Evolutionary biologists estimate that it was 1.5 billion years ago when plastids started to develop as the result of the engulfment of a cyanobacterium by a heterotrophic eukaryotic cell (Yoon et al., 2004). Later, it is widely accepted, this endosymbiosis process gave rise to chloroplasts in plants and algae (reviewed by Stadnichuk and Kusnetsov, 2021). The evolution of this endosymbiosis resulted in a drastic genome reduction of the originally engulfed cyanobacterium; 0.15 Mbp presently in chloroplasts vs. 3 Mbp in free-living cyanobacteria (Martin et al., 2002; Yagi and Shiina, 2014). While thousands of genes disappeared, most likely as the result of being redundant with the ones already present in the host, others were transferred and integrated into the host nuclear genome (Bock, 2017; De Marchis et al., 2019). As a result of this genome reduction, 2–5% of the genes found in free-living cyanobacteria remain in modern chloroplasts (Eckardt, 2006; Stegemann and Bock, 2006; Yagi and Shiina, 2014). This gene interchange is also made evident by the fact that 3.5–6% of protein-coding genes in the nuclear genome of Chlamydomonas and other Plantae have a cyanobacterial origin (Moustafa and Bhattacharya, 2008). This genetic rearrangement ultimately led to the chloroplasts as we know them today: semi-autonomous organelles that contain their own genome and the expression machinery for many genes involved in protein synthesis (including tRNAs and rRNAs) and photosynthesis (including proteins involved in light capture, CO₂ fixation, and ATP synthesis).

It has been estimated that around 93–99% of proteins functioning inside chloroplasts are encoded in the nucleus (Woodson and Chory, 2008), translated in the cytosol, and then transported into the chloroplast (Nakai, 2018). In photosynthetic organisms, the subunits of all photosynthetic complexes, the translation apparatus, some proteins of the chloroplast envelope, and the key enzyme in carbon fixation, RuBisCo, are encoded by genes located in both the nuclear and chloroplast genomes (Börner, 2017). For example, in Chlamydomonas reinhardtii, Photosystem I (PSI) is composed of 14 proteins, four of which are encoded in the chloroplast (PsaA, PsaB, PsaC, and PsaJ subunits) and the other 10 are nucleus-encoded (Harris, 2009). The assembly of the protein complexes of the photosystems I and II, the ATP synthase, cytochrome b₆ f complex, and RuBisCo is regulated by the control by epistasis of synthesis (CES) process, dependent on the regulation of translation. The synthesis of one subunit becomes dependent on the synthesis of another: when certain core subunits are absent, synthesis of other chloroplast-encoded subunits from the same protein complex is reduced (Choquet et al., 1998; Choquet and Wollman, 2009). Similarly, the cofactors that are accumulated in excess of their binding proteins can regulate their own synthesis and that of the proteins (Pogson et al., 2008). The expression of the divided encoded genetic information requires coordinated communication between chloroplast and nucleus to maintain cell homeostasis.

Cellular processes such as chloroplast biogenesis (reviewed by Jarvis and López-Juez, 2013; Yoo et al., 2020), plastid differentiation (reviewed by Sadali et al., 2019), photosynthesis including the assembly of photosynthetic apparatus and expression of photosynthetic genes (Lefebvre-Legendre et al., 2015, 2016; Douchi et al., 2016; Krupinska et al., 2019), pigment production (Terry and Bampton, 2019; Ganusova et al., 2020), and other metabolic processes such as starch and lipid biosynthesis, cell wall synthesis and/or modification, sugar transport, stress responses, and reactive oxygen species (ROS) responses are highly dependent on communication between the nucleus and the chloroplast (reviewed by Woodson and Chory, 2008; Börner, 2017; Piñas-Fernández and Strand, 2018). Trans-acting regulatory factors are generated in the nucleus and are essential for transcriptional, post-transcriptional, and translational regulation of chloroplast gene expression (Woodson and Chory, 2008; Piñas-Fernández and Strand, 2018). The regulation from the nucleus to the chloroplast is known as anterograde signaling. In contrast, retrograde signaling is when the signals originate from the organelles (chloroplasts and mitochondria) to influence the expression of nuclear genes, some of which, in turn, can alter the anterograde control (Hernández-Verdeja and Strand, 2018). Synchronization between anterograde and retrograde signaling allows the cell to survive against changes in the environment (Rochaix and Ramundo, 2017). These changes include temperature, light intensity, drought, and nutrient deficiency (Sun and Guo, 2016; Rochaix and Ramundo, 2017). As part of the anterograde signaling, in higher plants and microalgae, many nucleus-encoded proteins are RNA-binding factors for post-transcriptional regulation of gene expression, such as RNA stabilization, splicing, intercistronic processing of polycistronic RNAs, editing of chloroplast transcripts, and regulation of the translation (Lyska et al., 2013; Manavski et al., 2018; Zoschke and Bock, 2018; De Marchis et al., 2019).

RNA-binding proteins involved in post-transcriptional gene expression include the helical repeat protein family. This superfamily comprises the Tetra-tricopeptide repeat (TPR), Half-a-tetratricopeptide repeat (HAT), Penta-tricopeptide repeat (PPR), and Octo-tricopeptide repeat (OPR) proteins. TPR proteins are present in prokaryotic and eukaryotic organisms, where their principal function is the regulation of many cellular processes, including translation, by mediating protein–protein interactions (Bohne et al., 2016). PPR proteins are found in all eukaryotic organisms. In land plants, hundreds of PPR proteins have been identified, many of them even functionally characterized, determining that they are involved in RNA metabolism and translation regulation of organellar genes (Shikanai and Fujii, 2013; Barkan and Small, 2014; Rovira and Smith, 2019). OPR proteins are also found in prokaryotes and eukaryotes and similar functions have been attributed to them; however, although the number of OPR proteins is limited in land plants, in algae it is quite large (Bohne et al., 2009; Rahire et al., 2012).

Due to its ease of cultivation and its relative simplicity, the microalga C. reinhardtii is one of the best model organisms for the study of various cellular processes, including photosynthesis, flagellar function and pigment, biofuel, and recombinant proteins production (Salomé and Merchant, 2019). Furthermore, the study of the mechanisms regulating chloroplast gene expression in C. reinhardtii is crucial for the development of tools for the improvement of the production of biomass and bioactive compounds. In this paper, we first review the role of PPR, TPR (including HAT), and OPR proteins in chloroplast gene expression in C. reinhardtii and then consider the emerging tools being developed for biotechnological applications.

Penta-tricopeptide Repeat proteins are distributed in all eucaryotes, and due to their abundance and relevant functions, these proteins have been extensively studied in plants (Gutmann et al., 2019). These were initially identified in nuclear mutants with non-photosynthetic phenotypes and altered post-transcriptional processes (Fisk et al., 1999). Shortly after, the sequence of the A. thaliana genome revealed a new protein family that comprised hundreds of genes that until that moment had an unknown function (Aubourg et al., 2000). Later, Small and Peeters (2000) named them, PPR Proteins following their structural similarity with the previously described TPR motifs. Interestingly and surprisingly, Lurin et al. (2004) predicted that two-thirds of these proteins are targeted to mitochondria or chloroplasts in Arabidopsis.

These nucleus-encoded proteins are composed of degenerated motifs of 35 amino acids repeated in tandem (Small and Peeters, 2000). The PPR motif forms two antiparallel α-helices, which interact to produce a helix-turn-helix motif (Figures 2A,B). The series of helix-turn-helix motifs form an α-solenoid structure that interacts with nucleic acids and/or with other proteins (Barkan and Small, 2014; Wang et al., 2015). The bioinformatic analysis had predicted that each PPR-motif interacts with one ribonucleotide of their target RNA and that amino acids in precise positions determine the ribonucleotide-binding specificity (Figures 2B–D; Barkan et al., 2012; Shen et al., 2016).

In C. reinhardtii 14 PPR proteins have been identified (Tourasse et al., 2013), but only four have been experimentally characterized (Loiselay et al., 2008; Johnson et al., 2010; Jalal et al., 2015; Cavaiuolo et al., 2017). All four have been determined to be localized in the chloroplast and are involved in the stabilization, maturation, and/or translation of photosynthetic transcripts (Loiselay et al., 2008; Johnson et al., 2010; Jalal et al., 2015; Table 1). The first PPR protein identified in Chlamydomonas was MCA1 (PPR14), this protein binds to 21 nucleotides at the 5′-terminus of the chloroplast petA mRNA, protecting it from degradation by exonucleases and in consequence facilitating the synthesis of cytochrome f (Loiselay et al., 2008). MRL1 (Maturation factor of rbcL; PPR2), was the second PPR protein identified, and it is conserved in C. reinhardtii and Arabidopsis; this protein is involved in regulating RuBisCo large subunit (rbcL) transcript levels (Johnson et al., 2010). MRL1 binds to the 5′ end of the rbcL mRNA, stabilizing and protecting it from exonucleases degradation. Mutants deficient in MRL1 are deficient in RuBisCO large subunit, which results in a lack of the RuBisCO holoenzyme and thus yield a non-photosynthetic phenotype (Johnson et al., 2010; Johnson, 2011). Curiously, Arabidopsis mrl1 mutants still accumulate the unprocessed rbcL mRNA, can synthesize RuBisCo, and grow normally under photoautotrophic conditions. TCB1 (PPR1) is the ortholog of HCF152 from plants. Chlamydomonas mutants of ppr1 are non-phototrophic and the phenotype resembles that of cytochrome b₆f mutants. From small RNA profiling, it has been proposed that the role of PPR1 is mainly as a translation factor, hence the TCB1 name, and that in addition, it stabilizes petB (Cavaiuolo et al., 2017).

In contrast to MCA1 and MRL1, both of which have a single mRNA target, PPR7 protein, which is the most highly expressed PPR protein in Chlamydomonas, participates in regulating the expression of several chloroplast genes (Jalal et al., 2015). PPR7 is part of a ribonucleoprotein complex that contributes to the stabilization of rbcL, rpoC2, psbH, and tscA transcripts (Jalal et al., 2015). Additionally, PPR7 also participates in the processing and maturation of the following polycistronic transcripts: the gene cluster atpA-psbI-cemA-atpH, which encodes for the α-subunit of the ATP synthase; a small PSII subunit, a putative envelope membrane protein involved in inorganic carbon uptake; and the subunit III of chloroplast ATP synthase, respectively (Jalal et al., 2015). PPR7 also participates in the maturation of the co-transcript psbJ-atpI-psaJ-rps12, which encodes for PsbJ (a PSII reaction center protein), the subunit IV of ATP synthase; PsaJ (a PSI reaction center subunit), and the ribosomal protein S12. Additionally, PPR7 participates in processing the 16S rRNA, which is part of the rrn operon that encodes for the 16S, 7S, 3S, 23S, 5S rRNAs, and two tRNAs (trnI and trnA; Jalal et al., 2015). Detailed mechanisms of PPR7 in processing all these polycistronic transcripts are still scarce and unclear, particularly when compared to the level of details known for PPR proteins in plants (Pfalz et al., 2009; Stoppel and Meurer, 2012). A hypothesis is that PPR7 could recruit certain endonucleases or modify large secondary structures in large mRNAs to unmask nuclease sensitive targets. In any case, given the diversity of functions of the regulated genes, PPR7 is essential for the proper functioning of chloroplasts (Jalal et al., 2015).

Like PPR proteins, TPR proteins are helical repeat proteins characterized by the presence of 1–26 tandem repeats, each one of 34 amino acids that form an antiparallel α-helical hairpin (Bohne et al., 2016). The TPR motifs function as a scaffold for protein assembly in multi-subunit complexes and are involved in various processes ranging from transcriptional regulation and RNA metabolism to protein folding, chlorophyll synthesis, and transport (Table 2). Given the diversity of functions, it is not surprising that the location of TPR proteins is not limited to the chloroplast or the mitochondria. Two flagellar subunit proteins with TPR motifs, IFT139 and IFT88, have been identified in C. reinhardtii as part of the IFT-A and IFT-B subcomplex involved in intraflagellar transport (Pazour et al., 2000; Snell and Goodenough, 2009; Behal et al., 2012). IFT88-deficient C. reinhardtii mutants show a phenotype without flagella (Pazour et al., 2000). Because IFT88 is conserved in green algae, nematodes, and vertebrates, the analysis of C. reinhardtii IFT88 mutants has allowed the study of intra-flagellar transport in other species, for example, the study of its homologous genes in mice and humans called Tg737. Mice with Tg737 defects die shortly after birth from polycystic kidney disease (Pazour et al., 2000).

Tetra-tricopeptide repeat motifs can also act as sites for protein–protein interactions; in Chlamydomonas and tobacco, Ycf3, a chloroplasts-encoded TPR protein, is responsible for the initial assembly of PSI (Boudreau et al., 1997). The Ycf3 protein interacts with the PsaA and PsaB subunits forming the PSI reaction center subcomplex to which the Ycf4-module binds to provide stabilization and to facilitate the integration of the other central PSI subunits (PsaC, D, E, F, H, I, J, and L) to form the mature PSI–LHCI supercomplex (Naver et al., 2001; Nellaepalli et al., 2018, 2021). Ycf3 mutants display a light-sensitive phenotype, and they cannot grow photoautotrophically, demonstrating that Ycf3 is essential for photosynthesis. In addition to Ycf3, CGL71, another TPR protein, also participates in the biogenesis and stability of PSI, protecting it from oxidative disruption during its assembly (Heinnickel et al., 2016). FLP and Tic40 also participate in chloroplast biogenesis, the former in the regulation of chlorophyll biosynthesis and the latter as a component of the chloroplast import machinery (Falciatore et al., 2005; Kalanon and McFadden, 2008).

Another example is REP27, a TPR protein located on the thylakoid membrane, which participates in the PSII repair cycle (Park et al., 2007; Dewez et al., 2009). During oxygenic photosynthesis, the abundance of O₂ and the formation of oxidants lead to photo-oxidative damages causing irreversible inactivation of the D1 protein, impairing electron transport, and inhibiting the function of PSII (Park et al., 2007). REP27 participates by facilitating and regulating different stages of D1 biosynthesis de novo. It first acts as a factor for the initiation of psbA translation (D1 protein). Then it facilitates the assembly of the nascent D1 peptide. Finally, REP27 activates the D1 protein through post-translational modifications conferring it its functional state in the PSII complex (Dewez et al., 2009). Thus, REP27 plays a triple role, in the regulation of PSII turnover, assembly, and activation.

Like PPR proteins, TPR proteins are also involved in the metabolism of RNA, and this function has been associated with the HAT (half-a-TPR) motif, a subclass of TPR motifs found in several proteins that stabilize and/or activate the translation of specific chloroplast RNAs (Bohne et al., 2016). Unlike the TPR motif, the HAT motifs are characterized by a distinct pattern of conserved amino acid positions and by the presence of highly conserved aromatic residues, mainly tyrosine and tryptophan (Preker and Keller, 1998). In C. reinhardtii the existence of 10 HAT proteins has been estimated (Table 2; Bohne et al., 2016), from which three have been functionally characterized; NAC2 (also named MBD1; Kuchka et al., 1989, Boudreau et al., 2000; Schwarz et al., 2007), MAC1 (Douchi et al., 2016), and MBB1 (Vaistij et al., 2000a,b; Loizeau et al., 2014). NAC2 participates in the stabilization and processing of the 5′-UTRs of psbD (encoding the D2 subunit of the PSII reaction center), MAC1 for psaC (encoding the Photosystem I iron–sulfur protein), and MBB1 for psbB mRNA (encoding the PSII core antenna CP47). These factors bind to the 5′-UTR end of their target, protecting the transcript from exonucleases degradation. Additionally, MBB1, an orthologue of HCF107 (high chlorophyll fluorescence 107) protein in A. thaliana, participates in the processing of the psbB-psbT-psbH polycistronic transcript (Felder et al., 2001). Before translation, this mRNA must be divided into smaller transcripts, psbB-psbT, and psbH (Vaistij et al., 2000b). MBB1 binds to the 5′-UTR of psbB and psbH and it is speculated that this could recruit an endonuclease or modify the secondary structure of the mRNA exposing the sites for endonuclease cleavage. Once the polycistronic mRNA is cleaved, MBB1 stabilizes the monocistronic transcripts, protecting them from exonucleases (Loizeau et al., 2014).

Octo-tricopeptide repeat proteins are the most abundant helical repeat proteins in green algae. Their structure is characterized by the presence of motifs of 38–40 amino acids repeated in tandem, in the same way as in PPR and TPR proteins, where each motif forms a pair of antiparallel α-helices (Eberhard et al., 2011; Wang et al., 2015). Besides green algae, OPR proteins are present in some pathogenic bacteria (Coxiella burnetii; Rahire et al., 2012), symbiotic protozoa (e. g. Plasmodium falciparum, and Toxoplasma gondii; Hillebrand et al., 2018; Hollin et al., 2021) and the cercozoan amoeba Paulinella chromatophora (Oberleitner et al., 2020); but are rare in land plants. In A. thaliana, the presence of only one OPR protein has been reported, called RAP, which is involved in the processing of the 16S ribosomal RNA chloroplast gene (Kleinknecht et al., 2014). In contrast, analysis in silico has revealed that there are more than 120 members (Goodstein et al., 2012; Boulouis et al., 2015). The abundance of OPR proteins in the nuclear genome of Chlamydomonas shows not only a high prevalence but also a complex regulation of chloroplast gene expression, where these factors play an essential role. In C. reinhardtii, this family of proteins is extensively involved in the post-transcriptional control of chloroplast gene expression (Table 3). The first OPR described in C. reinhardtii was the chloroplast translation factor TBC2 (Auchincloss et al., 2002), which together with the nuclear proteins TBC1 and TBC3 interact with the 5′-UTR end of the psbC mRNA favoring its translation (Zerges et al., 2003). Like TBC2, TAB1 (Stampacchia et al., 1997; Rahire et al., 2012), TDA1 (Eberhard et al., 2011), and TAA1 (Lefebvre-Legendre et al., 2015; Reifschneider et al., 2016) also function as translation factors in the translation of psaB, atpA, and psaA mRNAs, respectively.

In addition to facilitating the translation of specific mRNAs, some OPR proteins can perform additional functions or even carry out dual functions (Eberhard et al., 2011; Ozawa et al., 2020). They can function as M (maturation) factors, protecting their mRNA target from degradation by exonucleases and/or participate in the processing of polycistronic mRNAs (Murakami et al., 2005; Wang et al., 2015; Viola et al., 2019; Ozawa et al., 2020), as well as acting as factors that participate in gene trans-splicing (Reifschneider et al., 2016). The coordinated interaction of these factors on maturation/stability and translation greatly affects organelle function and, consequently, photosynthesis, respiration, and environmental responses. Some of the OPR proteins functionally characterized as M factors comprise the proteins MBI1, MCG1, MTHI1, and MDA1, which stabilize the psbI, petG, atpH/atpI, and atpA transcripts, respectively (Wang et al., 2015; Viola et al., 2019; Ozawa et al., 2020). The binding of these proteins to the 5′ UTR protects transcripts against 5′-3′ exonucleolytic degradation by RNA exonucleases.

Interestingly, OPR proteins may also play a double role; in some cases, acting as T factors while also participating in the stabilization of their target transcript. The opposite may also take place, M factors contributing to translation of their target. Such is the case of TAA1 (Lefebvre-Legendre et al., 2015) a translation factor required for PsaA accumulation that also participates in preventing psaA degradation by exonucleases. Another example of a dual role is the MTHI1 protein; which, unlike TAA1, is involved in regulation of the expression of two different targets, atpH and atpI mRNAs (Ozawa et al., 2020). On the one hand, MTHI1 works as M factor for the atpH mRNA, allowing for the accumulation of a monocistronic transcript but also participating as translational enhancer. On the other hand, MTHI1 is required for translation activation of the atpI mRNA but not for its stabilization (Ozawa et al., 2020). Conversely, the expression of a single transcript can be regulated by more than one OPR protein; such is the case for the atpA transcript in the atpA-psbI-cemA-atpH gene cluster (Drapier et al., 1998). The accumulation of the AtpA protein depends on the maturation and translation activities of the MDA1 and TDA1 factors, respectively, both of which bind to the 5′-UTR of atpA. While MDA1 stabilizes and participates in processing the atpA transcript (Viola et al., 2019), TDA1 intervenes, facilitating the start of translation (Eberhard et al., 2011).

Another OPR protein is CCS2 which is required for the assembly of the cytochrome c complex. CCS2 contains an LWALAR consensus amino acids motif (Cline et al., 2017), which is different to the PPPEW motif, typical of other known OPR proteins such as TBC2, TDA1, and TAB1 (Auchincloss et al., 2002; Eberhard et al., 2011; Rahire et al., 2012). Due to its OPR motifs, CCS2 was initially suggested to participate in the maturation, stabilization, or translation of a chloroplast transcript involved in cytochrome c assembly. The transcript of the ccsA gene (a heme delivery factor required for cytochrome c maturation) has been suggested as the CCS2 target. Experimental evidence has shown that CCS2 is not required for stabilization of ccsA thus its role as a M factor can be discarded. However, CCS2 acting as T factor is a matter of consideration. An additional proposed mode of action, although less plausible, is that CCS2 could stabilize the CCS complex (composed of CCS1 and CcsA) via protein–protein interactions or by facilitating the recruitment of additional components to this complex (Cline et al., 2017).

Another role attributed to OPR proteins is participation in splicing processes. A clear example of this is the maturation of psaA mRNA, which requires two steps of trans-splicing (Herrin and Schmidt, 1988; Figure 1B). The psaA gene is encoded in three separate exons that are scattered throughout the chloroplast genome. Each of the exons is transcribed separately and has flanking sequences that allow its assembly by forming structures belonging to the group II introns (Kück et al., 1987; Choquet et al., 1988). Additionally, the trans-splicing chloroplast A (tscA) locus codes for a short non-coding RNA that completes the structure of the first tripartite group II intron (Goldschmidt-Clermont et al., 1991). At least 14 factors encoded in the nucleus participate in the splicing of psaA (Reifschneider et al., 2016). Although not all of them have been characterized, at least six of these factors contain OPR motifs: RAT2, RAA1, RAA3, RAA8 (also known as OPR120), OPR1 (Cre01.g001501), and OPR104 (Cre17.g698750; Merendino et al., 2006; Marx et al., 2015; Lefebvre-Legendre et al., 2016; Reifschneider et al., 2016; Figure 1B). All these factors are part of a post-transcriptional complex, presumably functioning as RNA interaction module (Lefebvre-Legendre et al., 2016; Reifschneider et al., 2016; Kück and Schmitt, 2021). On the one hand, RAT2 (RNA-maturation of psaA-tscA) is necessary to process tscA from a polycistronic precursor (Balczun et al., 2005). While RAA8 (RNA-maturation of psaA) participates in the splice junction of the first psaA intron (Marx et al., 2015), RAA1 (RNA-maturation of psaA1) is involved in the processing of the tscA mRNA and participates in the trans-splicing of introns 1 and 2 (Rochaix et al., 2004; Merendino et al., 2006). Like many of the nucleus-encoded proteins involved in the anterograde regulation, OPR proteins activity is regulated in response to environmental factors such as light induction (RAA1), light repression (RAA3), H₂O₂ suppression (RAA1; Wang et al., 2015), proteolytic degradation in response to nitrogen (MCA1 and TCA1; Wei et al., 2014), and iron deprivation (MAC1 and TAA1; Lefebvre-Legendre et al., 2015; Douchi et al., 2016).

Most functionally known OPR proteins have been identified by screening nuclear mutants with photosynthetic or respiratory defects after mutagenesis. However, Boulouis et al. (2015) characterized two nuclear spontaneous mutants with single amino acid substitutions in two distinct genes encoding OPR proteins: OPR87 (Cre15.g638950.t1) and OPR98 (Cre15.g640400.t1). These nuclear mutants, called ncc1 and ncc2, have expanded the recognition capacity of the OPR proteins, targeting the coding regions of two chloroplast transcripts, leading to the destabilization of atpA or petA transcripts, respectively (Drapier et al., 2002; Boulouis et al., 2015). NCC1 and NCC2 may recruit an endonuclease or may themselves carry on endo-nucleolytic activity and degrade their new targets (Boulouis et al., 2015). These proteins were classified as NCL (NCC-Like) proteins, a new subfamily of paralogs encoding OPR-RAP proteins, that comprise a highly conserved central region, containing 7–12 OPR repeats and a C-terminal RAP domain (Boulouis et al., 2015). The deeper we study these mutants and other OPR, PPR, and TPR proteins the better we will understand the importance and influence of anterograde control in organellar gene expression regulation. From a biotechnological point of view, elucidating how these proteins bind to nucleic acid sequences and understanding how changes in their motifs, as in the case of NCC1 and NCC2, alter their specificity will have an impact in the development of molecular tools. This will also allow us to skillfully tune the regulation of chloroplast metabolism to obtain new products, change the balance of chloroplast metabolites, and perhaps even achieve the long-sought improvement of photosynthesis.

The study of PPR proteins in plants has allowed us to elucidate the mechanism by which these proteins recognize and bind to their target mRNA. The level of details is such that it has been possible to postulate an RNA recognition code for PPR proteins (Barkan et al., 2012; Yagi et al., 2013, 2014). Considering that each PPR motif recognizes a ribonucleotide, it has been proposed that the amino acid residues at positions 1, 4, and ii (the penultimate residue) of each PPR motif are determinants for the recognition of their RNA bases. Being the residues 4 and ii, those with the greatest influence on ribonucleotide recognition (Barkan et al., 2012; Yagi et al., 2013; Figures 2C,D). Some of the recognition codes that have been proposed include the amino acids Val, Asn, and Ser at positions 1, 4, and ii, respectively, for cytosine recognition; Val, Asn, and Asp for uracil; Val, Thr, and Asp for guanine; and finally, Phe, Thr, and Asn for adenine (Barkan et al., 2012; Yagi and Shiina, 2014; Figure 2D). However, the bioinformatic analysis predicts that there are other combinations of the PPR code amino acids; in fact, a few of them have been explored (Barkan et al., 2012; Yin et al., 2013; Cheng et al., 2016; Yan et al., 2019). From a biotechnological point of view, elucidating the PPR code is an opportunity area for the design of new RNA-binding proteins with a particular motif or additional domains bound to specific targets for the identification or control of mRNA metabolism.

Imai et al. (2018) suggest using PPR motifs as scaffolds to develop designed RNA-binding proteins. The fusion of PPR motifs with other domains could allow the development of new tools for (a) RNA detection in vivo by assembling engineered-PPR (ePPR) to a reporter protein (e.g., GFP); (b) translation regulation by fusing ePPR motif to eIF4G (eukaryotic translation initiation factor 4G), allowing activation translation of the targeted mRNA; (c) development of a site-specific endonuclease, by fusing ePPR motif with an endonuclease, which allows recognizing and cleaving RNA in a specific sequence; (d) RNA editing, using an ePPR protein with cytidine deaminase domains to catalyze cytosine’s reaction to uracil at a specific position; and (e) finally, the use of engineered motifs targeted to otherwise naked or susceptible to degradation mRNA. This, depending on the nature of each original P/O/TPR protein, could be used to increase the half-life of the transcript, enhance translation, or even tune down transcript translatability. The use of designed PPR proteins has great potential in developing tools that require RNA binding or tagging applications. McDermott et al. (2019) recently demonstrated that an artificial protein constructed with PPR motifs targeting a specific RNA-target could specifically bind to it in vivo and have low levels of off-target bindings. This work was the first approach to developing engineered PPR proteins as tools to study RNA metabolism in plants.

Another opportunity for the biotechnological applications of PPR, TPR, and OPR proteins is their binding sites. Considering that natively these proteins bind to 5′- and 3′-UTR targets and function as M or T mRNA factors, it has been proposed to use them in the maturation-stabilization and translation of foreign genes with 5′-UTRs containing P/T/OPR proteins binding sites. Zhou et al. (2007), Legen et al. (2018), and Macedo-Osorio et al. (2018) have identified sequences called Intercistronic Expression Elements (IEEs), which contain TPR and PPR proteins binding sites, that have been used for the expression of heterologous polycistronic mRNA in tobacco (Zhou et al., 2007; Lu et al., 2013; Fuentes et al., 2016; Legen et al., 2018), tomato (Lu et al., 2013), and C. reinhardtii (Macedo-Osorio et al., 2018). IEEs were first described by Zhou et al. (2007), who identified a sequence of 50 bases on the 5′-UTR of the psbH gene in tobacco. It contains the binding site of the HAT protein HCF107; this element was recognized as an IEE. Under native conditions, HCF107 binds to the 5′-UTR of psbH and stabilizes the monocistronic transcript of psbH once it is cleaved from the bicistronic transcript psbT-psbH (Felder et al., 2001; Sane et al., 2005). When the IEE is inserted into the intergenic region of the bicistronic transcript yfp-nptII (yellow fluorescent protein and kanamycin resistance gene, respectively), it is able to stabilize and increase the translation of the monocistronic transcripts (Zhou et al., 2007). Its function has been attributed to the fact that it contains the binding site for HCF107 (Hammani et al., 2012). As expected, HCF107 joins to the 5′-UTR of the foreign gene, stabilizing and protecting the transcript from exonucleases and generating higher translated product (Zhou et al., 2007; Lu et al., 2013; Fuentes et al., 2016). This IEE has been successfully used to express genes involved in the tocochromanol biosynthesis pathway to produce vitamin E in tobacco and tomato (Lu et al., 2013) and for the expression of the core biosynthetic pathway of artemisinic acid production in tobacco chloroplasts (Fuentes et al., 2016). Although, these elements have great potential in metabolic pathway engineering, Legen et al. (2018) recently, demonstrated that when an IEE is used in the stabilization of foreign genes, there is a negative effect on the stabilization of the endogenous psbH transcript also, possibly due to a phenomenon of HCF107 titration for competition between the binding sites of the foreign transcript and the native psbH transcript. This problem can be solved by designing PPR and TPR proteins with specific binding sites to foreign genes. Additionally, Legen et al. (2018) showed that bindings sites for PPR10, HCF152, and CCR2 can be used as IEEs when they were evaluated in a synthetic neo-egfp bicistron in tobacco chloroplasts. Similar results were obtained by Macedo-Osorio et al. (2018) in C. reinhardtii when the intergenic region of psbN-psbH was used as IEE, which contains the anchoring site for the MBB1 protein, an ortholog of HCF107. This region, called IEE2, could stabilize the monocistronic transcript gfp on the bicistronic aphA6-gfp mRNA, allowing the translation and accumulation of the GFP protein. Similarly, in tobacco with HCF107, MBB1 could be binding to IEE2, functioning as an M-factor for the GFP monocistronic transcript in C. reinhardtii (Figure 3A).

Additionally, Rojas et al. (2019) developed an inducible switch to activate chloroplast transgenes’ expression in tobacco using an engineered PPR proteins. In native conditions, PPR10 binds to the 5′ end of the atpH mRNA protecting it from degradation by exonucleases and increasing its translational efficiency. In this case, Rojas et al. (2019) generated variants of PPR10 from maize, with modified amino acids that allow it to bind to a specific target sequence; the protein was expressed in the nucleus under the regulation of an inducible promoter. The protein once produced could bind to a specific target sequence, located in 5′-UTR of the GFP mRNA expressed in the chloroplast. The inducible system allows the regulated expression of chloroplast genes from the nucleus (Figure 3B).

Similarly, Carrera-Pacheco et al. (2020) developed an inducible gene expression system in the Chlamydomonas chloroplast. The OPR protein, TDA1 of Chlamydomonas was nuclear expressed under the regulation of light, and heat-inducible promoters, HSP70A-RBCS2, the generated protein is capable of specifically binding to the 5′-UTR of atpA used to regulate the expression of gfp in the chloroplast, showing a 1.9-fold increase in the production of the recombinant protein. A reduced number of examples, albeit clear and concise, have shown that it is possible to engineer and manipulate PPR proteins to modulate and rewire gene expression, particularly with a biotechnological application, that is, to express foreign proteins in the chloroplast of plants. Application of engineered PPR, OPR, and TPR proteins in the chloroplast of C. reinhardtii has yet to see the light.

Penta-tricopeptide repeat, TPR, and OPR proteins have been revealed as essential factors for the functioning of chloroplasts; they act as regulatory factors for maturation, stabilization, and translation of different transcripts coding for essential genes including those for photosynthesis. Although initial studies of PPR proteins in plants allowed the elucidation of their mechanisms of action, it is necessary to characterize additional PPR, TPR, and OPR proteins from C. reinhardtii to gain knowledge in their regulatory mechanisms in this important model organism. Understanding the way they control and regulate the expression of genes, mainly in the chloroplast, opens the door to generate new tools to improve not only the production levels of recombinant proteins and food and relevant pharmaceutical compounds but also to perform metabolic engineering aimed at enhancing the production of lipids for biofuels. A more complex task, though not unrealistic would be to remodel cell metabolism in Chlamydomonas to enhance photosynthesis to make the CO₂ fixation rate more efficient and thus contribute to the reduction of atmospheric CO₂ levels, while producing sustainable value-added products.

KM-O and JB-C: conceptualization. KM-O: investigation and writing of original draft. KM-O, AM-A, and JB-C: writing- reviewing and editing. AM-A: funding acquisition. All authors contributed to the article and approved the submitted version.

KM-O was financially supported by CONACYT through a postdoctoral fellowship. Research in the laboratory of JB-C is financially supported by Instituto Politécnico Nacional and CONACYT through project 286497.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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