How Does the Approach of Algal CCM Assist in Curbing CO₂ Pollution and Temperature?

Under the circumstances of high carbon dioxide levels and subsequent global warming, the Intergovernmental Panel on Climate Change (IPCC) disclosed two pathways to reduce the rate of high temperatures by about 1.5-2.0ºC per year. These pathways include reducing and removing CO_2. a. Reducing CO₂ involves limiting our reliance on

fossil fuels. b. Removing CO₂ involves methods that actively uptake

atmospheric CO_2.

The solution for carbon removal starts with photosynthetic organisms because they absorb atmospheric carbon dioxide through photosynthesis and convert it into organic carbon. Microalgae are considered highly efficient photosynthetic organisms because of their capacity to consume 2-10 times more CO₂ and produce 15-300 times more biomass efficiently than land-based plants [1]. This is because of the peculiar carbon fixation mechanism called CCM [23]. Microalgae with biophysical pyrenoid-based CCM plays a crucial role in assimilating ca. 40%-60% of global carbon dioxide each year [24]. This large amount of CO₂ fixation accounts for a large portion of global primary productivity [25]. CCM is an evolutionary adaptation acquired by microalgae and cyanobacteria to counter the negatives of RuBisco and the aquatic environment [26]. In aquatic environments, microalgae face a 10,000- fold slower diffusion rate of CO₂ compared to that of the terrestrial atmosphere [27, 28]. To get over this limited CO₂ environment, microalgae like Chlamydomonas and other aquatic photosynthetic plants actively capture the high amount of CO₂ in the form of inorganic carbon (C_i; CO₂ and HCO₃^-) into the cell through this unique carbon capture system and increase the level of CO₂ at the site of RuBisco [27].

As CCM-adapted microalgae are highly efficient in CO₂ fixation and biomass productivity, understanding how atmospheric CO₂ and temperature levels influence the mechanism and regulation of microalgal CCM at the cellular and molecular level is very predominant [26]. In addition, analyzing the adaptive response and recovery capacity of microalgae and plants to high temperatures probably extends our perception of thermostability in all plants and algae [29].

In this scenario, CCM gains significance in studying the obtaining efficiency of CO₂ in the cells under increased temperatures. Understanding the global climate change factors like CO₂ and temperature conjointly might give a broad scope of how these changes impact plant growth and metabolic networks.

Why Is an Optimum Temperature Considered to Be an Essential Factor in Living?

The majority of physiological functions and enzymatic activities in living organisms highly depend on temperature [30]. The changes in temperatures have an impact on cellular processes at different stages [31]. Photosynthesis was one of the cellular processes where the rate of RuBP carboxylation (V_cmax), electron transport/RuBP regeneration (J _max), and the ratio between V_cmax & J _max altered according to the growth temperatures [32] (Fig. 1). Similarly, at high temperatures in the diatom, Phaeodactylum tricornutum uptake of C_i and concentration of C_i at the site of RuBisco and the kinetics of CCM components change [33]. However, these responses are specific to particular species at their particular growth temperature. Plant cells thrive in the above-optimum range temperatures by adapting some acclimatory mechanisms. Chlamydomonas cells, as an acclimation process, activate heat shock chaperons, proteins (HSP & HSF), upregulate photosynthesis and CCM [34], and thermotaxis genes [35]. The temperature sensitivity of CCM components varies according to imbalanced C_i fluxes resulting from temperature fluctuations. Therefore, microalgae maintain balanced C_i levels by synchronizing their CCM components to temperature variations, supporting highly efficient photosynthesis [33].

How Do Chlamydomonas Cells Sense Temperature?

Plants and microalgae sense changes in surrounding temperatures via a network of molecular detectors located in different cell compartments, i.e., plasma membrane (PM) sensing via the influx of calcium (Ca⁺²), unfolded proteins (UP) in the endoplasmic reticulum, cytoplasm, and chloroplast, and increased membrane fluidity [36, 37]. This heat stress signaling process was activated primarily when the photosynthetic apparatus of the chloroplast was subjected to damage by thermal stress [38].

A combination of electrophysiology, reporter gene assays, and biochemical measurements revealed that Ca⁺² channels sense a mild increase in temperature at the PM. These channels open due to increased membrane fluidity and activate the influx of Ca⁺² ions [37, 39, 40].

Fig. 1.

Bar chart representing the variations in photosynthetic rate (PR), RUBP regeneration (J _max), RUBP carboxylation (V _max) at 350 μmol mol^⁻ ¹ CO₂, 15ºC (a) and 30ºC (b) in Quercus myrsinaefolia (redrawn from Hikosaka et al., 2006).

Studies with Arabidopsis report that the PM cyclic nucleotide-gated channels (CNGC) are well-known transport channels. These channels are activated when the membrane fluidity/cAMP levels increase due to heat stress-activated adenylyl cyclase leading to Ca⁺² influx. Knockdown of the cngc6 gene in Arabidopsis shows no active calcium influx followed by reduced thermotolerance [37]. Thus, the accumulation of intracellular Ca⁺² in the cell during heat stress ensures that the calcium-reliance signaling system plays a signature task in response to thermal stimuli [41]. Recently, a putative Ca⁺² voltage-gated channel gene (CAV4) was identified in Chlamydomonas, which was supposed to be activated at high temperatures of 35ºC [34].

Another temperature sensor in Chlamydomonas is a cation-selective sensory ion channel called transient receptor potential channel TRP1. Upon heat stress, the opening of the TRP1 channel was mediated by increased levels of phosphatidylinositol-4,5-bisphosphate (PIP2) in the cell. These TRP channels were also known to regulate cellular Ca⁺² uptake in Ulva compressa and Ectocarpus siliculosus [42]. However, Ca⁺² influx through this TRP1 channel in Chlamydomonas was understudied.

Interestingly, CCM gene regulation by Ca⁺²-mediated retrograde signaling from chloroplast to nucleus was discovered recently [1, 2]. Since Ca⁺² is involved in sensing both CO₂ and temperatures, the functioning of Ca⁺²-mediated channels and their regulation during fluctuating temperatures and CO₂ levels need to be examined.

Temperature-Induced Primary Protective Mechanism Adopted by Chlamydomonas

Chlamydomonas and other photoautotrophic organisms in the wild are exposed to environmental stress, like fluctuations in the surrounding temperature. Plants recognize temperatures exceeding the optimum range as heat stress (HS) [43]. This kind of exposure to high temperatures disturbs many cellular processes, such as disturbances in assembling protein complexes, protein folding, cell membrane fluidity, cellular metabolism, enzymatic reactions, cell division, DNA replication, and repair. These disturbances trigger the production of molecular chaperones called heat shock proteins (HSP) [37, 44, 45].

HS response was regulated at the transcriptional level by transcription factors called heat shock factors (HSFs). In plants, at optimum temperatures, heat shock protein (HSP) gene transcription by heat shock transcription factors was kept inactivated by bound HSP70-HSP90 chaperones in the cytosol and enwrapped HSP genes by histone proteins in the nucleus. Upon mild warming (priming), thermo-sensors signal to activate the transcription of HSP genes by dissociating the inhibitory HSP70-HSP90 chaperones from HSF. In Chlamydomonas, of the two HSFs, HSF1 & HSF2, HSF1 is a canonical HSF [37, 39] that is structurally trimeric and undergoes phosphorylation at the time of heat stress, binds to the promoter region of HSP genes, and initiates transcription leading to the proliferation of HSP transcripts [37] (Fig. 2). The most abundant HS chaperones expressed were cytosolic chaperones – HSP90A, HSP22A, and HSP70A; Plastid chaperones – HSP70B, HSP90C; and mitochondrial chaperones HSP70C and ER luminal – HSP90B [37].

During heat stress, increased levels of Ca⁺² and UP in the cell activate the stress kinases. These active stress kinases trigger the activation of HSF, which in turn promotes HSP gene expression in the nucleus [37, 46, 47]. In Chlamydomonas, unfolded protein accumulation along with Ca⁺² and calmodulin-binding kinase (CBK3) activate inactive stress kinase mitogen-activated protein kinase (MAPK), which leads to the activation of HSF [48, 49] (Fig. 2). This was supported by the work done with Chlamydomonas, where the cells treated with BAPTA, a Ca⁺² chelator, exhibited a low and delayed expression of the HSP gene and decreased thermotolerance under heat stress [37]. Even though Ca⁺² chelators inhibit Ca⁺² influx from the exterior, the heat stress response was induced in the cell by the intracellular Ca⁺²/calmodulin-dependent HS signaling mechanism [50]. Hence, both extra and intracellular calcium levels influence heat sensing.

Chloroplast heat shock chaperones/HSPs play an essential role in obtaining thermotolerance. They protect and stabilize the photosynthetic mechanism and the apparatus under increased temperatures [38]. In chloroplasts, the heat stress state signal is coordinated at HSF1, where heat stress is sensed and transferred to the nucleus to generate HSF and HSPs. During heat stress, chloroplast protein (Clp) protease degradation in chloroplasts led to misfolded proteins. This downside action was detected and sequestered by small HSPs (HSP22E/F) and vesicle-inducing plastid proteins (VIPP1/VIPP2) located in the chloroplast membrane. This process triggered a retrograde signal to the nucleus through cytosolic mutant affecting retrograde signaling 1 (MARS1) kinase, leading to the upregulation of the HSPs [48] (Fig. 2). This finding is supported by the work on the HSF1-RNA i strain, a mutant lacking the HSF1 gene, which completely lacks transcripts of the chloroplast chaperone HSP90C [40]. Chloroplast stromal HSP70B in C. reinhardtii and Dunaliella was reportedly involved in the molecular protection and repair of photosystem II (PSII) [36]. Small heat shock proteins Hsp22E/F are noticed upon interacting with RuBisco activase, RCA1, under heat stress for optimal activity [47]. Another retrograde signaling system that initiates during HS is in the ER. In the ER, unfolded protein accumulation at the time of HS leads to the activation of inositol-requiring enzyme I (IRE1) RNase activity. This active IRE1 splices the mRNA of the basic leucine zipper 1 (bZIP1) transcription factor. bZIP1 produced from the spliced mRNA acts as a retrograde signal in the nucleus, upregulating the HSPs [48] (Fig. 2).

Fig. 2.

Heat stress response at high temperature in Chlamydomonas reinharditii. FP-folded protein; UP-unfolded protein; HSE-heat stress element; bZIP1 TF - bZIP1 transcription factor; ER-endoplasmic reticulum; CP-chloroplast; MT-mitochondria; N-nucleus.

Influence of Temperature on Photosynthetic Light Machinery and Their Reciprocal Acclimatory Reactions

Photosynthesis is one of the most important physiochemical processes of both higher plants and algae. These autotrophic organisms carry photosynthetic apparatus that exhibits a characteristic feature of sensing different environmental changes such as temperature, CO₂ levels, nutrient availability, light quality, and quantity by rapid adjustment or acclimation [51]. Thus, the enzymes and the genes involved in the functioning of the photosynthetic apparatus and their mechanisms are highly susceptible to different stresses [36]. Although photosynthesis is purely a light-dependent process, it requires sufficient temperature because all a cell’s metabolic and enzymatic activities depend on the optimum temperature [52]. Since the greenhouse effect, plants face additional temperatures up to 45ºC; these high temperatures cause a large depletion in plant yield [53]. The growth of photosynthetic organisms may increase with increased temperatures to a certain degree; later on, the growth decreases when the temperature surpasses a certain heat threshold. This decrease in growth is due to the decreasing photosynthetic efficiency. Efficient photosynthesis occurs only when electron transport and thylakoid membrane complexes coordinate collectively during the light reaction and carbon fixation cycle, respectively [31]. Disturbance in the light reactions indirectly affects the carbon fixation cycle by the inefficient production of ATP and NADPH during abiotic stress conditions. Therefore, the photosynthetic apparatus (PA) within the chloroplast is considered the primary structure prone to thermal stress damage [38]. This damage leads to improper carbon fixation, ion leakage, increased thylakoid membrane fluidity followed by the unordered arrangement of PS-II, PS-І, the cyt b6f complex, and ATP synthase, and finally, disturbances in energy distribution between photosystems, which eventually reduces the rate of electron transport followed by decreased yield [54].

Photosystems are the major target of high temperatures. Of the two photosystems (PS-II/PS- І), the reaction center (RC) PS-II is considered the most thermosensitive complex and the foremost target of heat inactivation of photosynthetic light reactions [55]. The imbalance between light absorption and electron transport during high temperatures leads to the over-excitation of PS-II RC. In an over-excited state, chlorophylls’ high energy reacts with molecular oxygen to release reactive oxygen species (ROS). These products damage the PS-II protein subunit D1, leading to photoinhibition [31] along with the respective detachment of Mg²⁺ ions, extrinsic proteins of the oxygen-evolving complex/water oxidizing complex (OEC/WOC) [55]. In addition, electron transfer kinetics between Q_A & Q_B on the PS-II donor and acceptor sides play an essential role in PS-II photoprotection and the electron transport chain [56]. Though PS-II reaction center protein subunit D1 is the main target of light-induced damage, changes in amino acids of D1 protein and regulation of D1 protein synthesis during elevated temperatures were reported [36]. Work with spinach thylakoids under heat stress reports that the loss of PS-II activity occurs due to the cleavage of D1/PsbA protein and the release of extrinsic proteins of PS-II. PsbO/OEE1 is one such extrinsic protein of PS-II that aids in stabilizing the manganese cluster, whose loss results in destabilization and degradation of the entire PS-II complex. Tolerance of spinach PS-II to high temperatures was achieved by replacing mesophilic PsbO with a PsbO of thermophilic cyanobacteria [3].

Although high temperatures distract the photosynthetic machinery of photosynthetic organisms acclimated to such conditions, they express enhanced thermal stability [57]. This stability phenomenon has been observed in several species of higher plants and cyanobacteria. Recent studies report upregulating about 200 chloroplast-allied genes at high temperatures in plants [58]. Some genes that were upregulated during elevated temperatures in Chlamydomonas are listed in Table 1. Temperature-acclimated cells display a highly thermostable PS-II reaction center because of the high stability of oxygen-evolving machinery [59]. This stable activity of WOC/OEC is due to its extrinsic proteins PsbO, PsbP, and PsbQ; these proteins act as a shield over the Mn cluster of OEC and improve the O₂-evolving efficiency of PSII. Along with these extrinsic proteins, OEC is supported by intrinsic luminal heterodimer proteins D1 and D2 and chlorophyll-binding proteins CP43 and CP47. Also, seven amino acids, six from D1 and one from CP43, coordinate the Mn cluster with PS-II [60, 61]. Even the lack of a single protein causes remarkable conformational changes. Cyanobacterium synechococcus sp. mutant with a defective PsbU gene could not increase the thermal stability of oxygen-evolving machinery and cellular thermotolerance upon acclimation to high temperatures. This inactivation of oxygen-evolving machinery was stabilized by replacing cytochrome (Cyt) C₅₅₀ and PsbU proteins of PS-II [62, 63].

The robust nature of the thylakoid membrane protects photosynthetic apparatus from the damage caused by abiotic stress. This is due to peculiar actions like alteration of the electron transport pathway, light-harvesting complex II (LHC-II) rearrangement, and PS-II/PS-І stoichiometry [55]. Though photosynthetic algae and plants regulate their photosynthetic antennae according to light, temperature shifts often alter their light-harvesting antennae and LHC expression [64]. The state transition is a major mechanistic response that alters during abiotic stress. The state transition is a reversible excitation balancing mechanism that operates within minutes to balance the electron transport between PS-І and P [13]. Recent studies with Chlamydomonas revealed that the mechanism of light-induced state transitions varies from temperature-prompted state transitions. Under elevated temperatures, the decreased carbon fixation process at the PS-I acceptor side causes over-excitation of PS-II, reducing the plastoquinone (PQ) pool. This PQ pool reduction phosphorylates LHC-II by activating STT7/STN7 kinase. In the dark at high temperatures, phosphorylated-LHC-II detaches from PS-II and does not bind to any photosystems, but on recovery, a fraction of P-LHC-II binds to PS-І. In the light-induced state transition, the phosphorylated LHC-II of PS-II immediately detaches and binds to PS-I. Other aggregates, such as PS-II-LHC-II-PS-I and PS-I-Cytb₆f, were also observed during light-induced state transition. P-LHC-II allocation during state transition might prevent photo-oxidative damage from combined high light and temperature intensities [55]. Recent reports with Chlamydomonas disclosed that high temperature (40ºC) and light accelerate the formation of the PS-I cytochrome b₆f complex and elevate cyclic electron flow (CEF) activity. This CEF maintains the equilibrium of ATP and NADPH, develops a proton motive force, and safeguards both PS-I and PS-II from photo-oxidative damage [34].

Temperature Versus Carbon Dioxide Fixation and Acclimatory Responses

The reduced rate of photosynthesis during heat stress is more likely due to reduced capacities of downstream reactions rather than damage to PS-II. One of the downstream reactions affected was the Calvin cycle/ photosynthetic carbon reduction cycle. In the aquatic environment, increasing temperatures cause the upper mixed layer of water to become shallower. This results in an increased mean flux of PAR (photosynthetically active radiation) and UV-B, decreasing the supply of nutrients, including inorganic carbon [26]. A sufficient concentration of carbon source is necessary for a successful Calvin cycle. To avoid CO₂ deficiency, aquatic plants and microalgae actively uptake C_i (CO₂ and HCO₃^-) and transport them to the pyrenoid, which is rich with the RuBisco enzyme, through CCM. This active intake and transport is accelerated by the action of Ci transporters and carbonic anhydrases (CA) [23, 28, 65]. Hence, photosynthetic organisms are scheduled to have adequate CCM activity to a greater extent for effective photosynthesis [65]. Increased temperatures boost the CO₂ unavailability in the local environment and subsequently decrease the RuBisco affinity to CO_2, resulting in reduced photosynthetic efficiency [66]. Due to the C_i fluctuations caused by high temperatures, microalgae have to regulate the CCM accordingly to maintain balanced C_i levels and photosynthetic efficiency.

The RuBisco/RuBisco activase is a highly thermolabile enzyme complex, and its affinity for CO₂ decreases at high temperatures [21]. Work with spinach reports that an 80% reduction in CO₂ fixation rate under heat stress occurs due to the thermolabile nature of RuBisco [37]. However, a recent report discloses that RCA1 protein interacts with Hsp22E/F to support optimal RuBisco activity during high temperatures [47]. Because RuBisco is heat sensitive, heat shock proteins detect the unfolded proteins and fold them during stress as a primary protective mechanism to safeguard the activity of RuBisco and the subsequent CO₂ fixation cycle.

Carbonic anhydrases are crucial metalloenzymes that catalyze the interconversion of CO₂/HCO₃^-. These enzymes aid in the rapid supply of carbon dioxide and bicarbonate for various metabolic pathways such as photosynthesis; they are also involved in ion exchange, pH regulation, and C_i diffusion (carboxylation) [67]. During the daytime, due to high irradiance and high temperatures above 45ºC, plants face dual stress, leading to the fall of pH up to 5.5 in the thylakoid lumen, inhibiting PS-II activity [68]. In Chlamydomonas, maintenance of a functional CCM depends on the pH gradient that occurs during the light reactions. Thus, the formed pH gradient results in the basic stroma with pH 7.5-8.0, while the thylakoid lumen becomes acidic with pH 5.8-6.5. This acid pH range in the thylakoid lumen favors the CA to convert HCO₃^- (C_i) to CO₂ [67]. Hence, the occurrence of low pH in the thylakoid lumen during high temperatures of up to 40ºC probably supports CCM by increasing the CA activity for sustainable and efficient photosynthesis. Further research is needed to understand how various environmental stresses, such as CO₂, temperature, light, pH, etc., regulate the CAs.

As discussed earlier, light reaction and the processes of CO₂ fixation work together in an interconnected manner. PS-II oxygen evolutionary machinery exhibited a close association with carbonic anhydrase activity and bicarbonate ions. HCO₃^- is said to be one of the ionic cofactors involved in splitting water molecules. This anion is produced by PS-II from H₂O and CO₂, confirming that PS-II has the CA activity. Which component of PS-II involved in CA activity was analyzed by immunoblotting assay? This assay revealed that the CA gene sequence that migrated on SDS PAGE was positioned equally to the OEC33, also known as the 33 KDa extrinsic protein/manganese-stabilizing protein. So, from the above work, it can be concluded that the source of PS-II CA activity would be the OEC33. This is evidenced by analyzing the CA activity of purified OEC33 protein in Escherichia coli cells. These cells lack CA activity in the presence of Zn²⁺ while exhibiting CA activity in the presence of Mn²⁺. Since the manganese ion is an active site of OEC, PS II-OEC is proven to possess CA activity. The CA activity that was noticed here could be the thylakoid lumen protein CAH3 because this CAH3 protein is said to be associated with the donor side of the PSII complex, and it is essential for the stability of the Mn cluster and WOC [1]. The bicarbonate ion released by the enzymatic action of CA activity of CAH3 protein accelerates the formation of Mn clusters to attain stability. OEC33/OEC is highly heat stable; it can still reengineer the oxygen evolution after heating to 50ºC for 15 min [69]. The major action noticed was that even at these high temperatures, CA activity was not less, yet it became higher after heating than before [70]. This increased CA at high temperatures might be due to decreased pH levels in the thylakoid lumen. The acidic range of 4.5-5.7 supports the oxygen-evolving activity [71]. Although pH levels decrease at high temperatures, the oxygen-evolving activity remains active in acidic conditions, as noted above. This consistent activity supports the stable CA activity of PS-II. As evidence for the above statement, system-wide analysis of mixotrophically grown Chlamydomonas cells during acute temperatures up to 40ºC exhibits that most of the transcripts of CCM were upregulated as a consequence of maintaining the sufficient amount of CO₂ concentration at the site of RuBisco in the pyrenoid [34]. Henceforth, at high temperatures (38 and 40ºC), the acclimatory responses, such as stabilized PS II-OEC activity and increased CA activity in the thylakoid lumen, manage stable CCM and increase the CO₂ levels at RuBisco to maximize the photosynthetic efficiency because the availability of CO₂ decreases with increased temperatures. In Chlorella species, the DIC-dependent photosynthetic O₂ evolution rate (i.e., the capacity of DIC utilization) and the value of Pmax were greatly enhanced in the M4 (thermo-tolerant) mutant grown under 40ºC, 200 μmol/m²/s⁻¹, and 6% CO₂ conditions compared to the wild type (exhibiting decreased PS-II activity). This enhanced high thermotolerant mechanism is probably due to the gene mutations in their related components, such as genes involved in enhanced Pmax value (enlarged PS-II number) and CO₂ fixation process (the function of CO₂ utilization and Calvin cycle) under elevated temperatures [72].

This review highlights the various acclimatory responses that play an important role at each stage of the photosynthetic carbon fixation process, enabling stable photosynthesis during high temperatures. More work is needed to understand how these processes are interrelated and to identify the genes involved in acclimatory responses to abiotic stresses such as light, temperature, and CO₂ levels.

Future Perspectives

Chlamydomonas is a suitable model organism for studying the system-wide changes that occur during various environmental stresses. Recent advances at the molecular level have provided significant insights into the mechanistic detail of heat stress response and CCM. However, there is a lack of detailed information regarding carbon uptake, transport, and other cellular metabolic interactions at different temperatures. Therefore, further research is needed to provide a comprehensive understanding that could address this gap. Below are some questions that future research should aim to answer: