In the present work, DOE coupled to standard least squares multiple regression have been used to model the dependence of different respiratory (CR, MACYT, MAALT) and photosynthetic (P800, ΦPSII800, NPQ800) responses upon the concomitant modulation of light, carbon and inorganic nitrogen sources in the culture medium of C. reinhardtii. This methodology was applied to characterize the extent to which the different environmental factors contribute to bioenergetic plasticity (through a 1st-round of modeling) as well as the mathematical profile of their influence for those accounting for most of the response variability (through a 2nd-round of modeling). Altogether, these analyses provide an overview of the bioenergetic adaptations resulting from global changes in culture conditions. This type of sequential statistical approach, which is commonly undertaken for the optimization of industrial production yields and the design and analysis of “-omics” experiments, had never been used to characterize the bioenergetic plasticity of photosynthetic cells. The individual influence exerted by one or a few environmental factor(s) (maintaining the others constant) on the cellular bioenergetics and metabolism had extensively been studied independently, but little information was available concerning their cumulative effect and their relative contribution to bioenergetic plasticity in a context in which they vary concomitantly in the medium.
The present analyses demonstrate that maximum 3 environmental factors over 5 are sufficient to explain most of the response variability (Table 3) and remarkably evidence squared effects and second-order interactions in some cases (Figure 6; Figure 7). As shown in Figure 4, comparatively to the other responses, lower R2 and R2 adjusted characterize the 2nd-round models obtained for NPQ800 (this difference was also noticed in the 1st-round models; see Table 3). Such discrepancies could (at least partly) be attributable to the higher experimental error inherent to NPQ800 measurements (see in Table 4 and Figure 8b the large standard deviations among the 3 experimental replicates of the random combinations tested for model validation: on average, RSD = 19.7% for NPQ800 and ≤9.0% for the other responses).
In order to check whether the 2nd-round models could also be used to predict the responses associated with any combination of factors within the range of DOE (Table 1), k-fold cross-validation and experimental validation tests on new random combinations have been undertaken. The similarity between the deviation of the validation points (quantified using MAECV/MAEEV and RMSECV/RMSEEV) and the analytical error of the training or 2nd-round models (for cross-validation and experimental validation, respectively) tend to confirm the predictive ability of the 2nd-round models (Table 4). It must nevertheless be emphasized that deviations of 26 to 36% of the response average scale (in terms of MAEF) or 34 to 47% (in terms of RMSEF) corresponding to the analytical error of the 2nd-round models are inherent to the predictions.
In literature, O2 evolution commonly appears to be normalized in terms of chlorophyll concentration. However, in the present analyses, it was rather chosen to use protein concentration because of the high dependence of C. reinhardtii pigment content upon culture conditions, particularly light and acetate [40–42]. Our unconventional normalization strategy could therefore generate apparent discrepancies between previously reported studies and the present results in some cases.
In the following sections, the authors will attempt to propose literature-based hypotheses addressing the possible biological implications of their observations. They insist on emphasizing that these hypotheses must not be considered as firm assertions, but rather aim to provide tracks for future in-depth molecular investigations.
Light stimulates CO2 fixation through the Calvin cycle and provides mitochondrial respiration with oxidizable substrates
The gross photosynthetic O2 evolution and the quantum yield of photosystem II of C. reinhardtii cells adapted to moderate light intensities (0–200 μmolphotons.m−2.s−1) have been measured under 800 μmolphotons.m−2.s−1. Under such a so-called “saturating” intensity, the electron transport rate (ETR) is not limited by light availability but rather by the capacity of downstream metabolic pathways that consume photo-generated reductant and ATP (such as the Calvin cycle). In these conditions, the gross O2 evolution (which is partly mediated by the rate of water photolysis) can primarily be considered as representative of the capacity of these pathways, even if numerous studies indicate that complex photosynthesis-associated O2-consuming processes (particularly PTOX chlororespiration and Mehler reaction) can also importantly contribute in some circumstances to modulate this response in C. reinhardtii .
The present analyses indicate that light intensity exerts a positive linear influence on ΦPSII800 and P800 (Figure 6d and f). Accordingly, the maximal gross O2 evolution was previously reported to be doubled in C. reinhardtii cells grown under illumination of 400 μmolphotons.m−2.s−1 comparatively to a lower illumination of 50 μmolphotons.m−2.s−1 . These observations could (at least partly) be attributable to the well-known stimulation of the expression and activity of Calvin cycle enzymes by light , in good agreement with the higher CO2 fixation rates observed upon increasing illumination in C. reinhardtii . This improvement of CO2 fixation by light was reported to be correlated to higher cellular metabolite content, respiratory O2 consumption and TCA cycle-mediated CO2 production, in line with the linear stimulation of CR, MACYT and MAALT by light which could also be detected here (Figure 6a to c). For MACYT and MAALT, the term “apparent” is used because measurements were carried out on entire cells but not on isolated mitochondria. The availability of respiratory substrates could therefore not be directly controlled, so that the measured maximal activities could have been underestimated comparatively to the actual capacities if the intracellular reductant concentration was insufficient to saturate the mitochondrial electron transport chain in the presence of KCN or salicylhydroxamic acid (SHAM).
In apparent contradiction with these considerations, the present analyses did not retain CO2 concentration as a major explanatory factor of bioenergetic plasticity (Table 3). Such an absence of influence had already been highlighted for the maximal gross O2 evolution in a previous study, in which the sum between the net O2 evolution monitored under 600 μmolphotons.m−2.s−1 and the dark respiration measured before illumination was shown to be similar in low and high CO2-grown C. reinhardtii cells . These observations could be explained by the existence of a low CO2-inducible CCM in C. reinhardtii, by which a high CO2 availability for Rubisco is maintained in low CO2 condition. Several transcriptomic analyses demonstrated that adaptation to different CO2 concentrations mainly occurs through the regulation of the genetic expression of CCM components but not Calvin cycle enzymes in C. reinhardtii [47–49]. Moreover, transferring C. reinhardtii cells from high to low CO2 external concentration was shown to result in a transient decrease of the amount of the small and large Rubisco subunits before returning (within the time period required to induce CCM) to the levels characterizing high CO2-grown cells . Altogether, these different observations and the present ones tend to indicate that adaptation to low CO2 environment in C. reinhardtii principally occurs through CCM induction but not Calvin cycle regulation.
Acetate down-regulates the capacity of the Calvin cycle and promotes its own uptake and storage to counteract the osmotic stress associated with high extracellular acetate concentrations
The present analyses indicate that P800 depends on acetate concentration following a quadratic convex profile with a minimal value for 0.497 g.L−1 (Figure 6f). In a previous study, the net O2 evolution measured under 600 μmolphotons.m−2.s−1 was shown to decrease with acetate concentrations ranging from 0 to 1.75 g.L−1, but rates had been normalized in terms of chlorophyll concentration and cultures conducted under the same saturating light intensity than that of measurements (600 μmolphotons.m−2.s−1) .
In C. reinhardtii and Chlorogonium elongatum (a closely related unicellular green alga), acetate is known to repress the expression of the genes encoding the small and large Rubisco subunits (rbcS and rbcL, respectively), thereby lowering the capacity for CO2 fixation through the Calvin cycle [51–53]. In this context, carbon originating from acetate can substitute for up to half the photoautotrophically-generated biomass content [51,54], and light-driven photosynthetic reactions importantly contribute to provide reductant and ATP for biosynthetic acetate assimilation (as shown in Chlamydomonas mundane) . In heterotrophically-grown C. reinhardtii cells, acetate storage as starch is also known to be promoted through the improvement of the expression and activity of enzymes of the glyoxylate cycle (as isocitrate lyase, ICL) and gluconeogenesis [56,57]. In parallel to its influence on carbon metabolism, acetate inhibits C. reinhardtii heterotrophic growth beyond 0.4 g.L−1 in the medium (“substrate inhibition”) . From this concentration, the osmotic potential reaches a critical value beyond which active transport processes are impaired and energy requirements for cellular maintenance are considerably heightened. Interestingly, for P800, the present analyses point out a “concentration of inflexion” (0.5 g.L−1 approximately) which is very close to the critical substrate inhibition concentration of 0.4 g.L−1 (Figure 6f). This observation tends to indicate that P800 could be influenced by 2 independent acetate-responsive metabolic processes consuming photo-generated reductant and ATP: the Calvin cycle (repressed while increasing acetate concentration due to Rubisco down-regulation) and the biosynthetic assimilation of acetate (stimulated while increasing acetate concentration, especially beyond 0.5 g.L−1, to promote acetate uptake and storage in order to attenuate the osmotic stress).
Acetate stimulates mitochondrial respiration by heightening the intracellular reductant content and the capacity of the cytochrome pathway
The present analyses indicate that acetate concentration exerts a positive linear influence on CR and MACYT (Figure 6a and b). Accordingly, when grown in an acetate-containing medium, C. reinhardtii cells were previously shown to exhibit a twice-enhanced respiratory rate (partly due to the improvement of the intracellular reductant content in mixotrophic condition)  as well as increased transcript levels for diverse components of oxidative phosphorylation, suggesting a higher capacity of the cytochrome pathway . In parallel, MAALT depends on acetate concentration following a quadratic concave profile with an optimum for 0.623 g.L−1. As illustrated in Figure 6c, this response can nevertheless be considered as linearly stimulated up to 0.5 g.L−1 acetate without further increase beyond this concentration. This observation tends to indicate that substrate-saturation of the alternative pathway in the presence of KCN could be reached beyond 0.5 g.L−1 acetate, which would imply that AOX capacity is not responsive to acetate concentration. Accordingly, enhancement of the capacity of the cytochrome pathway was already suggested to contribute to the acetate-induced improvement of dark respiration without concomitant modification of AOX capacity .
Acetate inhibits NPQ through repression of the LHCSR3-dependent qE component
The present analyses demonstrate that acetate concentration exerts a negative linear influence on NPQ800 (Figure 6e), but only in case of high light intensity (Figure 7c). Interestingly, the extent of qE has recently emerged as being dramatically lowered by the presence of acetate in the growth medium, as notably evidenced by Finazzi and co-workers who demonstrated that qT is the major contributor to the global NPQ in mixotrophically-grown C. reinhardtii cells . Even if the molecular mechanisms underlying the functional relationship between NPQ and acetate are not yet understood, the present results tend to confirm these findings and indicate that the magnitude of the inhibitory effect of acetate on qE could depend on its external concentration. Recently, qE has been proposed to be mediated by LHCSR3, a light-harvesting complex orthologue which is only expressed upon high irradiance [63,64]. NPQ plasticity induced in response to changing environmental conditions (such as different acetate concentrations) could therefore be disabled in the dark and low light intensities due to the down-regulation of LHCSR3. These interpretations must be considered with caution due to the impossibility to distinguish the contributions of qE and qT to the global NPQ here.
Mitochondrial respiration contributes to provide nitrate assimilation with reductant through the acetate-dependent activity of AOX
Ammonium concentration is shown here to exert a negative linear influence on CR, MACYT and MAALT (Figure 6a to c). In Selenastrum minutum (another green alga), mitochondrial respiration was proposed to play a role in nitrate assimilation by acting as a trigger factor for the TCA cycle. This would in turn promote the production and export of reductant in the cytoplasm and the chloroplast to support nitrate reduction . In C. reinhardtii, the enzymatic activity and genetic expression of proteins involved in nitrate assimilation are known to be repressed by ammonium [20,21]. These regulatory events are responsible for a strict control of inorganic nitrogen uptake and assimilation by ammonium availability and enable to preferentially exploit this reduced N form if nitrate is also present in the medium . Such a primary control of nitrate assimilation by ammonium could rationalize the present observations with regards to the postulated role of mitochondrial respiration in this metabolic process.
For MAALT, ammonium concentration is the factor which explains the highest proportion of response variability (β = −0.450/p < 0.0001; Figure 6c). Interestingly, the gene encoding AOX (Aox1) is known to be located within a gene cluster which also encodes components of the nitrate assimilatory pathway and is tightly regulated by the nitrogen source ; consequently, AOX expression and capacity were shown to be induced by nitrate and repressed by ammonium in a concentration-dependent manner . With regards to the peculiar genetic localization and regulation of Aox1, the postulated role of mitochondrial respiration in nitrate assimilation was proposed to be essentially mediated by AOX, as also indicated by a recent comparative proteomic study published by our group . The present results are in good agreement with these findings.
Interestingly, a mutual influence could be detected between the individual effects of acetate and ammonium concentrations for CR and MAALT (second-order interactions). As illustrated in Figure 7a and b (left panels), the effect of acetate concentration is attenuated by ammonium. For MAALT, there is also a displacement of the optimal acetate concentration toward smaller values with increasing ammonium concentration (0.727 and 0.514 g.L−1 for 0 and 15 mM ammonium, respectively). These results are consistent with a negative influence of ammonium concentration on AOX capacity. They also tend to confirm that the involvement of mitochondrial respiration in nitrate assimilation is essentially mediated by AOX since no relevant second-order interaction was retained for MACYT. Reciprocally, as illustrated in Figure 7a and b (right panels), ammonium concentration exerts a relevant influence on CR and MAALT only upon high acetate concentration (these responses exhibit a basal ammonium-independent value in the absence of acetate). This observation tends to indicate that acetate assimilation could provide the TCA cycle with oxidizable substrates to support the involvement of AOX in nitrate assimilation.
Nitrate assimilation is retro-inhibited to prevent the deleterious effects of nitrite and ammonium intracellular accumulation
The present analyses demonstrate that nitrate concentration exerts a quadratic concave influence on P800 with an optimum for 10.48 mM (Figure 6f). In C. reinhardtii, photosynthesis is known to contribute to provide nitrate reduction with electrons (together with mitochondrial respiration as stated beyond) [22,23,67], so that the rate of nitrate assimilation can influence P800 in the same way as for the Calvin cycle. The effect of nitrate on P800 can therefore be thought to result (such as for acetate) from 2 distinct metabolic processes of which the relative importance varies with nitrate concentration: substrate stimulation of reductase activity (predominant from 0 to 10 mM) and retro-inhibition of nitrate reduction by nitrate-derived intracellular ammonium (predominant beyond 10 mM). Such a retro-inhibition could attenuate the production of nitrite and ammonium (despite the higher nitrate availability) and prevent the deleterious effects which would result from their intracellular accumulation (nitric oxide overproduction and buffering disturbance, respectively) [20,68,69].
Similarly to P800, NPQ800 also depends on nitrate concentration following a quadratic concave profile with an optimum for 9.09 mM (Figure 6e). Assuming that the NADPH-to-ATP stoechiometric ratio of nitrate assimilation is superior to the yield of photosynthesis, the reoxidation of photo-generated reductant may not be paralleled with ATP turnover. This feature could result in heightening ΔpH across the thylakoid membrane, which would in turn stimulate high energy state chlorophyll de-excitation (qE) in an extent depending on the rate of nitrate assimilation.