Probing the power grid of C₄ plants: how the cyclic electron transport fuels and protects photosynthesis

Light that reaches chloroplasts drives the production of ATP and NADPH, which are required for CO₂ fixation during photosynthesis. Such light-dependent reactions occur in the system of membranes embedded in chloroplasts known as thylakoids. Thylakoid membranes harbor two types of light-harvesting protein complexes, namely photosystems (PS) I and II. In PSII, light energy drives the oxidation of water, releasing electrons that pass through a series of redox reactions via a protein complex known as cytochrome b₆f, and PSI, ultimately reducing NADP⁺ into NADPH. This pathway, known as linear electron transport (LET), also generates a proton motive force (pmf) in the lumen of thylakoids that fuels ATP production via ATP synthase activity (Fig. 1). The redox reactions in thylakoids therefore work as a power transmission line that has to be tightly regulated not only to match the energetic demand of carbon fixation reactions of photosynthesis, but also to avoid overload when this demand decreases, for example when stomata close and photosynthesis is limited by CO₂ influx. Such downregulation is fundamental in preventing the formation of reactive oxygen species (ROS), which can damage the photosynthetic apparatus, reducing the photosynthetic rate and growth (Krause 1988).

Figure 1.

Electron transport pathways in thylakoid membranes. Blue and red lines indicate linear electron transport (LET) and cyclic electron transport around photosystem I (CET-PSI). Selected components of thylakoid membrane are indicated: photosystem II (PSII), plastoquinone (PQ), plastocyanin (PC), ferredoxin (Fd), ferredoxin-NADP⁺ reductase (FNR), PROTON-GRADIENT REGULATION 5 (PGR5)/PGR5-like photosynthetic phenotype 1 (PGRL1), chloroplast NADH dehydrogenase-like (NDH). Modified from Nakamura et al. (this issue). Schematic representation of a thylakoid membrane showing components of the electron transport flow.

Thylakoid membranes also support an alternative energetic pathway known as the cyclic electron transport around PSI (CET-PSI; Arnon et al. 1967). In this route, the electron that flows to PSI is redirected to cytochrome b₆f, which addresses it back to PSI, releasing protons in the thylakoid lumen during this process and contributing to ATP formation that is decoupled from NADPH production (Fig. 1). The CET-PSI is particularly important for C₄ plants, which have higher ATP demand than C₃ plants (Yin and Struik 2018). In C₄ leaves, CO₂ is initially fixed in mesophyll cells (MC) into a C₄ acid that is shuttled to the adjacent bundle sheath cells (BSC), where it is decarboxylated, releasing CO₂ that is fixed by Rubisco. The extra ATP required by C₄ plants is used for the regeneration of the initial CO₂ acceptor in the MC. In some C₄ species, NADP-malic enzyme (NADP-ME) is used for decarboxylating the C₄ acid in the BSC, and this forms NADPH as a byproduct. This additional source of NADPH translates into a smaller LET demand in the BSC, and indeed some C₄ NADP-ME species have agranal chloroplasts in the BSC, with reduced PSII and LET activity (Woo et al. 1970). The differential control of light-dependent reactions in MC and BSC chloroplasts has recently regained attention due to its relevance for understanding how the C₄ machinery evolved (Wang et al. 2026) and for C₄ engineering efforts (Ermakova et al. 2020). In this issue of Plant Physiology, Nakamura and colleagues (2026) provide new mechanistic insights into how the CET-PSI pathway contributes to photosynthesis and photoprotection in C₄ plants using the model plant Flaveria bidentis.

The CET-PSI has two known routes for passing electrons to cytochrome b₆f. One depends on PROTON-GRADIENT REGULATION 5 (PGR5)/PGR5-like photosynthetic phenotype 1 (PGRL1), and a second relies on the chloroplast NADH dehydrogenase-like (NDH) complex. During CET-PSI, an electron is transferred from PSI to ferredoxin (Fd), which then passes it to plastoquinone (PQ) either via PGR5/PGRL1 or NDH (Fig. 1). PQ then reduces plastocyanin (PC) via cytochrome b₆f, releasing two (PGR5/PGRL1 pathway) or four (NDH pathway) protons in the thylakoid lumen, while PC restarts the CET by reducing Fd via PSI. The specific roles of PGR5/PGRL1 and NDH have been studied in C₃ and C₄ species. In C₃ plants, the PGR5/PGRL1-dependent pathway contributes mainly to photoprotection by preventing PSI overreduction (Suorsa et al. 2012), while in some C₄ plants the NDH-dependent route mainly contributes to ATP formation (Takabayashi et al. 2005). However, the mechanism by which CET-PSI protects C₄ leaves from photoinhibition, and the specific role of PGR5/PGRL1 and NDH in MC and BSC remained unclear.

Nakamura et al. address these gaps using RNAi knockdown (KD) lines of F. bidentis. By crossing PGR5/PGRL1- and NDH-RNAi KD lines together, they generated double KD offspring with drastically impaired CET-PSI, and conducted growth, physiological, and biochemical analyses to determine the contribution of each CET-PSI route to photosynthesis and photoprotection. They report severe growth reduction in the double KD line as a result of reduced net CO₂ assimilation and electron transport rates, which were lower than both the single KD lines and the wild type. To elucidate the mechanistic link between CET-PSI and impaired photosynthesis, the authors performed detailed assays probing chlorophyll fluorescence in isolated thylakoids. Among their results is the finding that the reaction center of PSI (P700) is over-reduced in the KD lines, which limits the capacity of PSI to accept electrons from PC, therefore impairing the capacity of photochemical energy dissipation. To test the hypothesis that CET-PSI impaired lines would be particularly sensitive to high light, they grew plants at low light levels (80–100 μmol m⁻² s⁻¹) and assessed the amount of oxidized P700 before and after exposing the plants to a 15-min pulse of saturating light (2,200 μmol m⁻² s⁻¹) followed by dark acclimation. They observed that, while wild-type plants could recover normal levels of oxidized P700 after dark acclimation, CET-PSI deficient lines could not, with double KD lines recovering only 30% of pre-exposure levels, which are lower values than either parental KD line. Their results support the idea that both CET-PSI routes are necessary not only for energy production in C₄ plants, but also for photoprotection, through preventing overreduction of thylakoid membrane components when light energy input surpasses the photosynthetic demand.

Nakamura et al. provide a detailed mechanistic explanation for the regulatory mechanisms underlying the notable efficiency of the photosynthetic apparatus of C₄ plants. Some questions, however, deserve further investigation. Agranal BSC chloroplasts as those in F. bidentis were also observed in other NADP-ME species, but they are not the rule in C₄ plants (Ueno et al. 2005). Determining if agranal BSC results from lineage-specific contingencies or adaptation to particular environments would help us better understand how cell energetic functions are partitioned between MC and BSC during C₄ evolution. This could in turn have important implications for creating optimized designs of C₄-engineered crops.

Recent research articles in Plant Physiology:

Peltier et al. (2024) investigate the contribution of alternative electron transport pathways to energy production required for CO₂ fixation in the model microalgae Chlamydomonas reinhardtii, providing a perspective of the importance of the cyclic electron transport in the context of another CO₂-concentrating mechanism.

Lauterbach et al. (2025) provide insights into how the C₄ machinery evolved from a C₃ ancestral by conducting a detailed comparative study of leaf transcriptomes in closely related C₃, C₃-C₄ intermediate, and C₄ species of the grass genus Neurachne.

Funding

M.E.B. is supported by the Horizon Europe programme (Marie Skłodowska-Curie Actions Postdoctoral Fellowshipgrant 101105838).

Data availability

No new data were generated or analysed in support of this News and Views article.