Regulation of cyclic electron flow by chloroplast NADPH‐dependent thioredoxin system

Abstract Linear electron transport in the thylakoid membrane drives photosynthetic NADPH and ATP production, while cyclic electron flow (CEF) around photosystem I only promotes the translocation of protons from stroma to thylakoid lumen. The chloroplast NADH dehydrogenase‐like complex (NDH) participates in one CEF route transferring electrons from ferredoxin back to the plastoquinone pool with concomitant proton pumping to the lumen. CEF has been proposed to balance the ratio of ATP/NADPH production and to control the redox poise particularly in fluctuating light conditions, but the mechanisms regulating the NDH complex remain unknown. We have investigated potential regulation of the CEF pathways by the chloroplast NADPH‐thioredoxin reductase (NTRC) in vivo by using an Arabidopsis knockout line of NTRC as well as lines overexpressing NTRC. Here, we present biochemical and biophysical evidence showing that NTRC stimulates the activity of NDH‐dependent CEF and is involved in the regulation of generation of proton motive force, thylakoid conductivity to protons, and redox balance between the thylakoid electron transfer chain and the stroma during changes in light conditions. Furthermore, protein–protein interaction assays suggest a putative thioredoxin‐target site in close proximity to the ferredoxin‐binding domain of NDH, thus providing a plausible mechanism for redox regulation of the NDH ferredoxin:plastoquinone oxidoreductase activity.

• How exactly do the authors imagine a limitation of electron flow at Cyt b6f in ntrc? If this was photosynthetic control for example, one would expect an exaggerated ΔpH in comparison to WT, which was not observed at least in growth and high light. Response: Limitation of electron flow between PQ pool and PSI in ntrc might be related to the same PGR5-dependent mechanism that prevents OE-NTRC pgr5 from inducing photosynthetic control in high light despite recovery of WT-level pmf. If the relevant function of PGR5 is dependent on chloroplast thiol redox state, the 40 % decrease in PGR5 content in ntrc may occur to inhibit an overactive (due to altered stromal redox state) mechanism of photosynthetic control. However, this discussion is so far mostly speculative, falls outside the scope of the current study, and it would require further experiments and lengthy discussion to adequately verify and explain. Therefore we would here prefer to merely point out this observation. "Possibly at Cyt b6f" has been removed from the sentence in the manuscript on page 11.
• How do the authors explain the extremely elevated pmf in ntrc compared to WT in low light conditions ( Fig. 5e)? (Impaired CF1γ reduction?) Response: This question was discussed in the original manuscript on page 12: "In ntrc, high steady state pmf under low light intensity (Fig. 7) was likely caused by impaired activation of the chloroplast ATP synthase (= impaired CF1γ reduction ) and the Calvin-Benson cycle as previously reported (Nikkanen et al. 2016, Carrillo et al. 2016. Furthermore, exceptionally high NPQ was recorded in the ntrc line, especially at low light (Suppl. Fig. S5)." This chapter is on page 13 in revised manuscript.
• It is still not clear to me how NDH is supposed to significantly contribute to CEF during the induction phase of photosynthesis. Being highly substoichiometric with regard to PSI, it would have to sustain very high rates, which cannot be mediated by a diffusion limited PSI-NDH complex and which also contradicts the very low NDH rates measured in vivo (Trouillard et al. 2012). Response: Of course this is a tricky question to which no one seems to have final concluding answer. As far as there is no direct assay available to measure the activity chloroplast NDH complex, it is not possible to unambiguously demonstrate its activity in vivo (high or low rate). Also In the paper by Trouillard et al. 2012 the activity of NDH was estimated indirectly: They wrote "From the kinetic analysis of the profiles shown in Fig. 2 (presenting fluorescence induction kinetics measured in tomato leaves), it is possible to derive quantitative information of the influx and outflux of electron undergone by the PQ pool, i.e. on the activity of the NDH and PTOX enzymes." It is, however, difficult to validate, how well this influx and outflux of electrons represent specifically and only the activities of NDH and PTOX enzymes in vivo? However, it was recently demonstrated by electron microscope analysis of single particle projections of supercomplexes that a single NDH complex can bind up to six PSI complexes (Yadav et al., 2017). If this is the case, it will solve, at least to some extent, the stoichiometric differences in the amount of NDH and PSI complexes. It might be that NDH complex is mostly needed at dark-to-light transitions as well as during fast fluctuations in light intensity, when other PSI acceptors are less active, like suggested in the current paper as well as in another recent paper by Shimakawa & Miyake (2018). This is now discussed in more detail on page 19 of the revised manuscript.

Results:
• Please consistently add letters when referring to subfigures, this would facilitate readability a lot (e.g. Fig. 1a instead of just Fig. 1). Response: Subfigure letters have been added throughout the revised manuscript.
• The order of figures and subfigures is sometimes not matching their first mention in the text. Response: The order of figures is matched with the text. In some cases the subfigures are not always mentioned in the same order in the text that they are presented in the figures. This is the case with the figures containing large datasets and subfigures (Fig. 5, 6, 7, 8) from the experiments made with lines having both ndho and pgr5 mutant background. These mutants have different WT background and for clarity and for easy comparison the results are presented in different subfigures side by side (e.g. pmf (A and B), gH+ (C and D), etc.). Therefore sometimes e.g. Fig. 5C is mentioned before Fig.  5B.
• It should be mentioned and discussed that LHCII phosphorylation in HL is more pronounced in ntrc compared to WT (Fig. 4h). Response: This is now mentioned on page 9 of the revised manuscript.
• Suppl. Table S1 could be described and discussed in more detail. Response: Table S1 is now described on page 7 of the revised manuscript.
• There is a wrong reference to Fig. 4 in the paragraph describing the pmf kinetics, it should probably be Fig. 5 (page 9). Response: This is now corrected on page 10 of the revised manuscript.
• There is a wrong reference to Fig. 5 and 6 regarding the P700 oxidation, it should probably be Fig. 6 and 8 (page 10) Response: This is now corrected on page 10 of the revised manuscript. • Figure 4a to f: Maybe add a legend and format consistently regarding the black-red overlay. Response: It is now indicated in Fig. 4A that black and red curves represent dark-adapted and FR-preilluminated leaves, respectively.