The lumenal domain of Cyt b 559 interacting with extrinsic subunits is crucial for accumulation of functional photosystem II

The lumenal domain of Cyt b 559 interacting with extrinsic subunits is crucial for accumulation of functional photosystem II | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The lumenal domain of Cyt b 559 interacting with extrinsic subunits is crucial for accumulation of functional photosystem II Ko Imaizumi, Shin-ichi Arimura, Kentaro Ifuku This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6005678/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jun, 2025 Read the published version in Photosynthesis Research → Version 1 posted 11 You are reading this latest preprint version Abstract Cytochrome b 559 (Cyt b 559 ) is an essential component of the photosystem II (PSII) reaction center core. It consists of two subunits, PsbE and PsbF, which together coordinate a redox-active heme. While extensive studies have revealed the importance of Cyt b 559 , its structural and functional roles are not fully understood. Previous studies have implied that the lumenal region of Cyt b 559 , interacting with the PSII extrinsic subunit PsbP in green plant PSII, may have important roles. However, few studies have investigated its lumenal region. Here, we have focused on a well-conserved lumenal region of PsbE, which was found to interact with the N-terminal region of PsbP in green-lineage PSII (from green algae and land plants). In red-lineage PSII (from red algae and algae possessing red algal-derived plastids), very similar interactions were observed between the same lumenal region of PsbE and the N-terminal region of PsbQ′. We generated Arabidopsis thaliana mutants harboring mutations in the well-conserved lumenal region of PsbE through targeted base editing of the plastid genome by ptpTALECD. The mutations led to strong growth defects and extremely low F v / F m . This study suggests the importance of the lumenal regions of Cyt b 559 , and gives insight into possible structural and functional compensation between the N-terminal regions of PsbP in green-lineage PSII and PsbQ′ in red-lineage PSII. Photosystem II Cytochrome b559 Extrinsic subunits PsbP PsbQ′ Base editing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Photosystem II (PSII), the light-driven water:plastoquinone oxidoreductase, is a multi-subunit pigment–protein complex embedded in the thylakoid membranes of oxygenic photosynthetic organisms, including plants, algae, and cyanobacteria (Shen 2015; Shevela et al. 2023). Using light energy, PSII oxidizes water to molecular oxygen at its oxygen-evolving center (OEC) and reduces plastoquinone (PQ) to plastoquinol at its acceptor side. PSII is composed of more than 20 subunits, including 17–19 transmembrane subunits and 3–5 membrane extrinsic subunits binding to PSII on the thylakoid lumenal side. While many transmembrane subunits are conserved among the diverse oxygenic photosynthetic organisms, the compositions of extrinsic subunits have largely changed during the course of evolution (Roose et al. 2016; Ifuku and Nagao 2021; Imaizumi and Ifuku 2022). Cyanobacterial PSII possesses PsbO, PsbV, PsbU, and CyanoQ as extrinsic subunits. Red-lineage PSII (PSII in red algae and algae possessing red algal-derived plastids) has PsbO, PsbV, PsbU, and PsbQ′. An additional subunit, Psb31, is also found in various species in the red lineage. Green-lineage PSII (PSII in green plants: land plants and green algae) binds PsbO, PsbP, and PsbQ, and PsbTn is additionally found in different land plants. The roles and importance of the extrinsic subunits have been intensively studied. However, it is not yet fully understood how the different sets of extrinsic subunits functionally compensate for each other among different PSII types. The reaction center complex of PSII contains D1, D2, cytochrome b 559 (Cyt b 559 ), and PsbI (Nanba and Satoh 1987; Webber et al. 1989). Cyt b 559 consists of two subunits: PsbE (α subunit) and PsbF (β subunit). PsbE and PsbF each have a single transmembrane helix, and PsbE also has a lumenal domain (Fig. 1 ). A redox-active heme cofactor is coordinated in between the transmembrane helices, near the stromal side of the thylakoid membrane. Despite extensive studies on Cyt b 559 (Shinopoulos and Brudvig 2012; Chu and Chiu 2016; Chiu and Chu 2022), its role remains enigmatic (Cramer and Zakharov 2022). It has been shown that Cyt b 559 has essential structural roles, and it may also have physiological roles involving electron transport. Accumulation of Cyt b 559 is a prerequisite for PSII assembly, which initiates with the formation of the D2 module (D2 mod ) consisting of Cyt b 559 and D2 (Nickelsen and Rengstl 2013; Komenda et al. 2024). Cyt b 559 has been found to accumulate in etioplasts, where PSII assembly does not take place yet (Müller and Eichacker 1999; Kanervo et al. 2008; Plöscher et al. 2009; Li et al. 2024b), and in mutants lacking Cyt b 559 , D2 cannot accumulate and PSII cannot be assembled (Pakrasi et al. 1988; Morais et al. 1998; Swiatek et al. 2003; Komenda et al. 2004). Cyt b 559 is also thought to oxidize reduced PQ in the PQ pool (Bondarava et al. 2003, 2010) and to mediate cyclic electron flow within PSII for photoprotection of mature PSII (Takagi et al. 2019) as well as PSII during its assembly or repair processes (García-Cerdán et al. 2019; Li et al. 2024a). However, the physiological roles of Cyt b 559 involving its redox activeness remains controversial (Gates et al. 2022; Cramer and Zakharov 2022). While the heme, stromal region, and transmembrane helices of Cyt b 559 have been intensively studied, few studies have focused on the lumenal region of Cyt b 559 (Shinopoulos and Brudvig 2012; Sugiura et al. 2015; Chu and Chiu 2016; Chiu and Chu 2022). Here, we investigated the lumenal domains of Cyt b 559 in silico , and found a well-conserved region that interacts with the extrinsic subunits of PSII. Through targeted base editing of the psbE gene in the Arabidopsis thaliana plastid genome using ptpTALECD (Nakazato et al. 2021), we confirmed the importance of this well-conserved region within the lumenal domain of PsbE. The results give insight into possible structural and functional roles of the lumenal domains of Cyt b 559 , as well as the structural and functional compensation between the N-terminal regions of PsbP in green-lineage PSII and PsbQ′ in red-lineage PSII. Results Well-conserved regions in the lumenal domain of Cyt b 559 To investigate whether there are well-conserved regions within the lumenal domains of Cyt b 559 , we first conducted multiple sequence alignments of PsbE and PsbF using amino acid sequences from various photosynthetic organisms including photosynthetic green-lineage and red-lineage organisms and cyanobacteria. The N-terminal region and the transmembrane region of PsbE are highly conserved among the wide range of photosynthetic organisms (Fig. 2 a). In comparison, the C-terminal region located at the lumenal side of the thylakoid membrane is less well-conserved. This was expected, as the lumenal domain of PsbE is located in a less-crowded peripheral region not near the oxygen-evolving center of PSII. In fact, it had been pointed out previously that the amino acid sequence of the lumenal domain of PsbE was somewhat different among land plants, cyanobacteria, and glaucophytes (Pakrasi et al. 1988; Vallon et al. 1989), and even between land plants and green algae (Enami et al. 2008). However, we noticed a very well conserved region within the lumenal domain of PsbE: Gly48-(Ser/Thr)49-Pro50-Arg51-Pro52-(Asn/Asp)53. This region is found near the transmembrane helix of PsbE (Fig. 1 ). As for PsbF, there were some variations near the N-terminus, but the sequence of the N-terminal region (Tyr7–Phe10) near the transmembrane helix and that of the transmembrane helix were highly conserved (Fig. 2 b). PsbF only has a few residues on the stromal side of the transmembrane helix, and those were also very well conserved. These highly conserved regions in PsbE and PsbF can be speculated to be structurally and/or functionally important for PSII. Interaction of the lumenal domain of Cyt b 559 with PsbP in green-lineage PSII and with PsbQ′ in red-lineage PSII Having observed that the region, Gly48–(Asn/Asp)53 of PsbE is particularly well-conserved within its lumenal domain, we have further investigated this region. Earlier, we noticed that the N-terminal region of PsbP in the PSII structure from the green plant Pisum sativum (Su et al. 2017) and the N-terminal region of PsbQ′ in the PSII structure from the diatom Chaetoceros gracilis (Pi et al. 2019) both interacted with the lumenal domain of PsbE at a similar position (Imaizumi and Ifuku 2022). Taking a closer look around these interaction sites, the PsbP N-terminus and PsbQ′ N-terminus were both located adjacent to the well-conserved Gly48–(Asn/Asp)53 region of PsbE. PsbP is a PSII membrane extrinsic subunit unique to green-lineage PSII, whereas PsbQ′ is an extrinsic subunit unique to red-lineage PSII (Ifuku and Nagao 2021; Imaizumi and Ifuku 2022). As PSII structures from various photosynthetic green-lineage and red-lineage organisms have been reported recently, we have further investigated whether these interactions are also observed in these various PSII structures. The N-terminal regions of PsbP from the land plant Pisum sativum (Su et al. 2017), and green algae Chlamydomonas reinhardtii (Sheng et al. 2019) and Dunaliella salina (Caspy et al. 2023), and the N-terminal regions of PsbQ′ from diatoms Chaetoceros gracilis (Pi et al. 2019) and Thalassiosira pseudonana (Feng et al. 2023), the cryptophyte Chroomonas placoidea (Zhang et al. 2024c), and the red alga Porphyridium purpureum (You et al. 2023) were all located at very similar positions adjacent to the well-conserved region in the PsbE-lumenal domain (Fig. 3 ). This suggests that there may be conserved interactions of the lumenal domain of PsbE with the N-terminal region of PsbP in green-lineage PSII, and with the N-terminal region of PsbQ′ in red-lineage PSII. If the N-terminal regions of PsbP and PsbQ′ similarly interact with the well-conserved region in the PsbE lumenal domain, those N-terminal regions are likely to have similar amino acid sequences. Therefore, we next compared the amino acid sequences of the N-terminal regions of PsbP and PsbQ′. Although PsbP and PsbQ′ are not thought to share a common evolutionary origin, the N-terminal regions consisting of the first 10 to 12 amino acids located near the lumenal domain of Cyt b 559 were similar among PsbP and PsbQ′ from various organisms (Fig. 4 ), supporting the presence of conserved interactions of the N-terminal regions of these extrinsic subunits with the lumenal domain of PsbE. Only the above-mentioned amino acid sequence near the N-terminus is similar, and the sequence afterwards is not similar at all. Detailed structural comparisons suggest that the N-terminal regions of PsbP and PsbQ′ both can make multiple interactions with the well-conserved lumenal region of PsbE as well as with the C-terminal residues of PsbF. Although the resolution in this region may not be sufficient to determine the exact conformations of the side chains, PsbP-Ala1 (or PsbP-Tyr2) and PsbP-(Glu/Asp)4 can interact with PsbE-Arg51 in green-lineage PSII, and similarly, PsbQ′-Ala1 and PsbQ′-(Glu/Asp)4 can interact with PsbE-Arg52 (corresponding to green plant PsbE-Arg51) in red-lineage PSII (Fig. 5 ). In addition, the N-terminal regions of PsbP and PsbQ′ both seem to also interact similarly with the C-terminal residues of PsbF (PsbF-Phe36, Gln38, and Arg39 numbered based on Arabidopsis PsbF) ( Supplementary Fig. 1 ). Interaction of these extrinsic subunits with both PsbE and PsbF may affect the heme environment in between these two Cyt b 559 subunits, possibly explaining the effect of PsbP on the redox potential of Cyt b 559 (Ghanotakis et al. 1986; de Paula et al. 1986; Thompson et al. 1989; Nishimura et al. 2016). Comparison of the N terminal regions of PsbQ and PsbQ′ PsbP, unique to green-lineage PSII, is suggested to have evolved from the cyanobacterial CyanoP (De Las Rivas et al. 2004; De Las Rivas and Roman 2005; Ishihara et al. 2007), whereas PsbQ′, unique to red-lineage PSII, is thought to have evolved from the cyanobacterial CyanoQ (Ohta et al. 2003; De Las Rivas and Roman 2005). In green-lineage PSII, PsbQ, which is also considered to have evolved from CyanoQ (Kashino et al. 2002; De Las Rivas et al. 2004; De Las Rivas and Roman 2005), is present instead of PsbQ′. PsbQ, PsbQ′, and CyanoQ all share a structurally similar four-helix bundle core, with which they bind to PSII at the same location (the lumenal surface of CP43) (Gisriel and Brudvig 2022; Imaizumi and Ifuku 2022; Gisriel et al. 2022). In addition to the four-helix bundle core, PsbQ′ and PsbQ have a long N-terminal loop region, whereas CyanoQ has a shorter N-terminal loop with an N-terminal lipid modification (Thornton et al. 2004). To confirm whether or not the N-terminal sequence of PsbQ′, which we found to be similar to that of PsbP, is also found in PsbQ and/or CyanoQ, we compared the amino acid sequences of the N-terminal loop of PsbQ, PsbQ′, and CyanoQ, from the (predicted) N-terminus up to the region near the N-terminal end of the four-helix bundle core. The amino acid sequences of the N-terminal loops of PsbQ, PsbQ′, and CyanoQ were very different from each other (Fig. 6 a). We did not observe any regions in the N-terminal loops of PsbQ or CyanoQ with sequences similar to that of the N-terminal end of PsbQ′ and PsbP. Furthermore, the amino acid sequence alignment of the full-length mature PsbQ′ from various red-lineage PSII revealed that, despite having a structurally conserved four-helix bundle core, the N-terminal region was the most well-conserved region within PsbQ′ (Fig. 6 b). These results suggest that PsbQ′ and PsbP most likely independently acquired the similar N-terminal amino acid sequence to interact with the lumenal domain of Cyt b 559 . Moreover, the results imply that the interaction with the Cyt b 559 lumenal domain is likely one of the major roles of PsbQ′ in red-lineage PSII, and that the PsbP N-terminus is responsible for this role in green-lineage PSII. Importance of the conserved lumenal region in PsbE suggested from Arabidopsis mutants generated by genome editing using TALECD The above observations suggested that the well-conserved region, PsbE-Gly48–(Asn/Asp)53, in the lumenal domain of PsbE may have important functions in PSII. Therefore, we have investigated this by generating Arabidopsis thaliana mutants harboring mutations in this region. PsbE is encoded in the psbEFLJ operon in the plastid genome, and PsbE-deficient plants are incapable of photoautotrophic growth. In order to introduce mutations in the well-conserved PsbE lumenal region without knocking out the psbE gene, we conducted targeted base editing in the plastid genome using ptpTALECD (plastid-targeted platinum transcription activator-like effector cytidine deaminase) (Nakazato et al. 2021). TALECD mainly consists of the DNA binding domain (TALE domain) of TALEN, the N-terminal or C-terminal half of a split cytidine deaminase (CD), and an uracil glycosylase inhibitor (UGI). When a pair of TALECD bind to their target DNA, the reassembled CD converts C to U, resulting in targeted C/G-to-T/A conversions (Mok et al. 2020; Arimura and Nakazato 2024; Nakazato and Arimura 2024). Using this genome editing strategy, we attempted to substitute Arg with Cys at residue 51 of PsbE, as PsbE-Arg51 seemed to be a key residue for interaction with PsbP (Fig. 5 ), but we could not obtain a PsbE-R51C mutant; this may have been due to the targeted DNA sequence, while it might also be possible that the single PsbE-R51C mutation was lethal. However, we successfully obtained two independent Arabidopsis thaliana mutant lines with homoplasmic PsbE-G48E/R51C/P52S mutations. All three amino acid substitutions, G48E, R51C, and P52S occurred within the well-conserved Gly48–(Asn/Asp)53 region in the lumenal domain of PsbE. The Arabidopsis PsbE-G48E/R51C/P52S mutants showed severe growth defects (Fig. 7 and Fig. 8 a). Although the plants were grown on sucrose-supplemented medium for two weeks before transferring to soil, the mutants managed to survive, but only showed minimal growth with pale green leaves. The maximal quantum yield of PSII ( F v / F m ) was extremely low in the PsbE-G48E/R51C/P52S mutants ( F v / F m = 0.2–0.3) due to high F o (dark-adapted minimal fluorescence) (Fig. 8 ). These results reveal that the well-conserved lumenal region of PsbE is critical for the accumulation of functional PSII. Unfortunately, due to the strong effects of the deleterious mutation, we were unable to conduct further detailed analyses. Discussion In this study, we have investigated the lumenal regions of Cyt b 559 . Numerous studies have investigated the role of Cyt b 559 using Synechocystis sp. PCC 6803, Thermosynechococcus vestitus , Chlamydomonas reinhardtii , Arabidopsis thaliana , or Nicotiana tabacum mutants with various mutations in PsbE or PsbF (Table 1 ) (Pakrasi et al. 1991; Tae and Cramer 1992; Bock et al. 1994; Morais et al. 2001; Bondarava et al. 2003, p. 200, 2010; Hung et al. 2007, 2010; Ma et al. 2007; Cai et al. 2009; Chiu et al. 2009, 2013, 2022; Yagi et al. 2013, p. 20; Guerrero et al. 2014; Hamilton et al. 2014; Sugiura et al. 2015; Huang et al. 2016; Endo et al. 2019; Nakamura et al. 2019; Che et al. 2024). However, almost all of these mutations were introduced in the stromal (N-terminal) region or within the transmembrane helix of PsbE or PsbF, and very few studies have focused on the lumenal regions of Cyt b 559 (Table 1 and Fig. 9 ). While R59Q, R68Q, and R59Q/R68Q Synechocystis sp. PCC 6803 mutants (corresponding to R60Q, R69Q, and R60Q/R69Q in Arabidopsis ) have been constructed, these mutations did not show apparent effects on PSII (Tae and Cramer 1992). In Arabidopsis thaliana , psbE transcripts undergo RNA editing mediated by the PPR (pentatricopeptide repeat) protein CREF3 (Chloroplast RNA Editing Factor 3) (Yagi et al. 2013). Through RNA editing, the genomically encoded CCU (proline) codon is post-transcriptionally edited to a UCU (serine) codon (Yagi et al. 2013), resulting in a Ser residue at position 72, which is conserved among land plants (Fig. 2 a). Consequently, the CREF3-deficient Arabidopsis thaliana mutant cref3 has a PsbE-S72P mutation in the lumenal domain of PsbE. However, no apparent effects on PSII have been observed by this mutation either (Yagi et al. 2013; Che et al. 2024). Meanwhile, it has been reported that the deletion of 12, 22, or 31 C-terminal residues of PsbE, all included within the lumenal domain, results in decreased PSII function (Tae and Cramer 1992). Especially, when 31 C-terminal residues were deleted, PSII assembly was largely diminished. Also, we have previously reported that the interaction of the N-terminal region of PsbP with the lumenal domain of PsbE modulates the redox potential of Cyt b 559 (Nishimura et al. 2016). These reports implied that the lumenal region of PsbE might have both structural and functional roles. Table 1 List of amino acid residues of PsbE and PsbF that have been studied previously by substitution mutations. Substituted residue a Organism (Reference) b PsbE-Thr5 S. 6803 (Endo et al. 2019) PsbE-Arg8 S. 6803 (Arg7) (Chiu et al. 2009) PsbE-Ala11* S. 6803 (Ser11) (Endo et al. 2019) PsbE-Ile14 T. vestitus (Guerrero et al. 2014) PsbE-Arg18 C. reinhardtii (Ma et al. 2007) S. 6803 (Arg17) (Chiu et al. 2013) T. vestitus (Guerrero et al. 2014) PsbE-Tyr19 S. 6803 (Tyr18) (Hung et al. 2010) T. vestitus (Sugiura et al. 2015) T. vestisus (Nakamura et al. 2019) PsbE-His23 C. reinhardtii (Morais et al. 2001) C. reinhardtii (Hamilton et al. 2014) S. 6803 (His22) (Pakrasi et al. 1991) S. 6803 (His22) (Hung et al. 2007) S. 6803 (His22) (Hung et al. 2010) S. 6803 (His22) (Chiu et al. 2022) T. vestitus (Sugiura et al. 2015) T. vestitus (Nakamura et al. 2019) PsbE-Ser24 S. 6803 (Ser23) (Huang et al. 2016) PsbE-Thr26 T. vestitus (Sugiura et al. 2015) T. vestitus (Nakamura et al. 2019) PsbE-Ile27 T. vestitus (Guerrero et al. 2014) PsbE-Arg60 S. 6803 (Arg59) (Tae and Cramer 1992) PsbE-Arg69 S. 6803 (Arg68) (Tae and Cramer 1992) PsbE-Ser72 A. thaliana (Yagi et al. 2013) c A. thaliana (Che et al. 2024) c PsbF-Arg13 S. 6803 (Arg17) (Chiu et al. 2013) PsbF-His18 S. 6803 (His22) (Pakrasi et al. 1991) S. 6803 (His22) (Hung et al. 2007) S. 6803 (His22) (Chiu et al. 2022) PsbF-Thr24* S. 6803 (Ser28) (Huang et al. 2016) PsbF-Phe26 N. tabacum (Bock et al. 1994) N. tabacum (Bondarava et al. 2003) N. tabacum (Bondarava et al. 2010) A. thaliana (Cai et al. 2009) d T. vestitus (Phe32) (Guerrero et al. 2014) PsbF-Leu28* S. 6803 (Val32) (Huang et al. 2016) a, Amino acid residues and residue numbers are based on Arabidopsis . Asterisks (*) indicate that the amino acid residue in Arabidopsis is different from that studied in the cited literature. b, The studied amino acid residues are shown in brackets after the species, when the corresponding residue or residue number is different from that in Arabidopsis . c, A study using the cref3 mutant, deficient of RNA editing of PsbE. d, A study using the lpa66 mutant, deficient of RNA editing of PsbF. Our results with the PsbE-G48E/R51C/P52S mutant Arabidopsis reveals that the lumenal region of Cyt b 559 , particularly the region PsbE-Gly48–(Asn/Asp)53 that we have found to be well-conserved, is indeed important. The drastic effects of the PsbE-G48E/R51C/P52S mutation on plant growth (Fig. 7 ) and on PSII (Fig. 8 ) shows that this well-conserved lumenal region of PsbE is critical for the accumulation of functional PSII. The pale-green leaf color and very low F v / F m due to dramatically increased F o have been observed in various mutants with low levels of PSII (Bondarava et al. 2003; Cai et al. 2009; Armbruster et al. 2010; Cecchin et al. 2021; Zhang et al. 2024a; Che et al. 2024). The protein structure of wild-type Arabidopsis thaliana PsbE predicted by AlphaFold2 (Jumper et al. 2021; Mirdita et al. 2022) was similar to the cryo-electron microscopy (cryo-EM) structure of the PsbE subunit in the PSII supercomplex from Pisum sativum ( Supplementary Fig. 2a ). In contrast, the predicted protein structure of G48E/R51C/P52S mutant PsbE had a partially loosened C-terminal domain ( Supplementary Fig. 2b, c ), seemingly due to instability of the loop region containing the well-conserved lumenal region, considering from the low pLDDT structure confidence score in this region ( Supplementary Fig. 3 ). This structural perturbation may have inhibited the proper assembly of PSII. It is also possible that the mutations led to an instable PSII core, disabling the stable accumulation of PSII complexes. Meanwhile, it is not likely that the mutations affected the stable accumulation of Cyt b 559 itself, as even large deletions of the PsbE lumenal domain (from Arg51 to the C-terminus numbered based on Arabidopsis PsbE) has little effect on the accumulation of Cyt b 559 (Tae and Cramer 1992). Although the strongly deleterious effects of the mutation hindered further experiments in Arabidopsis thaliana , further investigations on this conserved region in organisms with mixotrophic capabilities may clarify the detailed roles of the lumenal domain of PsbE. In addition to structural roles, the well-conserved PsbE-Gly48–(Asn/Asp)53 region is likely to also have physiological roles. In green plant PSII, this region in PsbE is the site with which the N-terminal region of PsbP interacts. Interaction of PsbP with the lumenal domain of PsbE (Nagao et al. 2010), particularly by its N-terminal region (Ido et al. 2012, 2014; Nishimura et al. 2016) had already been observed before it was confirmed in cryo-EM structures of green plant PSII (Su et al. 2017; Sheng et al. 2019; Shan et al. 2024). In red-lineage PSII, PsbQ′ extends its long N-terminal loop to the same site as the N-terminal region of PsbP in green-lineage PSII to interact with the well-conserved lumenal region of PsbE (Imaizumi and Ifuku 2022) (Fig. 3 ). The observations that the amino acid sequences of the N-terminal regions of PsbP and PsbQ′ are similar (Fig. 4 ) and that the N-terminal region of PsbQ′ is the most well-conserved region within the full-length mature PsbQ′ (Fig. 6 b) even though similar sequences are not observed in N-terminal loops of the cyanobacterial CyanoQ or green plant PsbQ (Fig. 6 a), suggest that there is a conserved interaction between extrinsic subunits (PsbP or PsbQ′) and the PsbE lumenal region, and that the interaction of the N-terminal region of PsbQ′ with the lumenal domain of PsbE can be related to one of the major roles of PsbQ′. In green plant PSII, this interaction between the N-terminal region of PsbP and the conserved lumenal region of PsbE on the lumenal side of the thylakoid membrane modulates the redox potential of the heme of Cyt b 559 , located near the stromal side (Nishimura et al. 2016). Such transmembrane effects of PsbP have also been observed on the electron transport from Q A to Q B or the redox potential of Q A (Ono and Inoue 1986; Yi et al. 2007; Ido et al. 2009; Roose et al. 2010; Semin et al. 2018; Kato and Noguchi 2021). Interestingly, PsbQ′, bound to the lumenal side of PSII, has similar transmembrane effects on the redox potential of Q A as PsbP does (Yamada et al. 2018). Considering that PsbP in green-lineage PSII and PsbQ′ in red-lineage PSII only overlap with their N-terminal regions adjacent to the lumenal domain of Cyt b 559 (Fig. 3 ), that green plant PsbQ is reported to have little effect on the PSII acceptor side under normal growth conditions (Yi et al. 2006), and that some mutations in Cyt b 559 (Hamilton et al. 2014; Nakamura et al. 2019) and compounds that affect the properties of Cyt b 559 (Takagi et al. 2019; Imaizumi et al. 2024) also affect the electron transport from Q A to Q B or the redox potential of Q A (Hamilton et al. 2014; Nakamura et al. 2019), it is possible that the N-terminal regions of PsbP and PsbQ′ similarly modulate the acceptor side of green-lineage PSII and red-lineage PSII, respectively (Imaizumi and Ifuku 2022). These effects of PsbP and PsbQ′ on the redox potential of Q A have both been proposed to have photoprotective roles (Yamada et al. 2018; Kato and Noguchi 2022). While, both structurally and functionally, PsbP in green-lineage PSII mostly replaces PsbV in red-lineage PSII (Ifuku and Nagao 2021), we have recently shown that the Loop 4 region of PsbP, critical for Cl − retention in the OEC, replaces the C-terminus of PsbU (Imaizumi et al. 2022). Our current results, suggesting that the N-terminal region of PsbP replaces the N-terminal region of PsbQ′, gives further insight into the complexed functional replacement of the different sets of extrinsic subunits between green- and red-lineage PSII. In cyanobacteria, CyanoP and CyanoQ are present instead of PsbP and PsbQ (or PsbQ′). However, CyanoP is an assembly factor of PSII rather than an extrinsic subunit (Cormann et al. 2014; Knoppová et al. 2016). Moreover, although CyanoQ is found in PSII structures from mesophilic cyanobacteria (Gisriel et al. 2022; Zhang et al. 2024b), its N-terminal loop seems to be too short to interact with Cyt b 559 . The N-terminus of PsbV in cyanobacterial PSII as well as in red-lineage PSII does interact with the lumenal domain of PsbE at a position different from the N-terminal regions of PsbP or PsbQ′ ( Supplementary Fig. 4 ). This may partially compensate for the absence of extrinsic subunits interacting with the well-conserved lumenal region of PsbE, but it is also possible that this interaction is not required in cyanobacterial PSII. In fact, although Cyt b 559 in native Thermosynechococcus PSII is mostly in a high-potential form as in PSII from land pants, Cyt b 559 in Synechocystis PSII is known to have a lower redox potential (Chiu and Chu 2022). In addition to giving insight into the importance of the lumenal region of PsbE, this study also illustrates the powerfulness of TALECD enabling targeted base editing in the plastid genome (Kang et al. 2021; Nakazato et al. 2021). Although improvements have been made recently (Ruf et al. 2019), plastid transformation remains challenging in many land plants including Arabidopsis thaliana (Maliga 2022). In fact, in land plants, i n vivo mutagenesis studies on Cyt b 559 , whose subunits are encoded in the plastid genome, had only been conducted in tobacco, in which plastid transformation can be routinely conducted (apart from studies using Arabidopsis mutants deficient of RNA editing of psbE or psbF ). The psbE gene is encoded within the psbEFLJ operon together with the psbF , psbL , and psbJ genes. All four subunits are important for PSII; PsbE and PsbF are essential for the assembly of PSII (Pakrasi et al. 1988; Morais et al. 1998), PsbL and PsbJ are required for stable assembly of functional PSII (Anbudurai and Pakrasi 1993; Lind et al. 1993; Kitamura et al. 1994; Regel et al. 2001; Hager et al. 2002), and all four subunits are required for photoautotrophic growth in tobacco (Swiatek et al. 2003). Therefore, in order to introduce substitution mutations into a specific region within the psbE gene in Arabidopsis , a targeted genome editing strategy with high specificity and a broad targeting range and that does not affect the flanking regions is preferable, and TALECD fulfills these requirements (Arimura and Nakazato 2024; Nakazato and Arimura 2024). Materials and Methods Multiple sequence alignments The N-terminus of the amino acid sequences after removal of the transit peptides were decided based on prediction using TargetP-2.0 (Almagro Armenteros et al. 2019), with the support of multiple sequence alignments when necessary. The N-terminal residue of CyanoQ proteins were predicted in a previous report (Michoux et al. 2014). Multiple sequence alignments were conducted with MAFFT v7.511 using the L-INS-i algorithm (Katoh et al. 2019). In all figures of multiple sequence alignments, asterisks (*), colons (:), and dots (.) below the aligned sequences indicate identical, conserved, and semi-conserved amino acids, respectively. Identical amino acids are shown with black backgrounds, conserved amino acids matching the consensus sequence are shown with gray backgrounds, and conserved amino acids that do not match the consensus sequence, semi-conserved amino acids, and non-conserved amino acids matching the consensus sequence are shown with light gray backgrounds. Protein structure prediction and visualization Protein structure predictions were performed by AlphaFold2 (Jumper et al. 2021) using ColabFold v1.5.5 (Mirdita et al. 2022), and top-ranked models were used. All protein structures were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC.). Vector construction The ptpTALECD expression binary vectors were constructed following the method described by Nakazato et al. (2021). The base editing target, a cytosine-to-thymine (C-to-T) conversion resulting in the R51C mutation, was positioned at either the 8th or 10th C within the 14-bp target window between the TALE recognition sequences of ptpTALECD1333NC or ptpTALECD1397NC. The TALE left and right recognition sequences were as follows: 1397NC—TTAGCTTACGATGTGTTC (left) and CTGTAAAATACTCGTTT (right); 1333NC—TACGATGTGTTCGG (left) and CTCTGTAAAATACTCG (right). All intermediate plasmid components used for vector assembly, including the Platinum TALEN assembly kit, are available from Addgene ( https://www.addgene.org ). The assembled TALE repeats were verified by Sanger sequencing, and the final tandem-expression binary vectors—ptpTALECD1397NC (22,018 bp) and ptpTALECD1333NC (21,508 bp)—were confirmed by multiple restriction enzyme digestion patterns. Plant transformation, screening of transformants, and genotyping The TALECD vectors were introduced into Agrobacterium tumefaciens strain GV3101 (pMP90), and Arabidopsis thaliana wild-type (Col-0; Columbia-0) plants were transformed by the floral dip method. To select transformants, the collected seeds were sown on half-strength Murashige and Skoog (MS) medium supplemented with 1.5% sucrose and 0.9% agar and containing kanamycin (50 µg/mL) and cefotaxime (100 µg/mL). Kanamycin resistant T1 seedlings were transplanted to half-strength MS agar plates with 1.5% sucrose but without kanamycin, and grown for 1–2 more weeks before transplanting to soil. Total DNA was extracted from leaves, genomic DNA regions including the target window was amplified by PCR, and purified PCR products were subjected to Sanger sequencing by Eurofins Genomics. Plant materials and growth conditions Arabidopsis thaliana seeds were sterilized and sown on half-strength MS agar plates supplemented with 1.5% sucrose. After stratification at 4 ºC for 3 days in the dark, plants were grown at 22 ºC under long-day condition (16 hours light/8 hours dark) at a light intensity of 80 µmol photons m − 2 s − 1 . 2 weeks after germination, plants were transferred to soil, and were grown at the same condition. F v / F m measurements F v / F m of 17-day-old plants were measured using JUNIOR-PAM/W fluorometer (Walz) after one hour of dark adaptation or using the chlorophyll fluorescence imaging device FluorCam 800MF (Photon Systems Instruments) after 15 minutes of dark adaptation. Declarations Competing Interests: The authors declare that they have no conflict of interest. Acknowledgements This work was supported in part by JSPS Grant-in-Aid for JSPS Fellows JP23KJ1361 to K. Imaizumi, for Challenging Research (Exploratory) JP24K21968 to K. Ifuku, and for Transformative Research Areas (A) 24H02271 to S. Arimura. Author contributions K. Imaizumi and K. Ifuku conceived the project; S. 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Biochimica et Biophysica Acta (BBA) - Bioenergetics 1817:66–75. https://doi.org/10.1016/j.bbabio.2011.08.002 Su X, Ma J, Wei X, et al (2017) Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex. Science 357:815–820. https://doi.org/10.1126/science.aan0327 Sugiura M, Nakamura M, Koyama K, Boussac A (2015) Assembly of oxygen-evolving Photosystem II efficiently occurs with the apo-Cytb559 but the holo-Cytb559 accelerates the recovery of a functional enzyme upon photoinhibition. Biochimica et Biophysica Acta - Bioenergetics 1847:276–285. https://doi.org/10.1016/j.bbabio.2014.11.009 Swiatek M, Regel RE, Meurer J, et al (2003) Effects of selective inactivation of individual genes for low-molecular-mass subunits on the assembly of photosystem II, as revealed by chloroplast transformation: the psbEFLJ operon in Nicotiana tabacum. Molecular Genetics and Genomics 268:699–710. https://doi.org/10.1007/s00438-002-0791-1 Tae GS, Cramer WA (1992) Truncation of the carboxy-terminal domain of the psbE gene product in Synechocystis sp. PCC 6803: requirements for photosystem II assembly and function. Biochemistry 31:4066–4074. https://doi.org/10.1021/bi00131a024 Takagi D, Ifuku K, Nishimura T, Miyake C (2019) Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II. Photosynthesis Research 139:487–498. https://doi.org/10.1007/s11120-018-0519-7 Thompson LK, Miller AF, Buser CA, et al (1989) Characterization of the multiple forms of cytochrome b559 in photosystem II. Biochemistry 28:8048–8056. https://doi.org/10.1021/bi00446a012 Thornton LE, Ohkawa H, Roose JL, et al (2004) Homologs of Plant PsbP and PsbQ Proteins Are Necessary for Regulation of Photosystem II Activity in the Cyanobacterium Synechocystis 6803[W]. The Plant Cell 16:2164–2175. https://doi.org/10.1105/tpc.104.023515 Vallon O, Tae G-S, Cramer WA, et al (1989) Visualization of antibody binding to the photosynthetic membrane: The transmembrane orientation of cytochrome b-559. Biochimica et Biophysica Acta (BBA) - Bioenergetics 975:132–141. https://doi.org/10.1016/S0005-2728(89)80211-7 Webber AN, Packman L, Chapman DJ, et al (1989) A fifth chloroplast-encoded polypeptide is present in the photosystem II reaction centre complex. FEBS Letters 242:259–262. https://doi.org/10.1016/0014-5793(89)80481-8 Yagi Y, Tachikawa M, Noguchi H, et al (2013) Pentatricopeptide repeat proteins involved in plant organellar RNA editing. RNA Biology 10:1419–1425. https://doi.org/10.4161/rna.24908 Yamada M, Nagao R, Iwai M, et al (2018) The PsbQ’ protein affects the redox potential of the Q A in photosystem II. 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Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 10 Jun, 2025 Read the published version in Photosynthesis Research → Version 1 posted Editorial decision: Revision requested 14 Mar, 2025 Reviews received at journal 14 Mar, 2025 Reviews received at journal 12 Mar, 2025 Reviews received at journal 04 Mar, 2025 Reviewers agreed at journal 03 Mar, 2025 Reviewers agreed at journal 27 Feb, 2025 Reviewers agreed at journal 17 Feb, 2025 Reviewers invited by journal 17 Feb, 2025 Editor assigned by journal 17 Feb, 2025 Submission checks completed at journal 12 Feb, 2025 First submitted to journal 11 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6005678","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":432129731,"identity":"e498d8af-4228-41d9-8353-27e821856f0a","order_by":0,"name":"Ko Imaizumi","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Ko","middleName":"","lastName":"Imaizumi","suffix":""},{"id":432129732,"identity":"4468d374-dee7-4af6-9c83-b0fce8a5d2a9","order_by":1,"name":"Shin-ichi Arimura","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Shin-ichi","middleName":"","lastName":"Arimura","suffix":""},{"id":432129733,"identity":"bf3ab323-1dd4-471e-a33f-5b36c6f8be94","order_by":2,"name":"Kentaro Ifuku","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABMElEQVRIie2RsUrEQBCG5whsLFbTbgrNK2xYUA58E5uEQKpslWY7A0KqQNoIPkQkcFhGArFZ8RUiB1ddEfEBzuTulDsvEUuR/ZqZYfn4Z3cBFIo/CNJKrSvO53wJBt49JwMKcrYKWs8+mElXyh8U+KZUQOWuMsAJwoxh8M8urJtF24J2xaS0560AN9KrBqYPA4th5mEI2DRG7DYDxGfPCaOl7BTsUzDloWIlrMIg3LwGpmHAfPaCz8ljvHIjCADMeDClV67zWn/vFMKLdK10KcZyVOkXc2iN+xTK8+Nkq5CxFBTad9S38zoIJxl1eCbrkEoJLCYLWg7cxYq1giyFZ9HqqYBWrHiaePeNEHCaGt7r3Dx8sQ10rznajP1HVWY0ouyjN1/t5O13ikKhUPxrPgBdU17xOBetPAAAAABJRU5ErkJggg==","orcid":"","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Kentaro","middleName":"","lastName":"Ifuku","suffix":""}],"badges":[],"createdAt":"2025-02-11 09:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6005678/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6005678/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11120-025-01157-2","type":"published","date":"2025-06-10T15:58:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80329917,"identity":"330bc7d8-2d8b-47ad-8bac-08b316180e46","added_by":"auto","created_at":"2025-04-10 15:08:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1419138,"visible":true,"origin":"","legend":"\u003cp\u003eOverall structure of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e in PSII and the PsbE lumenal region investigated in this study. Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e in the PSII structure from \u003cem\u003ePisum sativum\u003c/em\u003e (PDB ID: 5XNL) is shown in cartoon view with PsbE in blue, and PsbF in green. The heme in Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e is shown as an orange stick model. The lumenal region of PsbE investigated in this study is indicated with a black box, with Gly48, Arg51, and Pro52 of PsbE shown as stick models. Note that some N-terminal residues of PsbE and PsbF are not observed in this structure.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/d14af23e13c11fab7e1a339f.png"},{"id":80329916,"identity":"9e623e8a-209e-475c-af91-88128a1cb1ba","added_by":"auto","created_at":"2025-04-10 15:08:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1848746,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignments of (\u003cstrong\u003ea\u003c/strong\u003e) PsbE and (\u003cstrong\u003eb\u003c/strong\u003e) PsbF from various species conducted with MAFFT v7.511. The black asterisks (*), colons (:), and dots (.) indicate identical, conserved, and semi-conserved amino acids, respectively. Helical regions are shown with a blue box and the regions at the lumenal side after the transmembrane helix are indicated by arrows. Residue numbers are based on \u003cem\u003eArabidopsis\u003c/em\u003esequences. (\u003cstrong\u003ea\u003c/strong\u003e) The sequences used are as follows: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (5MXD_e), \u003cem\u003ePisum sativum\u003c/em\u003e (5XNL_E), \u003cem\u003eSpinacia oleracea\u003c/em\u003e(3JCU_E), \u003cem\u003eOryza sativa\u003c/em\u003e Japonica Group (NP_039403.1), \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e (6KAC_E), \u003cem\u003eDunaliella salina\u003c/em\u003e (YP_005089824.1), \u003cem\u003ePorphyridium purpureum\u003c/em\u003e (7Y5E_eL), \u003cem\u003eCyanidium caldarium\u003c/em\u003e (4YUU_e1), \u003cem\u003eChaetoceros gracilis\u003c/em\u003e (6J3Y_E), \u003cem\u003eThalassiosira pseudonana\u003c/em\u003e (8IWH_E), \u003cem\u003eChroomonas placoidea\u003c/em\u003e (8XR6_E), \u003cem\u003eThermostichus vulcanus\u003c/em\u003e (P12238.3), and \u003cem\u003eSynechocystis\u003c/em\u003esp. PCC 6803 (7N8O_E). Ser72 in \u003cem\u003eA. thaliana\u003c/em\u003e PsbE shown in bold is the RNA edited site (Pro-to-Ser) by CREF3 (Yagi et al. 2013). The red asterisks indicate Gly48, Arg51, and Pro52 numbered based on \u003cem\u003eA. thaliana\u003c/em\u003e PsbE. (\u003cstrong\u003eb\u003c/strong\u003e) The sequences used are as follows: \u003cem\u003eA. thaliana\u003c/em\u003e (NP_051075.1 with S26 changed to F26 ), \u003cem\u003eP. sativum\u003c/em\u003e (5XNL_F), \u003cem\u003eS. oleracea\u003c/em\u003e (3JCU_F), \u003cem\u003eO. sativa\u003c/em\u003eJaponica Group (NP_039402.1), \u003cem\u003eC. reinhardtii\u003c/em\u003e (6KAC_F), \u003cem\u003eD. salina\u003c/em\u003e(YP_005089841.1), \u003cem\u003eP. purpureum\u003c/em\u003e (7Y5E_fL), \u003cem\u003eC. caldarium\u003c/em\u003e (4YUU_f1), \u003cem\u003eC. gracilis\u003c/em\u003e (6J3Y_F), \u003cem\u003eT. pseudonana\u003c/em\u003e (8IWH_F), \u003cem\u003eC. placoidea\u003c/em\u003e(8XR6_F), \u003cem\u003eT. vulcanus\u003c/em\u003e (P12239.3), and \u003cem\u003eS.\u003c/em\u003e PCC 6803 (7N8O_F). Phe26 in \u003cem\u003eA. thaliana\u003c/em\u003e PsbF shown in bold is the RNA edited site (Ser-to-Phe) by LPA66 (Cai et al. 2009). Phe26 in \u003cem\u003eP. sativum\u003c/em\u003e and \u003cem\u003eS. oleracea\u003c/em\u003e seem to undergo similar S-to-F editing, based on comparisons with other \u003cem\u003eP. sativum\u003c/em\u003e and \u003cem\u003eS. oleracea\u003c/em\u003e PsbF sequences with Ser26 (YP_003587552.1 for \u003cem\u003eP. sativum\u003c/em\u003e and NP_054951.1 for \u003cem\u003eS. oleracea\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/ce10aa393c4ef28cfa474ba8.png"},{"id":80329921,"identity":"069e3dbf-c503-4941-aaea-2ce0174a528c","added_by":"auto","created_at":"2025-04-10 15:08:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4898772,"visible":true,"origin":"","legend":"\u003cp\u003eStructural comparison of interactions between extrinsic subunits and Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e in green-lineage and red-lineage PSII. (\u003cstrong\u003ea\u003c/strong\u003e­–\u003cstrong\u003eg\u003c/strong\u003e) PSII structures from (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eP. sativum\u003c/em\u003e (PDB ID: 5XNL), (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eC. reinhardtii\u003c/em\u003e (PDB ID: 6KAC), (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eD. salina\u003c/em\u003e (PDB ID: 7PI5), (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eC. gracilis\u003c/em\u003e (PDB ID: 6JLU), (\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eT. pseudonana\u003c/em\u003e (PDB ID: 8IWH), (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eC. placoidea\u003c/em\u003e (PDB ID: 8XR6), and (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eP. purpureum\u003c/em\u003e (PDB ID: 7Y5E). PsbE, PsbF, PsbP (in green-lineage PSII), and PsbQ′ (in red-lineage PSII) are shown as blue, green, orange, and red cartoon views, respectively. (\u003cstrong\u003eh\u003c/strong\u003e) Superposition of panels \u003cstrong\u003ea\u003c/strong\u003e to \u003cstrong\u003eg\u003c/strong\u003e (with the colors for PsbP and PsbQ′ changed) with an enlarged view of the interaction site between Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e and the N-terminal regions of PsbP or PsbQ′.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/19267e0fcaf0de2d7bbd55e8.png"},{"id":80331067,"identity":"5d1a5d0d-9483-4787-b874-47218586bf0e","added_by":"auto","created_at":"2025-04-10 15:16:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":516225,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of the N-terminal regions of PsbP and PsbQ′. Multiple sequence alignment was conducted with MAFFT v7.511. The asterisks (*), colons (:), and dots (.) indicate identical, conserved, and semi-conserved amino acids, respectively. Blue box indicates the conserved region similar between PsbP and PsbQ′. Amino acid residues shown in green and red indicate residues conserved only in PsbP and PsbQ′, respectively.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/1e5b85874057391a8a5e58f7.png"},{"id":80331065,"identity":"353436f0-3899-4c21-96af-e377e462061e","added_by":"auto","created_at":"2025-04-10 15:16:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2246356,"visible":true,"origin":"","legend":"\u003cp\u003ePossible interactions between the well-conserved lumenal region of PsbE and the N-terminal regions of PsbP or PsbQ′ in (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003ePisum sativum\u003c/em\u003e (PDB ID: 5XNL), (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e (PDB ID: 6KAC), and (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eThalassiosira pseudonana\u003c/em\u003e (PDB ID: 8IWH). PsbE, PsbF, PsbP (in green-lineage PSII), and PsbQ′ (in red-lineage PSII) are shown as cyan, green, orange, and pink cartoon views, respectively. Residues that can be involved in the interactions are shown as stick models, and the black numbers indicate the N–O distances (Å).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/312184472e02c18000add708.png"},{"id":80329930,"identity":"7aad87ae-8995-4919-84ca-c03689b43a81","added_by":"auto","created_at":"2025-04-10 15:08:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1482305,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of amino acid sequences of PsbQ′ and homologous extrinsic subunits in other species. Multiple sequence alignments of (\u003cstrong\u003ea\u003c/strong\u003e) the N-terminal loop regions of PsbQ, PsbQ′, and CyanoQ, and (\u003cstrong\u003eb\u003c/strong\u003e) full-length PsbQ′ conducted with MAFFT v7.511. (\u003cstrong\u003ea\u003c/strong\u003e) The N-terminal regions from the (predicted) N-terminal residue to the residue near the N-terminal end of the conserved four-helix bundle core are compared. The following amino acid sequences were used: \u003cem\u003ePisum sativum\u003c/em\u003e (XP_050874474.1), \u003cem\u003eSpinacia oleracea\u003c/em\u003e (NP_001413330.1), \u003cem\u003eOryza sativa\u003c/em\u003e Japonica Group (NP_001390036.1), \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e (XP_042922059.1), \u003cem\u003eDunaliella salina\u003c/em\u003e (KAF5832886.1), \u003cem\u003eBigelowiella natans\u003c/em\u003e (AAP79208.1), \u003cem\u003ePorphyridium purpureum\u003c/em\u003e (7Y5E_qL), \u003cem\u003eCyanidium caldarium\u003c/em\u003e (4YUU_q1), \u003cem\u003eChaetoceros gracilis\u003c/em\u003e (BAG85211.1), \u003cem\u003eThalassiosira pseudonana\u003c/em\u003e (XP_002286571.1), \u003cem\u003eChroomonas placoidea\u003c/em\u003e (XCO00667.1), \u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e (WP_011057892.1), and \u003cem\u003eSynechocystis\u003c/em\u003e (multispecies) (WP_010871697.1). No conserved amino acids were found. (\u003cstrong\u003eb\u003c/strong\u003e) The following amino acid sequences were used: \u003cem\u003eP. purpureum\u003c/em\u003e (7Y5E_qL), \u003cem\u003eC. caldarium\u003c/em\u003e (4YUU_q1), \u003cem\u003eC. gracilis\u003c/em\u003e (BAG85211.1), \u003cem\u003eT. pseudonana\u003c/em\u003e (XP_002286571.1), and \u003cem\u003eC. placoidea\u003c/em\u003e (XCO00667.1). The asterisks (*), colons (:), and dots (.) below the sequences indicate identical, conserved, and semi-conserved amino acids, respectively.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/4da28a5fc75c38829817ae04.png"},{"id":80329927,"identity":"5392be62-65d1-43ed-8f72-5a00baea1e30","added_by":"auto","created_at":"2025-04-10 15:08:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1178236,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of wild-type and PsbE-G48E/R51C/P52S mutant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Phenotypes of (\u003cstrong\u003ea\u003c/strong\u003e) 20-day-old and (\u003cstrong\u003eb\u003c/strong\u003e) 45-day-old Col-0 and PsbE-G48E/R51C/P52S mutant plants grown under a standard condition (long-day condition (16 hours light/8 hours dark) at a light intensity of 80 µmol photons m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e). Scale bars, (\u003cstrong\u003ea\u003c/strong\u003e) 2 cm and (\u003cstrong\u003eb\u003c/strong\u003e) 10 cm. (\u003cstrong\u003ec\u003c/strong\u003e) Shoot length of 55-day-old Col-0 and PsbE-G48E/R51C/P52S mutant plants grown under a standard condition. Data are mean ± SD (\u003cem\u003en\u003c/em\u003e = 3–4, biological replicates). Different lowercase letters above bars indicate a statistically significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Tukey’s HSD test).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/ccedf400d4c44498e07dc903.png"},{"id":80329935,"identity":"d022fe2b-7997-4436-bafe-cabfbab92514","added_by":"auto","created_at":"2025-04-10 15:08:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1895010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of wild-type and PsbE-G48E/R51C/P52S mutant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. (\u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ed\u003c/strong\u003e) (\u003cstrong\u003ea\u003c/strong\u003e) Phenotypes, (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eF\u003c/em\u003e\u003csub\u003eo\u003c/sub\u003e levels, and (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e levels of 17-day-old Col-0 and PsbE-G48E/R51C/P52S mutant plants grown under a standard condition (long-day condition (16 hours light/8 hours dark) at a light intensity of 80 µmol photons m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e). Scale bar, 2 cm. \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, \u003cem\u003eF\u003c/em\u003e\u003csub\u003eo\u003c/sub\u003e levels, and \u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e levels were analyzed using the chlorophyll fluorescence imaging device FluorCam 800MF. The ranges of color scales in \u003cstrong\u003ec\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e are different from each other for clarity. (\u003cstrong\u003ee\u003c/strong\u003e–\u003cstrong\u003eg\u003c/strong\u003e) (\u003cstrong\u003ee\u003c/strong\u003e) \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e, (\u003cstrong\u003ef\u003c/strong\u003e) \u003cem\u003eF\u003c/em\u003e\u003csub\u003eo\u003c/sub\u003e levels, and (\u003cstrong\u003eg\u003c/strong\u003e) \u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e levels of 17-days-old Col-0 and PsbE-G48E/R51C/P52S mutant plants grown under a standard condition, measured using the JUNIOR-PAM fluorometer. Data are mean ± SD (\u003cem\u003en\u003c/em\u003e = 3–4, biological replicates). Different lowercase letters above bars indicate a statistically significant difference (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Tukey’s HSD test).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/15e4f3f6ae6c6527d8d22917.png"},{"id":80331068,"identity":"6c9003e2-427b-48f0-b20f-e5e266336e2c","added_by":"auto","created_at":"2025-04-10 15:16:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":884902,"visible":true,"origin":"","legend":"\u003cp\u003eLocations of the various amino acid residues of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e studied previously. Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e from \u003cem\u003eSpinacia oleracea\u003c/em\u003e PSII (PDB ID: 8Z9D) is shown with PsbE in blue cartoon view, PsbF in green cartoon view, and the heme as a black stick model. The amino acid residues previously investigated by substitution mutations in PsbE and in PsbF are shown as cyan and green stick models, respectively. The three amino acid residues mutated in this study is shown as magenta stick models.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/aca6198d204fc9853fe01053.png"},{"id":84726571,"identity":"89a8f3d0-3cb9-410c-8f95-af14121d962a","added_by":"auto","created_at":"2025-06-16 16:07:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17558939,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/bd82f554-b7b6-4249-9263-db7924138897.pdf"},{"id":80329920,"identity":"d681a620-08a1-44e2-91ed-cdb184f06804","added_by":"auto","created_at":"2025-04-10 15:08:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1710632,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6005678/v1/a928c78f4b471cfcab179ad9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The lumenal domain of Cyt b 559 interacting with extrinsic subunits is crucial for accumulation of functional photosystem II","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePhotosystem II (PSII), the light-driven water:plastoquinone oxidoreductase, is a multi-subunit pigment\u0026ndash;protein complex embedded in the thylakoid membranes of oxygenic photosynthetic organisms, including plants, algae, and cyanobacteria (Shen 2015; Shevela et al. 2023). Using light energy, PSII oxidizes water to molecular oxygen at its oxygen-evolving center (OEC) and reduces plastoquinone (PQ) to plastoquinol at its acceptor side. PSII is composed of more than 20 subunits, including 17\u0026ndash;19 transmembrane subunits and 3\u0026ndash;5 membrane extrinsic subunits binding to PSII on the thylakoid lumenal side. While many transmembrane subunits are conserved among the diverse oxygenic photosynthetic organisms, the compositions of extrinsic subunits have largely changed during the course of evolution (Roose et al. 2016; Ifuku and Nagao 2021; Imaizumi and Ifuku 2022). Cyanobacterial PSII possesses PsbO, PsbV, PsbU, and CyanoQ as extrinsic subunits. Red-lineage PSII (PSII in red algae and algae possessing red algal-derived plastids) has PsbO, PsbV, PsbU, and PsbQ\u0026prime;. An additional subunit, Psb31, is also found in various species in the red lineage. Green-lineage PSII (PSII in green plants: land plants and green algae) binds PsbO, PsbP, and PsbQ, and PsbTn is additionally found in different land plants. The roles and importance of the extrinsic subunits have been intensively studied. However, it is not yet fully understood how the different sets of extrinsic subunits functionally compensate for each other among different PSII types.\u003c/p\u003e \u003cp\u003eThe reaction center complex of PSII contains D1, D2, cytochrome \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e), and PsbI (Nanba and Satoh 1987; Webber et al. 1989). Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e consists of two subunits: PsbE (α subunit) and PsbF (β subunit). PsbE and PsbF each have a single transmembrane helix, and PsbE also has a lumenal domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A redox-active heme cofactor is coordinated in between the transmembrane helices, near the stromal side of the thylakoid membrane. Despite extensive studies on Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Shinopoulos and Brudvig 2012; Chu and Chiu 2016; Chiu and Chu 2022), its role remains enigmatic (Cramer and Zakharov 2022). It has been shown that Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e has essential structural roles, and it may also have physiological roles involving electron transport. Accumulation of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e is a prerequisite for PSII assembly, which initiates with the formation of the D2 module (D2\u003csub\u003emod\u003c/sub\u003e) consisting of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e and D2 (Nickelsen and Rengstl 2013; Komenda et al. 2024). Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e has been found to accumulate in etioplasts, where PSII assembly does not take place yet (M\u0026uuml;ller and Eichacker 1999; Kanervo et al. 2008; Pl\u0026ouml;scher et al. 2009; Li et al. 2024b), and in mutants lacking Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, D2 cannot accumulate and PSII cannot be assembled (Pakrasi et al. 1988; Morais et al. 1998; Swiatek et al. 2003; Komenda et al. 2004). Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e is also thought to oxidize reduced PQ in the PQ pool (Bondarava et al. 2003, 2010) and to mediate cyclic electron flow within PSII for photoprotection of mature PSII (Takagi et al. 2019) as well as PSII during its assembly or repair processes (Garc\u0026iacute;a-Cerd\u0026aacute;n et al. 2019; Li et al. 2024a). However, the physiological roles of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e involving its redox activeness remains controversial (Gates et al. 2022; Cramer and Zakharov 2022).\u003c/p\u003e \u003cp\u003eWhile the heme, stromal region, and transmembrane helices of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e have been intensively studied, few studies have focused on the lumenal region of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Shinopoulos and Brudvig 2012; Sugiura et al. 2015; Chu and Chiu 2016; Chiu and Chu 2022). Here, we investigated the lumenal domains of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e \u003cem\u003ein silico\u003c/em\u003e, and found a well-conserved region that interacts with the extrinsic subunits of PSII. Through targeted base editing of the \u003cem\u003epsbE\u003c/em\u003e gene in the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plastid genome using ptpTALECD (Nakazato et al. 2021), we confirmed the importance of this well-conserved region within the lumenal domain of PsbE. The results give insight into possible structural and functional roles of the lumenal domains of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, as well as the structural and functional compensation between the N-terminal regions of PsbP in green-lineage PSII and PsbQ\u0026prime; in red-lineage PSII.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eWell-conserved regions in the lumenal domain of Cyt\u003c/b\u003e \u003cb\u003eb\u003c/b\u003e\u003csub\u003e\u003cb\u003e559\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eTo investigate whether there are well-conserved regions within the lumenal domains of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, we first conducted multiple sequence alignments of PsbE and PsbF using amino acid sequences from various photosynthetic organisms including photosynthetic green-lineage and red-lineage organisms and cyanobacteria. The N-terminal region and the transmembrane region of PsbE are highly conserved among the wide range of photosynthetic organisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In comparison, the C-terminal region located at the lumenal side of the thylakoid membrane is less well-conserved. This was expected, as the lumenal domain of PsbE is located in a less-crowded peripheral region not near the oxygen-evolving center of PSII. In fact, it had been pointed out previously that the amino acid sequence of the lumenal domain of PsbE was somewhat different among land plants, cyanobacteria, and glaucophytes (Pakrasi et al. 1988; Vallon et al. 1989), and even between land plants and green algae (Enami et al. 2008). However, we noticed a very well conserved region within the lumenal domain of PsbE: Gly48-(Ser/Thr)49-Pro50-Arg51-Pro52-(Asn/Asp)53. This region is found near the transmembrane helix of PsbE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As for PsbF, there were some variations near the N-terminus, but the sequence of the N-terminal region (Tyr7\u0026ndash;Phe10) near the transmembrane helix and that of the transmembrane helix were highly conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). PsbF only has a few residues on the stromal side of the transmembrane helix, and those were also very well conserved. These highly conserved regions in PsbE and PsbF can be speculated to be structurally and/or functionally important for PSII.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInteraction of the lumenal domain of Cyt\u003c/b\u003e \u003cb\u003eb\u003c/b\u003e\u003csub\u003e\u003cb\u003e559\u003c/b\u003e\u003c/sub\u003e \u003cb\u003ewith PsbP in green-lineage PSII and with PsbQ\u0026prime; in red-lineage PSII\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHaving observed that the region, Gly48\u0026ndash;(Asn/Asp)53 of PsbE is particularly well-conserved within its lumenal domain, we have further investigated this region. Earlier, we noticed that the N-terminal region of PsbP in the PSII structure from the green plant \u003cem\u003ePisum sativum\u003c/em\u003e (Su et al. 2017) and the N-terminal region of PsbQ\u0026prime; in the PSII structure from the diatom \u003cem\u003eChaetoceros gracilis\u003c/em\u003e (Pi et al. 2019) both interacted with the lumenal domain of PsbE at a similar position (Imaizumi and Ifuku 2022). Taking a closer look around these interaction sites, the PsbP N-terminus and PsbQ\u0026prime; N-terminus were both located adjacent to the well-conserved Gly48\u0026ndash;(Asn/Asp)53 region of PsbE. PsbP is a PSII membrane extrinsic subunit unique to green-lineage PSII, whereas PsbQ\u0026prime; is an extrinsic subunit unique to red-lineage PSII (Ifuku and Nagao 2021; Imaizumi and Ifuku 2022). As PSII structures from various photosynthetic green-lineage and red-lineage organisms have been reported recently, we have further investigated whether these interactions are also observed in these various PSII structures. The N-terminal regions of PsbP from the land plant \u003cem\u003ePisum sativum\u003c/em\u003e (Su et al. 2017), and green algae \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e (Sheng et al. 2019) and \u003cem\u003eDunaliella salina\u003c/em\u003e (Caspy et al. 2023), and the N-terminal regions of PsbQ\u0026prime; from diatoms \u003cem\u003eChaetoceros gracilis\u003c/em\u003e (Pi et al. 2019) and \u003cem\u003eThalassiosira pseudonana\u003c/em\u003e (Feng et al. 2023), the cryptophyte \u003cem\u003eChroomonas placoidea\u003c/em\u003e (Zhang et al. 2024c), and the red alga \u003cem\u003ePorphyridium purpureum\u003c/em\u003e (You et al. 2023) were all located at very similar positions adjacent to the well-conserved region in the PsbE-lumenal domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This suggests that there may be conserved interactions of the lumenal domain of PsbE with the N-terminal region of PsbP in green-lineage PSII, and with the N-terminal region of PsbQ\u0026prime; in red-lineage PSII.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIf the N-terminal regions of PsbP and PsbQ\u0026prime; similarly interact with the well-conserved region in the PsbE lumenal domain, those N-terminal regions are likely to have similar amino acid sequences. Therefore, we next compared the amino acid sequences of the N-terminal regions of PsbP and PsbQ\u0026prime;. Although PsbP and PsbQ\u0026prime; are not thought to share a common evolutionary origin, the N-terminal regions consisting of the first 10 to 12 amino acids located near the lumenal domain of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e were similar among PsbP and PsbQ\u0026prime; from various organisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), supporting the presence of conserved interactions of the N-terminal regions of these extrinsic subunits with the lumenal domain of PsbE. Only the above-mentioned amino acid sequence near the N-terminus is similar, and the sequence afterwards is not similar at all.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDetailed structural comparisons suggest that the N-terminal regions of PsbP and PsbQ\u0026prime; both can make multiple interactions with the well-conserved lumenal region of PsbE as well as with the C-terminal residues of PsbF. Although the resolution in this region may not be sufficient to determine the exact conformations of the side chains, PsbP-Ala1 (or PsbP-Tyr2) and PsbP-(Glu/Asp)4 can interact with PsbE-Arg51 in green-lineage PSII, and similarly, PsbQ\u0026prime;-Ala1 and PsbQ\u0026prime;-(Glu/Asp)4 can interact with PsbE-Arg52 (corresponding to green plant PsbE-Arg51) in red-lineage PSII (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, the N-terminal regions of PsbP and PsbQ\u0026prime; both seem to also interact similarly with the C-terminal residues of PsbF (PsbF-Phe36, Gln38, and Arg39 numbered based on \u003cem\u003eArabidopsis\u003c/em\u003e PsbF) (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Interaction of these extrinsic subunits with both PsbE and PsbF may affect the heme environment in between these two Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e subunits, possibly explaining the effect of PsbP on the redox potential of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Ghanotakis et al. 1986; de Paula et al. 1986; Thompson et al. 1989; Nishimura et al. 2016).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eComparison of the N terminal regions of PsbQ and PsbQ\u0026prime;\u003c/h2\u003e \u003cp\u003ePsbP, unique to green-lineage PSII, is suggested to have evolved from the cyanobacterial CyanoP (De Las Rivas et al. 2004; De Las Rivas and Roman 2005; Ishihara et al. 2007), whereas PsbQ\u0026prime;, unique to red-lineage PSII, is thought to have evolved from the cyanobacterial CyanoQ (Ohta et al. 2003; De Las Rivas and Roman 2005). In green-lineage PSII, PsbQ, which is also considered to have evolved from CyanoQ (Kashino et al. 2002; De Las Rivas et al. 2004; De Las Rivas and Roman 2005), is present instead of PsbQ\u0026prime;. PsbQ, PsbQ\u0026prime;, and CyanoQ all share a structurally similar four-helix bundle core, with which they bind to PSII at the same location (the lumenal surface of CP43) (Gisriel and Brudvig 2022; Imaizumi and Ifuku 2022; Gisriel et al. 2022). In addition to the four-helix bundle core, PsbQ\u0026prime; and PsbQ have a long N-terminal loop region, whereas CyanoQ has a shorter N-terminal loop with an N-terminal lipid modification (Thornton et al. 2004).\u003c/p\u003e \u003cp\u003eTo confirm whether or not the N-terminal sequence of PsbQ\u0026prime;, which we found to be similar to that of PsbP, is also found in PsbQ and/or CyanoQ, we compared the amino acid sequences of the N-terminal loop of PsbQ, PsbQ\u0026prime;, and CyanoQ, from the (predicted) N-terminus up to the region near the N-terminal end of the four-helix bundle core. The amino acid sequences of the N-terminal loops of PsbQ, PsbQ\u0026prime;, and CyanoQ were very different from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We did not observe any regions in the N-terminal loops of PsbQ or CyanoQ with sequences similar to that of the N-terminal end of PsbQ\u0026prime; and PsbP. Furthermore, the amino acid sequence alignment of the full-length mature PsbQ\u0026prime; from various red-lineage PSII revealed that, despite having a structurally conserved four-helix bundle core, the N-terminal region was the most well-conserved region within PsbQ\u0026prime; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). These results suggest that PsbQ\u0026prime; and PsbP most likely independently acquired the similar N-terminal amino acid sequence to interact with the lumenal domain of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e. Moreover, the results imply that the interaction with the Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e lumenal domain is likely one of the major roles of PsbQ\u0026prime; in red-lineage PSII, and that the PsbP N-terminus is responsible for this role in green-lineage PSII.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImportance of the conserved lumenal region in PsbE suggested from\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e \u003cb\u003emutants generated by genome editing using TALECD\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe above observations suggested that the well-conserved region, PsbE-Gly48\u0026ndash;(Asn/Asp)53, in the lumenal domain of PsbE may have important functions in PSII. Therefore, we have investigated this by generating \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutants harboring mutations in this region. PsbE is encoded in the \u003cem\u003epsbEFLJ\u003c/em\u003e operon in the plastid genome, and PsbE-deficient plants are incapable of photoautotrophic growth. In order to introduce mutations in the well-conserved PsbE lumenal region without knocking out the \u003cem\u003epsbE\u003c/em\u003e gene, we conducted targeted base editing in the plastid genome using ptpTALECD (plastid-targeted platinum transcription activator-like effector cytidine deaminase) (Nakazato et al. 2021). TALECD mainly consists of the DNA binding domain (TALE domain) of TALEN, the N-terminal or C-terminal half of a split cytidine deaminase (CD), and an uracil glycosylase inhibitor (UGI). When a pair of TALECD bind to their target DNA, the reassembled CD converts C to U, resulting in targeted C/G-to-T/A conversions (Mok et al. 2020; Arimura and Nakazato 2024; Nakazato and Arimura 2024).\u003c/p\u003e \u003cp\u003eUsing this genome editing strategy, we attempted to substitute Arg with Cys at residue 51 of PsbE, as PsbE-Arg51 seemed to be a key residue for interaction with PsbP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), but we could not obtain a PsbE-R51C mutant; this may have been due to the targeted DNA sequence, while it might also be possible that the single PsbE-R51C mutation was lethal. However, we successfully obtained two independent \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutant lines with homoplasmic PsbE-G48E/R51C/P52S mutations. All three amino acid substitutions, G48E, R51C, and P52S occurred within the well-conserved Gly48\u0026ndash;(Asn/Asp)53 region in the lumenal domain of PsbE. The \u003cem\u003eArabidopsis\u003c/em\u003e PsbE-G48E/R51C/P52S mutants showed severe growth defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Although the plants were grown on sucrose-supplemented medium for two weeks before transferring to soil, the mutants managed to survive, but only showed minimal growth with pale green leaves. The maximal quantum yield of PSII (\u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e) was extremely low in the PsbE-G48E/R51C/P52S mutants (\u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e = 0.2\u0026ndash;0.3) due to high \u003cem\u003eF\u003c/em\u003e\u003csub\u003eo\u003c/sub\u003e (dark-adapted minimal fluorescence) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results reveal that the well-conserved lumenal region of PsbE is critical for the accumulation of functional PSII. Unfortunately, due to the strong effects of the deleterious mutation, we were unable to conduct further detailed analyses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we have investigated the lumenal regions of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e. Numerous studies have investigated the role of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e using \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803, \u003cem\u003eThermosynechococcus vestitus\u003c/em\u003e, \u003cem\u003eChlamydomonas reinhardtii\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, or \u003cem\u003eNicotiana tabacum\u003c/em\u003e mutants with various mutations in PsbE or PsbF (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Pakrasi et al. 1991; Tae and Cramer 1992; Bock et al. 1994; Morais et al. 2001; Bondarava et al. 2003, p. 200, 2010; Hung et al. 2007, 2010; Ma et al. 2007; Cai et al. 2009; Chiu et al. 2009, 2013, 2022; Yagi et al. 2013, p. 20; Guerrero et al. 2014; Hamilton et al. 2014; Sugiura et al. 2015; Huang et al. 2016; Endo et al. 2019; Nakamura et al. 2019; Che et al. 2024). However, almost all of these mutations were introduced in the stromal (N-terminal) region or within the transmembrane helix of PsbE or PsbF, and very few studies have focused on the lumenal regions of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). While R59Q, R68Q, and R59Q/R68Q \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 mutants (corresponding to R60Q, R69Q, and R60Q/R69Q in \u003cem\u003eArabidopsis\u003c/em\u003e) have been constructed, these mutations did not show apparent effects on PSII (Tae and Cramer 1992). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003epsbE\u003c/em\u003e transcripts undergo RNA editing mediated by the PPR (pentatricopeptide repeat) protein CREF3 (Chloroplast RNA Editing Factor 3) (Yagi et al. 2013). Through RNA editing, the genomically encoded CCU (proline) codon is post-transcriptionally edited to a UCU (serine) codon (Yagi et al. 2013), resulting in a Ser residue at position 72, which is conserved among land plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Consequently, the CREF3-deficient \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutant \u003cem\u003ecref3\u003c/em\u003e has a PsbE-S72P mutation in the lumenal domain of PsbE. However, no apparent effects on PSII have been observed by this mutation either (Yagi et al. 2013; Che et al. 2024). Meanwhile, it has been reported that the deletion of 12, 22, or 31 C-terminal residues of PsbE, all included within the lumenal domain, results in decreased PSII function (Tae and Cramer 1992). Especially, when 31 C-terminal residues were deleted, PSII assembly was largely diminished. Also, we have previously reported that the interaction of the N-terminal region of PsbP with the lumenal domain of PsbE modulates the redox potential of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Nishimura et al. 2016). These reports implied that the lumenal region of PsbE might have both structural and functional roles.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eList of amino acid residues of PsbE and PsbF that have been studied previously by substitution mutations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstituted residue\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOrganism (Reference)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Thr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Endo et al. 2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Arg8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Arg7) (Chiu et al. 2009)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Ala11*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Ser11) (Endo et al. 2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Ile14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Guerrero et al. 2014)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Arg18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. reinhardtii\u003c/em\u003e (Ma et al. 2007)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Arg17) (Chiu et al. 2013)\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Guerrero et al. 2014)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Tyr19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Tyr18) (Hung et al. 2010)\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Sugiura et al. 2015)\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestisus\u003c/em\u003e (Nakamura et al. 2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-His23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eC. reinhardtii\u003c/em\u003e (Morais et al. 2001)\u003c/p\u003e \u003cp\u003e\u003cem\u003eC. reinhardtii\u003c/em\u003e (Hamilton et al. 2014)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Pakrasi et al. 1991)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Hung et al. 2007)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Hung et al. 2010)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Chiu et al. 2022)\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Sugiura et al. 2015)\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Nakamura et al. 2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Ser24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Ser23) (Huang et al. 2016)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Thr26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Sugiura et al. 2015)\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Nakamura et al. 2019)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Ile27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Guerrero et al. 2014)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Arg60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Arg59) (Tae and Cramer 1992)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Arg69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Arg68) (Tae and Cramer 1992)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbE-Ser72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. thaliana\u003c/em\u003e (Yagi et al. 2013)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eA. thaliana\u003c/em\u003e (Che et al. 2024)\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbF-Arg13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Arg17) (Chiu et al. 2013)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbF-His18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Pakrasi et al. 1991)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Hung et al. 2007)\u003c/p\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (His22) (Chiu et al. 2022)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbF-Thr24*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Ser28) (Huang et al. 2016)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbF-Phe26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eN. tabacum\u003c/em\u003e (Bock et al. 1994)\u003c/p\u003e \u003cp\u003e\u003cem\u003eN. tabacum\u003c/em\u003e (Bondarava et al. 2003)\u003c/p\u003e \u003cp\u003e\u003cem\u003eN. tabacum\u003c/em\u003e (Bondarava et al. 2010)\u003c/p\u003e \u003cp\u003e\u003cem\u003eA. thaliana\u003c/em\u003e (Cai et al. 2009)\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eT. vestitus\u003c/em\u003e (Phe32) (Guerrero et al. 2014)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePsbF-Leu28*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eS.\u003c/em\u003e 6803 (Val32) (Huang et al. 2016)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea, Amino acid residues and residue numbers are based on \u003cem\u003eArabidopsis\u003c/em\u003e. Asterisks (*) indicate that the amino acid residue in \u003cem\u003eArabidopsis\u003c/em\u003e is different from that studied in the cited literature.\u003c/p\u003e \u003cp\u003eb, The studied amino acid residues are shown in brackets after the species, when the corresponding residue or residue number is different from that in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ec, A study using the \u003cem\u003ecref3\u003c/em\u003e mutant, deficient of RNA editing of PsbE.\u003c/p\u003e \u003cp\u003ed, A study using the \u003cem\u003elpa66\u003c/em\u003e mutant, deficient of RNA editing of PsbF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur results with the PsbE-G48E/R51C/P52S mutant \u003cem\u003eArabidopsis\u003c/em\u003e reveals that the lumenal region of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, particularly the region PsbE-Gly48\u0026ndash;(Asn/Asp)53 that we have found to be well-conserved, is indeed important. The drastic effects of the PsbE-G48E/R51C/P52S mutation on plant growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and on PSII (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e) shows that this well-conserved lumenal region of PsbE is critical for the accumulation of functional PSII. The pale-green leaf color and very low \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e due to dramatically increased \u003cem\u003eF\u003c/em\u003e\u003csub\u003eo\u003c/sub\u003e have been observed in various mutants with low levels of PSII (Bondarava et al. 2003; Cai et al. 2009; Armbruster et al. 2010; Cecchin et al. 2021; Zhang et al. 2024a; Che et al. 2024). The protein structure of wild-type \u003cem\u003eArabidopsis thaliana\u003c/em\u003e PsbE predicted by AlphaFold2 (Jumper et al. 2021; Mirdita et al. 2022) was similar to the cryo-electron microscopy (cryo-EM) structure of the PsbE subunit in the PSII supercomplex from \u003cem\u003ePisum sativum\u003c/em\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a\u003c/b\u003e). In contrast, the predicted protein structure of G48E/R51C/P52S mutant PsbE had a partially loosened C-terminal domain (\u003cb\u003eSupplementary Fig.\u0026nbsp;2b, c\u003c/b\u003e), seemingly due to instability of the loop region containing the well-conserved lumenal region, considering from the low pLDDT structure confidence score in this region (\u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). This structural perturbation may have inhibited the proper assembly of PSII. It is also possible that the mutations led to an instable PSII core, disabling the stable accumulation of PSII complexes. Meanwhile, it is not likely that the mutations affected the stable accumulation of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e itself, as even large deletions of the PsbE lumenal domain (from Arg51 to the C-terminus numbered based on \u003cem\u003eArabidopsis\u003c/em\u003e PsbE) has little effect on the accumulation of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Tae and Cramer 1992). Although the strongly deleterious effects of the mutation hindered further experiments in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, further investigations on this conserved region in organisms with mixotrophic capabilities may clarify the detailed roles of the lumenal domain of PsbE.\u003c/p\u003e \u003cp\u003eIn addition to structural roles, the well-conserved PsbE-Gly48\u0026ndash;(Asn/Asp)53 region is likely to also have physiological roles. In green plant PSII, this region in PsbE is the site with which the N-terminal region of PsbP interacts. Interaction of PsbP with the lumenal domain of PsbE (Nagao et al. 2010), particularly by its N-terminal region (Ido et al. 2012, 2014; Nishimura et al. 2016) had already been observed before it was confirmed in cryo-EM structures of green plant PSII (Su et al. 2017; Sheng et al. 2019; Shan et al. 2024). In red-lineage PSII, PsbQ\u0026prime; extends its long N-terminal loop to the same site as the N-terminal region of PsbP in green-lineage PSII to interact with the well-conserved lumenal region of PsbE (Imaizumi and Ifuku 2022) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The observations that the amino acid sequences of the N-terminal regions of PsbP and PsbQ\u0026prime; are similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and that the N-terminal region of PsbQ\u0026prime; is the most well-conserved region within the full-length mature PsbQ\u0026prime; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) even though similar sequences are not observed in N-terminal loops of the cyanobacterial CyanoQ or green plant PsbQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), suggest that there is a conserved interaction between extrinsic subunits (PsbP or PsbQ\u0026prime;) and the PsbE lumenal region, and that the interaction of the N-terminal region of PsbQ\u0026prime; with the lumenal domain of PsbE can be related to one of the major roles of PsbQ\u0026prime;. In green plant PSII, this interaction between the N-terminal region of PsbP and the conserved lumenal region of PsbE on the lumenal side of the thylakoid membrane modulates the redox potential of the heme of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, located near the stromal side (Nishimura et al. 2016). Such transmembrane effects of PsbP have also been observed on the electron transport from Q\u003csub\u003eA\u003c/sub\u003e to Q\u003csub\u003eB\u003c/sub\u003e or the redox potential of Q\u003csub\u003eA\u003c/sub\u003e (Ono and Inoue 1986; Yi et al. 2007; Ido et al. 2009; Roose et al. 2010; Semin et al. 2018; Kato and Noguchi 2021). Interestingly, PsbQ\u0026prime;, bound to the lumenal side of PSII, has similar transmembrane effects on the redox potential of Q\u003csub\u003eA\u003c/sub\u003e as PsbP does (Yamada et al. 2018). Considering that PsbP in green-lineage PSII and PsbQ\u0026prime; in red-lineage PSII only overlap with their N-terminal regions adjacent to the lumenal domain of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), that green plant PsbQ is reported to have little effect on the PSII acceptor side under normal growth conditions (Yi et al. 2006), and that some mutations in Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Hamilton et al. 2014; Nakamura et al. 2019) and compounds that affect the properties of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e (Takagi et al. 2019; Imaizumi et al. 2024) also affect the electron transport from Q\u003csub\u003eA\u003c/sub\u003e to Q\u003csub\u003eB\u003c/sub\u003e or the redox potential of Q\u003csub\u003eA\u003c/sub\u003e (Hamilton et al. 2014; Nakamura et al. 2019), it is possible that the N-terminal regions of PsbP and PsbQ\u0026prime; similarly modulate the acceptor side of green-lineage PSII and red-lineage PSII, respectively (Imaizumi and Ifuku 2022). These effects of PsbP and PsbQ\u0026prime; on the redox potential of Q\u003csub\u003eA\u003c/sub\u003e have both been proposed to have photoprotective roles (Yamada et al. 2018; Kato and Noguchi 2022). While, both structurally and functionally, PsbP in green-lineage PSII mostly replaces PsbV in red-lineage PSII (Ifuku and Nagao 2021), we have recently shown that the Loop 4 region of PsbP, critical for Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e retention in the OEC, replaces the C-terminus of PsbU (Imaizumi et al. 2022). Our current results, suggesting that the N-terminal region of PsbP replaces the N-terminal region of PsbQ\u0026prime;, gives further insight into the complexed functional replacement of the different sets of extrinsic subunits between green- and red-lineage PSII.\u003c/p\u003e \u003cp\u003eIn cyanobacteria, CyanoP and CyanoQ are present instead of PsbP and PsbQ (or PsbQ\u0026prime;). However, CyanoP is an assembly factor of PSII rather than an extrinsic subunit (Cormann et al. 2014; Knoppov\u0026aacute; et al. 2016). Moreover, although CyanoQ is found in PSII structures from mesophilic cyanobacteria (Gisriel et al. 2022; Zhang et al. 2024b), its N-terminal loop seems to be too short to interact with Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e. The N-terminus of PsbV in cyanobacterial PSII as well as in red-lineage PSII does interact with the lumenal domain of PsbE at a position different from the N-terminal regions of PsbP or PsbQ\u0026prime; (\u003cb\u003eSupplementary Fig.\u0026nbsp;4\u003c/b\u003e). This may partially compensate for the absence of extrinsic subunits interacting with the well-conserved lumenal region of PsbE, but it is also possible that this interaction is not required in cyanobacterial PSII. In fact, although Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e in native \u003cem\u003eThermosynechococcus\u003c/em\u003e PSII is mostly in a high-potential form as in PSII from land pants, Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e in \u003cem\u003eSynechocystis\u003c/em\u003e PSII is known to have a lower redox potential (Chiu and Chu 2022).\u003c/p\u003e \u003cp\u003eIn addition to giving insight into the importance of the lumenal region of PsbE, this study also illustrates the powerfulness of TALECD enabling targeted base editing in the plastid genome (Kang et al. 2021; Nakazato et al. 2021). Although improvements have been made recently (Ruf et al. 2019), plastid transformation remains challenging in many land plants including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Maliga 2022). In fact, in land plants, i\u003cem\u003en vivo\u003c/em\u003e mutagenesis studies on Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, whose subunits are encoded in the plastid genome, had only been conducted in tobacco, in which plastid transformation can be routinely conducted (apart from studies using \u003cem\u003eArabidopsis\u003c/em\u003e mutants deficient of RNA editing of \u003cem\u003epsbE\u003c/em\u003e or \u003cem\u003epsbF\u003c/em\u003e). The \u003cem\u003epsbE\u003c/em\u003e gene is encoded within the \u003cem\u003epsbEFLJ\u003c/em\u003e operon together with the \u003cem\u003epsbF\u003c/em\u003e, \u003cem\u003epsbL\u003c/em\u003e, and \u003cem\u003epsbJ\u003c/em\u003e genes. All four subunits are important for PSII; PsbE and PsbF are essential for the assembly of PSII (Pakrasi et al. 1988; Morais et al. 1998), PsbL and PsbJ are required for stable assembly of functional PSII (Anbudurai and Pakrasi 1993; Lind et al. 1993; Kitamura et al. 1994; Regel et al. 2001; Hager et al. 2002), and all four subunits are required for photoautotrophic growth in tobacco (Swiatek et al. 2003). Therefore, in order to introduce substitution mutations into a specific region within the \u003cem\u003epsbE\u003c/em\u003e gene in \u003cem\u003eArabidopsis\u003c/em\u003e, a targeted genome editing strategy with high specificity and a broad targeting range and that does not affect the flanking regions is preferable, and TALECD fulfills these requirements (Arimura and Nakazato 2024; Nakazato and Arimura 2024).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMultiple sequence alignments\u003c/h2\u003e \u003cp\u003eThe N-terminus of the amino acid sequences after removal of the transit peptides were decided based on prediction using TargetP-2.0 (Almagro Armenteros et al. 2019), with the support of multiple sequence alignments when necessary. The N-terminal residue of CyanoQ proteins were predicted in a previous report (Michoux et al. 2014). Multiple sequence alignments were conducted with MAFFT v7.511 using the L-INS-i algorithm (Katoh et al. 2019). In all figures of multiple sequence alignments, asterisks (*), colons (:), and dots (.) below the aligned sequences indicate identical, conserved, and semi-conserved amino acids, respectively. Identical amino acids are shown with black backgrounds, conserved amino acids matching the consensus sequence are shown with gray backgrounds, and conserved amino acids that do not match the consensus sequence, semi-conserved amino acids, and non-conserved amino acids matching the consensus sequence are shown with light gray backgrounds.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtein structure prediction and visualization\u003c/h3\u003e\n\u003cp\u003eProtein structure predictions were performed by AlphaFold2 (Jumper et al. 2021) using ColabFold v1.5.5 (Mirdita et al. 2022), and top-ranked models were used. All protein structures were visualized using PyMOL (The PyMOL Molecular Graphics System, Version 3.0 Schr\u0026ouml;dinger, LLC.).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eVector construction\u003c/h2\u003e \u003cp\u003eThe ptpTALECD expression binary vectors were constructed following the method described by Nakazato et al. (2021). The base editing target, a cytosine-to-thymine (C-to-T) conversion resulting in the R51C mutation, was positioned at either the 8th or 10th C within the 14-bp target window between the TALE recognition sequences of ptpTALECD1333NC or ptpTALECD1397NC. The TALE left and right recognition sequences were as follows: 1397NC\u0026mdash;TTAGCTTACGATGTGTTC (left) and CTGTAAAATACTCGTTT (right); 1333NC\u0026mdash;TACGATGTGTTCGG (left) and CTCTGTAAAATACTCG (right). All intermediate plasmid components used for vector assembly, including the Platinum TALEN assembly kit, are available from Addgene (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.addgene.org\u003c/span\u003e\u003cspan address=\"https://www.addgene.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The assembled TALE repeats were verified by Sanger sequencing, and the final tandem-expression binary vectors\u0026mdash;ptpTALECD1397NC (22,018 bp) and ptpTALECD1333NC (21,508 bp)\u0026mdash;were confirmed by multiple restriction enzyme digestion patterns.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant transformation, screening of transformants, and genotyping\u003c/h3\u003e\n\u003cp\u003eThe TALECD vectors were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 (pMP90), and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e wild-type (Col-0; Columbia-0) plants were transformed by the floral dip method. To select transformants, the collected seeds were sown on half-strength Murashige and Skoog (MS) medium supplemented with 1.5% sucrose and 0.9% agar and containing kanamycin (50 \u0026micro;g/mL) and cefotaxime (100 \u0026micro;g/mL). Kanamycin resistant T1 seedlings were transplanted to half-strength MS agar plates with 1.5% sucrose but without kanamycin, and grown for 1\u0026ndash;2 more weeks before transplanting to soil. Total DNA was extracted from leaves, genomic DNA regions including the target window was amplified by PCR, and purified PCR products were subjected to Sanger sequencing by Eurofins Genomics.\u003c/p\u003e\n\u003ch3\u003ePlant materials and growth conditions\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seeds were sterilized and sown on half-strength MS agar plates supplemented with 1.5% sucrose. After stratification at 4 \u0026ordm;C for 3 days in the dark, plants were grown at 22 \u0026ordm;C under long-day condition (16 hours light/8 hours dark) at a light intensity of 80 \u0026micro;mol photons m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. 2 weeks after germination, plants were transferred to soil, and were grown at the same condition.\u003c/p\u003e \u003cp\u003e \u003cb\u003eF\u003c/b\u003e \u003csub\u003e \u003cb\u003ev\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e/\u003c/b\u003e \u003cb\u003eF\u003c/b\u003e \u003csub\u003e \u003cb\u003em\u003c/b\u003e \u003c/sub\u003e \u003cb\u003emeasurements\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eF\u003c/em\u003e \u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e of 17-day-old plants were measured using JUNIOR-PAM/W fluorometer (Walz) after one hour of dark adaptation or using the chlorophyll fluorescence imaging device FluorCam 800MF (Photon Systems Instruments) after 15 minutes of dark adaptation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported in part by JSPS Grant-in-Aid for JSPS Fellows JP23KJ1361 to K. Imaizumi, for Challenging Research (Exploratory) JP24K21968 to K. Ifuku, and for Transformative Research Areas (A) 24H02271 to S. Arimura.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK. Imaizumi and K. Ifuku conceived the project; S. Arimura provided the system for the targeted base editing of the plastid genome. K. Imaizumi performed all analyses and drafted the original manuscript; K. Imaizumi and K. Ifuku revised the manuscript and wrote the final manuscript, and all authors joined the discussion of the results.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlmagro Armenteros JJ, Salvatore M, Emanuelsson O, et al (2019) Detecting sequence signals in targeting peptides using deep learning. 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It consists of two subunits, PsbE and PsbF, which together coordinate a redox-active heme. While extensive studies have revealed the importance of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, its structural and functional roles are not fully understood. Previous studies have implied that the lumenal region of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, interacting with the PSII extrinsic subunit PsbP in green plant PSII, may have important roles. However, few studies have investigated its lumenal region. Here, we have focused on a well-conserved lumenal region of PsbE, which was found to interact with the N-terminal region of PsbP in green-lineage PSII (from green algae and land plants). In red-lineage PSII (from red algae and algae possessing red algal-derived plastids), very similar interactions were observed between the same lumenal region of PsbE and the N-terminal region of PsbQ\u0026prime;. We generated \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutants harboring mutations in the well-conserved lumenal region of PsbE through targeted base editing of the plastid genome by ptpTALECD. The mutations led to strong growth defects and extremely low \u003cem\u003eF\u003c/em\u003e\u003csub\u003ev\u003c/sub\u003e/\u003cem\u003eF\u003c/em\u003e\u003csub\u003em\u003c/sub\u003e. This study suggests the importance of the lumenal regions of Cyt \u003cem\u003eb\u003c/em\u003e\u003csub\u003e559\u003c/sub\u003e, and gives insight into possible structural and functional compensation between the N-terminal regions of PsbP in green-lineage PSII and PsbQ\u0026prime; in red-lineage PSII.\u003c/p\u003e","manuscriptTitle":"The lumenal domain of Cyt b 559 interacting with extrinsic subunits is crucial for accumulation of functional photosystem II","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 15:08:03","doi":"10.21203/rs.3.rs-6005678/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-14T19:34:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-14T12:35:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-12T15:40:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-04T12:39:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"168912897212517289091714413862605172874","date":"2025-03-03T09:39:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115175177073061864874137074348627497968","date":"2025-02-27T16:29:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71574429293623863805109722898799411121","date":"2025-02-17T19:42:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-17T10:31:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-17T08:51:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-12T16:46:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photosynthesis Research","date":"2025-02-11T09:06:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ce689237-6208-4cc8-93ed-f19fd050d0af","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:02:56+00:00","versionOfRecord":{"articleIdentity":"rs-6005678","link":"https://doi.org/10.1007/s11120-025-01157-2","journal":{"identity":"photosynthesis-research","isVorOnly":false,"title":"Photosynthesis Research"},"publishedOn":"2025-06-10 15:58:02","publishedOnDateReadable":"June 10th, 2025"},"versionCreatedAt":"2025-04-10 15:08:03","video":"","vorDoi":"10.1007/s11120-025-01157-2","vorDoiUrl":"https://doi.org/10.1007/s11120-025-01157-2","workflowStages":[]},"version":"v1","identity":"rs-6005678","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6005678","identity":"rs-6005678","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}