Cyclic electron flow A to Z

Oxygen-evolving photosynthesis involve the electron-transport part and CO₂ assimilation part. While the main electron-transport pathway provides NADPH and ATP (linear electron flow, LEF), one of the alternative electron-transport mechanism is the cyclic electron transport around photosystem (PS) I, which provides only ATP (cyclic electron flow, CEF). The phenomenon of CEF was discovered by Arnon et al. (1954). CEF balances the ATP: NADPH ratio in the chloroplast by building a proton motive force (pmf) across the thylakoid membranes, which drives ATP synthesis. Lumenal acidification obtained by the formation of pmf induces the nonphotochemical quenching (NPQ), which contributes to the photoprotection of PSII (Muller et al. 2001). To protect PSI, the oxidation of P700 (the reaction center of PSI) is crucial to help avoid the overexcitation of P700, resulting in PSI photoinhibition (Miyake 2020). CEF functions to oxidize P700 at the donor side of PSI by pmf dependent downregulation of photosynthetic electron transport (photosynthetic control), and at the acceptor side of PSI by recycling the electrons from PSI to PSI (Yamori and Shikanai 2016). Therefore, CEF is an important mechanism for the photoprotection of photosystems. In a JPR symposium titled “Cyclic Electron Flow A to Z”, the symposiasts presented their work related to the regulation of CEF (Dr. Okegawa and Dr. Buchert) and the diversity of CEF (Dr. Bailleul, Mr. Furutani, and Dr. Shimakawa). Dr. Bailleul and Mr. Furutani also highlighted the limitations of the methodology used to measure the CEF by monitoring P700 redox state. In this special issue, Dr. Okegawa and the colleagues further analyzed thioredoxins mutants in Arabidopsis thaliana. Dr. Shimakawa and the colleagues investigated the electron flow into PSI in Synechocystis sp. PCC 6803. Mr. Furutani and Prof. Miyake's group demonstrated that the misestimation of the photo-oxidaizable P700 occurred due to the limitation at the PSI donor side using Arabidopsis thaliana and Triticum aestivum. Here I introduce three valuable articles in this special issue along with a briefly summarized background of the CEF pathways and related measurement methods.

In oxygen-evolving photosynthetic organisms, two pathways are known to contribute to CEF (Shikanai 2014). The NDH pathway mediates electron flow from ferredoxin to the plastoquinone pool via the NAD(P)H dehydrogenase-like (NDH) complex in the thylakoid membranes (Peltier et al. 2016). Because the NDH complex is a homologue of complex I in the respiratory chain, it functions as a proton pump across the thylakoid membrane. While NADH is the electron donor in complex I of the respiratory chain, ferredoxin is the electron donor in the NDH complex (Schuller et al. 2019; Yamamoto et al. 2011). Electron transfer from ferredoxin to the complex was demonstrated using an in vitro assay (Schuller et al. 2019; Yamamoto and Shikanai 2013).

Another CEF pathway is the PGR pathway, which mediated by the proton gradient regulation 5 (PGR5) protein and likely its partner protein(s) (Shikanai 2014). PGR5 is a small extrinsic thylakoid membrane protein with no cofactors or domains (Munekage et al. 2002). PGR5-deficient Arabidopsis thaliana showed lower levels of NPQ, and a lower ratio of oxidized P700 to the total amount of P700 under the illumination, meaning that the electron flow was easily limited at PSI acceptor side (Johnson et al. 2014; Munekage et al. 2002). Although the electron derived from ferredoxin is thought to reduce plastoquinone in the PGR pathway, it remains unclear whether ferredoxin directly reduces plastoquinone or reduces plastoquinone through the cytochrome b₆f complex. Recently, Buchert et al. demonstrated that PGR5 mediates the Q-cycle at the cytochrome b₆f complex in Chlamydomonas reinhardtii (Buchert et al. 2020).

PGRL1 (PGR5-like photosynthetic phenotype 1) has been identified as a binding partner of PGR5 (DalCorso et al. 2008). Hertle et al. (2013) demonstrated that PGR5 and PGRL1 form a hetero dimer to drive CEF. Although PGRL1 seems to be crucial in driving CEF, PGRL1 has recently been proposed to stabilize PGR5 rather than drive CEF in Arabidopsis thaliana (Rühle et al. 2021). Rühle et al. (2021) reported that PGR5 accumulation is stabilized by interacting with PGRL1, while the PGRL1-homolog PGRL2 promotes the degradation of PGR5. Because PGRL2 was not present in Physcomitrella patens and Chlamydomonas reinhardtii (Rühle et al. 2021), aquatic photosynthetic organisms seem to use different regulatory mechanisms in the PGR pathway.

The PGR pathway is upregulated during the induction of photosynthesis in a redox-dependent manner in Arabidopsis thaliana (Okegawa and Motohashi 2020; Wolf et al. 2020). f-type and m-type thioredoxins help activate Calvin-Benson-Bassham cycle enzymes (Geigenberger and Fernie 2014). Okegawa and Motohashi demonstrated that one of the m-type thioredoxins, Trx m4, interacted with PGRL1 through its cysteine residue in the N-terminal region, and they proposed a model in which Trx m4 negatively regulates the PGR pathway (Okegawa and Motohashi 2020). According to this model, Trx m4 suppresses the PGR pathway under a steady state by the formation of a hetero dimer with PGRL1 (Okegawa and Motohashi 2020), to avoid the overactivation of the PGR pathway that triggers the growth retardation (Okegawa et al. 2007). Negative regulations of the PGR pathway by PGRL2 or Trx m4 might be crucial for the distributions of the reductants derived from photosynthetic electron transport to the CO₂ assimilation process and the Trx system in the land plants. The PGR pathway is upregulating by dissociating the Trx m4-PGRL1 complex during the induction of photosynthesis, in which the reductants hyperaccumulate in the downstream of PSI (Okegawa and Motohashi 2020). The activation of Calvin-Benson-Bassham cycle enzymes by the f-type thioredoxins and PGR5-dependent formation of pmf also contribute to the efficient induction of photosynthesis (Okegawa et al. 2020). In this special issue, Okegawa et al. further analyzed using trx f1f2 m124-2 quintuple mutants. Their results suggested that the m-type and f-type thioredoxins cooperatively function to induce photosynthesis, whereas the m-type thioredoxins solely regulate the PGR pathway (Okegawa et al. 2022).

Evaluating the activity of CEF is crucial in CEF research. However, the methods used to measure CEF are various and ambiguous. It is likely due to the fact that PSI has no evident activity, unlike the oxygen-evolving activity of PSII. To date, several methods have been utilized for the evaluating CEF: the redox state of P700, electrochromic shift, and chlorophyll fluorescence measurements (Kou et al. 2013). The redox state of P700 is monitored as the light-induced absorbance change (Alric 2010; Klughammer and Schreiber 1994). The redox change in P700 reflects the electron flow into PSI in both the LFF and CEF. Therefore, CEF is measured under far-red illumination to excite preferentially PSI, and/or in the presence of DCMU, to inhibit the electron flow from PSII. In cyanobacteria, the redox change of P700 is also affected by the electron flow derived from the respiration. Kusama et al. (2022) measured the CEF rate at PSI using the inhibitors of PSII, the oxidative pentose phosphate (OPP) pathway, and the respiratory terminal oxidases and in the corresponding mutants. The specificity of the inhibitors was evaluated by monitoring O₂ evolution and uptake, and the NAPD(P)H redox state. Subsequently, they investigated the efficiency of re-reduction of P700 in the absence and presence of the inhibitors. Their results showed that the re-reduction rate of P700 was extremely slow under the condition in which PSII, the OPP pathway and the respiratory terminal oxidases were inhibited, suggesting that the proportion of the electron injection derived from CEF into PSI is less than 1% of the total injection (Kusama et al. 2022).

The maximal level of photo-oxidizable P700 under dark-adapted conditions reflects the total amount of PSI reaction centers (Pm), which, under actinic light, reflects the amount of functional PSI reaction centers (Pm’) (Klughammer and Schreiber 1994). The Pm and Pm’ values are determined by flashing a saturation pulse of illumination and are corrected using oxidized plastocyanin. These values are also used to determine the parameter Y(I), which defines the effective quantum yield of PSI. Principally, Y(I) and Y(II), the effective quantum yield of PSII, are supposed to show the same level. However, there was a tendency for higher Y(I) than Y(II) when observed under the conditions in which the P700 oxidation level was increased, and Furutani et al. assumed that higher Y(I) values were caused by the charge recombination of P700 (Kadota et al. 2019) or over estimation of Y(I) (Furutani et al. 2021). To address this issue, they mimicked a PSI-donor limiting situation and analyzed the kinetics of the redox changes of P700, plastocyanin and ferredoxin using Dual-KLAS/NIR. They also conducted the same series of measurement in the plastocyanin-knockdown mutant and PGR5-defective mutant. They demonstrated that the Y(I) value could easily be overestimated due to the large proportion of oxidized plastocyanin when PSI donor side limitation occurred (Furutani et al. 2021).

Although the CEF-research field has been advanced using biochemistry, biophysics, and molecular genetics, some aspects of the pathway remain elusive, and methodologies to evaluate the activity of CEF have been under debated. Three articles in this issue illustrate the regulation of CEF and related methodology, and I hope that these articles will facilitate further research on CEF.