Comparison of the intrinsic disorder propensities of the RuBisCO activase enzyme from the motile and non-motile oceanic green microalgae

ABSTRACT

Green oceanic microalgae are efficient converters of solar energy into the biomass via the photosynthesis process, with the first step of carbon fixation in the photosynthesis being controlled by the enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO), which is a large proteinaceous machine composed of large (L, 52 kDa) and small (S, 12 kDa) subunits arranged as a L₈S₈ hexadecamer that catalyzes the formation of 2 phosphoglyceric acid molecules from one ribulose 1,5-bisphosphate (RuBP) molecule and one of carbon dioxide (CO₂) and that is considered as the most abundant protein on Earth. The catalytic efficiency of this protein is controlled by the RuBisCO activase (RCA) that interacts with RuBisCO and promotes the CO₂ entrance to the active site of RuBisCO by removing RuBP. One of the peculiar features of RCA is the presence of functional disordered tails that might play a role in RCA-RuBisCO interaction. Based on their ability to move, microalgae are grouped into 2 major class, motile and non-motile. Motile microalgae have an obvious advantage over their non-motile counterparts because of their ability to actively migrate within the water column to find the most optimal environmental conditions. We hypothesizes that the RCA could be functionally different in the non-motile and motile microalgae. To check this hypothesis, we conducted a comparative computational analysis of the RCAs from the representatives of the non-motile (Ostreococcus tauri) and motile (Tetraselmis sp. GSL018) green oceanic microalgae.

KEYWORDS:

• green oceanic microalgae
• intrinsically disordered proteins
• photosynthesis
• protein-protein interactions
• protein function
• protein structure
• RuBisCO
• RuBisCO activase

Introduction

Green algae photosynthesis

Green algae are eukaryotic organisms that obtain chemical energy (is stored in carbohydrate molecules) from energy of light via photosynthesis, the process described by the following general reaction:$6\mathrm{C}{\mathrm{O}}_2+6{\mathrm{H}}_2\mathrm{O}+\mathrm{l}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\to {\mathrm{C}}_2{\mathrm{H}}_{12}{\mathrm{O}}_6+6{\mathrm{O}}_2,$where C₆H₁₂O₆ is glucose, which is subsequently transformed into other sugars, cellulose, lignin, etc. Green microalgae are typically more efficient than higher plants in transforming solar energy to biomass because of their simple cellular structure, fast growth rates, and abundance of nutrients and CO₂ dissolved in water.¹ In eukaryotes, photosynthesis occur in the chloroplasts in the 2 set of reactions: light-dependent reaction (light reaction) that occurs specifically in the thylakoid membrane through photosystem I and photosystem II, and the Calvin cycle (dark reaction) that occur in the stroma (see Fig. 1). During the light dependent reaction, adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate reduced form (NADPH) are formed:

Figure 1. Oversimplified schematic representation of the photosynthesis process (light reaction and Calvin cycle) that also shows a central role of RuBisCo in carbon fixation.

$2{\mathrm{H}}_2\mathrm{O}+2\mathrm{N}\mathrm{A}\mathrm{D}{\mathrm{P}}^{+}+3\mathrm{A}\mathrm{D}\mathrm{P}+3\mathrm{P}\mathrm{i}+\mathrm{l}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\to 2\mathrm{N}\mathrm{A}\mathrm{D}\mathrm{P}\mathrm{H}+2{\mathrm{H}}^{+}+3\mathrm{A}\mathrm{T}\mathrm{P}+{\mathrm{O}}_2.$

Then, NADPH and ATP are released in the stroma to fuel the Calvin cycle, which consist of series of reactions to produce Glyceraldehyde 3-phosphate (G3P):$3\mathrm{C}{\mathrm{O}}_2+9\mathrm{A}\mathrm{T}\mathrm{P}+6\mathrm{N}\mathrm{A}\mathrm{D}\mathrm{P}\mathrm{H}+6{\mathrm{H}}^{+}\to {\mathrm{C}}_3{\mathrm{H}}_7{\mathrm{O}}_6\mathrm{P}+9\mathrm{A}\mathrm{D}\mathrm{P}+8{\mathrm{P}}_{\mathrm{i}}+6\mathrm{N}\mathrm{A}\mathrm{D}{\mathrm{P}}^{+}+3{\mathrm{H}}_2\mathrm{O}.$

Although, Calvin cycle is also known as light independent reaction or dark reaction, this process occurs only in presence of light. The Calvin cycle consist of 3 major steps: carbon fixation, reduction, and, regeneration of ribulose-1,5-bisphosphate, a 5-carbon sugar (RuBP). The first major step of carbon fixation in the photosynthesis process occurs when the enzyme Ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the carboxylation of RuBP. This enzyme is one of the most abundant protein on Earth and is considered by many as one of the most essential factors for all life on earth. It is composed of the large chain (L, ∼55,000 Da) and the small chain (S, ∼13,000 Da) subunits arranged as a L₈S₈ heterohexadecamer (∼540,000 Da). To illustrate an intricate packing of the RuBisCO subunits, Fig. 2 represents half of this large protein complex (PDB ID: 4MKV). RuBisCO is active when a lysine residue inside of its active site is carbamylated (a proton is lost from the amino group and the change in composition is NHCO) and bind a Mg²⁺ ion.²

RuBisCO and RuBisCo activase (RCA)

Analysis of the concentrations of intermediates found in Calvin cycle combined with the calculations of free energy differences of reactions included in this cycle and evaluation of the control coefficients of the enzymes participating in this pathway provided strong evidence that RuBisCo might represent the main limitation on photosynthesis in C₃ plants (which are the most common and the most efficient at photosynthesis in cool, wet climates) and algae at limiting CO₂ concentrations and saturating irradiance.^3-6 However, RuBisCO is rather inefficient enzyme and is rapidly self-inactivated under the physiological conditions, because its active site can undergo spontaneous decarbamylation (loosing CO₂) and strongly binds RuBP or any of the sugar inhibitor analogs such us 3-ketoarabinitol-1,5-bisphosphate (KABP), D-xylulose-1,5-bisphosphate (XuBP), D-glycero-2,3-pentodiulose-1,5-bisphosphate (PDBP), 2-carboxytetritol- 1,4-bisphosphate (CTBP), and 2-carboxy-D-arabinitol 1-phosphate (CA1P). Therefore, the rate of CO₂ fixation and the rate of photosynthetic electron transport depend on the RuBisCO regulation.⁷ As a result, RuBisCO is a highly regulated machine used to control carbon fixation reaction in response of environmental fluctuations.⁸

RuBisCO activase (RCA) is an enzyme responsible for the RuBisCO activation by uncoupling RuBisCO and RuBP interaction or releasing the bound RuBP or its analog from RuBisCO.⁹ It allows the rapid formation of the critical carbamate in the active site of RuBisCO by binding the activator CO₂.¹⁰ It is believed that the presence of RCAs defines the RuBisCO ability to function at its maximal capacity in sub-optimal CO₂ concentration that would normally not permit carbamylation in vivo.¹¹ Although microalgae and cyanobacteria contain a CO₂-concentrating mechanism and therefore can efficiently increase CO₂ concentration,(Aizawa and Miyachi, 1986) the presence RCA in these organisms was shown to be crucial to sustain high-CO₂ stress, which dramatically increased expression levels of this protein.¹² Fig. 3 provides a schematic representation of the RuBisCO activation by RCA.¹³ Only the active form of RuBisCO is capable of catalyzing CO₂ fixation, and the proportion of RuBisCO that is active depends on RCA effectiveness in removing bound RuBP.¹⁴

Figure 2. Crystal structure of the half of the RuBisCO hexadecamer (L₄S₄ complex) from Pisum sativum (PDB ID: 4MKV). To illustrate an intricate packing of the RuBisCO subunits, top (A), side (B), and bottom views (C) of this L₄S₄ complex are shown.

Figure 3. Schematic representation of the RuBisCO activation by RCA (modified from ref. 13). In this scheme, a RuBisCO active site that has spontaneously lost CO₂ (was decarbamylated) binds a sugar phosphate RuBP, causing RuBisCO to undergo conformational changes leading to the inactivation of this protein. RCA interacts directly with the inactive RuBisCO, altering its conformation. ATP hydrolysis by RCA is required for these conformational changes. These conformational changes allow RuBP to dissociate and the CO₂ molecule to enter the active RuBisCO site.

Mechanistically, RCA-driven release of the RuBisCO inhibition is based on the induction of ATP-dependent (RCA is a member of the AAA+ family of proteins, which are ATPases associated with diverse cellular activities) conformational changes that open closed RuBisCO sites, increase their accessibility to solvents, and facilitate dissociation of inhibitory sugar phosphates.¹⁵ Transition of the RuBisCO structure from the closed to the open state involves rotation of the N-terminal domains that can proceed only in an RCA- RuBisCO super-complex comprising the RuBisCO holoenzyme with its 8 active sites encircled by 16 (or possibly 8) RCA subunits.¹⁵ The torque needed to move the N-terminal domains of RuBisCO is provided by the concerted movement of the RCA subunits, the energy for which is provided by ATP hydrolysis.¹⁵

The RCA activity is regulated by the ADP/ATP ratio in the stroma and by the ambient redox status.⁸ In other words, the activity of RCA is principally dependent on the light intensity variation and the movement of protons into thylakoids that define the changes in the relative amounts of active and inactive forms of the enzyme. In addition, RCA activity can also be affected by high temperature, because it has been shown that RCA aggregates at high temperatures and can no longer activate RuBisCO diminishing carboxylation capacity.¹⁶

Intrinsically disordered regions (IDRs) and RuBisCO activase (RCA)

Most of the eukaryotic proteins contain both structured and disordered regions. Intrinsically disordered regions (IDRs) are polypeptide segments that lack a defined 3D structure, existing as highly dynamic ensembles of flexible conformations,^17-24 and that have numerous important biological functions.^19,20,24-34 The high flexibility, decreased content of hydrophobic residues, and increased content of polar and charge residues (A, E, G, K, P, Q, R, and S) in the disordered regions allow them to interact with more than one partner and facilitate their multiple contacts. This ability to bind various partners makes intrinsically disorder proteins (IDPs) and IDRs important regulators capable of modulating the activity of several proteins in a coordinated way, playing an important role in various regulatory pathways.^23,35-38

The number of studies dedicated to the discovery and characterization of IDPs and IDRs has increased exponentially in the last years.²³ However, little is currently known about intrinsic disorder status of proteins in photosynthetic organism. The most recent studies of protein disorder in plants have shown that IDPs play central role in many responses such as stress (biotic and abiotic), development and metabolism regulation.^39-42 Yruela and Contreras-Moreira (2012) analyzed 12 plants genomes and concluded that the nuclear-encoded proteins follow the same trend that other multi-cellular eukaryotes and show a higher disorder in the internal part of nuclear-encoded proteomes rather than at their extremities, in contrast to the chloroplast- and mitochondrion- encoded proteomes.⁴³

RuBisCO activase (RCA) might be present in 2 different isoforms: α-isoform and β-isoform in photosynthetic organism,⁴⁴ but not all species appear to have both isoforms (e.g., Nicotiana tabacum has one RCA isoform).^45-47 Fig. 4 shows low resolution 3D-structure of the Nicotiana tabacum RCA complex obtained by electron microscopy (PDB ID: 3ZW6).⁴⁸ The α-isoform differ from the β-isoform by the presence of a short C-terminal disordered extension that in most cases contains 2 cysteine residues, and is light dependent.⁴⁹ It was also pointed out that of these 2 isoforms, the longer RCA variant possesses higher ADP/ATP sensitivity,⁵⁰ but this high sensitivity is mostly eliminated via thioredoxin-f-induced reduction likely leading to the formation of a disulfide bridge between the C-terminal cysteine residues.⁴⁹ Therefore, although both RCA isoforms are capable of activating RuBisCO, only the large isoform is redox regulated.⁴⁹ This opens an intriguing possibility that changes in the expression pattern of RCAs (α-isoform vs. β-isoform) might represent a specific means to control and regulate the activity of activase. In other words, it is likely that the alternative splicing of RCA could serve as a means for the carbon assimilation optimization. ⁵¹ This hypothesis is partially supported by the results of a recent analysis of the relationships between RuBisCO activity, the expression of 2 RCA splicing variants, photoacclimation, and pre-hardening (15°C) and cold acclimation (4°C) of Lolium perenne genotypes with contrasting levels of freezing tolerance to low temperature.⁵¹ This study revealed that only a highly freezing-tolerant genotype showed an induction of RuBisCO activity at both 15°C and 4°C. Curiously, noticeably enhanced RuBisCO activity after pre-hardening correlated with the increased expression of the long splicing variant (α-isoform) of RCA, whereas during cold acclimation, RuBisCO activity was increased due to the expression of both alternatively spliced variants.⁵¹

Figure 4. Low resolution 3D-structure of the Nicotiana tabacum RCA complex obtained by the electron microscopy (PDB ID: 3ZW6).⁴⁸

Zhang et al. (2001) indicated that RCAs are functional heteromers (α_nβ_n) and not homomers (α_n or β_n),⁴⁶ and Stotz et al. (2011) who worked with Nicotiana tabacum RCA found that the active state of this protein is a hexamer, where the helical insertion of the C-terminal is implicated in RuBisCO recognition and the Loop segments exposed toward the central pore of the hexamer required for the ATP-dependent remodeling of RuBisCO.⁴⁸ Based on this observations, Thieulin-Pardo et al. (2015) proposed a model for RCA in for A.thaliana.¹³ They indicated that RCA is the α₃β₃ heterohexamers, and that one of its flat faces is involved in interaction with RuBisCO, whereas an exposed loop of the RuBisCO protein could fit into the central hole of RCA.¹³

Motile microalgae have the ability to migrate vertically in the water column in order to exploit nutrients sources, to avoid predators, low and high irradiance, UV and energetic turbulence (temperature, sediments, etc.) near the surface.^52,53 The mobility allow them to adjust for optimal irradiance and maximize photosynthetic efficiency offering potential advantages over passive microalgae (non-motile) that are also exposed to all the aforementioned factors.⁵² Therefore, motility in microalgae can be seen as an indirect mechanism that optimize irradiance condition for the photosynthesis. In this sense, non-motile microalgae can be more exposed to higher and lower levels of irradiance than the motile microalgae. It is likely that RuBisCo might be differently regulated in motile and non-motile algae. Although such regulation could act on many different levels (ranging from different expression of key metabolic enzymes (such as RuBisCo itself and other enzymes participating in Calvin cycle) to different regulation of transporters, etc.), we hypothesizes that the RCA, which is responsible for the RuBisCO activation through physical interaction with the disorder regions of this proteinaceous machine and whose activity is determined by the light irradiance (as well as ATP/ADP ratio and the ambient redox status), could be functionally different in the non-motile and motile microalgae. Therefore, there must be differences in the sequences, amino acid compositions, and content of functional intrinsic disorder in RCAs from these 2 microalgae.

Materials and methods

To test our hypothesis, 2 oceanic green microalgae were selected 2 species, Tetraselmis sp GSL018 and Ostreococcus tauri (O. tauri) that belong to the chlorophyta phylum. Tetraselmis sp. GSL018 is a eukaryotic unicellular flagellate microalgae that belongs to the Chlorodendraceae class, has 4 flagella, possesses phototaxis (light-dependent behavior, either toward the source of light or away from it),⁵⁴ and is of ecological and economic importance. On the other hand, O. tauri is the smallest unicellular non-motile eukaryotic microalgae known, and is the main component of the phytoplankton.⁵⁵ Aligned amino acid sequences of the corresponding RCA proteins are shown in Fig. 6C.

Figure 5. Intrinsic disorder profiles of the RCA from O. tauri (A) and Tetraselmis sp. (B) generated by PONDR-FIT, PONDR® VLXT, PONDR® VSL2, and PONDR® VL3, IUPred_short, IUPred_long, DISOPRED3, and RONN v3.2. Bold dark pink dashed line shows the mean disorder propensity calculated by averaging disorder profiles of individual predictors. Light pink shadow around the PONDR® FIT curves shows error distribution. The amino acids in the input sequence are considered disordered when the corresponding disorder propensity curve is located above the 0.5 threshold.

Figure 6. (A) Aligned mean disorder profiles of the RCA proteins from O. tauri (solid black line) and Tetraselmis sp (dashed red line). Gaps in the corresponding curves correspond to the insertion found via ClustalW-based sequence alignment (see plot C). (B) Aligned ANCHOR profiles of the RCA proteins from O. tauri (solid black line) and Tetraselmis sp. (dashed red line). Corresponding outputs of DISOPRED3 for the position of disordered binding regions are shown at the top of the plot by light gray and pink curves for the O. tauri and Tetraselmis sp TCAs, respectively. (C) Sequence alignment of the RCAs from O. tauri (query sequence of 407 residues) and Tetraselmis sp. (subject sequence of 416 residues) by ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Although these 2 proteins show high overall sequence identity, the sequence similarity is low at their termini, where the IDRs are located. This is especially the case for the N-termini of these RCAs.

Intrinsic disorder propensities of target proteins were evaluated using 4 algorithms from the PONDR family, PONDR-FIT, PONDR® VLXT, PONDR® VSL2, and PONDR® VL3,^56-61 as well as the IUPred web server,⁶² DISOPRED3,⁶³ and RONN v3.2.⁶⁴ For each protein, after obtaining an average disorder score by each predictor, all predictor-specific average scores were averaged again to generate an average per-protein intrinsic disorder score. Use of consensus for evaluation of intrinsic disorder is motivated by empirical observations that this approach usually increases the predictive performance compared to the use of a single predictor.^65-67

Potential disorder-based protein binding sites of query proteins (molecular recognition features, MoRFs) were identified by the ANCHOR algorithm.^68,69 This algorithm utilizes the pair-wise energy estimation approach originally used by IUPred^62,70 and acts on the hypothesis that long regions of disorder include localized potential binding sites which are not capable of folding on their own due to not being able to form enough favorable intrachain interactions, but can obtain the energy to stabilize via interaction with a globular protein partner.^68,69 We also used an SVM based classifier incorporated into the DISOPRED3 to identify IDRs that are predicted as protein binding sites based on their profile data and additional sequence-derived features.⁶³

Results and discussion

The molecular weights of the RCAs from O. tauri (407 amino acids) and Tetraselmis sp (416 amino acids) were 44.5 KDa and 45.3 kDa, respectively. RCAs are globular proteins that possess disordered tails.¹³ Based on the results obtained by the sequences analysis using a wide range of disorder predictors (PONDR-FIT, PONDR® VLXT, PONDR® VSL2, and PONDR® VL3, IUPred_short, IUPred_long, DISOPRED3, and RONN v3.2) and the mean disorder propensity calculated by averaging disorder profiles of individual predictors, the 2 RCAs analyzed in our study are characterized by different disorder propensities (estimated as percentage of intrinsically disordered residues). In fact, Fig. 5 shows that the percentage of intrinsically disordered residues in the sequence of the Tetraselmis sp RCA and its protein-average disorder scores are a bit higher than those of the O. tauri protein. This idea is further illustrated by Fig. 6A that shows the aligned mean disorder profiles of these 2 proteins. In fact, based on the analysis of disorder profiles shown in Fig. 6A, the Tetraselmis sp. RCA has 21.9% disordered residues and is characterized by the protein-average disorder score of 0.36±0.09, whereas the RCA from O. tauri contains 17.9% disordered residues and has the protein-average disorder score of 0.32 ± 0.08.

The intrinsic disorder profiles of RCA for O. tauri and Tetraselmis sp are shown in Figs. 5 and 6. Profiles clearly show that the RCA enzymes from both microalgae possess IDRs at their extremities, suggesting that the N- and C-termini of these proteins can be considered as disordered tails. Additionally, the RCA of Tetraselmis has a short disordered region at the central part of its sequence. For both RCAs, the C-terminal tails are noticeably shorter than corresponding N-tails. Since the C-terminal regions of these 2 RCAs do not have cysteine residues, these proteins cannot be considered as α-RCA isoforms, which, according Zhang and Portis classification,⁴⁹ should contain 2 cysteines within the C-termini. It is known that the functionality of both α- and β-isoforms of RCAs depends on the ADP/ATP ratio. However, it has been also demonstrated that α-RCAs is additionally dependent on the light because of the thioredoxin action (redox response) on its 2 cysteine residues present on its C-terminal. Curiously, not all species appear to have both isoforms, and only one RCA was found in tobacco, maize, and the fresh water microalgae Chlamydomonas.^45,46

Figure 6C represents results of the ClustalW-based sequence alignment of RCAs from O. tauri and Tetraselmis sp The sequence alignment showed a 68% identity, which indicates high structural and functional similarity of these proteins. However, despite this high overall identity levels, Fig. 6C shows that the intrinsically disordered N- and C-termini of these proteins are characterized by low sequence similarity.

According to Thieulin-Pardo et al. (2015),¹³ RCAs probably acquired their disorder regions during evolution to gain new regulation opportunities, such as enhanced responsiveness to the environmental factors and/or the ability to be engaged in new protein-protein interactions. In addition, a positive correlation has been found between the increase of disorder content and the increase in the biological complexity during evolution.^63,65,71-75 The microalgae O. tauri is the smallest and simplest photosynthetic eukaryotic organism described until now, which belongs to the earlier branch of the chlorophyte (Prasinophyceae), whereas Tetraselmis sp. belongs to the Chlorodendrophyceae branch. Therefore, it is possible that this differences in the total content of predicted intrinsic disorder in the RCAs of these microalgae and low sequence similarity of their N- and C-tails can be due to the evolutionary diversification of these proteins, leading to the development of different regulatory responses or different modes of protein-protein interactions.

To check this hypothesis, we evaluated the presence of disorder-based binding sites in the RCA proteins from 2 microalgae. Results of this analysis are shown in Fig. 6B where the outputs of ANCHOR algorithm and functional annotation of DISOPRED3 are compared. According to ANCHOR, the O. tauri RCA has an N-terminally located 12 residue-long disorder-based binding site (residues 12-23) and a short binding site within the C-terminal tail (residues 385-386). The RCA from Tetraselmis sp has a short N-terminal binding site (residues 1-4) followed by 16 residue-long binding site (residues 18-33) and another short disorder-based binding site located at the 205-207 region (see Fig. 6B). DISOPRED3, which generates precise assignments of disordered protein binding regions,⁶³ indicated that both, the RCAs have 5 disordered binding regions, residues 1-23, 34-38, 45-58, 392-401, and 403-407 in the O. tauri protein and residues 3-5, 21-23, 245-250, 401-404, and 407-409 in the Tetraselmis sp. RCA.

Providing annotations of IDRs, secondary structure elements, and binding sites, DISOPRED3 gives a possibility to compare distribution of these features within the query proteins. Fig. 7 shows the differences between the DISOPRED3 profiles of the RCA from both microalgae. Particularly, Figs. 6B and 7 show that although the N- and C-tails of both RCAs contain disorder-based protein binding sites and although the overall level of predicted disorder is somewhat higher for the Tetraselmis RCA, the amount of binding-related disorder is higher for the O. tauri protein than for the RCA from Tetraselmis sp Comparing ANCHOR and DISOPRED data for these 2 proteins it is clear that in addition to the N- and C-terminal disorder-based binding sites the Tetraselmis protein has extra binding regions located within its central part. These differences in distribution of the disorder-based binding sites probably indicate that the ways in which the protein-protein interactions occur between the RCAs and their partners are different in different microalgae.

Figure 7. DISOPRED3 outputs for the RCA proteins from O. tauri (A) and Tetraselmis sp (B).

According to Parry et al.⁸ the C-terminus of RCA is important for the RuBisCO recognition and for the ATP hydrolysis, whereas the N-terminus of this protein is necessary for RuBisCO activation.⁸ However, the functional role and potential targets of the short disordered binding region at the central part of the Tetraselmis RCA are unknown as of yet. The presence of a remarkable sequence diversity in functional N- and C-terminal regions of these RCAs provides a support to the RuBisCO activation is carried out in different ways by these microalgae because the RCA binding disorder residues that interact with RuBisCO are not the same. Also, since C-tails of analyzed RCAs from both algae do not have characteristic cysteine residues, it is likely that these activases are not redox-sensitive.

Tetraselmis sp. has positive phototaxis (movement toward the light source) at lower light intensity and negative phototaxis (movement away from light source) at higher light intensity.⁵⁴ This kind of response is observed in Tetraselmis because it has a photoreceptor apparatus (stigma) that perceive a modulated light signal from the environment, which controls flagellar beat frequency and direction until the cell is aligned with the incoming light rays.⁷⁶ Tetraselmis motility and photosynthesis rate had shown to be conditioned by the quality and quantity of radiation (light intensity), so for this microalgae both process photosynthesis and motility are tightly coupled.⁵⁴ In other words, being a representative of the motile microalgae, Tetraselmis sp can actively search for the most optimal conditions of its growth, such as sufficient nutrients sources, optimal irradiance, temperature, etc. On the other hand, O. tauri is a passive (non-motile) microalgae and therefore has to use motility- and migration-unrelated means to adjust to changes in its environment. Therefore, it is expected that these 2 microalgae would utilize different approaches to maximize their photosynthetic efficiency. It is likely that one of the means to control the photosynthetic efficiency in motile Tetraselmis sp. is linked to its ability to actively migrate to the most optimal and “comfortable” environment. This, actually, decreases evolutionary pressure on the optimization of the RCA-driven regulation of RuBisCo activity (even a relatively lousy farmer can have great harvest if the soil is rich). On the contrary, the non-motile microalgae O. tauri likely to experience strong evolutionary pressure to optimize its RCA-RuBisCO system for maximal efficiency under the uncontrolled and likely non-optimized irradiance condition for the photosynthesis (if soil is poor, the ability to get a good harvest depends on specific skills of a farmer). In line with this hypothesis, we found several small, but important differences between the intrinsic disorder predisposition and presence and distribution disordered binding regions in RCAs from O. tauri and Tetraselmis sp Collectively, the differences in the total percentages of intrinsic disorder, in the presence of disordered biding and non-binding regions, and in the amino acid compositions of the IDRs of these 2 proteins, although been small, suggest that RCA, this important enzyme responsible for the RuBisCO activation in both microalgae, probably utilizes different modes of interaction with RuBisCO. This disorder-based functional difference is most likely due to the differences in the degree of variability in the irradiance conditions to which these microalgae are exposed as a consequence of their motility behavior.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.