Light, CO2, and carbon storage in microalgae

Yasuyo Yamaoka, Dimitris Petroutsos, Sujeong Je, Takashi Yamano, Yonghua

Li-Beisson

► To cite this version:

Yasuyo Yamaoka, Dimitris Petroutsos, Sujeong Je, Takashi Yamano, Yonghua Li-Beisson. Light, CO2, and carbon storage in microalgae. Current Opinion in Plant Biology, 2025, 84, pp.102696. 10.1016/j.pbi.2025.102696. hal-04964370

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Distributed under a Creative Commons Attribution 4.0 International License
1 Light, CO₂, and carbon metabolism in microalgae

2

3 Yasuyo Yamaoka¹*, Dimitris Petroutsos², Sujeong Je¹, Takashi Yamano³, Yonghua Li-

4 Beisson⁴

5

6 ¹Division of Biotechnology, The Catholic University of Korea, Bucheon, 14662, Republic of

7 Korea

8 ²Department of Organismal Biology, Uppsala University, 75236, Uppsala, Sweden

9 ³Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502, Japan

10 ⁴CEA, CNRS, Aix-Marseille University, Institute of Biosciences and Biotechnologies of Aix-

11 Marseille (BIAM), UMR7265, CEA Cadarache, Saint-Paul-lez-Durance, 13108, France

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13 Email contacts of all authors: yasuyoyamaoka@gmail.com; dimitris.petroutsos@ebc.uu.se;

14 yamano.takashi.5v@kyoto-u.ac.jp; sujeongh.je@gmail.com; yonghua.li@cea.fr

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16 *Corresponding author: Yasuyo Yamaoka (yasuyoyamaoka@gmail.com)

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18 Key words: CO₂ concentrating mechanism; light spectrum; triacylglycerol; redox regulation;

19 photoreceptors; starch; inter-organelle communication

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21 Conflict of interest statement: Nothing declared

22

23 Highlight:

24 ● Light plays a dual role in dictating physiology and metabolism in photosynthetic
25 organisms.

26 ● The balance of light and CO₂ contributes to energy homeostasis.

27 ● Light spectrum influences carbon partitioning.

28 ● Energy crosstalk between subcellular organelles affect carbon storage and partitioning.

29 ● CO₂ availability plays a role in carbon partitioning.

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**Abstract:**

Microalgae exhibit remarkable adaptability to environmental changes by integrating light and CO₂ signals into regulatory networks that govern energy conversion, carbon fixation, and storage. Light serves not only as an energy source for photosynthesis but also as a regulatory signal mediated by photoreceptors. Specific light spectra distinctly influence carbon allocation, driving lipid or starch biosynthesis by altering transcriptional and metabolic pathways. ATP/NADPH imbalances significantly impact carbon allocation toward lipid or starch production. To maintain this balance, alternative electron flow pathways play critical roles, while inter-organelle redox exchanges regulate cellular energy states to support efficient carbon storage. The CO₂-concentrating mechanism (CCM) enhances photosynthetic efficiency by concentrating CO₂ at Rubisco, energized by ATP from photosynthetic electron transport. This review examines how light receptors, energy-producing pathways, and the CCM interact to regulate carbon metabolism in microalgae, emphasizing their collective roles in balancing energy supply and carbon storage.

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1. Introduction

Light and CO2 are fundamental regulators of photosynthetic metabolism. These factors
influence not only the efficiency of organic carbon and energy production, as well as cellular
energy homeostasis and cell fitness (Figure 1). Moreover, light plays a dual role: it is an energy
source indispensable for photosynthesis, driving the primary production of organic carbon, and
also serves as a pivotal regulatory signal controlling key cellular functions including
metabolism. As an energy source, light plays an essential role in the biosynthesis of lipids and
starch within chloroplasts. Additionally, a balanced supply of ATP and NADPH is necessary
for the production of these compounds. Recent studies have revealed that disruptions in the
energy balance between ATP and NADPH significantly impact lipid and starch biosynthesis
[1,2]. Beyond its role as a general energy source, light intensity and spectra further regulate
carbon storage pathways, influencing TAG and starch synthesis. For example, *Chlamydomonas
reinhardtii* exhibits light-induced expression of the plastidial enzyme diacylglycerol
acyltransferase 3 (DGAT3), which contributes to TAG accumulation [3]. Investigations into
light spectra reveals that blue-green light promotes fatty acid unsaturation and pigment
concentrations in *Monoraphidium braunii* [4], while red light induces starch accumulation in
*Chlorella vulgaris* [5] and blue light suppresses starch accumulation in *Chlamydomonas* [6].
Moreover, research on the blue-light-sensing transcription factor NobZIP77 has been shown to
enhance TAG productivity in *Nannochloropsis oceanica*, as discussed in the section below [7].
Transcriptomic studies further revealed the influence of light on lipid metabolism, as
demonstrated in *Chlorella vulgaris* under high light conditions [8] and in *Eustigmatos cf.
Polyphem* under varying light conditions [9].

The regulation of the CO₂-Concentrating Mechanism (CCM) in microalgae is tightly
influenced by light and atmospheric CO₂ concentration. The transcriptional regulator LCR1
(low-CO₂ stress response 1) coordinates both CCM and photoprotection mechanisms under low
CO₂ and light conditions [10,11], while the blue light photoreceptor PHOT (PHOTOTROPIN)
suppresses the expression of CCM and photoprotection genes [11]. Intriguingly, these genes
can also be activated in complete darkness if CO₂ is removed from the medium [12]. The
expression of *HLA3* (*High-light activated 3*) and *LCI1* (*Low CO₂-inducible 1*) under low CO₂
conditions is regulated by CAS-mediated Ca²⁺ signaling [13], which also controls the
expression of the photoprotective gene *LHCSR3* (*Light-harvesting complex stress-related
protein 3*) under high light [14]. These findings highlight the intricate interplay between light,
atmospheric CO₂, and carbon concentration, providing critical insights for enhancing
microalgal productivity under varying conditions.
In this review, we explore the latest advances in understanding how light and CO₂ levels regulates energy conversion, CCM, and carbon storage processes in microalgae. First, we discuss the effect of light spectrum and utilisation on cell physiology and carbon metabolism. We then review current knowledge on energy management pathways as well as the importance of energy trafficking between subcellular compartments and discuss the consequence on carbon storage when above mechanisms are absent. Finally, we explain how CO₂ availability could affect metabolic pathway remodeling, and what is the overall energetic cost for cells to maintain CCM.

## 2. Light signaling and its impact on metabolism

In plants and algae, light serves as both the energy source for photosynthesis and a biological signal that triggers cellular responses through specific sensory photoreceptors. Red light is detected by bilin-containing phytochromes, while blue light is perceived by flavin-containing cryptochromes and/or PHOTs, the latter characterized by two photosensory light, oxygen, or voltage (LOV) domains [15].

While algal phytochromes are not restricted to red and far-red light responses; instead, they display diverse spectral sensitivities, with some capable of sensing orange, green, and even blue light [16], Chlamydomonas notably lacks phytochromes in its genome [17]. Nevertheless, this microalga has evolved a rich repertoire of photoreceptors, including a single-copy PHOT, four cryptochromes, eight rhodopsin-like proteins, and the UV-B photoreceptor UVR8 (Figure 2a). These photoreceptors enable Chlamydomonas to accurately sense and respond to light, regulating key cellular functions such as gene expression, the sexual life cycle, phototaxis, photosynthesis, and photoprotection [18]. Among the various blue light photoreceptors found in algae, a particularly intriguing group of blue light photoreceptors are the aureochromes, which uniquely combine a LOV domain for light perception with a basic region leucine zipper (bZIP) domain for DNA binding, functioning as light-driven transcription factors. Aureochromes have been identified exclusively in photosynthetic stramenopiles, a single algal group, and are absent in other prokaryotic or eukaryotic organisms [19].

Although there are clear indications in the literature linking light quality to metabolism (e.g., see references [4,9]), mechanistic insights into how light perception translates into specific metabolic outputs remain largely elusive. In *Nannochloropsis oceanica*, the blue-light sensor aureochrome NobZIP77 plays a regulatory role in lipid metabolism [7]. Under nitrogen (N)-replete conditions, NobZIP77 represses the transcription of the *NoDGAT2B* gene, which encodes a type-2 diacylglycerol acyltransferase (Figure 2b). However, under N-depleted
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conditions, this repression is lifted, activating *NoDGAT2B* expression and promoting TAG synthesis, preferentially from C16:1 fatty acids. Blue light further reduces the binding of NobZIP77 to the *NoDGAT2B* promoter, relieving inhibition and enhancing TAG accumulation under N-depleted conditions. NobZIP77 specifically binds to an ACGT-containing sequence in the *NoDGAT2B* promoter, directly linking light perception and transcriptional regulation of lipid metabolism [7].

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More recently, another example of the link between blue light perception and metabolic regulation, specifically in starch metabolism, was reported in Chlamydomonas [6]. PHOTOTROPIN-MEDIATED SIGNALLING KINASE 1 (PMSK1) was identified as a key regulator in this process, linking blue-light perception to carbon metabolism. Blue light triggers the dephosphorylation of PMSK1, which in turn reduces the mRNA levels of GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAP1), a key Calvin cycle enzyme now recognized as essential for starch synthesis. In its phosphorylated state at a critical serine residue (S120), PMSK1 activates GAP1, thereby promoting starch production (Figure 2b). Therefore, blue light, via PHOT, represses starch accumulation by favoring the dephosphorylated (inactive) form of PMSK1 at S120. This regulation offers an explanation for the long-standing observation that starch accumulation predominantly occurs under red light in green algae [20]. It is important to note that the *phot* mutant was shown to accumulate higher starch levels under low light without compromising growth or photosynthesis, underscoring the efficiency of this pathway in balancing resource allocation between starch storage and cellular growth [6]. These findings align well with observations in *Chlorella* which accumulated high levels of starch when grown under red light without any compromise in growth under nutrient replete conditions [5].

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### 3. Subcellular energetic management and carbon storage

TAG and starch are primary reservoirs of cellular carbon and energy in green photosynthetic cells. Their accumulation is often triggered by changing environmental conditions [21], while their rapid degradation release organic carbon and energy for anabolic reactions. One of the driving forces underlying the biosynthesis or the degradation of TAG and starch is the need to satisfy cellular energy homeostasis in terms of ATP and NAD(P)H balancing. The equilibrium between the two is essential for cell fitness, because when they are disproportionately produced or not well consumed/dissimilated, reactive oxygen species (ROS) could be generated eventually leading to oxidative stress [22].
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Photosynthetic cells produce ATP and NADPH through the linear electron flow of photosynthesis, a relatively rigid process. To adapt to varying metabolic needs as a result of ever-changing environmental situations, microalgae have evolved a diverse set of alternative electron management pathways to enable efficient coupling of ATP and NAD(P)H production with downstream carbon metabolism [23]. The currently known mechanisms include cyclic electron flow (CEF), pseudo-cyclic electron flow (PCEF), energy trafficking pathways between subcellular energy-producing organelles such as the chloroplast, mitochondria, and peroxisome, as well as the biosynthesis of TAG and starch [24,25]. These responses differ in their relative importance, in their nature as ‘sustainable” (e.g., energy-conserving processes like TAG and starch formation) or “non-sustainable” (e.g., energy loss when water acts as an electron acceptor) as well as in terms of the time scale the response occur [23]. Below we discuss some of latest evidences on the interplay between energetic management pathways and carbon storage.

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### Chloroplast-based energy management pathways

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de novo fatty acid biosynthesis and starch biosynthesis both occur in the chloroplast. In addition to carbon precursors, they both require ATP and NADPH in a stoichiometric balance (Figure 3a; [26]). Lipid biosynthesis demands more NAD(P)H than starch biosynthesis. Indeed, changes in the chloroplast ATP/NAD(P)H poise has been shown to affect not only carbon storage but also influence carbon allocation between TAG and starch (Figure 3b). For example, in the absence of either one of the major components of CEF (PGR5 or PGRL1), an over-production of TAGs or total lipids is observed in the respective single mutant under photoautotrophic N starvation [1,2]. Interestingly, although the *pgr5* mutant was reported to make less than 50% starch than its parental strain, the *pgr11* mutant made similar amount of starch, pointing to potential differences in the regulatory mechanism involving PGR5 or PGRL1. The situation with the flavodiiron (FLV)-based PCEF pathway is again different. The FLV deficient mutants were found to accumulate 20% less TAG without affecting starch amount, highlighting unique features between CEF and PCEF [1]. Interestingly, knocking out FLV in the *pgr11* background restored a phenotype similar to the parental strain in terms of carbon storage during photoautotrophic N starvation [1]. This is in line with their respective photosynthetic capacities.

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### Inter-organelle energy exchange
In addition to chloroplast-based electron management pathways, chloroplast redox balance can also be modulated through energy exchange with other subcellular organelles, particularly mitochondria and peroxisome. Because NAD(P)H cannot permeable to cellular membranes, energy exchanges between subcellular compartments is achieved through malate shuttles and triose phosphate transporters (TPTs). For example, it was observed that knock-out of the peroxisome-located malate dehydrogenase 2 (MDH2) enhanced fatty acid *de novo* biosynthesis and starch accumulation in the chloroplast, suggesting the occurrence of a redox communication between chloroplast and peroxisome [27]. Lately, the chloroplast triose phosphate transporter 3 (TPT3) is found to play a key role in dictating carbon allocation, in its absence, the mutant cells over-accumulated both TAG and starch [28]. In the absence of either MDH2 or TPT3, an over-accumulation of H₂O₂ is reported, suggesting a modified redox poise in the above said mutants. Taken together, subcellular energy state play a critical role in dictating carbon metabolism and allocation, emphasizing the importance of inter-organelle communication in maintaining cellular homeostasis. The landscape is even more complex during varying CO₂ concentration [29], which is discussed in the section below.

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## 4. CO₂ availability and its influence on carbon allocation

CO₂ availability regulates microalgal metabolism through complex mechanisms that control carbon allocation. Central to this regulation is the CO₂-concentrating mechanism (CCM), which enhances photosynthetic efficiency by elevating CO₂ levels around Rubisco in the chloroplast. The pyrenoid, a specialized organelle essential for CCM function, relies on Essential Pyrenoid Component 1 (EPYC1) to densely pack Rubisco optimizing carbon fixation under CO₂-limited conditions [30,31]. Remarkably, the pyrenoid exhibits liquid-like properties, enabling rapid reorganization in response to environmental changes [32]. This dynamic adaptability is crucial for maintaining optimal carbon fixation rates under fluctuating environmental conditions.

CO₂ levels and light intensity regulate the localization of CCM components, with the Low-CO₂-inducible protein B-C (LCIB-LCIC) complex relocating to the pyrenoid periphery under CO₂-limiting conditions (Figure 3c; [33]). The starch sheath surrounding the pyrenoid facilitates this localization [34], highlighting the intricate relationship between organelle structure and function. Notably, LCIB-LCIC relocalization occurs independently of light, *de novo* protein synthesis, or photosynthetic electron flow [35].

Under high CO₂ availability, Chlamydomonas downregulates CCM activity, reallocating energy and resources towards carbon assimilation and TAG accumulation (Figure 3d). This metabolic shift is accompanied by increased photosynthetic efficiency and strategic
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mitochondrial adjustments that help balance the elevated reducing power generated under these conditions [29]. Furthermore, during CO₂-limiting conditions, mitochondria undergo a CIA5/CCM1-regulated repositioning to the cell periphery along the apico-basal axis [36], suggesting a coordinated optimization of cellular energy metabolism.

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The interaction between CO₂ availability and light creates a sophisticated regulatory network governing CCM efficiency and photoprotection. Excess light depletes intracellular CO₂, triggering CCM gene expression, high CO₂ suppresses these genes via the CCM master regulator CIA5/CCM1, while depletion of CO₂ can trigger expression of CCM genes even in complete darkness [12]. This dual regulation highlights the dynamic interplay between light-driven energy supply and CO₂ availability in controlling carbon allocation. High CO₂ also enhances carbon fixation and biomass production through rearrangements in the photosynthetic machinery, illustrating the integrated role of light and CO₂ in microalgal metabolism [37].

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CCM efficiency undergoes further fine-tuning through modifications in photosynthetic electron transport chains. The activation of alternative photosynthetic pathways not only improves CO₂ assimilation efficiency but also affects the distribution of carbon towards storage compounds like starch and lipids [38]. This metabolic flexibility enables microalgae to optimize their metabolism under varying CO₂ conditions, demonstrating the sophisticated interplay between energy supply and carbon allocation. These insights into microalgal CO₂ response mechanisms continue to unveil new possibilities for biotechnological applications.

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## 5. Conclusion and future directions

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Microalgae exhibit remarkable adaptability to environmental changes, with sophisticated regulatory networks linking energy conversion, carbon fixation, and storage processes. This review highlights how light and CO₂ availability determine the capacity of cells to assimilate, fix and store carbon, and could further influence the balance between lipid and starch biosynthesis. Light functions not only as an energy source for photosynthesis but also as a regulatory signal through photoreceptors like phototropin and aureochromes. Specific light spectra, such as blue and red light, differentially affect carbon allocation, enhancing lipid or starch synthesis. Advances in engineering light-responsive transcription factors have further demonstrated potential for improving lipid productivity. Subcellular energy management also plays a crucial role, with pathways like cyclic electron flow and inter-organelle redox exchanges maintaining energy balance and regulating carbon storage. CO₂ availability influences the subcellular energetic state, as CCM energization relies on photosynthetic energy. Although it
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is clear that the CCM efficiently supports carbon fixation and allocation, its impact on carbon storage processes remains poorly understood.

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In summary, advancing our understanding of how photoreceptors and subcellular energy balance influence metabolic regulation, including CCM, will allow us to fully utilize the potential of light and microalgae to develop sustainable solutions for addressing global sustainability challenges.

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**Declaration of competing interest:**

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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**Acknowledgments:**

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Y.Y. was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (Grant Nos. 2022R1C1C1008690 and 2022M3A9I3018121). The lab of Y Li-Beisson is fully supported by the CEA and currently by a France 2030 PEPR grant AlgAdvance. D.P. was supported by grants from the Swedish Research Council (grant no. 2023-04397_VR), the Novo Nordisk Foundation (grant no. NNF23OC0085412), and the Horizon 2020 Framework Programme for Research and Innovation (grant agreement no. 101122224). T.Y. was supported by the Japan Society for the Promotion of Science (Grants Numbers JP24K01851), GteX Program Japan Grant Number JPMJGX23B0, and the Asahi Glass Foundation.

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**Data availability:**

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No data was used for the research described in the article.

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**Display element:**

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**Figure 1.** Schematic representation of how light and CO₂ influence storage molecule production in microalgae.

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**Figure 2.** Photoreceptors and their roles in light signaling and metabolic regulation in microalgae.

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**Figure 3.** Hypothetical model for subcellular energetic management for CO₂ concentration mechanisms (CCM) and carbon storage.

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* of special interest

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Figure legends:

<caption>Figure 1. Schematic representation of how light and CO₂ influence storage molecule production in microalgae.</caption>

Light and CO₂, as key environmental factors, regulate cellular systems including photoreceptors, energy pathways, and the CO₂ Concentrating Mechanism (CCM). These systems coordinate the interaction between environmental factors and cellular metabolism, driving the accumulation of storage molecules such as triacylglycerols (TAG) and starch. The diagram highlights the interconnected processes underlying these regulatory pathways within the microalgal cell.

<figure><img src="image_2.png" /><figcaption>Figure 1.</figcaption></figure>
<caption>Figure 2. Photoreceptors and their roles in light signaling and metabolic regulation in microalgae.</caption>

(a) Comparison of photoreceptors in plants and algae across the visible light spectrum. In plants, phytochromes detect red and far-red light, cryptochromes and phototropins respond to blue light, and UVR8 senses UV-B. Zeitlupe, unique to plants, is involved in circadian regulation. Algae share many of these photoreceptors but also possess unique ones, including aureochromes (blue light sensors with transcriptional regulation domains), neochromes (red and blue light sensitivities), and rhodopsins (broad spectral sensitivity).

(b) Regulation of lipid and starch metabolism by light and nitrogen availability in *Nannochloropsis oceanica* and *Chlamydomonas reinhardtii*. In Nannochloropsis, under nitrogen (N)-replete conditions and white light, the blue-light receptor aureochrome NobZIP77 represses *NoDGAT2B* transcription, inhibiting triacylglycerol (TAG) synthesis. In contrast, under blue light and N-depleted conditions, NobZIP77 binding to the *NoDGAT2B* promoter is relieved, allowing TAG accumulation. In Chlamydomonas, red light or white light under N-replete conditions leads to the phosphorylation of PHOTOTROPIN-MEDIATED SIGNALING KINASE 1 (PMSK1), activating GAP1, a key enzyme in the Calvin cycle, and promoting starch synthesis. Conversely, under blue light, phytotropin (PHOT)-mediated signaling dephosphorylates PMSK1, reducing GAP1 levels and repressing starch accumulation.

<figure><img src="image_3.png" /><figcaption>Figure 2.</figcaption></figure>
<caption>**Figure 3.** Hypothetical model for subcellular energetic management for CO₂ concentration mechanisms (CCM) and carbon storage.</caption>

(a) Under ambient CO₂ (0.03-0.5%), balanced NADPH-ATP production supports carbon metabolism. The photosynthetic electron transport chain, including photosystem II (PSII), photosystem I (PSI), and cytochrome b6f complex (Cyt b₆f), drives linear electron flow (LEF) to produce NADPH and ATP. Additional ATP is generated through cyclic electron flow (CEF) around PSI, while proton gradient cyclic electron flow (PCEF) and chloroplast-mediated electron flow (CMEF) provide further ATP fine-tuning. Under these conditions, CO₂ transport is primarily mediated by LCI1, while HLA3/LCIA transporters are inactive.

The transported CO₂ is processed by LCIB/C, and the Calvin-Benson-Bassham (CBB) cycle fixes CO₂ to produce G3P, which serves as a precursor for both starch synthesis and triacylglycerols (TAG) production through fatty acid synthesis (FAS), utilizing the generated NADPH and ATP.

(b) The impact of imbalanced NADPH and ATP production caused by disrupted CEF and PCEF on carbon allocation to starch and TAGs in various mutants. Mutants deficient only in CEF (ΔCEF *pgrl1* and ΔCEF *pgr5*) overproduce TAGs under nitrogen starvation, with *pgr5* producing less than 50% starch while *pgrl1* retains normal starch levels. In contrast, mutants deficient in PCEF (ΔPCEF *flv*) accumulate 20% less TAG without affecting starch levels.

Combined deficiencies in both CEF and PCEF (ΔCEF ΔPCEF *pgrl1 flv*) restore carbon storage phenotypes to the control, as indicated by arrows, reflecting distinct roles of CEF and PCEF in energy and carbon allocation.

(c) Under very-low CO₂ (<0.03%), HCO₃⁻ transport is primarily mediated by HLA3/LCIA transporters, which become actively engaged in response to the carbon-limiting conditions.

This represents a shift from the ambient CO₂ condition where LCI1 is the main transporter.

The system requires increased ATP production, as indicated by multiple ATP molecules shown at the bottom. LCIB/C proteins are strategically positioned around the CBB region to facilitate efficient CO₂ recapture. The enhanced CCM operation under these conditions necessitates additional energy investment, while maintaining the overall pathway from HCO₃⁻ transport through carbon fixation to TAG and starch production.
<figure><img src="image_4.png" /><figcaption>Figure 3.</figcaption></figure>