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

Abstract

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.

Keywords: Phaeodactylum tricornutum; analysis; diatom; energy metabolism; flux balance; genome-scale modeling; photorespiration.

Figure 1

Cellular composition changes as a function of irradiance as well as progression through the light period. (a) Confocal microscopy images of representative Phaeodactylum tricornutum cells acclimated to 15, 150 and 600 μmol photons m⁻² s⁻¹ of photosynthetically active radiation (PAR) (in the figure, μE = μmol photons). Chl autofluorescence is indicated in red. The scale bar in white is 5 μm. (b) Correlation between calculated chloroplast volume and experimental Chla concentration. Vertical error bars are the standard deviation of at least five individual cell measurements per PAR intensity. Horizontal error bars are the standard deviation of three biological replicates at each PAR intensity. (c) Biomass allocation of the major cellular components across the light period, determined in this study, for P. tricornutum cultured under a sinusoidal light regime at full solar irradiance derived from values reported in Jallet et al. (2016) and this study. Values were used to parameterize the simulation of sinusoidal circadian growth.

Figure 2

Model simulations vs experimental values for Phaeodactylum tricornutum cultured under a diurnal sinusoidal light regime. (a) Total organic carbon (TOC) content in the culture biomass. (b) Total nitrogen (TN) content in the culture biomass. (c) Total carbohydrate content in the culture biomass, including structural and storage carbohydrates. (d) Total Chla content in the culture biomass. (e) Total protein content in the culture biomass. (f) Total triacylglycerol (TAG) content in the culture biomass. Light and dark periods are indicated above the plot. Error bars on the experimental values are the standard deviations reported with the published data (Jallet et al., 2016).

Figure 3

Photoacclimation results in efficient photosynthesis across experimental photosynthetically active radiation (PAR) values. (a) Gross photosynthesis vs quantum flux (P vs QF) across the experimental PAR values. Experimental PAR values in units of μmol photon m⁻² s⁻¹ are indicated in the legend. Vertical dashed lines indicate the respective QF at each of the experimental PAR values. The black, open circles indicate the QF and photosynthetic rate used to calculate the experimental quantum requirement. The shaded region indicates the theoretical minimum quantum requirement lower bound of eight photons per oxygen evolved. (b) Expanded view of the initial slope of net P vs QF. Experimental PAR values are indicated in the legend. The shaded region indicates the theoretical minimum quantum requirement lower bound of eight photons per oxygen evolved. A line is displayed representing a minimum quantum requirement of 12 photons per oxygen evolved. (In the figure, μE = μmol photons.)

Figure 4

Biomass allocation dynamics for Phaeodactylum tricornutum acclimated to different light regimes. Fold‐change in Chla (a), protein (b) and fatty acid methyl esters (FAMEs) (c) over the light period for cells acclimated to a sinusoidal light regime at full solar irradiance (circles), and square‐light regimes at low (squares, 60 μmol photons m⁻² s⁻¹) and high (diamonds, 600 μmol photons m⁻² s⁻¹) irradiance. Error bars represent the standard deviation of three replicates for the square‐wave light regime and the standard deviations reported with the sinusoidal data (Jallet et al., 2016). (d) Biomass allocation of the major cellular components across the light period, determined in this study, for P. tricornutum cultured under a square‐wave light regime at a photosynthetically active radiation (PAR) value of 60 μmol photons m⁻² s⁻¹. Values were used to parameterize the genome‐scale model simulations. (e) Biomass allocation of the major cellular components across the light period, determined in this study, for P. tricornutum cultured under a square‐wave light regime at a PAR value of 600 μmol photons m⁻² s⁻¹. Values were used to parameterize the genome‐scale model simulations. (f) Mean transcript abundance, relative to maximum, for genes associated with fatty acid biosynthesis (open circles) and fatty acid degradation (closed squares) from P. tricornutum as reported in Smith et al. (2016). Maximum was T = 0 for biosynthesis genes and T = 17 for beta‐oxidation genes. Error bars are the standard deviation of at least three biological replicates as reported (Smith et al., 2016) of three representative genes for each pathway. FA biosynthesis‐Phatr3_Jdraft1143: 3‐hydroxyacyl‐[acyl‐carrier‐protein] dehydratase, Phatr3_J37367: 3‐oxoacyl‐[acyl‐carrier‐protein] synthase, Phatr3_J10068: enoyl‐[acyl‐carrier‐protein] reductase; FA degradation‐Phatr3_J11014: Acyl‐CoA dehydrogenase, Phatr3_J35240: 3‐hydroxacyl‐CoA dehydrogenase, Phatr3_J55192: Enoyl‐CoA hydratase. (In the figure, μE = μmol photons.)

Figure 5

Model simulations vs experimental values for Phaeodactylum tricornutum cultured under various photoacclimated photosynthetically active radiation (PAR) intensities. (a) Growth rate comparison, based on biomass accumulation, between measured (open markers) and simulated values (closed markers). PAR values are indicated in the legend. (In the figure, μE = μmol photons.) (b) Rate of biomass increase, relative to dawn, for the high‐light (600 μmol photons m⁻² s⁻¹) and low‐light (60 μmol photons m⁻² s⁻¹) cultures. Markers indicate experimental values, and the lines are simulation values. Error bars are the standard deviation of at least three biological replicates.

Figure 6

Model simulations vs experimental values for Phaeodactylum tricornutum biomass components cultured under various photoacclimated photosynthetically active radiation (PAR) intensities. (a) Particulate organic carbon (POC) content comparison between measured and simulated values in mass per culture volume. Error bars are the combined standard deviations of two technical replicates and cell count measurements. (b) Particulate organic nitrogen (PON) content comparison between measured and simulated values in mass per culture volume. Error bars are the combined standard deviations of two technical replicates and cell count measurements. (c) Chla content comparison between measured and simulated values in mass per culture volume. Error bars represent the standard deviation of cell count measurements. Photoacclimated PAR values are indicated by color and marker shape. The dashed line indicates the line of perfect agreement between the experimental and simulated values. The coefficient of determination (deviation from perfect agreement, R ²) is indicated for each biomass component. (In the figure, μE = μmol photons.)

Figure 7

Metabolic reconfiguration at various photoacclimated photosynthetically active radiation (PAR) values. (a) Overview of metabolic coupling between the mitochondria and chloroplast in Phaeodactylum tricornutum. Dashed lines between the compartments indicate transport of metabolites. The dotted line represents a disputed metabolic pathway (see main text). (b–e) Predicted metabolic pathway usage and reaction flux for cultures acclimated to various PAR intensities. Line thickness corresponds to metabolic reaction flux normalized to 100 units of Rubisco carboxylase flux. Color indicates the PAR value according to the figure legend. 2Pglycolate, 2‐phosophoglycolate; 3PG, 3‐phosphoglycerate; α‐KG, alpha‐ketoglutarate; ALA, alanine; BCAA, branched‐chain amino acid; CBB, Calvin–Bensen–Bassham; Cyto, cytochrome; ETC, electron transport chain; G3P, glyceraldehyde‐3‐phosphate; GSH, glutathione (oxidized); GSSG, glutathione (reduced); Ox, oxidation; Q9, Ubiquinone; PYR, pyruvate; R15BP, ribulose‐1,5‐bisphosphate; Red, reduction; TCA, tricarboxylic acid. Reaction abbreviations are in BiGG format (bigg.ucsd.edu) and correspond to the abbreviations used in the model (Supporting Information Dataset S1). A more complete metabolic map and flux data for all model reactions can be found in the Dataset S1. (In the figure, μE = μmol photons.)

Figure 8

Alternate electron flows (AEF) in Phaeodactylum tricornutum as a function of photoacclimated photosynthetically active radiation (PAR) values. (a) Carbon cycling between the chloroplast and mitochondria. Metabolites shuttled to the mitochondria are indicated in the left‐hand shaded region. Carbon recycling metabolites returned the chloroplast are indicated in the right‐hand shaded region. Units are given in mol carbon (100 mol CO ₂)^–1 fixed by Rubisco. Photoacclimated PAR values (μmol photons m⁻² s⁻¹) are indicated by bar color. (b) Mitochondrial consumption of photosynthetically generated electrons. Metabolites that reduce mitochondrial electron carriers are indicated on the left‐hand side of the vertical axis. Reactions that consume mitochondrial reductant are indicated on the right. Values are given in percent photo‐system I linear electron flow (%PSI LEF). PAR values (μmol photons m⁻² s⁻¹) are indicated by the shaded regions. AOX, alternative oxidase; BCAA, branched‐chain amino acid; CYOR, cytochrome c reductase; G3P, glyceraldehyde‐3‐phosphate; GSSG, glutathione reduction; Orn, ornithine; PR, photorespiration; Pyr, pyruvate; TCA, tricarboxylic acid cycle. Amino acids are shown using their three‐letter code. (In the figure, μE = μmol photons.)

Figure 9

Mitochondrial glycolate oxidase participates in photorespiration. Model‐predicted photorespiration reaction flux between the chloroplast and mitochondria in Phaeodactylum tricornutum, and the correlated gene expression levels (reads per kilobase of transcript per million mapped reads) during a diel cycle (Smith et al., 2016). Error bars represent the standard deviation of at least three biological replicates as reported in Smith et al. (2016). Reaction and metabolite abbreviations are in BiGG format (http://bigg.ucsd.edu/) and correspond to the abbreviations used in the model (Dataset S1). 2pglyc, 2‐phosphoglycolate; 3pg, 3‐phosphoglycerate; akg, 2‐oxoglutarate; ala, alanine; co2, carbon dioxide; GCS, glycine cleavage system; GHMT, glycine/serine hydroxymethyl transferase; glu, glutamate; glyclt, glycolate; glx, glyoxylate; GLYTA, glycine/alanine transaminase; gly, glycine; GOX, glycolate oxidase; h2o2, hydrogen peroxide; m, mitochondria localized; mlthf, methylene tetrahydrofolate; nh4, ammonium; o2, oxygen; PGLYCP, 2‐phosphoglycolate phosphatase; pi, inorganic phosphate; pyr, pyruvate; rb15bp, ribulose‐1,5‐bisphosphate; RUBISO, ribulose‐1,5‐bisphosphate oxidase; SAL, serine ammonia lyase; ser, serine; thf, tetrahydrofolate; x, peroxisome localized.