Alternative outlets for sustaining photosynthetic electron transport during dark-to-light transitions

Abstract

Environmental stresses dramatically impact the balance between the production of photosynthetically derived energetic electrons and Calvin-Benson-Bassham cycle (CBBC) activity; an imbalance promotes accumulation of reactive oxygen species and causes cell damage. Hence, photosynthetic organisms have developed several strategies to route electrons toward alternative outlets that allow for storage or harmless dissipation of their energy. In this work, we explore the activities of three essential outlets associated with Chlamydomonas reinhardtii photosynthetic electron transport: (i) reduction of O₂ to H₂O through flavodiiron proteins (FLVs) and (ii) plastid terminal oxidases (PTOX) and (iii) the synthesis of starch. Real-time measurements of O₂ exchange have demonstrated that FLVs immediately engage during dark-to-light transitions, allowing electron transport when the CBBC is not fully activated. Under these conditions, we quantified maximal FLV activity and its overall capacity to direct photosynthetic electrons toward O₂ reduction. However, when starch synthesis is compromised, a greater proportion of the electrons is directed toward O₂ reduction through both the FLVs and PTOX, suggesting an important role for starch synthesis in priming/regulating CBBC and electron transport. Moreover, partitioning energized electrons between sustainable (starch; energetic electrons are recaptured) and nonsustainable (H₂O; energetic electrons are not recaptured) outlets is part of the energy management strategy of photosynthetic organisms that allows them to cope with the fluctuating conditions encountered in nature. Finally, unmasking the repertoire and control of such energetic reactions offers new directions for rational redesign and optimization of photosynthesis to satisfy global demands for food and other resources.

Keywords: FLV; PTOX; alternative electron outlets; photosynthesis; starch metabolism.

Fig. 1.

Integration of photosynthetic light reactions and C metabolism. The light reactions include the reaction center/electron transport components PSI and PSII (green), cytochrome b₆f (b₆f) (orange), and ATP synthase (blue), with CBBC (cycle, Upper Right) and starch biosynthesis (pathway, Upper Left) using the products of these reactions. Metabolites: G3P, glyceraldehyde 3-phosphate; F6P, fructose 6-phosphate; G6P, glucose 6-phosphate; G1P, glucose 1-phosphate. C metabolism-related enzymes are highlighted in gold filled rectangles (Rubisco, ribulose bisphosphate carboxylase oxygenase; PGM, phosphoglucomutase; SS, starch synthase). Other enzymes/electron transfer components are PC, plastocyanin (gray-filled circle, involved in transferring electrons from cytochrome b₆f to PSI); PTOX and FLV (blue-filled ovals; involved in reducing O₂); NDA2, NADPH oxidoreductase (gray-filled rectangle; moves electrons into the PQ pool); FDX, ferredoxin; TRX, thioredoxin; FNR, flavin:NADPH reductase (gray-filled circles; moves electrons out of the electron transport chain).

Fig. 2.

Light-induced O₂ evolution and uptake. C6 (A) and sta6 (B) were cultured photoautotrophically, bubbled with 2% CO₂ in air, and maintained in continuous light of 100 μmol photons⋅m⁻²⋅s⁻¹. Gross O₂ production (¹⁶O₂ evolution, green curve) and light-induced uptake of O₂ (¹⁸O₂ uptake, red curve) were monitored for 6 min using MIMS at four different light intensities (50, 150, 300, and 1,000 μmol photons⋅m⁻²⋅s⁻¹, as indicated in the upper right of each graph). Before turning on the light, the cells were maintained for 5 min in the dark (black bars on the top, left of each graph), and for an additional 3 min in the dark following the illumination (black bar, on the top, right of each graph). Gross O₂ uptake and both gross and net (blue curve) O₂ production were calculated according to the formula given in Materials and Methods. Each trace represents an average of at least three biological replicates ± SE.

Fig. 3.

Maximal rates of light-induced O₂ production and uptake. Maximal values for gross and net O₂ production (green and blue, respectively) and gross O₂ uptake (red) for C6 (A) and sta6 (B) are plotted as a function of light intensity. The left-hand y axis gives gross and net O₂ evolution and gross O₂ uptake. The right-hand y axis gives the ratio of maximal gross O₂ uptake relative to maximal gross O₂ production; this value is plotted as a function of light intensity (black line with black squares). For both A and B, the left panel presents the data for the first 3 min of the light phase and the right panel presents the data for the final 3 min of the light phase (i.e., early and late periods during illumination, respectively; see main text for details). Net O₂ evolution was calculated as gross O₂ production – gross O₂ uptake. Each point is an average of at least three biological replicates ± SD.

Fig. 4.

Kinetics of light-induced O₂ production and uptake. (A) MIMS traces of ¹⁶O₂ evolution (gross production, dotted-dashed lines) and ¹⁸O₂ uptake (gross uptake, solid lines) during dark-to-light transition (300 μmol photons⋅m⁻²⋅s⁻¹) for C6 (black) and sta6 (red). Gross O₂ production (¹⁶O₂) data during the first 20 s of the light period was linearly fitted and the initial rates (ΔY/ΔX) calculated (black and red dash-dotted lines). The ¹⁸O₂ uptake curves were fitted to first-order exponential decay curves to determine the rate (τ) at which a peak of ¹⁸O₂ uptake for C6 and sta6 was attained (dashed black and red line for C6 and sta6, respectively). (Inset) Presentation of the MIMS traces for gross O₂ evolution and uptake during the entire light period (SE is only shown for gross O₂ uptake for both sta6 and C6). (B) The ratio of gross ¹⁸O₂ uptake to ¹⁶O₂ production during the light period (300 μmol photons⋅m⁻²⋅s⁻¹). Same conditions as in A. Gray box highlights the region in which the decline in the ratio for C6 and sta6 cells is similar. Each data line represents an average of three biological replicates.

Fig. 5.

Effect of a PTOX on O₂ production and light-induced O₂ uptake. C6 (A) and sta6 (B) were exposed to 300 μmol photons⋅m⁻²⋅s⁻¹ (white bars) in the presence of 2 mM PG and gas exchange was analyzed using MIMS, as in Fig. 3 (see Fig. 3 legend). Comparisons of the first 2 min of ¹⁸O₂ uptake for C6 (C) and sta6 (D) in the presence (dashed line with purple shading) and absence (solid line with red shading) of PG. The time (seconds) to achieve Umax for C6 and sta6 is highlighted by dotted red lines that extend from the x axis to the Umax peak. Each trace represents an average of at least three biological replicates with the shaded area associated with each trace representing the SE. (E) 1-qL values of PG-treated/untreated cells. The cells were PG-treated during the 5-min preincubation in the dark and then exposed to 300 μmol photons⋅m⁻²⋅s⁻¹ for 1 min. n = 3 ± SD * t test, P = 0.01. (F) The 1-qL values of PG-treated/untreated cells exposed to increasing light intensities. Cells were exposed to each intensity for 30 s before raising the intensity. n = 3 ± SD.

Fig. 6.

Conversion of F6P of CBBC to metabolites associated with starch synthesis: Metabolite profiling following exposure of C6 and sta6 cells (black and red bars of bar graphs, respectively) to light for 60 s. The pathway for starch biosynthesis is highlighted by green arrows and the CBBC is given as a cycle (CBBC enzymes in blue). Quantified metabolites are shown in black. The numbers next to each metabolite represent the concentration of the metabolite (nanograms per 10⁷ cells) following a 60-s exposure to light. Each bar graph represents four to six biological replicates ± SE. Metabolites: F6P; G6P; G1P; X5P; R5P; RuBP; 3PGA, 3-phosphglyceraldehyde; FBP, fructose 1,6- bisphosphate. Enzymes: PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; Trans., transaldolase; RPE, ribulose phosphate 3-epimerase.

Fig. 7.

O₂ Uptake attributed to FLV proteins. (A) O₂ uptake rate of C6 cells exposed to different light intensities (represented in different colors and indicated on the graph) are plotted during the first 2 min of the light period (300 to 420 s). The rates of baseline O₂ uptake (uptake in the light after the CBBC becomes active), calculated from the data in SI Appendix, Fig. S3, are shown as dashed lines in the color that corresponds to the measurements of total O₂ uptake. (B) Total O₂ uptake at each intensity that cannot be attributed to the basal rate of uptake was calculated by integrating the area under the curves at the different intensities (with baseline set by the rate of basal uptake at that intensity). (C) Rates of O₂ uptake attributed to the FLV proteins at the different light intensities. These values were calculated by subtracting the rate of O₂ uptake attributed to basal uptake from Umax depicted in the curve in A. For all panels n = 3 ± SE.