Toshiharu Shikanai

Summary: Chlorophagy removes photodamaged chloroplasts in plants, while individual chloroplasts are also protected by mechanisms that limit damage to the photosynthetic apparatus caused by excess light energy, known as photoinhibition. How chlorophagy is functionally coupled with chloroplast‐level protective mechanisms during acclimation to photoinhibition remains unclear. We investigated acclimatory responses to photoinhibition at the intracellular level in Arabidopsis thaliana through an integrative analysis combining Chl fluorescence measurements, microscopy imaging, and genetic analyses using mutants defective in nonphotochemical quenching (NPQ) or the chloroplast avoidance response. The functionality of individual chloroplasts was further assessed by Chl fluorescence lifetime imaging and probe‐based visualization of singlet oxygen and starch. Severe photoinhibition of photosystem II precedes the appearance of abnormally swollen chloroplasts, which are subsequently recognized by the autophagy machinery for degradation. The swollen chloroplasts represent irreversibly damaged chloroplasts that accumulate reactive oxygen species and lose photosynthetic function. The formation of these nonfunctional chloroplasts was enhanced in mutants defective in NPQ or the chloroplast avoidance response, and hyperaccumulation of nonfunctional chloroplasts led to necrotic cell death. Together, our findings illustrate how multilayered responses coordinate acclimation to photoinhibition: Chloroplast protective mechanisms minimize the formation of nonfunctional chloroplasts while chlorophagy removes them when they arise.

To avoid photodamage of photosystem I (PSI) under fluctuating light, plants have evolved multiple photoprotective mechanisms. One key mechanism is photosynthetic control, in which acidification of the thylakoid lumen downregulates electron transport through the cytochrome b6f (Cyt b6f) complex, thereby preventing over-reduction of PSI. The Arabidopsis proton gradient regulation 5 (pgr5) mutant, which is defective in cyclic electron transport around PSI, fails to induce photosynthetic control and consequently suffers severe PSI photodamage under fluctuating light. Previously, we showed that introduction of the pgr1 mutation, which enhances the pH sensitivity of the Cyt b6f complex, partially restored PSI oxidation and alleviated PSI photodamage in the pgr5 background. However, excessively strong photosynthetic control limits electron transport at relatively low light intensities. To investigate whether a milder enhancement of photosynthetic control can protect PSI without compromising photosynthetic performance, we introduced a series of amino acid substitutions into the Rieske subunit of the cytochrome b6f complex using Target-AID-mediated base editing. Among these, the E143K mutation partially oxidized PSI and improved photosynthetic induction in the pgr5-2 background more effectively than the pgr1 mutation. Although the E143K mutation had little effect on electron transport parameters in the wild-type background, it significantly reduced the proton motive force. The unexpected reduction in proton motive force suggests that moderately enhanced photosynthetic control can be accommodated without major impairment of photosynthetic electron transport.

C₄ photosynthesis requires more ATP than C₃ photosynthesis to drive the C₄ cycle, which concentrates CO₂ in the bundle sheath cells. This ATP demand is expected to be met by cyclic electron transport around photosystem I. Of the two cyclic electron transport pathways, PROTON GRADIENT REGULATION 5 (PGR5)/PGR5-like photosynthetic phenotype 1 (PGRL1)-dependent and chloroplast NADH dehydrogenase-like (NDH)-dependent, ATP is supplied primarily by the NDH-dependent pathway in NADP-malic enzyme-type C₄ plants. However, the roles of these pathways in photoprotection of photosystem I have not been fully evaluated in C₄ plants. In this study, we used Flaveria bidentis knockdown lines targeting PGRL1 or NdhO and their double-knockdown lines to investigate the redox state of the electron transport chain and high light sensitivity of photosystems. The double-knockdown line exhibited poorer growth than the FbNdhO-RNAi line, indicating that both pathways were essential for ATP production. The acceptor side of P700 was limited under high light conditions in the FbNdhO-RNAi line, while it was limited under lower to high light conditions in the FbPGRL1-RNAi and double-knockdown lines. Ferredoxin was reduced more in the FbPGRL1-RNAi line than in the wild type, and it was even more reduced in the double-knockdown line. Photoinhibition caused by high light exposure was more pronounced in photosystem I than in photosystem II in these lines. These results demonstrate that both cyclic electron transport pathways are essential not only for ATP production required for growth but also for protecting photosystem I from photoinhibition by alleviating the acceptor-side limitations of P700 in C₄ plants.

Toshiharu Shikanai; A New Player in Photosystem I Photoprotection under Fluctuating Light, Plant and Cell Physiology, , pcag021, https://doi.org/10.1093/pc

The chloroplast NADH dehydrogenase (NDH)-like complex facilitates the ferredoxin-dependent reduction of plastoquinone, coupled with proton translocation across the thylakoid membrane, thereby mediating cyclic electron transport around photosystem I (PSI). The NDH complex evolved from the cyanobacterial counterpart and it forms a large supercomplex with two copies of the PSI complex in angiosperms. In Arabidopsis, NDH-deficient mutants exhibit impaired oxidation of PSI during low-light phases under fluctuating light conditions. Despite its important physiological function clarified in angiosperms, the NDH complex has been lost in certain lineages of eukaryotic phototrophs, including some green and red algae, as well as specific gymnosperms. This loss is likely to be compensated by alternative regulatory mechanisms involving the PROTON GRADIENT REGULATION 5 protein or flavodiiron proteins (Flv). This review article provides an overview of the current knowledge on the evolution of the NDH-PSI supercomplex through the acquisition of new subunits. We also summarize the evolutional loss of the NDH complex, primarily focusing on monocotyledonous plants to extensively investigate the loss of the NDH complex in angiosperms, which had lost Flv genes early in their evolution. In monocots, loss of the NDH complex is relatively rare and occurred mostly in Orchidaceae (Asparagales) and among submerged aquatic plants in Alismatales. These findings support the idea that the NDH complex is crucial for maintaining optimal photosynthetic activity in terrestrial angiosperms exposed to harsh light environments.

SUMMARY: In angiosperms, cyclic electron transport around photosystem I (PSI) is mediated by two pathways that depend on the PROTON GRADIENT REGULATION 5 (PGR5) protein and the chloroplast NADH dehydrogenase‐like (NDH) complex, respectively. In the Arabidopsis double mutants defective in both pathways, plant growth and photosynthesis are impaired. The pgr5‐1 mutant used in the original study is a missense allele and accumulates low levels of PGR5 protein. In this study, we generated two knockout (KO) alleles, designated as pgr5‐5 and pgr5‐6, using the CRISPR‐Cas9 technology. Although both KO alleles showed a severe reduction in P700 similar to the pgr5‐1 allele, NPQ induction was less severely impaired in the KO alleles than in the pgr5‐1 allele. In the pgr5‐1 allele, the second mutation affecting NPQ size was mapped to ~21 cM south of the pgr5‐1 locus. Overexpression of the pgr5‐1 allele, encoding the glycine130‐to‐serine change, complemented the pgr5‐5 phenotype, suggesting that the pgr5‐1 mutation destabilizes PGR5 but that the mutant protein retains partial functionality. Using two KO alleles, we created the double mutants with two chlororespiratory reduction (crr) mutants defective in the NDH complex. The growth of the double mutants was notably impaired. In the double mutant seedlings that survived on the medium containing sucrose, PSI activity evaluated by the P700 oxidation was severely impaired, whereas PSII activity was only mildly impaired. Cyclic electron transport around PSI is required to maintain PSI activity.

Cyclic electron transport (CET) around PSI was discovered seven decades ago (Arnon et al., 1954). Nevertheless, its precise physiological function has only recently become the focus of investigation. PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET contributes to photoprotection by acidifying the thylakoid lumen (Munekage et al., 2002; Suorsa et al., 2012). While the generation of ∆pH concurrently stimulates ATP synthesis, it has not been experimentally proven how PGR5-dependent CET contributes to ATP synthesis during steady-state photosynthesis. Degen et al. (2024) have successfully monitored the contribution of PGR5-dependent CET to photosynthesis in planta by studying oscillations of photosynthesis.

The proton motive force (pmf) generated across the thylakoid membrane rotates the Fo-ring of ATP synthase in chloroplasts. The pmf comprises two components: membrane potential (∆Ψ) and proton concentration gradient (∆pH). Acidification of the thylakoid lumen resulting from ∆pH downregulates electron transport in the cytochrome b6f complex. This process, known as photosynthetic control, is crucial for protecting photosystem I (PSI) from photodamage in response to fluctuating light. To optimize the balance between efficient photosynthesis and photoprotection, it is necessary to regulate pmf. Cyclic electron transport around PSI and pseudo-cyclic electron transport involving flavodiiron proteins contribute to the modulation of pmf magnitude. By manipulating the ratio between the two components of pmf, it is possible to modify the extent of photosynthetic control without affecting the pmf size. This adjustment can be achieved by regulating the movement of ions (such as K+ and Cl−) across the thylakoid membrane. Since ATP synthase is the primary consumer of pmf in chloroplasts, its activity must be precisely regulated to accommodate other mechanisms involved in pmf optimization. Although fragments of information about each regulatory process have been accumulated, a comprehensive understanding of their interactions is lacking. Here, I summarize current knowledge of the network for pmf regulation, mainly based on genetic studies.

C₄ plants assimilate CO₂ more efficiently than C₃ plants because of their C₄ cycle that concentrates CO₂. However, the C₄ cycle requires additional ATP molecules, which may be supplied by cyclic electron flow (CEF) around photosystem I. One CEF route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C₄ plants despite the low activity in C₃ plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1) and is considered a major CEF route to generate the proton gradient across the thylakoid membrane in C₃ plants. However, its contribution to C₄ photosynthesis is still unclear. In this study, we investigated the contribution of the two CEF routes to the NADP-malic enzyme subtype of C₄ photosynthesis in Flaveria bidentis. We observed that suppressing the NDH-dependent route drastically delayed growth and decreased the CO₂ assimilation rate to approximately 30% of the wild-type rate. On the other hand, suppressing the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO₂ assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent CEF substantially contributes to the NADP-malic enzyme subtype of C₄ photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during C₄ evolution, photosynthetic electron flow may have been optimized to provide the energy required for C₄ photosynthesis.

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F₁F₀-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c₁₅-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.