Cross‐compartment metabolic coupling enables flexible photoprotective mechanisms in the diatom Phaeodactylum tricornutum

Summary Photoacclimation consists of short‐ and long‐term strategies used by photosynthetic organisms to adapt to dynamic light environments. Observable photophysiology changes resulting from these strategies have been used in coarse‐grained models to predict light‐dependent growth and photosynthetic rates. However, the contribution of the broader metabolic network, relevant to species‐specific strategies and fitness, is not accounted for in these simple models. We incorporated photophysiology experimental data with genome‐scale modeling to characterize organism‐level, light‐dependent metabolic changes in the model diatom Phaeodactylum tricornutum. Oxygen evolution and photon absorption rates were combined with condition‐specific biomass compositions to predict metabolic pathway usage for cells acclimated to four different light intensities. Photorespiration, an ornithine‐glutamine shunt, and branched‐chain amino acid metabolism were hypothesized as the primary intercompartment reductant shuttles for mediating excess light energy dissipation. Additionally, simulations suggested that carbon shunted through photorespiration is recycled back to the chloroplast as pyruvate, a mechanism distinct from known strategies in photosynthetic organisms. Our results suggest a flexible metabolic network in P. tricornutum that tunes intercompartment metabolism to optimize energy transport between the organelles, consuming excess energy as needed. Characterization of these intercompartment reductant shuttles broadens our understanding of energy partitioning strategies in this clade of ecologically important primary producers.

a white LED and the photon flux was determined with a LI-COR LI-250A light probe. Cultures were sampled in early-mid exponential phase, chlorophyll content determined, and imaged using a Leica TCS SP8 confocal microscope. Chlorophyll 61 a concentration was determined by pelleting cells by centrifugation and extracting pigments with 90% MeOH/10% DMSO. 62 Chlorophyll concentration was determined using the coefficients as reported in (6). For imaging,chlorophyll autofluorescence of 63 representative cells was detected at 640-750nm following laser excitation at 552nm. 64 Modeling the chloroplast as two ellipsoids, we derived an equation (1) that allows for the approximation of chloroplast 65 volume based on a single 2D image of chlorophyll auto-fluorescence, assuming the plane of the image approximately bisects the 66 cell.
V lobe is the total volume of a chloroplast lobe, of which there are two in P. tricorutum. L is the semi-major axis of the lobe and 68 W is the semi-minor axis. Consistent with the existing literature, we assumed the cell was a prolate sphere (7) (Table S8).

87
Methods S3: Photophysiology constraints. The biophysical constraints were based on an extension of our previous modeling 88 of photoautotrophy in cyanobacteria (10). Briefly, using the spectral distribution of photon flux for the given light source at 89 the experimental irradiance (E0(λ)), the chlorophyll a specific spectral absorption coefficient (a * λ ), and the biomass fraction of 90 chlorophyll a, the photon uptake flux (Ea) was determined using the following equation: In vivo whole cell absorption was determined at 1 nm intervals using a dual beam spectrophotometer equipped with an 92 integrating sphere. The chlorophyll-specific absorption coefficient was estimated using Equation (3), in which a * ph (λ) is the 93 chlorophyll a specific absorption coefficient, and a ph (λ) is the absorption coefficient. Both parameters are spectrally distributed.
To fully capture the wavelength specific light-pigment interactions, we switched from photosynthetically available radiance 95 (PAR) to quantum flux (QF) (11), which describes the total absorbed photon flux, as the fundamental variable the oxygen 96 evolution constraint: where E (λ) is the fraction of total PAR at a given wavelength λ. The measured photosynthetic rates (oxygen evolution in 98 this study) were then fitted to a Platt (5) equation for photosynthesis prediction (P), using quantum flux as the independent 99 variable.
Pmax is the maximum photosynthetic rate, α and β are the parameters that describe the initial slope of the curve, and the 101 photoinhibition (if present), respectively.

102
Methods S4: Photoautotrophic simulations of cellular growth. The P. tricornutum genome-scale model (GEM), iLB1025 (12), experimental values. From this the percentages used in the dynamic biomass accumulation were determined (Table S2).

126
For the sinusoidal light condition, the model was simulated under a 12 h : 12 h light : dark cycle, f/2 media, with 500 mL 127 total culture volume and light from a white LED placed above the culture as reported (13)  flux at each wavelength in the range 400-700 nm was determined using the relative spectral irradiance ( well as parameterized the simulation for the subsequent time interval. This process was repeated for the duration of the light interval (T=0 to T=720 minutes) with the output of one interval parameterizing the next.

165
Dark period simulations were carried out in a similar fashion. A new BOF named bof_dark_c was constructed that included 166 all biomass components except Chla since P. tricornutum lacks a light-independent protochlorophyllide oxidoreductase (17). 167 We assumed the biomass ratios at T=0 were the target ratios for cell division. Thus, the biomass component ratios at T=720 168 were parsed and compared with the desired ratios. The bof_dark_c stoichiometric coefficients were constructed to balance the 169 biomass component ratios to the T=0 values. Upon achieving the target ratios, biomass was allowed to accumulate at the 170 target ratios for the remainder of the simulation.  (Table S2).

211
All experiments were performed using a custom designed 2 ml glass cuvette equipped with a carbon stopper. Cells were 212 collected according to Methods S1 and supplemented with 20 mM HEPES buffer pH 7.5 and 5 mM sodium bicarbonate (Jallet       . The pigments fucoxanthin, diatoxanthin, diadinoxanthin and beta-carotene were taken from a culture in this study (Fig. S1). DNA and RNA were taken from the original GEM of P. tricornutum (12). Plastid and membrane lipids were determined from the chlorophyll a content per cell (Fig. 1b) and literature values (9) respectively.  Table S6. Predicted chloroplast resource allocation as a function of photoacclimated PAR. Pigment values were determined experimentally and the resulting Chla content was used to approximate the chloroplast volume (Fig. 1a). This volume was converted to chloroplast lipid content using the data reported in (9) (Table S8)
Average gross oxygen evolution (µmol O2 L -1 s -1 µg Chl a -1 ) Average gross oxygen consumption (µmol O2 L -1 s -1 µg Chl a -1 ) Average net photosynthesis (µmol O2 L -1 s -1 µg Chl a -1 )      The 2 electrons transferred at the glycine cleavage system is divided by two as 2 molecules of glycolate are required to generate the prerequisite 2 glycine molecules. (d) Branched chain amino acid (BCAA) degradation. Acyl-CoA generated by leucine degradation (3 moles acyl-CoA per mole amino acid) and isoleucine degradation (1 mole acyl-CoA per mole amino acid) is not included in the energy relocation capacity. Reaction and metabolite abbreviations are in BiGG format (bigg.ucsd.edu) and correspond to the abbreviations used in the model (Supporting Dataset S1).