Collaboration between NDH and KEA3 Allows Maximally Efficient Photosynthesis after a Long Dark Adaptation

In angiosperms, the NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around PSI (CET). K⁺ Efflux Antiporter3 (KEA3) is a putative thylakoid H⁺/K⁺ antiporter and allows an increase in membrane potential at the expense of the ∆pH component of the proton motive force. In this study, we discovered that the chlororespiratory reduction2-1 (crr2-1) mutation, which abolished NDH-dependent CET, enhanced the kea3-1 mutant phenotypes in Arabidopsis (Arabidopsis thaliana). The NDH complex pumps protons during CET, further enhancing ∆pH, but its physiological function has not been fully clarified. The observed effect only took place upon exposure to light of 110 µmol photons m⁻² s⁻¹ after overnight dark adaptation. We propose two distinct modes of NDH action. In the initial phase, within 1 min after the onset of actinic light, the NDH-dependent CET engages with KEA3 to enhance electron transport efficiency. In the subsequent phase, in which the ∆pH-dependent down-regulation of the electron transport is relaxed, the NDH complex engages with KEA3 to relax the large ∆pH formed during the initial phase. We observed a similar impact of the crr2-1 mutation in the genetic background of the PROTON GRADIENT REGULATION5 overexpression line, in which the size of ∆pH was enhanced. When photosynthesis was induced at 300 µmol photons m⁻² s⁻¹, the contribution of KEA3 was negligible in the initial phase and the ∆pH-dependent down-regulation was not relaxed in the second phase. In the crr2-1 kea3-1 double mutant, the induction of CO₂ fixation was delayed after overnight dark adaptation.

Photosynthesis consists of two sets of reactions, the light reactions and the Calvin-Benson cycle. It takes place in the chloroplast and fixes CO₂ into organic compounds using solar energy. In the light reactions, the absorption of photons activates electron transport in two photosystems. In linear electron transport (LET), PSII catalyzes the light-dependent oxidation of water, resulting in the release of oxygen and protons (H⁺) in the thylakoid lumen. The water-derived excised electrons are transferred to PSI through the cytochrome (Cyt) b₆f complex and ultimately to NADP⁺, producing NADPH. This electron transport is coupled with the translocation of H⁺ from the stroma to the thylakoid lumen via the quinone cycle at the Cyt b₆f complex, resulting in the formation of a proton concentration gradient across the thylakoid membrane. This ∆pH contributes to the formation of proton motive force (pmf) in addition to the membrane potential formed across the thylakoid membrane (∆ψ) that results from the uneven distribution of ions across the membrane. The pmf energizes ATP synthesis via F_oF₁-ATP synthase in chloroplasts (Kramer et al., 2003; Soga et al., 2017) and thus influences the efficiency of the light reactions.The Calvin-Benson cycle depends on NADPH and ATP produced by the light reactions. To fix a molecule of CO₂ into a carbohydrate, three molecules of ATP and two molecules of NADPH are needed. However, this ratio of ATP to NADPH (1.5) is not satisfied by LET (Shikanai, 2007). Photorespiration, which takes place due to the low specificity of Rubisco, the CO₂-fixing enzyme for CO₂, increases the energetic requirements in terms of ATP, raising the above ratio to 1.67. The additional ATP is thought to be supplied by cyclic electron transport around PSI (CET; Yamori and Shikanai, 2016). In contrast to LET, CET is driven solely by PSI and does not contribute to the net production of reducing power. CET recycles electrons from ferredoxin (Fd) to the plastoquinone (PQ) pool and contributes to the additional generation of ∆pH via the quinone cycle. As a result, CET balances the production ratio of ATP and NADPH. In angiosperms, CET has been proposed to consist of two pathways: the PROTON GRADIENT REGULATION5 (PGR5)/PGR5-like Photosynthetic Phenotype1 (PGRL1) protein-dependent, antimycin A-sensitive pathway and the NADH dehydrogenase-like (NDH) complex-dependent antimycin A-insensitive pathway (Munekage et al., 2004). The NDH complex pumps four protons, coupled with the movement of two electrons, from Fd to PQ, further increasing the efficiency of ∆pH formation (Strand et al., 2017).In addition to ATP synthesis, the ∆pH component of pmf also contributes to the down-regulation of electron transport (Shikanai, 2014). Acidification of the thylakoid lumen triggers the thermal dissipation of excessively absorbed light energy from the PSII antennae, a process that is monitored by nonphotochemical quenching (NPQ) of chlorophyll fluorescence (Müller et al., 2001). Low lumenal pH also down-regulates the activity of the Cyt b₆f complex, slowing down the rate of electron transport toward PSI (Stiehl and Witt, 1969). CET-dependent ∆pH formation is also necessary to induce the down-regulation of electron transport, as indicated by the phenotype of the pgr5 mutant. The Arabidopsis (Arabidopsis thaliana) pgr5 mutant cannot induce thermal dissipation under excessive light conditions (Munekage et al., 2002), suggesting that CET-generated ∆pH plays an important role in providing a sufficiently acidic lumen pH that can trigger NPQ. The pgr5 mutant is also defective in the down-regulation of Cyt b₆f activity, resulting in hypersensitivity of PSI to fluctuating light intensity (Tikkanen et al., 2010). Compared with the physiological function of the PGR5/PGRL1-dependent CET, the contribution of the NDH-dependent CET to photoprotection is somewhat minor, although clear phenotypes have been observed in these mutants at low light intensities and fluctuating light levels (Ueda et al., 2012; Yamori et al., 2015, 2016). Furthermore, the physiological function of the NDH complex has not been fully clarified.Both ∆pH and ∆ψ contribute to pmf, but only ∆pH down-regulates electron transport. To optimize the operation of the accelerator (ATP synthesis) and the brake on electron transport, it is necessary to precisely regulate the ratio of the two pmf components as well as the total size of pmf (Cruz et al., 2001; Kramer et al., 2003). Several channels and antiporters localized to the thylakoid membrane regulate the partitioning of the pmf components (Spetea et al., 2017). K⁺ Efflux Antiporter3 (KEA3) is thought to be an H⁺/K⁺ antiporter localized to the thylakoid membrane (Armbruster et al., 2014; Kunz et al., 2014), although its antiport activity has not been experimentally demonstrated (Tsujii et al., 2019). Based on its structure, topology, and the mutant phenotypes, KEA3 most likely moves H⁺ from the thylakoid lumen while taking up K⁺ as a counter ion. Consequently, KEA3 transforms ∆pH to ∆ψ and is necessary to rapidly relax the down-regulation of electron transport by raising the luminal pH (i.e. by alkalinizing the lumen). The C-terminal domain of KEA3, KTN (K⁺ transport/nucleotide binding), is exposed to the stroma (Wang et al., 2017) and is thought to regulate its activity by monitoring ATP or NADPH levels (Schlosser et al., 1993; Roosild et al., 2002). However, information on the regulation of KEA3 is limited. Armbruster et al. (2014) demonstrated that KEA3 contributes to efficient photosynthesis under fluctuating light conditions. The disturbed proton gradient regulation is a dominant mutant allele of KEA3, and its mutant phenotype is evident after a long period of dark adaptation (overnight; Wang et al., 2017). KEA3 is likely important during the induction of photosynthesis as well as under fluctuating light intensities. The similarity between the two conditions suggests that KEA3 is required for readjusting the ∆pH-dependent regulation immediately after any drastic change in light conditions.In this study, we characterized double mutants defective in the CET pathways and KEA3 to understand whether and how the synergy between CET and KEA3 in the regulatory network of photosynthesis affects this process. We focused on the contribution of NDH-dependent CET during the induction of photosynthesis after overnight dark adaptation in the kea3-1 mutant context. Based on our results, we propose a novel physiological function of the NDH complex: that of allowing flexibility of the regulatory network during the induction of photosynthesis.