CO 2 concentrations within the pyrenoid with increasing temperature. Total C i uptake increases at higher temperatures, which was largely driven by an increase in CO 2 uptake from 10 to 18 °C (Fig. 3a) and by an increase in HCO 3 − uptake from 18 to 25 °C (Fig. 3b). This results in the proportional supply of C i in Pt to be ~ 80% CO 2 at the lower temperatures of 10 and 18 °C, but only ~ 50% CO 2 supply at 25 °C (p ≤ 0.006, Fig. 3c).
Using 18 O-exchange method (Hopkinson et al. 2011;Li and Young 2022), we estimated the mass transfer coefficients of CO 2 and HCO 3 − (f c , f b respectively) in Pt at different temperatures. The permeability of CO 2 across the Pt cell membrane (and frustule), measured as f c , steadily increased with temperature (p ≤ 0.006 for each temperature pair, Fig. 4a). The permeability for HCO 3 − , indicated by f b , was three orders of magnitude lower than CO 2 and insensitive to temperature (Fig. 4a). Thus, as noted in previous studies (Hopkinson et al. 2011), the cell membrane can be treated as essentially impermeable for HCO 3 − . The increased f c at higher temperatures may facilitate the influx of CO 2 , where CO 2 uptake rates are faster at higher temperatures than at 10 °C (p ≤ 0.007, Fig. 3b). As discussed by Hopkinson (2014), the cytosolic CA can also facilitate CO 2 uptake. Like f c , the estimated catalytic rate of intracellular CA in Pt was higher at warmer temperatures (p < 0.02 for each temperature pair, Fig. 4b). Fig. 1 Pt net photosynthetic, respiratory and growth rate at different temperature. a. Net oxygen evolutionary rate in the light per cell across different temperatures. b. Oxygen consumption (dark respiratory) rate per cell across different temperatures. c. The correlation between growth rate and averaged daily primary production rate, presented as daily net O 2 evolution rate per cell. All box plots have whiskers extending maximum 1.5 times of IQR from box 1 3

C i uptake rate versus C i concentration
To understand the kinetics of C i uptake by Pt and to model C i fluxes during photosynthesis at different temperatures, we investigated the effect of [DIC] on net photosynthetic rates (Fig. 5a). As expected, lower [DIC] limited net photosynthetic rates across different temperatures. However, when C i uptake was analyzed, the limitation of photosynthesis was not from the limited HCO 3 − uptake under our experimental conditions. HCO 3 − uptake was not responsive to bulk [HCO 3 − ] in the range of 0.08-2 mM, regardless of temperature (Fig. 5b). This indicates that Pt has highly specific HCO 3 − transporters that operate with half saturation constants (K 0.5 ) < < 100 µM (agreeing with earlier research on Pt (Nakajima et al. 2013;Tsuji et al. 2017a;Nawaly et al. 2022). The observed insensitivity of HCO 3 − uptake to bulk [HCO 3 − ] (and [DIC]) suggests an alternative entry of C i into the cytosol (Fig. S4), i.e., through CO 2 diffusion, which causes the limit on photosynthesis at low [DIC].
The diffusion of CO 2 into the cell is driven by the CO 2 concentration gradient between the extracellular environment and the cytosol (Hopkinson 2014). C i from the cytosol is then concentrated within the chloroplast by the "chloroplast pump". The "chloroplast pump" as defined by Hopkinson (2014) intensively "pumps" HCO 3 − from the cytosol to the chloroplast, maintaining low HCO 3 − concentrations within the cytosol. Rapid cytosolic-CA-mediated net flux of CO 2 to HCO 3 − then drives the diffusive influx of CO 2 into the cytosol from outside the cell. With our measured f c , we estimated cytosolic CO 2 concentrations using Eq. 1, which allowed us to investigate the relationship between cytosolic CO 2 concentration and net photosynthetic rate (Fig. S5b). Fitting the net O 2 evolution rate versus extracellular and cytosolic CO 2 concentrations to the Michaelis-Menten curve separately revealed a strong "chloroplast pump", with average K 0.5 ≤ 1 µM for both extracellular and cytosolic CO 2 (Fig. S5). With the equilibrium constant of CO 2 /HCO 3 − interconversion being ~ 100, the K 0.5 for HCO 3 − transport from the cytosol to the chloroplast was estimated to be < 100 µM. With our experimental setup, the temperature impact on the affinity (K 0.5 ) of HCO 3 − /CO 2 at the chloroplast level was not distinguishable.

Modeling the CCM with temperature
Combining the quantitative data of RuBisCO kinetics, CA activities, net O 2 evolution, C i uptake rates and its kinetic properties, etc., we modeled the C i fluxes in Pt during photosynthesis at different temperatures (Fig. 6). At steady state in general, CO 2 diffuses from the bulk environment through the surface layer and cytoplasmic membrane into the cytosol, with additional CO 2 supplied to the cytosol from mitochondrial respiration and leakage from the chloroplast (gray arrows pointing at cytosolic CO 2 , Fig. 6). The concentrations of CO 2 and HCO 3 − in the cytosol favor the net conversion of CO 2 to HCO 3 − by cytosolic CA. The CA supplied HCO 3 − , combined with extracellular HCO 3 − uptake through cytoplasmic membrane, is pumped into the chloroplast by potential transporter(s). This allows the accumulation of high concentrations of HCO 3 − within chloroplast and subsequently in the pyrenoid, where HCO 3 − is converted to CO 2 by pyrenoid CA and fixed by RuBisCO (green arrows, Fig. 6). Due to the concentration gradient between pyrenoid and chloroplast stroma, some proportion of the CO 2 leaks out from the pyrenoid into the chloroplast stroma and subsequently into the cytosol. As discussed by Hopkinson (Hopkinson 2014), the lack of CAs in the chloroplast stroma allows the buildup of HCO 3 − , with a very small fraction converted to CO 2 before reaching the pyrenoid.
As temperature increases, the model was able to capture the experimentally observed fluxes of C i uptake and photosynthetic rates, including faster diffusive CO 2 uptake from the bulk environment to the cytosol at 18 °C compared with 10 °C, and enhanced HCO 3 − uptake into the cytosol at 25 °C  (Figs. 3, 6). The model also enabled visualization of the intracellular C i fluxes that are less accessible through experiments. All intracellular C i fluxes operate at faster rates as temperatures rise (Fig. 6). Notably the net fluxes of cytosolic CA-catalyzed conversion of CO 2 to HCO 3 − , HCO 3 − transport from the cytosol to the chloroplast, and the leakage of CO 2 from the chloroplast to the cytosol are faster at higher temperatures. For every mole of HCO 3 − transported into the chloroplast, there are 0.37, 0.45, and 0.43 mol of CO 2 leaking out of the chloroplast at 10, 18, and 25 °C, respectively. In other words, for every carbon fixed by RuBisCO, there are 1.6, 1.8 and 1.8 HCO 3 − molecules transported into the chloroplast at 10, 18, and 25 °C, respectively. When HCO 3 − uptake from the surface/bulk environment into the cytosol is included (i.e., total active HCO 3 − transport), the ratio of active HCO 3 − transport to carboxylation rate at RuBisCO is approximately 2:1 for all three temperatures. The HCO 3 − concentrations within the chloroplast are ~ 20 times of that within cytosol at 10 °C, and ~ 40 times at 18 and 25 °C. This is the result of cytosolic HCO 3 − concentrations remaining ~ 0.7 mM across all temperatures, whereas the chloroplast HCO 3 − concentration increased from 15 mM at 10 °C to ~ 30 mM at 18 and 25 °C.

Discussion
The CCM of Pt is essential to supply elevated CO 2 concentrations around RuBisCO (Hopkinson et al. 2011;Hopkinson 2014). While there has been much focus on the response of diatom carbon fixation to ocean acidification (Wu et al. 2014;Shi et al. 2019;Wada et al. 2021), less is known about how marine heatwaves and rising sea surface temperatures (SST) may impact the diatom CCM and ultimately the ability of diatoms to sequester carbon.

CCM activity increases with temperature
Our experimental conditions of nutrient replete, saturating light resulted in increasing carbon fixation rates in Pt as temperatures increased. Concomitantly, there was also an increase in CCM activity to support the increased demand of CO 2 for fixation, overcoming the challenges at higher temperatures of lower CO 2 solubility in the bulk medium, higher half saturation constants for CO 2 by RuBisCO and lower CO 2 /O 2 specificity of RuBisCO (Galmés et al. 2016). Increased activity of the CCM occurred due to an overall increase in catalytic rates of CAs and HCO 3 − transporters, as well as enhanced membrane permeability to CO 2 and increased rates of CO 2 diffusion. As temperatures increased, we observed a shift towards a larger proportion of HCO 3 − to CO 2 uptake from the bulk media, and a slight lowering of RuBisCO saturation state (from 81 to 73%). Modeling of the CCM revealed that Pt initially increased CO 2 supply to RuBisCO by enhancing its "chloroplast pump" (from 10 to 18 °C). This involved increasing HCO 3 − transport across the chloroplast membranes. The rapid removal of inorganic carbon from the cytosol was quickly replaced by predominantly diffusive entry of CO 2 but also active HCO 3 − uptake into the cell. The ratio of cellular CO 2 : HCO 3 − uptake did not change between 10 and 18 °C (Fig. 3c). However, between 18 and 25 °C it appeared that the "chloroplast pump" cannot Uptake rate of CO 2 (U CO2 ) and HCO 3 − (U HCO3-) per cell basis. c. Fraction of CO 2 supply normalized to O 2 evolution be further enhanced, either unable to elevate chloroplast HCO 3 − concentrations above 30 mM or unable to maintain a chloroplast-to-cytosol HCO 3 − gradient greater than ~ 40 fold. Once this limit had been reached, the increased demand for inorganic carbon was supplied by enhancing the uptake of HCO 3 − from the bulk media. A comparable intracellular HCO 3 − concentration ~ 30 mM has been estimated in cyanobacteria previously (Woodger et al. 2005;Mangan and Brenner 2014).

Temperature effects on kinetics or molecular regulation on CCM components?
As temperature increases, catalytic activity, diffusion rates, and membrane fluidity also increase until an optimum is reached. Observed increased rates of CA activity, HCO 3 − transport, and CO 2 carboxylation at RuBisCO are of magnitudes that could be explained by the direct impact of temperature (for enzymes assuming a Q 10 around 2) without changes in enzyme abundance. Likewise, the differences in the magnitudes of various C i fluxes (Fig. 6b, c) between 10 and 18 °C could also be entirely due to different temperature sensitivities of CCM components. However, at 25 °C the shifted ratio of CO 2 to HCO 3 − uptake fluxes (Fig. 6a) and the upheld saturation state of RuBisCO suggest that there is a temperature-driven physiological and molecular regulation of CCM components. Further investigations (e.g., transcriptional studies) beyond our current model are needed to test whether Pt regulates its CCM through gene expression in response to changing temperatures. Previous studies have demonstrated that CCM components (e.g., CA, HCO 3 − transporters) are transcriptionally regulated via CO 2 availability (Harada et al. 2006;Ohno et al. 2012;Nakajima et al. 2013;Hennon et al. 2015) indicating that the CCM is dynamically able to respond to changing environmental conditions.

Temperature impacts on the energy cost of the Pt CCM
The energetic cost of the CCM is thought to be primarily driven by the active uptake of HCO 3 − across membranes and to be strongly correlated with the amount of CO 2 leaked from the pyrenoid. While CO 2 leaks from the pyrenoid and chloroplast in the Pt CCM model, it is recycled in the cytosol by CA into HCO 3 − . This "chloroplast pump" model in Pt (Hopkinson et al. 2011;Hopkinson 2014) revealed the dependence on active pumping of HCO 3 − from the cytosol into the chloroplast to maintain a low cytosolic CO 2 concentration. This low cytosolic CO 2 concentration drives the net influx of CO 2 from the bulk environment into the cytosol, along with rapid recycling of CO 2 respired from the mitochondria or leaked from the chloroplast. Below we discuss the energy costs of this model in the context of temperature but acknowledge that other diatoms have different CCM strategies such as relying on external CA for extracellular HCO 3 uptake while CO 2 could leak from the cell (Tsuji et al. 2017a;Li and Young 2022).

Fig. 4 Temperature impact on
Pt inorganic carbon mass transfer coefficient (a) and intracellular CA catalytic rate (b) Fig. 5 The impact of DIC (a) and HCO 3 − (b) concentration on net O 2 evolution rate and uptake rate of HCO 3 − respectively. Data points were pooled from 4, 3, 5 independent cultures for temperature at 10, 18, 25 °C respectively Active transport of HCO 3 − across the cytoplasmic membrane in Pt is done by the Na + -dependent SLC4-type transporters while the "chloroplast pump" HCO 3 − transporters are yet to be identified (Nakajima et al. 2013;Tsuji et al. 2017b;Nawaly et al. 2022). Without direct measurements, HCO 3 − transport across the plasmalemma or chloroplast envelope membranes was estimated to require 0.5 or 1 ATP for each HCO 3 − transported (Hopkinson et al. 2011;Raven et al. 2014). Considering the HCO 3 − concentration gradient between the chloroplast and cytosol was ~ 40 fold at maximum (Fig. 6), and factoring in the ~ 20 mV chloroplast envelope membrane potential under light (Demmig and Gimmler 1983;Pottosin and Dobrovinskaya 2015), the energy input required would be ~ 11 kJ/mol HCO 3 − transported from the cytosol to the chloroplast stroma, which is considerably lower than the −51 kJ/mol for the hydrolysis of ATP (estimated from chloroplast ATP/ADP concentrations (Heineke et al. 1991)). This indicates that a relatively energy efficient "chloroplast pump" is possible, so the estimate of 0.5 ATP for each HCO 3 − transported can hold. In contrast to HCO 3 − transport into the chloroplast, the cytoplasmic uptake of HCO 3 − has a favorable concentration gradient, i.e., the cytosolic HCO 3 − concentration is lower than the bulk environment. This favorable concentration gradient, together with the unfavorable cytoplasmic membrane potential −84 mV (resting potential, oscillating up to −40 mV) measured from the diatom Odontella sinensis (Taylor 2009), points to an estimated energy requirement of ~ 5.5 kJ/mol for HCO 3 − transport across the cytoplasmic membrane into the cytosol. Thus, HCO 3 − transporters (such as the SLC4-type) on the cytoplasmic membrane can operate efficiently using 0.5 or less ATP per HCO 3 − uptake into the cytosol. The ratio of total active HCO 3 − transport to CO 2 fixed at RuBisCO remained at 2:1 across all temperatures (Fig. 6) indicating that the energy cost of the Pt CCM per CO 2 fixed does not change with temperature unless the energetic costs for chloroplast and cytoplasmic HCO 3 − uptake are different. Nevertheless, higher photosynthetic rates at higher temperatures (under nutrient replete conditions) will need a higher total energy input, requiring higher saturating light intensities.
In addition to the energetic cost of HCO 3 − transport, energy costs and resource allocation of the CCM must also take into consideration the synthesis and maintenance of the CCM biophysical components (Raven et al. 2014). To compensate for slow catalytic rates at cold temperatures, many CCM components and RuBisCO are likely increased in abundance Li and Young 2022), this would require a larger investment in resources, such as nitrogen. While the abundance of other CCM components were not directly measured in this study, RuBisCO abundance did not change with temperature. Fig. 6 CCM models of Pt carbon (Ci) fluxes during photosynthesis at different temperatures. Gray, black, blue, and green arrows along with net flux rates (unit in 10 -18 mol cell −1 s −1 ) represent diffusive, interconversion, active uptake, and fixation of inorganic carbon species, respectively. Arrow widths are drawn proportional to flux rates. The concentrations of CO 2 and HCO 3 − are labeled in each compartment with units. The "Surface" compartment (Hopkinson et al. 2013) denotes the cellular layer outside the cytoplasmic membrane, including periplasmic space and frustule

Combined effects of increasing SST and ocean acidification
Climate change will lead to both increasing SST and lowering of ocean pH. While warming SST leads to lower solubility of CO 2 in seawater with a given pH, increasing atmospheric CO 2 leads to elevating dissolved CO 2 and HCO 3 − in the ocean, decreasing the oceanic pH (Zeebe 2012;Cheng et al. 2022). We hypothesized that the effect of rising temperatures would put additional pressure on the CCM, negating any relaxation of the CCM due to increasing CO 2 concentrations. Our study showed that Pt increases CCM activity with increasing temperature, maintaining RuBisCO saturation above 70%. However, the increase in CCM activity is in pace with faster carboxylation rates, so that the estimated cost of the CCM does not increase per carbon fixed. Our study was conducted under nutrient replete conditions over a temperature range that led to increased carbon fixation rates with temperature. Nutrient limitation is predicted to become more prevalent in conjunction with warming SST due to enhanced stratification and could counter any temperature-driven increases in growth rate (Marinov et al. 2010). How the CCM functions under higher temperatures when nutrient limitation restricts carbon fixation rates has yet to be elucidated though there is evidence of either the Calvin cycle or CCM being downregulated in phytoplankton under phosphate limitation (Beardall et al. 2005;Brembu et al. 2017). Our study also highlighted the dependence of HCO 3 − transport in Pt's "chloroplast pump" and an upper limit of either chloroplast HCO 3 − concentrations or chloroplast-to-cytosol gradients. At this limit (above 18 °C in this study), the CCM is no longer sensitive to CO 2 availability and ocean acidification would not affect carbon fixation rates under these experimental conditions.