Title: The ferredoxin/thioredoxin pathway constitutes an indispensable redox-signaling cascade for light-dependent reduction of chloroplast stromal proteins
Abstract: To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH–Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin–Benson cycle enzymes, NADP–malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is exceptionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chloroplast redox-regulatory system operates in vivo. To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date. Especially, recent identifications of multiple redox-related factors in chloroplasts, as represented by the NADPH–Trx reductase C, have raised a controversial proposal that other redox pathways work redundantly with the Fd/Trx pathway. To address these issues directly, we used CRISPR/Cas9 gene editing to create Arabidopsis mutant plants in which the activity of the Fd/Trx pathway was completely defective. The mutants generated showed severe growth inhibition. Importantly, these mutants almost entirely lost the ability to reduce several redox-sensitive proteins in chloroplast stroma, including four Calvin–Benson cycle enzymes, NADP–malate dehydrogenase, and Rubisco activase, under light conditions. These striking phenotypes were further accompanied by abnormally developed chloroplasts and a drastic decline in photosynthetic efficiency. These results indicate that the Fd/Trx pathway is indispensable for the light-responsive activation of diverse stromal proteins and photoautotrophic growth of plants. Our data also suggest that the ATP synthase is exceptionally reduced by other pathways in a redundant manner. This study provides an important insight into how the chloroplast redox-regulatory system operates in vivo. Thiol-based redox regulation is a post-translational modification that controls protein function by switching the oxidation/reduction states of Cys residues (e.g., formation/cleavage of disulfide bonds). As a mediator of reducing power, the small ubiquitous protein thioredoxin (Trx) plays a pivotal role in redox regulation. Trx contains a highly conserved amino acid sequence WCGPC at its active site. Using the two Cys residues in this sequence, Trx catalyzes a dithiol–disulfide exchange reaction with its target proteins, allowing their activities to be modulated. Trx is thus key to sensing local redox environments and tuning cell physiology accordingly (1Holmgren A. Thioredoxin.Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 2Jacquot J.P. Lancelin J.M. Meyer Y. Thioredoxins: structure and function in plant cells.New Phytol. 1997; 136: 543-570Crossref PubMed Scopus (161) Google Scholar). Trx-mediated redox regulation has important implication for plants that live under fluctuating light conditions. Although this regulatory system is preserved in all organisms, its mode of action in plant chloroplasts is unique in terms of being linked with light (3Buchanan B.B. Schurmann P. Wolosiuk R.A. Jacquot J.P. The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.Photosynth. Res. 2002; 73: 215-222Crossref PubMed Scopus (117) Google Scholar, 4Buchanan B.B. The path to thioredoxin and redox regulation in chloroplasts.Annu. Rev. Plant Biol. 2016; 67: 1-24Crossref PubMed Scopus (84) Google Scholar). The thylakoid membrane converts light energy to reducing power through a series of photosynthetic electron transport reactions. Trx receives some of the reducing power from photosynthetically reduced ferredoxin (Fd) via Fd–Trx reductase (FTR) and then transfers it to several Trx-targeted proteins. In most cases, these Trx-targeted proteins are switched from inactive to active forms upon the reductive cleavage of specific disulfide bonds. Therefore, the redox cascade via the Fd/Trx pathway allows the activation of chloroplast proteins in concert with the excitation of photosynthetic electron transport and, thereby, light availability. The Fd/Trx pathway was identified about half a century ago and, since then, has been recognized as the hallmark of the redox-regulatory system in chloroplasts (3Buchanan B.B. Schurmann P. Wolosiuk R.A. Jacquot J.P. The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.Photosynth. Res. 2002; 73: 215-222Crossref PubMed Scopus (117) Google Scholar, 4Buchanan B.B. The path to thioredoxin and redox regulation in chloroplasts.Annu. Rev. Plant Biol. 2016; 67: 1-24Crossref PubMed Scopus (84) Google Scholar). It is known that diverse chloroplast enzymes, including four Calvin–Benson cycle enzymes (Glyceraldehyde 3-phosphate dehydrogenase [GAPDH], fructose 1,6-bisphosphatase [FBPase], sedoheptulose 1,7-bisphosphatase [SBPase], and phosphoribulokinase [PRK]), ATP synthase, NADP–malate dehydrogenase (MDH), and Rubisco activase (RCA), are subject to redox regulation (3Buchanan B.B. Schurmann P. Wolosiuk R.A. Jacquot J.P. The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.Photosynth. Res. 2002; 73: 215-222Crossref PubMed Scopus (117) Google Scholar, 4Buchanan B.B. The path to thioredoxin and redox regulation in chloroplasts.Annu. Rev. Plant Biol. 2016; 67: 1-24Crossref PubMed Scopus (84) Google Scholar, 5Miginiac-Maslow M. Lancelin J.M. Intrasteric inhibition in redox signalling: light activation of NADP-malate dehydrogenase.Photosynth. Res. 2002; 72: 1-12Crossref PubMed Scopus (39) Google Scholar, 6Hisabori T. Sunamura E. Kim Y. Konno H. 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NADPH–Trx reductase C (NTRC) is the most well-known example; this protein harbors both an NADPH–Trx reductase domain and a Trx domain in a single polypeptide and can use NADPH directly for redox regulation (18Serrato A.J. Perez-Ruiz J.M. Spinola M.C. Cejudo F.J. A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana.J. Biol. Chem. 2004; 279: 43821-43827Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 19Cejudo F.J. Ferrandez J. Cano B. Puerto-Galan L. Guinea M. The function of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system in plastid redox regulation and signalling.FEBS Lett. 2012; 586: 2974-2980Crossref PubMed Scopus (54) Google Scholar). These advances have raised the hypothesis that plants have acquired a complex redox network in chloroplasts, enabling the flexible and sophisticated regulation of chloroplast functions. 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As mentioned above, it has been generally accepted that the Fd/Trx pathway serves to activate various chloroplast enzymes in light (3Buchanan B.B. Schurmann P. Wolosiuk R.A. Jacquot J.P. The ferredoxin/thioredoxin system: from discovery to molecular structures and beyond.Photosynth. Res. 2002; 73: 215-222Crossref PubMed Scopus (117) Google Scholar, 4Buchanan B.B. The path to thioredoxin and redox regulation in chloroplasts.Annu. Rev. Plant Biol. 2016; 67: 1-24Crossref PubMed Scopus (84) Google Scholar); however, this concept has been established mainly by in vitro reconstitution experiments, and its physiological relevance remains largely elusive. In particular, given the diversity of redox-related factors in chloroplasts, we must consider the possibility that other redox pathways may also participate in light-dependent redox regulation, thereby complementing the function of the Fd/Trx pathway. Indeed, NTRC was recently suggested to have an ability to regulate Calvin–Benson cycle enzymes and ATP synthase (42Nikkanen L. Rintamaki E. Chloroplast thioredoxin systems dynamically regulate photosynthesis in plants.Biochem. J. 2019; 476: 1159-1172Crossref PubMed Scopus (66) Google Scholar, 48Thormahlen I. Meitzel T. Groysman J. Ochsner A.B. von Roepenack-Lahaye E. Naranjo B. et al.Thioredoxin f1 and NADPH-dependent thioredoxin reductase C have overlapping functions in regulating photosynthetic metabolism and plant growth in response to varying light conditions.Plant Physiol. 2015; 169: 1766-1786PubMed Google Scholar, 49Carrillo L.R. Froehlich J.E. Cruz J.A. Savage L. Kramer D.M. Multi-level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance.Plant J. 2016; 87: 654-663Crossref PubMed Scopus (71) Google Scholar, 50Naranjo B. Mignee C. Krieger-Liszkay A. Hornero-Mendez D. 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Keech O. Droux M. et al.Identification of plant glutaredoxin targets.Antioxid. Redox Signal. 2005; 7: 919-929Crossref PubMed Scopus (165) Google Scholar). Revisiting the role of the Fd/Trx pathway is, therefore, key to better understanding the redox-regulatory system in chloroplasts. In this study, we have addressed this issue by taking advantage of gene editing technology. FTR is a heterodimer protein composed of a catalytic subunit (FTRc) and a variable subunit. FTRc contains a [4Fe–4S] cluster and a redox-active disulfide bond, both of which are essential to catalysis (54Dai S. Johansson K. Miginiac-Maslow M. Schurmann P. Eklund H. Structural basis of redox signaling in photosynthesis: structure and function of ferredoxin:thioredoxin reductase and target enzymes.Photosynth. Res. 2004; 79: 233-248Crossref PubMed Scopus (41) Google Scholar). FTR can transfer Fd-derived reducing power to all the Trx isoforms in chloroplasts (17Yoshida K. Hisabori T. Distinct electron transfer from ferredoxin-thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts.Biochem. J. 2017; 474: 1347-1360Crossref PubMed Scopus (45) Google Scholar); therefore, FTR acts as a critical signaling hub in the Fd/Trx pathway. In addition, unlike plastid-targeted Trx, which is encoded by a total of 10 genes, FTRc is encoded by a single gene (FTRB) in Arabidopsis. Based on these facts, we focused on FTR as a target for disrupting the Fd/Trx pathway. Using CRISPR/Cas9 gene editing, we introduced a point mutation into the third exon of the FTRB gene in Arabidopsis (Fig. S1A). Consequently, we were able to isolate two types of mutants, designated as ftrb-CR1 and ftrb-CR2 ("CR" refers to CRISPR/Cas9). The ftrb-CR1 mutant contained a T insertion, whereas the ftrb-CR2 mutant contained a TCAT deletion (Fig. S1B). In both cases, a frameshift mutation was caused, leading to disruption of the FTRB gene. When grown in a sucrose-supplemented Murashige and Skoog medium, the ftrb-CR mutants showed severe growth phenotypes with pale-green leaves (Fig. 1A). The fresh weight of the aerial parts and leaf chlorophyll content were largely lowered in the ftrb-CR mutants (Fig. S2). We confirmed by immunoblotting analyses that the FTRc protein was undetectable in the ftrb-CR mutants (Figs. 1B and S3). On the other hand, other redox-related proteins, including the Trx subtypes and NTRC, did not show large changes in their accumulation levels (Fig. 1B). When grown in soil, the growth phenotypes of the ftrb-CR mutants became more marked (Fig. S4). These results indicate that FTR is an essential factor in plant autotrophic growth. To check the impact of FTR deficiency on the growth phenotype, we transformed the wildtype FTRB gene under the control of the cauliflower mosaic virus 35S promoter into the ftrb-CR mutant background. In these FTRB-complemented plants (ftrb-CR1_comp and ftrb-CR2_comp), the mutations in the intrinsic FTRB gene were maintained, but the FTRc protein accumulated because of the exogenously introduced FTRB gene (Figs. S1B and S3). The growth phenotypes were completely recovered in the ftrb-CR_comp plants (Figs. 1A, S2 and S4), confirming that FTR deficiency is responsible for growth inhibition in the ftrb-CR mutants. We analyzed the light-responsive changes in the protein redox states using a thiol-modifying reagent (55Yoshida K. Hisabori T. Simple method to determine protein redox state in Arabidopsis thaliana.Bio Protoc. 2019; 9e3250Crossref Scopus (8) Google Scholar). Wildtype and ftrb-CR mutant plants were irradiated at several light intensities (0, 10, 80, and 800 μmol photons m−2 s−1) (Fig. 2A). In the wildtype plants, four Calvin–Benson cycle enzymes, including FBPase, SBPase, GAPDH (redox-sensitive GAPB isoform), and PRK, were shifted from the oxidized to reduced forms in response to increased light intensity. Similarly, MDH and RCA (redox-sensitive RCAα isoform) were reduced in a light-dependent manner. By contrast, these stromal proteins could not be reduced under any light conditions in the ftrb-CR mutants. We then investigated the time course of the protein redox states after irradiation (Fig. 2B). We have previously demonstrated the different reduction kinetics of FBPase, SBPase, and RCA (56Yoshida K. Hisabori T. Determining the rate-limiting step for light-responsive redox regulation in chloroplasts.Antioxidants (Basel). 2018; 7: 153Crossref PubMed Scopus (20) Google Scholar). The present study clarified the distinct redox responses more comprehensively. SBPase and GAPDH showed relatively slow reduction patterns, whereas PRK rapidly reached a fully reduced state; FBPase, MDH, and RCA were reduced at intermediate rates. Recently, Zimmer et al. (57Zimmer D. Swart C. Graf A. Arrivault S. Tillich M. Proost S. et al.Topology of the redox network during induction of photosynthesis as revealed by time-resolved proteomics in tobacco.Sci. Adv. 2021; 7eabi8307Crossref Scopus (16) Google Scholar) reported the protein redox responses during dark to light transitions at the proteome level; our present data (e.g., rapid reduction of PRK) appear to be in line with their observations and further highlight the dynamics of chloroplast redox regulation in light. These redox responses were not detected in the ftrb-CR mutants, indicating that FTR is essential for the light-dependent reductive activation of various stromal proteins. We also assessed the redox states of Trx-f and Trx-m. It is known that these Trx subtypes are mainly involved in the activation of several metabolic enzymes (e.g., Calvin–Benson cycle enzymes) (13Collin V. Issakidis-Bourguet E. Marchand C. Hirasawa M. Lancelin J.M. Knaff D.B. et al.The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity.J. Biol. Chem. 2003; 278: 23747-23752Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 16Yoshida K. Hara S. Hisabori T. Thioredoxin selectivity for thiol-based redox regulation of target proteins in chloroplasts.J. Biol. Chem. 2015; 290: 14278-14288Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In the wildtype plants, both Trx-f and Trx-m were reduced under strong light conditions (800 μmol photons m−2 s−1) (Fig. 2A). They were apparently present in the oxidized states under weak (10 μmol photons m−2 s−1) and growth (80 μmol photons m−2 s−1) light conditions, which was possibly because of higher rates of oxidation by their target proteins than those of reduction. Trx reduction responses were quite rapid; both Trx-f and Trx-m reached stably reduced states within 30 s after irradiation (Fig. 2B). In the ft