In chloroplasts from Embryophyta, dark-to-light transitions are accompanied by a shift of the chloroplast internal pH from 7 in the dark to 8 in the light (Werdan and Heldt, 1973; Hauser et al., 1995). These changes directly regulate photosynthesis since many key chloroplastic enzymes have optimal activity at pH 8 and are much less active at pH 7 [reviewed in Gontero et al. (2007)]. In diatoms, pH responses have mainly been studied for external/environmental, rather than internal, pH. External pH can affect growth rate, silicon metabolism and biomineralization of Conticribra weissflogii (formerly known as Thalassiosira weissflogii) as well as its intracellular/cytoplasmic pH homeostasis (Herve et al., 2012). For other photosynthetic organisms, it is not the extracellular pH, but the intracellular pH in the chloroplast that is the critical factor for regulation of carbon acquisition, transport capacity and other metabolic processes. To our knowledge, internal pH has only been measured for a small number of diatoms. For P. tricornutum and Cyclotella sp. the pH was around 7 in the dark and 7.5 in the light (Colman and Rotatore, 1995), and for Navicula pelliculosa, it was 7.4 in the dark and 7.6 in the light (Colman and Rotatore, 1988). We found no published values for the pH within diatom chloroplasts stroma. One of the few studies of the effect of pH on enzyme activity in chromalveolates is for the chloroplastic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that catalyzes the reversible reduction and dephosphorylation of 1,3-bisphosphoglycerate to produce glyceraldehyde-3-phosphate and inorganic phosphate. Avilan et al. (Avilan et al., 2012) compared the optimal pH of GAPDH in the freshwater diatom, Asterionella formosa, the freshwater eustigmatophyte, Pseudocharaciopsis ovalis, and the model green alga, C. reinhardtii. In A. formosa, GAPDH was still active at the pH occurring in the dark, assuming that the internal pH (pH 7) reflects the one in the chloroplast. This suggests that GAPDH is regulated by factors other than pH in this diatom, unlike in the green algal enzyme that was down-regulated at the pH that occurs in the dark. The response of GAPDH from the eustigmatophyte P. ovalis was similar to that of the green alga C. reinhardtii. We do not know the internal chloroplast pH for P. ovalis but if the dark-to-light pH transition in this species is similar to that of C. reinhardtii, GAPDH could be partly regulated by pH under dark-light transitions. The different regulation of GAPDH by pH in the two heterokonts, A. formosa and P. ovalis, might be the result of the diverse evolutionary history of chromalveolates. Another example of regulation by pH is the lumenal enzyme violaxanthin de-epoxidase [VDE, (Lavaud et al., 2012)] that is involved in dissipating excess light energy (Lohr and Wilhelm, 1999).

Beyond photosynthesis, the carbon metabolism of the marine diatom Skeletonema costatum is regulated by the pH of the growth medium (Taraldsvik and Myklestad, 2000). The content of the carbohydrate storage compound, chrysolaminarin (β-1,3 linked glucan) decreased from 7.1 mg.L⁻¹ at pH 6.5 to 0.2 mg.L⁻¹ at pH 9.4 and concomitantly, the total organic carbon as glucan also decreased from 60 to 10%. The total amino acid content also decreased from 7.41 to 2.51 fmol.cell⁻¹ when the pH of the growth medium increased (Taraldsvik and Myklestad, 2000). It is unclear if these are direct effects on carbon and nitrogen metabolism of external or internal pH or indirect effects linked to the greater external concentration of CO₂ at pH 6.5 than at pH 9.4. Nevertheless, to relate these physiological responses to enzyme activity regulation, the authors report results from a Norwegian PhD thesis (Kirkvold, 1994) that showed that the specific activity of glutamine synthetase, a key enzyme in the metabolic pathway of glutamine and glutamate synthesis, also decreased with increasing pH when measured in vitro.

Studies on the effect of pH on activity should be expanded to more enzymes and their optimal pH compared to the internal pH in dark and light in order to determine if enzyme activity is regulated by internal pH. The difficulty of working with enzymes from diatoms and from chromalveolates in general, is probably responsible for the lack of data for this important group. For instance, in order to extract proteins from diatoms, litres of culture are required and it is not always easy to measure activity. Expressing recombinant diatom enzymes in heterologous systems is also challenging with many enzymes found in the insoluble fractions (B. Gontero, personal communication). Measurement of internal pH is also an experimental tour de force. Colman and Rotatore used the 5,5-dimethyl-2,4-oxazolidinadione distribution method that distributes between the medium and the cell as a function of their respective pH (Colman and Rotatore, 1988; Colman and Rotatore, 1995). However, this method does not distinguish between the pH in the cytoplasm, chloroplast stroma or thylakoid lumen.

The redox control of enzyme activity in the Viridiplantae is primarily mediated by small proteins, thioredoxins, that are oxidized in the dark and reduced in the light (Buchanan et al., 1980; Buchanan, 2017). This regulation avoids futile cycles between the CBB and the OPP pathway, since both occur within the chloroplast, with enzymes from the CBB being active in the light and those from OPP being active in the dark. In contrast, in diatom plastids the OPP is incomplete, and presumably lacking (Kroth et al., 2008), and accordingly the regulation of their metabolism is different (Jensen et al., 2017). Moreover, diatoms have a high stromal reductant pressure and in contrast to Viridiplantae, metabolic activity in long dark periods leads to an enhanced reduction state of the plastoquinone pool. In the dark, since the plastoquinone pool is reduced, it may regulate redox-sensitive enzymes as is the case for algal nitrate reductase (Giordano et al., 2005). This avoids reducing equivalents to accumulate maintaining cellular redox poise (Wilhelm et al., 2006). In Viridiplantae in contrast, oxidizing conditions prevail in the dark, therefore suggesting that redox control may be different. Because of this unusual redox control, the redox regulation of diatom enzymes has been questioned (Wilhelm et al., 2006). However, diatoms possess many different thioredoxins, each encoded by a specific gene and located in different compartments, including the chloroplast. Most thioredoxins contain the regulatory cysteine residue in the conserved motif, WCGPC (Weber et al., 2009), thus they are likely to have specific regulation targets and some targets have been identified such as two CAs in P. tricornutum (Kikutani et al., 2012). The relatively few targets of thioredoxins currently identified in diatoms, contrasts with the 1188 targets found by combining qualitative and quantitative proteomic analyses in the C. reinhardtii thioredoxome (Perez-Perez et al., 2017).

Using a redox proteomics approach on P. tricornutum, Rosenwasser et al. identified the “redoxome”, or in other words the redox-sensitive proteins, and demonstrated its involvement in photosynthesis, photorespiration, lipid biosynthesis, and nitrogen metabolism (Rosenwasser et al., 2014). In that case, however, the redox-sensitivity is a response to oxidative stress rather than light-dark transition even though reactive oxygen species are photo-induced, and increase in parallel to glutathione (GSH). GSH is a low-molecular-weight tripeptide that consists of cysteine (Cys), glutamic acid (Glu), and glycine (Gly) and is present in microorganisms, plants, and mammals (Zaffagnini et al., 2012). It can regulate protein activity by forming a mixed disulfide bridge between the thiol group of its Cys and an accessible free thiol on a protein, a process known as protein S-glutathionylation (Zaffagnini et al., 2012; Marri et al., 2014). This post-translational modification can protect specific Cys residues from irreversible oxidation but can also modulate protein activities (Zaffagnini et al., 2012; Marri et al., 2014; Thieulin-Pardo et al., 2015). In T. pseudonana, a diurnal redox-related pattern has been observed in which GSH accumulates in the light, and decreases upon darkness, (Dupont et al., 2004) (Figure 2). However, direct regulation of the enzyme activities by glutathionylation in diatoms, or in other chromalveolates, in contrast to Viridiplantae, has not yet been studied, to the best of our knowledge.

Although the effect of glutathionylation on enzymes has not been studied in diatoms, the effect of other reducing agents such as dithiothreitol has been investigated, though understudied as compared to other photosynthetic organisms such as Cyanobacteria, Chlorophyta, Rhodophyta, and Embryophyta. The chloroplastic phosphoglycerate kinase belonging to the CBB cycle, catalyzes the ATP-Mg²⁺-dependent phosphorylation of 3-phosphoglycerate (3-PGA) to 1,3-bisphosphoglycerate, in a reversible reaction and was redox-regulated in P. tricornutum (Belen Bosco et al., 2012). However, in our hands, PGK was not redox-regulated, as was also the case in T. pseudonana, in Navicula pelliculosa grown with sea water and fresh water medium and in a freshwater diatom, A. formosa (Jensen et al., 2019b). In contrast to the Viridiplantae, two enzymes that are unique to the CBB were not redox regulated (Michels et al., 2005; Maberly et al., 2010; Jensen et al., 2017). These include, sedoheptulose 1,7-bisphosphatase that irreversibly catalyzes the dephosphorylation of sedoheptulose-1,7-bisphosphate producing sedoheptulose-7-phosphate, and phosphoribulokinase (PRK) that irreversibly catalyzes the ATP-Mg²⁺-dependent phosphorylation of ribulose-5-phosphate into ribulose-1,5-phosphate. The general lack or weak redox regulation of PRK in the chromalveolates (diatoms and other groups) seems to be related to its sequence, where the connectivity between two regulatory cysteine residues is crucial [at position 16 and 55 in C. reinhardtii (Maberly et al., 2010)]. In many photosynthetic organisms PRK can also be sequestered, and inactivated, in a PRK-GAPDH-CP12 complex, that has not yet been found in diatoms. The absence of the ternary complex with GAPDH in diatoms has been attributed to the absence of two cysteine residues on PRK (at position 243 and 249 numbered from the enzyme from C. reinhardtii) that are present in Cyanobacteria, Chlorophyta, Rhodophyta, and Embryophyta where the complex has been identified (Thieulin-Pardo et al., 2015). In contrast however, a ferredoxin-NADP reductase (FNR)-GAPDH-CP12 complex has been found in A. formosa (Mekhalfi et al., 2014). For the chloroplastic GAPDH, the regulation is more complex as discussed above in the pH regulation section, but in many diatoms, this enzyme seems to be redox regulated (Maberly et al., 2010; Mekhalfi et al., 2012; Mekhalfi et al., 2014; Jensen et al., 2019b).

Regulation at the transcriptional level by light-dark transitions, occurs in Viridiplantae, and also in diatoms (Sun et al., 2003; Fey et al., 2005). In T. pseudonana, after 12 h of light, 1,859 genes were upregulated compared to cells exposed to 12 hours of dark, and inversely, after 12h of dark, 1,326 genes were up-regulated compared to cells exposed to 12 h of light (Ashworth et al., 2013). Among the most highly expressed genes after 12 h of light were the ones encoding enzymes for cell division, DNA replication and repair, carbon metabolism and oxidative phosphorylation while after 12 h of dark, the most highly expressed genes were those encoding ribosomal biogenesis, aminoacyl-tRNA and key photosynthetic enzymes (Figure 2). Some genes, such as that encoding RuBisCO, anticipates the diurnal cycle and is up-regulated before the onset of light. The dark-light expression pattern of genes was affected by growth phase (exponential vs. stationary). In the stationary phase, the expression of only a few genes fluctuated under dark-light transitions (Ashworth et al., 2013). One of these genes encodes a putative pyruvate carboxylase suggesting a switch toward other types of metabolism such as gluconeogenesis and lipid biosynthesis. This might be explained by the hypothesis raised by Norici et al. in S. marinoi (Norici et al., 2011) who postulated that the diatom re-routes its metabolism toward lipid biosynthesis, because of the relatively high volume-based energy content of lipids in an organism in which size decreases with vegetative cell divisions, thus requiring carbon allocation into more energy-compact compounds.

In the light, more than 4,500 transcripts were differentially expressed in P. tricornutum, including genes such as the one encoding for pyruvate transporter that had never been previously described in this organism (Chauton et al., 2013). This work shows that transcriptional regulation of carbohydrate and lipid metabolism occurs in diatoms (Figure 2). Indeed, the content of soluble glucans and lipids decreased in the dark and fatty acid biosynthesis genes were up-regulated within 30 min of a switch from dark to light. Fatty acid biosynthesis and the tricarboxylic acid (TCA) cycle are also tightly co-ordinated (Chauton et al., 2013). During the day, carbon skeletons are produced within the chloroplast while in the night these carbon-rich compounds are broken down in the mitochondria and the cytosol.

Interestingly, four carbon fixation enzymes were co-regulated in P. tricornutum: PGK, GAPDH, triose phosphate isomerase/GAPDH and PRK. Their mRNAs were all highest at the beginning of the light period (dawn) and lowest at the beginning of the dark period (dusk). Bai et al. (2016) showed however that the expression of PRK increased after 4 days of dark treatment using a proteomic approach. Similarly, the expression of phosphoribose isomerase, the enzyme that produces PRK substrate, as well as the expression of transaldolase and glyoxylase, increased after dark treatment.

Redox regulation of PRK is observed and well-studied in Viridiplantae, but in contrast in diatoms and other chromalveolates, as mentioned above, the activity of PRK was affected neither by oxidizing nor by reducing conditions (Maberly et al., 2010). Therefore, for enzymes that are not redox-regulated, regulation at the transcriptional level could allow a dark-light regulation of the CBB. The expression pattern of the gene of fructose-1,6-bisphosphatase (FBPase) that catalyzes the dephosphorylation of fructose-1,6-bisphosphate into fructose-6-phosphate and inorganic phosphate, was not induced to the same degree by light as other CBB enzymes. Since this enzyme can be redox regulated (Michels et al., 2005; Mekhalfi et al., 2012), its regulation could occur both at the level of activity and transcription.

Carbon acquisition is also stopped at night since in P. tricornutum, mRNA encoding for bicarbonate transporters of the Solute Carrier family 4 (SLC4) and for alpha-carbonic anhydrases, especially α-CA-VII, were much less abundant in the dark than in the light (Chauton et al., 2013). Recently, in the same organism, the pattern of mRNA levels at a photon irradiance of 30,300, 1,000 µmol photon m⁻² s⁻¹ differed at the lag, exponential and stationary phases of growth (Heydarizadeh et al., 2019).

Dark-treated P. tricornutum cells preferentially utilize carbon and nitrogen obtained from protein breakdown to increase lipid cell quotas at low cost (Bai et al., 2016). Long-term dark stress inhibited several key proteins involved in nitrogen assimilation and in the synthesis of the photosynthetic machinery. Simultaneously, key enzymes of glycolysis and the synthesis of fatty acids were induced apparently to assimilate the excess of C and N from protein breakdown. Uptake of other resources for growth are also light-regulated: transporters for uptake of phosphate and silica are higher in the light and nitrate and ammonium transporters are higher in the dark (Ashworth et al., 2013) (Figure 2). Data from the literature showing differential expression of proteins, transcripts and metabolites in light and dark are summarized in Figure 2.

Gene expression gives important clues on how light (quality, irradiance, duration) affects metabolism. However, it is also necessary to measure enzyme activity and metabolite concentration as these are the ultimate response to environmental change. For instance, an excess of light modifies lipid biosynthesis in the coastal marine diatom, Skeletonema marinoi (Norici et al., 2011). In S. costatum, carbohydrate increased with irradiance (Hitchcock, 1980) while lipids increased in Chaetoceros calcitrans (Harrison et al., 1990) and therefore the carbon allocation seems to be different and species-specific. Under different light-regimes, different species behave differently and the amount of essential fatty acids with growth irradiance is also species-specific. These examples illustrate that although enzyme activities have been measured, the mechanism underlying the change in activity is unknown. For example, PEPCK that converts oxaloacetate into PEP and CO₂ in gluconeogenesis, increased 2.5-fold in cells of S. marinoi grown under low light (25 µmol.photon.m⁻².s⁻¹) vs. high light (250 µmol.photon.m⁻².s⁻¹). The authors suggested that this enzyme might be involved in the conversion of lipid to carbohydrates especially under low light (Norici et al., 2011). In contrast, since energy demand for lipid synthesis is much higher than for carbohydrate synthesis (Raven et al., 2005; Subramanian et al., 2013) under excess light, lipids represent a better sink for excess energy. As a consequence, lipid accumulation in high irradiance was observed in S. marinoi, although this is not always the case. In this species, other enzymes are probably not regulated by light since their activity remains unchanged. For instance, the activity of PEPC, that catalyzes the addition of bicarbonate (HCO₃⁻) to PEP to produce oxaloacetate, was similar for cells grown at low or high light. This enzyme is involved in C4 metabolism and in anaplerotic reactions. Similarly, the activity of glutamine synthetase that is involved in photorespiration did not change in cells grown at low or high light.

Many related metabolic pathways are affected by CO₂ in addition to CCM expression. Gamma CA and the NADH-ubiquinone oxidoreductase complex (C1) are associated in many organisms (Cardol, 2011) and in N. oceanica, at very low [CO₂] (100 ppm), the genes coding for these two mitochondrial enzymes were up-regulated (Figure 4). Since they can facilitate transport of CO₂ produced by the TCA cycle and photorespiration toward the chloroplast, in the form of bicarbonate, they contribute to what is called a basal CCM (Wei et al., 2019). At the enzyme activity level, T. pseudonana cells grown at high CO₂ concentration (20,000 ppm) vs. atmospheric (400 ppm) displayed higher NADPH-dependent GAPDH and FBPase activity (Figure 4) indicating that the CBB cycle could be affected even though PRK activity was unaltered (Clement et al., 2017b). The activity of pyruvate kinase, a glycolytic enzyme, was also strongly stimulated when cells were grown at 20,000 ppm (Figure 4) while two other glycolytic enzymes, NADH-dependent GAPDH and glucose-6-phosphate dehydrogenase (G6PDH) were unaffected (Clement et al., 2017b). In this diatom, a model summarizing the effect of inorganic carbon limitation, based both on activities and protein expression profiles has been elaborated (Clement et al., 2017b). This model shows the remodelling of metabolism with a diversion of energy and resources toward carbon metabolism at high CO₂ and toward carbon capture at low CO₂. An increase in δ-CA gene expression, to capture as much CO₂ as possible, was also observed by Kutska et al. (2014) in T. pseudonana. Metabolism enzymes, as well as enzymes responsible for pigment synthesis and indirectly in light capture (Figure 4), were less abundant, while enzymes involved in reactive oxygen species (ROS) defence increased in order to avoid accumulation of ROS that might occur when energy is in excess. The increased activity of pyruvate kinase (Figure 4), observed in T. pseudonana seems to be shared by other diatoms, as it was observed in many diatoms from freshwater to seawater species grown under high CO₂ (20,000 ppm) vs. atmospheric CO₂ (400 ppm) (Jensen et al., 2019b). In addition, a modification of gene expression has been described that allows synthesis of either PEP or pyruvate under carbon shortage, indicating that pyruvate is an important hub in these organisms (Heydarizadeh et al., 2017; Heydarizadeh et al., 2019) (Figure 4).