The NADPH-Dependent thioredoxin reductase C-2-Cys peroxiredoxin redox system modulates the activity of thioredoxin x in arabidopsis chloroplasts

The NADPH-Dependent Thioredoxin Reductase C–2-Cys Peroxiredoxin Redox System Modulates the Activity of Thioredoxin xin Arabidopsis Chloroplasts Valle Ojeda 1,2 , Juan M. Pe ´rez-Ruiz 1,2 and Francisco J. Cejudo 1, * 1 Instituto de Bioquı ´mica Vegetal y Fotosı ´ntesis, Universidad de Sevilla-Consejo Superior de Investigaciones Cientı ´ficas, 41092 Sevilla, Spain 2 These authors contributed equally to this work. *Corresponding author: E-mail, [email protected]; Fax, +34-954460165. (Received February 9, 2018; Revised July 9, 2018) The chloroplast redox network is composed of a complex set of thioredoxins (Trxs), reduced by ferredoxin (Fdx) via a Fdxdependent Trx reductase (FTR), and an NADPH-dependent Trx reductase with a joint Trx domain, NTRC, which efficiently reduces 2-Cys peroxiredoxins (2-Cys Prxs). Recently, it was proposed that the redox balance of 2-Cys Prxs maintains the redox state of f-type Trxs, thus allowing the proper redox regulation of Calvin–Benson cycle enzymes such as fructose 1,6-bisphosphatase (FBPase). Here, we have addressed whether the action of 2-Cys Prxs is also exerted on Trx x. To that end, an Arabidopsis thaliana quadruple mutant, ntrc-trxx-D2cp, which is knocked out for NTRC and Trx x, and contains severely decreased levels of 2-Cys Prxs, was generated. In contrast to ntrc-trxx, which showed a severe growth inhibition phenotype and poor photosynthetic performance, the ntrc-trxx-D2cp mutant showed a significant recovery of growth rate and photosynthetic efficiency, indicating that the content of 2-Cys Prxs is critical for the performance of plants lacking both NTRC and Trx x. Light-dependent reduction of FBPase was severely impaired in mutant plants lacking NTRC or NTRC plus Trx x, despite the fact that neither NTRC nor Trx xis an effective reductant of this enzyme. However, FBPase reduction was recovered in the ntrc-trxx-D2cp mutant. Our results show that the redox balance of 2-Cys Prxs, which is mostly dependent on NTRC, modulates the activity of Trx xin a similar way as f-type Trxs, thus suggesting that the activity of these Trxs is highly interconnected. Keywords: Arabidopsis Chloroplast Peroxiredoxin  Redox regulation Thioredoxin. Abbreviations: ETR, electron transport rate; FBPase, fructose 1,6-bisphosphatase; Fdx, ferredoxin; FTR, ferredoxin-depend- ent thioredoxin reductase; NPQ, non-photochemical quenching; NTRC, NADPH-dependent thioredoxin reductase C; Prx, peroxiredoxin; Trx, thioredoxin. Introduction Thiol-dependent redox regulation of enzyme activity plays an essential role in the ability of chloroplast metabolism to respond rapidly to unpredictable changes in light intensity. This regulatory mechanism is largely based on the protein disulfide reductase activity of thioredoxins (Trxs). In clear contrast to heterotrophic organisms, which contain a small number of Trxs, the number of these enzymes in chloroplasts is much larger (Meyer et al. 2012, Geigenberger et al. 2017). The first chloroplast Trxs identified were assigned the types fand m, based on their ability to reduce fructose 1,6-bisphosphatase (FBPase) and NADP-malate dehydrogenase (NADP-MDH), respectively (Meyer et al. 2012, Buchanan 2016). Although f-type Trxs have been considered to have a prevalent role in lightdependent redox regulation of enzymes of the Calvin–Benson cycle, recent evidence indicated that m-type Trxs also play an important role in the redox regulation of this important metabolic pathway (Okegawa and Motohashi 2015). Indeed, the light-dependent redox regulation of several chloroplast enzymes including FBPase and Trxs f2 and m2 has recently been reported (Yoshida et al. 2014). The availability of the genome sequence from Arabidopsis and rice led to the identification of three additional types of Trxs termed x,yand z. Initial in vitro analyses revealed better efficiency of Trxs xand yfor 2-Cys peroxiredoxin (2-Cys Prx) reduction, as compared with Trxs mand f, and thus an antioxidant function was assigned for these Trxs (Collin et al. 2003, Collin et al. 2004), a notion further supported by the finding that type yTrxs are efficient reductants of methionine sulfoxide reductase (Vieira Dos Santos et al. 2007) and Prx Q (Yoshida et al. 2015). However, more recent analyses showed similar efficiencies of Trx f1, Trx m2 and Trx x for 2-Cys Prx reduction (Yoshida and Hisabori 2016). Finally, although it was shown that Trx zis a component of the plastidencoded RNA polymerase (PEP) complex playing an essential role in chloroplast development (Arsova et al. 2010), this Trx is also able to reduce Prx Q in vitro (Yoshida et al. 2015). The source of reducing power for chloroplast Trxs is ferredoxin (Fdx) reduced by the photosynthetic electron transport chain with the participation of a plastid-specific Fdx-dependent Trx reductase (FTR), forming the FTR–Trxs redox system, which thus links chloroplast redox regulation to light (Schu ¨rmann and Buchanan 2008). In addition to this redox system, chloroplasts harbor an NADPH-dependent Trx reductase with a joint Trx domain, termed NTRC (Serrato et al. 2004). Initially, it was proposed Plant Cell Physiol. 59(10): 2155–2164 (2018) doi:10.1093/pcp/pcy134, Advance Access publication on 14 July 2018, available online at www.pcp.oxfordjournals.org !The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Regular Paper Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 that NTRC exerts an antioxidant function since it is an efficient reductant of 2-Cys Prxs, enzymes with thiol-dependent peroxidase activity (Moon et al. 2006, Pe ´rez-Ruiz et al. 2006, Alkhalfioui et al. 2007, Pe ´rez-Ruiz and Cejudo 2009). However, further analyses of an Arabidopsis thaliana mutant knockout for NTRC shows the participation of this enzyme in the redox regulation of chloroplast metabolic pathways previously known to be Trx dependent, which include starch (Michalska et al. 2009, Lepisto¨ et al. 2013) and tetrapyrrole (Stenbaek et al. 2008, Richter et al. 2013, Pe ´rez-Ruiz et al. 2014) biosynthesis. Furthermore, the deficiency of NTRC exerts a severe impairment of the efficiency of light energy utilization (Carrillo et al. 2016, Naranjo et al. 2016a), which might, at least in part, explain the growth inhibition phenotype of the ntrc mutant. Altogether, these findings indicate that chloroplast redox regulation is more complex than previously anticipated, being controlled by two redox systems, the classical FTR–Trxs system, which relies on reduced Fdx, hence being light dependent, and NTRC, which relies on NADPH and might be operative also during the night using NADPH generated from sugars via the oxidative pentose phosphate pathway (Spı ´nola et al. 2008, Cejudo et al. 2012). Thus, a relevant issue in chloroplast redox biology is to establish the relationship between the FTR–Trxs and NTRC redox systems. While Arabidopsis mutants knocked out for f-type Trxs show a phenotype very similar to the wild type (Yoshida et al. 2015, Naranjo et al. 2016b), mutants simultaneously devoid of NTRC and Trxs fshow severe growth inhibition and very decreased photosynthetic performance (Thorma¨hlen et al. 2015, Ojeda et al. 2017), indicating that NTRC and Trxs fact together, a notion further supported by the finding that mutants simultaneously devoid of NTRC and FTR are inviable when grown under autotrophic conditions (Yoshida and Hisabori 2016). An additional issue to be taken into account is that NTRC is an efficient reductant of 2-Cys Prxs, suggesting a possible relationship between antioxidant mechanisms and redox regulation. In a recent report (Pe ´rez- Ruiz et al. 2017), we have shown that decreased levels of 2-Cys Prxs exert a suppressor effect on the phenotypes of the ntrc and ntrc-trxf1f2 mutants, leading to the proposal that NTRC participates in the redox regulation of chloroplast enzymes that depend on f-type Trxs through the control of the redox balance of 2-Cys Prxs. Despite the fact that in vitro assays with purified proteins showed the activity of Trx xas a reductant of 2-Cys Prxs (Collin et al. 2003), an Arabidopsis mutant knocked out for Trx xshows a wild-type phenotype and no alteration of the redox state of 2-Cys Prxs (Pulido et al. 2010). However, an Arabidopsis mutant simultaneously devoid of Trx xand NTRC, the ntrc-trxx double mutant, shows a severe growth inhibition phenotype and strongly impaired photosynthetic performance, which is critical during early stages of plant development (Ojeda et al. 2017), thus resembling the phenotype of the ntrc-trxf1f2 triple mutant. These results suggest that the regulatory action of NTRC is exerted not only on f-type Trxs, but also on Trx x. However, the mechanism that allows this ubiquitous function of NTRC remains poorly understood. Here, we have addressed this issue by the generation and characterization of the Arabidopsis mutant ntrc-trxx-D2cp, which is knocked out for NTRC and Trx xand contains decreased levels of 2-Cys Prxs. Our results show that the NTRC–2-Cys Prxs system modulates the redox state of different Trx types, and support the central function of 2-Cys Prxs in chloroplast redox regulation. Results Decreased contents of 2-Cys Prxs suppress the phenotype of mutant plants lacking NTRC and Trx x In a previous report (Pe ´rez-Ruiz et al. 2017), we have shown that the phenotype of Arabidopsis mutant plants devoid of NTRC or simultaneously devoid of NTRC and f-type Trxs is highly dependent on the contents of 2-Cys Prxs. With the purpose of testing whether the redox balance of 2-Cys Prxs might act as a central hub in chloroplasts able to maintain the reducing capacity of different Trxs, we have extended these analyses to plants simultaneously deficient in NTRC and Trx x, which also show a severe growth inhibition phenotype (Ojeda et al. 2017). To that end, the Arabidopsis ntrc-trxx-D2cp quadruple mutant was generated (Fig. 1A). This quadruple mutant is knocked out for NTRC, Trx xand 2-Cys Prx B but contains a low amount of 2-Cys Prx A (6% of the content of 2-Cys Prxs in the wild type), as determined by Western blot (Fig. 1B, C). Unfortunately, no anti-Trx xantibodies are available and, thus, the lack of Trx xwas tested by quantitative reverse transcription–PCR (RT–qPCR) analysis, which confirmed the absence of transcripts of the gene in the trxx mutant backgrounds (Fig. 1D). Compared with the D2cp mutant, the levels of 2CPA transcripts (Fig. 1D) and protein (Fig. 1B, C) are increased in the ntrc-trxx-D2cp mutant, indicating that the absence of NTRC and Trx xexerts a positive effect on the expression of this gene in a D2cp genetic background. Interestingly, the performance of the ntrc-trxx-D2cp mutant is much better than that of the ntrc-trxx mutant, since this mutant recovered a growth phenotype similar to that of the D2cp mutant (Fig. 1A), as shown by the rosette fresh weight (Fig. 2A) and leaf Chl content (Fig. 2B). The suppressor effect is also exerted at early stages of plant development as seedlings of the ntrctrxx-D2cp mutant recovered the wild-type rate of root growth in synthetic media lacking an exogenous carbon source (Fig. 2C), and the timing of appearance of true leaves, which was clearly delayed in the ntrc-trxx mutant (Fig. 2D). Therefore, these results show that decreased contents of 2- Cys Prxs suppress the severe growth inhibition phenotype of plants simultaneously lacking NTRC and Trx x. The analysis of the photochemical parameters indicates that the recovery of the growth phenotype of the ntrc-trxx-D2cp mutant is in agreement with the recovery of the photosynthetic performance in this line. The ntrc-trxx double mutant shows even lower efficiency of light energy utilization than the ntrc mutant, as revealed by the severely decreased photosynthetic electron transport rate [ETR(II)] (Fig. 3A), reduced quantum yield of PSII photochemistry, Y(II) (Fig. 3B, C) and increased non-photochemical quenching [Y(NPQ)] (Fig. 3B, D). While 2156 V. Ojeda et al. | The central role of 2-Cys Prxs in redox regulation Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 the ntrc-trxx-D2cp mutant showed higher Y(II) and lower Y(NPQ) than the ntrc mutant, the photosynthetic ETR at growth light intensities (125 mEm –2 s –1 ) was similar to that observed in the ntrc mutant (Fig. 3A). Interestingly, the photosynthetic performance was improved in the D2cp mutant and diminished in the trxx mutant when compared with wild-type plants as shown by the ETR (Fig. 3A), Y(II) (Fig. 3B, C) and Y(NPQ) (Fig. 3B, D). Finally, the ratio of variable fluorescence to maximal fluorescence (F v /F m ) was clearly reduced in the ntrc and, to a higher extent, in ntrc-trxx mutants, but not in the ntrctrxx-D2cp suppressed line (Table 1), further indicating the suppressor effect of decreased contents of 2-Cys Prxs on the phenotype of plants simultaneously devoid of NTRC and Trx x. Overall, these results indicate that the dose of 2-Cys Prxs is critical for the phenotype of NTRC-deficient plants, which is not strictly dependent on the photosynthetic performance. A B Ponceau 2-Cys Prx WT trxx D2cp ntrc ntrc-trxx ntrc-trxx-D2cp NTRC 1/41/2 1 WT ntrcD2cptrxx ntrctrxx ntrctrxx- D2cp D 0 50 100 NTRC 2CPA 2CPBTRXX Relative expression (%) C 0 25 50 75 100 2-Cys Prxs (% of WT) Fig. 1 The phenotype of the Arabidopsis ntrc-trxx mutant is partially suppressed by a decreased content of 2-Cys Prxs. (A) Plants of the wild type and mutant lines, as indicated, grown under long-day conditions for 4 weeks. (B) Western blot analysis of the levels of NTRC and 2-Cys Prxs. Protein extracts were obtained from leaves of plants grown as stated in (A) and aliquots (3 mg of protein) from wild-type (WT) (1) and mutant lines, as well as 1/2 and 1/4 dilutions from the WT extract, as indicated, were subjected to SDS–PAGE under reducing conditions, transferred to nitrocellulose filters and probed with anti-NTRC or anti-2-Cys Prx antibodies. (C) Band intensities of 2-Cys Prxs and Ponceau were quantified (ScionImage) and the ratios between them, referred to the WT sample of 3 mg (arbitrarily assigned a value of 100), are indicated as the average ± SD of four determinations (two technical replicates of two independent biological replicates). (D) The levels of transcripts of the genes encoding NTRC, Trx x, 2-Cys Prx A and 2-Cys Prx B were determined by RT–qPCR using the oligonucleotides indicated in Supplementary Table S3 and normalized against two reference genes (as indicated in the Materials and Methods). Relative expression was referred to the level of transcripts of each of the genes in wild-type plants, arbitrarily considered as 100%. Determinations were performed three times and mean values ±SEM are represented. 2157 Plant Cell Physiol. 59(10): 2155–2164 (2018) doi:10.1093/pcp/pcy134 Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 For further testing the relevance of the content of 2-Cys Prxs for the function of the chloroplast redox systems, the Arabidopsis ntrc-trxx-D2cp mutant was transformed with a construct driving the expression of a cDNA encoding 2-Cys Prx A by the Cauliflower mosaic virus (CaMV) 35S promoter. Two independent transgenic lines with increased contents of 2-Cys Prx A in the ntrc-trxx-D2cp mutant background (Fig. 4A– C) partially restored the growth inhibition phenotype displayed by the ntrc-trxx line, as shown by the reduced rosette size (Fig. 4A), decreased Y(II) and increased Y(NPQ) (Fig. 4D)in these transgenic lines. Therefore, these results further confirm that the contents of 2-Cys Prxs determine the phenotypic traits of plants devoid of NTRC and Trx x, in line with the previously reported effect on the phenotype of plants lacking NTRC and f-type Trxs (Pe ´rez-Ruiz et al. 2017) and allow the conclusion that the regulatory action of 2-Cys Prxs is exerted on both types of Trx. The redox balance of 2-Cys Prxs underlies the regulatory function of Trxs xand f To gain more insight into the functional relationship of NTRC, Trx xand f-type Trxs in the redox regulation of chloroplast enzymes, we analyzed the in vivo redox state of FBPase, a well-established redox-regulated enzyme of the Calvin– Benson cycle which is known to be target of f-type Trxs, but not of Trx x(Serrato et al. 2013). In agreement with previous results (Naranjo et al. 2016b), labeling of the reduced form of FBPase with the alkylating agent methyl maleimide polyethylene glycol [(MM(PEG 24 )] showed that this enzyme is fully oxidized in dark-adapted leaves and becomes partially reduced after 30 min of illumination at a growth light intensity of 125 mEm –2 s –1 (Fig. 5A, B). Compared with the wild type, the level of light-dependent reduction of FBPase, which was slightly lower (though not statistically significant) in the trxx mutant, was clearly increased in the D2cp mutant (Fig. 5A, B). Mutant plants lacking NTRC or NTRC plus Trx xshowed a very severe impairment of light-dependent FBPase reduction, which was recovered up to trxx levels in the quadruple mutant ntrc-trxx- D2cp (Fig. 5A, B). Likewise, the degree of reduction of f-type Trxs in response to light reflected that of FBPase in all lines analyzed. It was slightly decreased and enhanced, respectively, in the trxx and D2cp mutants, severely impaired in the ntrc and ntrc-trxx mutants, and partially recovered in the ntrc-trxx-D2cp mutant (Fig. 5A, B). Since in vitro assays showed that FBPase is 34567 0 10 20 30 days Root length (mm) CWT trxx ∆2cp ntrc ntrc-trxx ntrc-trxx-∆2cp D True leaves (%) days 0481216202428 0 20 40 60 80 100 WT trxx ∆2cp ntrc ntrc-trxx ntrc-trxx-∆2c p A Rosette fresh weight (g) 0 0.1 0.2 0.3 a a bb c d WT Δ2cp ntrc ntrctrxx trxx ntrctrxx- Δ2cp 0.0 0.5 1.0 Chl (mg/g fresh weight) WT Δ2cp ntrc ntrctrxx trxx ntrctrxx- Δ2cp aaaa bb B Fig. 2 The suppressor effect of decreased contents of 2-Cys Prxs is exerted during early stages of plant development. (A) The weight of the rosette from 11 plants per line grown under long-day conditions for 4 weeks, is represented as average values ±SEM, with letters indicating significant differences (Student’s t-test at a 95% confidence interval). (B) Chl contents determined from the same plants are represented as average values ±SEM. Letters indicate significant differences with the Tukey test at a 99% confidence interval. (C) Seedlings of the wild type and mutant lines, as indicated, were grown for 7 d on MS synthetic medium in the absence of sucrose under continuous light (125 mEm –2 s –1 ). Root growth was monitored at the indicated time and mean values ±SEM from three replicates are represented. (D) Seeds of the wild type and mutant lines were allowed to germinate on soil. For each line, the percentage of seedlings with emerging true leaves on the indicated day were determined and represented as means values ±SEM from three replicates. 2158 V. Ojeda et al. | The central role of 2-Cys Prxs in redox regulation Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 not reduced by NTRC and poorly reduced by Trx x(Ojeda et al. 2017), the severe impairment of the light-dependent redox regulation of FBPase, and f-type Trxs, in the ntrc and ntrc-trxx mutants and to a lower extent in the trxx mutant, suggests an indirect effect of NTRC on the redox regulation of FBPase via f-type Trxs. Remarkably, decreased contents of 2-Cys Prxs show a positive effect on the level of reduced FBPase and f-type Trxs in response to light, as occurs in the D2cp and the ntrc-trxx-D2cp mutants when compared with the wild type and ntrc-trxx mutant, respectively (Fig. 5A, B). These pieces of evidence suggest an important function of 2-Cys Prxs in the light-dependent redox regulation of these chloroplast enzymes. As suggested by our previous proposal (Pe ´rez-Ruiz et al. 2017), a likely possibility is that the content of 2-Cys Prxs determines the extent of the draining of reducing equivalents from the pool of Trxs, hence affecting the redox state of Trxs f. To test this possibility, the changes in the in vivo redox state of 2-Cys Prxs were determined in dark–light transitions. Alkylation experiments identified two shifted bands of 2-Cys Prxs (Fig. 6A). Arabidopsis 2-Cys Prxs A and B contain exclusively the two cysteine residues, peroxidatic and resolving, that form part of their active site. The catalytic form of the enzyme is arranged as a head-to-tail homodimer, which in its fully oxidized form is linked by two intermolecular disulfide bridges involving the peroxidatic cysteine of one subunit with the resolving cysteine of the other subunit (Puerto-Gala ´n et al. 2013), and no shifted bands are expected in alkylation assays. In the fully reduced form of the enzyme, the two disulfides are reduced; thus, the two cysteine residues in each monomer are in its thiolic form (2SH) and are labeled by the alkylating agent, hence producing a shifted band with the lowest electrophoretic mobility. In addition, the enzyme can be detected in half reduced form, when only one of the disulfides is reduced; thus, one cysteien residue in each B ntrctrxx ntrcΔ2cptrxxWT Y(NPQ) Y(II) 01 ntrctrxx Δ2cp 0.2 0.4 0.6 0.8 A WT trxx D2cp ntrc ntrc-trxx ntrc-trxx-D2cp 0 50 100 150 0 10 20 30 40 PAR ETR (II) WT trxx D2cp ntrc ntrc-trxx ntrc-trxx-D2cp C Y(II) 0 200 400 600 800 0 0.2 0.4 0.6 0.8 Time (s) D Y(NPQ) 200 400 600 800 0 0.2 0.4 0.6 0.8 Time (s) Fig. 3 Photosynthetic performance in Arabidopsis wild type and mutant lines. (A) The linear photosynthetic electron transport rate, ETR(II), was determined at increasing photosynthetically active radiation (PAR). Each value is the average of four determinations, except for ntrc-trxx which is the average of five determinations, and the SEM is represented as error bars. (B) False-color images representing quantum yields of nonphotochemical quenching, Y(NPQ), and PSII photochemistry, Y(II), of plants of the wild type and mutant lines grown under long-day conditions for 4 weeks. For each parameter, signal intensities (from 0 to 1.0) are indicated according to the color scale bar. (C) Quantum yields of PSII photochemistry, Y(II) and (D) non-photochemical quenching, Y(NPQ), were measured in whole plants grown at 125 mEm –2 s –1 under long-day conditions and adapted to darkness. Determinations were performed three times, except for mutant ntrc-trxx which were performed 15 times, and each data point is the mean ±SEM. White and black blocks indicate periods of illumination with actinic light (81 mEm –2 s –1 ) and darkness, respectively. Table 1 Effect of the combined deficiencies of NTRC, Trx xand 2-Cys Prxs on F v /F m WT trxx D2cp ntrc ntrc-trxx ntrc-trxx-D2cp F v /F m 0.76 ±0.01a 0.76 ±0.01a 0.76 ±0.01a 0.70 ±0.02b 0.67 ±0.01c 0.76 ±0.01a The maximum PSII quantum yield was determined as the ratio of variable fluorescence (F v ) to maximal fluorescence (F m ), F v /F m , in dark-adapted leaves of plants grown under long-day conditions. The F v /F m values (±SD) are the average of 12 measurements. Letters indicate significant differences with the Student’s t-test at a confidence interval of 99.9%. 2159 Plant Cell Physiol. 59(10): 2155–2164 (2018) doi:10.1093/pcp/pcy134 Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 monomer is in its thiolic form (1SH), producing a shifted band of intermediate mobility in alkylation assays. Under dark and light conditions, NTRC-deficient plants exhibited significantly lower levels of the reduced form of 2-Cys Prxs (1SH plus 2SH forms) as compared with the wild type, which showed similar levels to those in trxx (Fig. 6A, B). The dark–light transition led to increased levels of reduced 2-Cys Prxs in all lines under analysis, the increase being higher in the ntrc and ntrc-trxx-D2cp plants. Discussion In a recent report, we have shown that the activity of the FTR– Trxs fand NTRC redox systems is integrated by the redox balance of the 2-Cys Prxs, which controls the redox regulatory network of the chloroplast (Pe ´rez-Ruiz et al. 2017). This proposal is based on the finding that the deficiency of 2- Cys Prxs exerts a suppressor effect of the ntrc and ntrc-trxf1f2 phenotypes. Similar to the ntrc-trxf1f2 mutant, the ntrc-trxx mutant also shows a very severe growth inhibition phenotype and impairment of photosynthetic performance (Ojeda et al. 2017), suggesting that the regulatory action of NTRC is not only exerted on f-type Trxs, but also on Trx x. Since it is considered that Trxs fand xhave different functions in chloroplast redox regulation, the finding that the lack of either Trx type exerts such a severe phenotypic effect in the absence of NTRC suggests that the activities of these Trxs are somehow interconnected by an NTRC-dependent way. According to our model of redox regulation in chloroplasts, the most likely candidates to exert this interconnecting function are 2-Cys Prxs, the redox balance of which is mainly controlled by NTRC (Kirchsteiger et al. 2009). Here, we have addressed this possibility by the generation of the Arabidopsis ntrc-trxx-D2cp quadruple mutant. This mutant, which is devoid of NTRC and Trx xand contains decreased levels of 2-Cys Prxs (Fig. 1B–D), shows a much better growth rate than the ntrc-trxx or even the ntrc mutant (Figs. 1A, 2A, B), and a better performance also at early stages of plant development (Fig. 2C, D). The suppressor effect of the ntrc-trxx phenotype by decreased contents of 2-Cys Prxs in the ntrc-trxx-D2cp mutant is in agreement with the better efficiency of light energy utilization by this mutant (Fig. 3A–D). The ETR of the suppressed line, ntrc-trxx-D2cp, is similar to that in the ntrc mutant (Fig. 3A), thus indicating partial recovery of this photosynthetic parameter. Therefore, the partial recovery of the efficiency of light energy utilization of the ntrc-trxx-D2cp mutant is sufficient to nearly restore the wild-type growth rate, at least under the controlled growth conditions used in this work. Altogether, these results further confirm the central role of 2-Cys Prxs in chloroplast redox regulation and, consequently, in plant performance. The results reported here showing the effect of the dose of 2- Cys Prxs on the phenotype of the ntrc-trxx mutant extend previous results showing a similar effect on the phenotype of the ntrc and ntrc-trxf1f2 mutants (Pe ´rez-Ruiz et al. 2017) and indicate that the redox balance of 2-Cys Prxs modulates the activity of Trxs fand x. Furthermore, since Trxs fand xare considered to perform different functions in chloroplast A WT ntrctrxx ntrctrxx- D2cp ntrc-trxx-D2cp/ 35S-2CPA #1 #2 B ntrc-trxx ntrc-trxx-D2cp ponceau 2-Cys Prx NTRC ntrc-trxx-D2cp/ 35S-2CPA #1 #2 WT 1/4 1/2 1 D WT ntrctrxx ntrctrxx- D2cp ntrc-trxx-D2cp / 35S-2CPA #1 #2 Y(II) Y(NPQ) 010.2 0.4 0.6 0.8 C #1 #2 0 25 50 75 100 ntrc-trxx-D2cp/ 35S-2CPA Fig. 4 The effect of the overexpression of 2-Cys Prx A on the ntrc-trxx- D2cp suppressed line. (A) Wild type, mutant lines and two independent transgenic lines (T 1 generation), #1 and #2, expressing 2-Cys Prx A under the CaMV 35S constitutive promoter in the ntrc-trxx-D2cp genetic background were grown under a long-day photoperiod for 4 weeks. (B) Western blot analysis of the contents of NTRC and 2-Cys Prxs in the indicated lines. Protein extracts were obtained from leaves, and aliquots (3 mg of protein, corresponding to dilution 1) from wildtype and mutant lines were loaded. In addition, 1/2 and 1/4 dilutions from the wild-type extracts, as indicated, were loaded and samples were subjected to SDS–PAGE under reducing conditions. (C) Band intensities of 2-Cys Prxs and Ponceau were quantified (ScionImage) and the ratios between them, referred to the wild-type sample of 3 mg (arbitrarily assigned a value of 100), are indicated as the average ±SD of two independent experiments. (D) False-color images representing quantum yields of non-photochemical quenching, Y(NPQ), and PSII photochemistry, Y(II). For each parameter, signal intensities (from 0 to 1.0) are indicated according to the color scale bar. 2160 V. Ojeda et al. | The central role of 2-Cys Prxs in redox regulation Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 redox regulation, these results suggest that the activity of these Trxs is somehow interdependent. In this regard, the analysis of the light-dependent redox state of FBPase in the lines under study revealed that Trxs fand Trx xexert a concerted action that affects the redox regulation of the enzyme in plants devoid of NTRC. In contrast with mutant plants lacking f-type Trxs, which show impaired light-dependent FBPase reduction (Naranjo et al. 2016b), the level of FBPase reduction in plants lacking Trx xis nearly similar to that in wild-type plants (Fig. 5A, B). These results are in full agreement with in vitro analyses showing that FBPase is efficiently reduced by Trxs f1 and f2 but not by Trx x, which is a poor reductant of the enzyme (Yoshida et al. 2015, Ojeda et al. 2017). However, the level of light-dependent reduction of FBPase was severely impaired in the ntrc mutant, as it was in the ntrc-trxx mutant (Fig. 5A, B), suggesting that NTRC and Trx xaffect the FBPase redox state indirectly. The light-dependent reduction of Trxs fparallels that of FBPase in all lines analyzed (Fig. 5A, B), indicating that the deficiencies of NTRC or NTRC plus Trx xaffects the redox state of f-type Trxs, thereby affecting the redox state of FBPase. The relevant role of NTRC as a reductant of 2-Cys Prxs was confirmed by the lower level of reduction of these enzymes in mutant lines devoid of NTRC, as compared with the wild type and the trxx mutant (Fig. 6A, B). Though at variable levels, all lines show increased reduction of 2-Cys Prxs upon illumination (Fig. 6), a result which is not fully consistent with our previous results (Pe ´rez-Ruiz et al. 2017). This inconsistency is most probably due to the different growth conditions, short-day photoperiod in the previous study (Pe ´rez-Ruiz et al. 2017) and long-day photoperiod in the present study due to the reduced viability of the ntrc-trxx mutant under the short-day photoperiod (Ojeda et al. 2017). The fact that the light-depend- ent reduction of f-type Trxs and FBPase was affected in the ntrc and ntrc-trxx mutants, but not in the ntrc-trxx-D2cp mutant (Fig. 5A, B), which contains decreased contents of 2-Cys Prxs, supports the notion that, in plants devoid of NTRC or NTRC plus Trx x, the drainage effect exerted by 2-Cys Prxs affects the light-dependent reduction of f-type Trxs and, consequently, of FBPase, in line with the relevant role proposed for 2-Cys in chloroplast redox regulation (Pe ´rez-Ruiz et al. 2017). The suppressor effect observed in the ntrc-trxx-D2cp mutant indicates that decreased contents of 2-Cys Prxs alleviate the drainage of reducing power, thus restoring the redox state of f-type Trxs and FBPase. In support of this notion, transgenic lines recovering the expression of 2-Cys Prx A in the ntrc-trxx-D2cp mutant recover the mutant phenotype (Fig. 4A–C). The results reported in this study confirm the essential function of 2-Cys Prxs in chloroplast redox regulation, extending the A D WT LD trxx LD D2cp LD ntrc LD ntrctrxx LD ntrctrxx- D2cp L FBPase Trxs f red oxi BWT D2cp ntrc ntrc-trxx trxx ntrc-trxx-D2cp Reduced Enzyme (%) FBPase Trxs f 0 25 50 75 100 ab abc bc a c c a a b cc a red oxi ns Fig. 5 In vivo redox state of FBPase and f-type Trxs in dark–light transitions. (A) The in vivo redox state of FBPase and f-type Trxs were determined at the end of the dark period (D), and after 30 min of illumination at 125 mEm –2 s –1 (L) by labeling of the thiol groups with the alkylating agent MM(PEG) 24 . (B) Band intensities were quantified (ScionImage) and the percentage of reduction, as the ratio between the reduced form and the sum of reduced and oxidized forms, of FBPase and f-type Trxs is represented as the mean of three independent experiments ±SEM. Letters indicate significant differences between lines (Tukey test at a 95% confidence interval). red, reduced: oxi, oxidized; ns, non-specific band. The vertical lines mark different filters. B A mock D WT LD trxx LD D2cp LD ntrc LD ntrctrxx LD ntrctrxx- D2cp L oxi 1SH 2SH 2-Cys Prxs WT trxx ntrc ntrctrxx ntrctrxx- D2cp 0 50 100 Reduced 2-Cys Prx (%) DARK LIGHT ab a bc c c AAB BC CC Fig. 6 In vivo redox state of 2-Cys Prxs in dark–light transitions. (A) The in vivo redox state of 2-Cys Prxs was determined at the end of the dark period (D), and after 30 min of illumination at 125 mEm –2 s –1 (L) by labeling of the thiol groups with the alkylating agent MM(PEG) 24 . (B) Band intensities were quantified (ScionImage) and the level of reduction is the ratio between the sums of the half-reduced (1SH) and fully reduced (2SH), and the sum of reduced and oxidized (oxi) forms is shown as the mean of four (WT, trxx, ntrc and ntrc-trxx) or three (ntrc-trxx-D2cp) independent experiments ±SEM. Letters above bars indicate significant differences between lines in darkness (lower case letters) and in light (upper case letters) with Tukey test at a 95% confidence interval. Oxi, oxidized; 1SH and 2SH indicate reduction of one or two cysteines, respectively. 2161 Plant Cell Physiol. 59(10): 2155–2164 (2018) doi:10.1093/pcp/pcy134 Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 action of this hydrogen peroxide-scavenging enzyme to the function of different Trxs such as those of the types xand f. Moreover, the finding that tetrapyrrole biosynthesis is severely affected in Arabidopsis plants simultaneously deficient in mtype Trxs and NTRC (Da et al. 2017) further suggests that the NTRC-dependent redox balance of 2-Cys Prxs may also modulate the function of m-type Trxs. Remarkably, the D2cp mutant, which contains severely decreased levels of 2-Cys Prxs (6% of the wild type), showed slightly better efficiency of light energy utilization than the wild type (Fig. 3A–D) and higher level of light-dependent reduction of f-type Trxs and FBPase (Fig. 5A, B). These results suggest that 2-Cys Prxs, which are among the more abundant proteins of the chloroplast (Dietz 2011), exert an oxidizing effect on the pool of Trxs. The reductive activity of each chloroplast Trx is dependent on their redox potential. The f-type Trxs are more reducing than Trx x(Yoshida et al. 2015), hence some redundancy of these Trxs cannot be excluded. In this regard, several types of chloroplast Trxs are able to reduce 2-Cys Prxs with different efficiencies (Collin et al. 2003, Collin et al. 2004, Yoshida et al. 2015, Yoshida and Hisabori 2016). Moreover, atypical chloroplast Trxs ACHT1 (Dangoor et al. 2012) and ACHT4 (Eliyahu et al. 2015), and the Trx-like CDSP32 (Broin et al. 2003) are also reductants of 2-Cys Prxs. Based on these results, we propose that the NTRC–2-Cys Prxs redox system acts as a central hub playing a critical role in the maintenance of the reducing capacity of the pool of chloroplast Trxs, hence suggesting that the activities of the different types of Trxs of the chloroplast are highly interconnected. Materials and Methods Biological material and growth conditions Arabidopsis thaliana wild-type (ecotype Columbia) and mutant plants (Supplementary Table S1) were routinely grown in soil in growth chambers under long days (16 h light/8 h darkness) at 22 and 20C during light and dark periods, respectively, and a light intensity of 125 mEm –2 s –1 . Arabidopsis mutants ntrc (Serrato et al. 2004), D2cp,trxx (Pulido et al. 2010) and ntrc-trxx (Ojeda et al. 2017) were described previously. To generate the ntrc-trxx-D2cp quadruple mutant, the ntrc-trxx and D2cp mutants were manually crossed and seeds resulting from this cross were checked for heterozygosity of the T-DNA insertions in the NTRC,TRX X, 2CPA and 2CPB genes. Plants were then self-pollinated and the quadruple homozygous line was identified in the progeny by PCR analysis of genomic DNA with oligonucleotides listed in Supplementary Table S2. For the generation of Arabidopsis transgenic lines, plasmids for overexpressing 2-Cys Prx A driven by the CaMV 35S promoter (Pe ´rez-Ruiz et al. 2017) were transformed into the Agrobacterium tumefaciens strain GV301, which were then used for plant transformation by the floral dip method (Clough and Bent 1998). Root growth assays were performed as previously reported (Ojeda et al. 2017) on Murashige and Skoog medium containing 0.35% (w/v) Gelrite (Duchefa). For seedling development experiments, the formation of true (post-embryonic) leaves was monitored as previously described (Ojeda et al. 2017). In brief, three sets of at least 60 seeds of each line were sown on soil and grown during 28 d under long-day conditions. True leaf formation was scored at 3–4 d intervals and quantified to determine the rate of seedlings reaching the true leaf stage. RT–qPCR analysis Total RNA was isolated with Trizol reagent (Invitrogen) and cDNA was synthesized with the Maxima first strand cDNA synthesis kit (Fermentas). Gene expression analyses were performed using an IQ5 system (Bio-Rad) with oligonucleotides listed in Supplementary Table S3 and a standard thermal profile (95C, 3 min; 40 cycles at 95C for 10 s, and 60C for 30 s). After the PCR, a melting curve analysis (55–94C at 0.5C/30 s) was performed to confirm the specificity of the amplicon. Relative contents of transcripts were normalized using AT1G13320 and AT5G25760 as reference genes (Czechowski et al. 2005). Protein extraction, alkylation assays and Western blot analysis Plant tissues were ground under liquid nitrogen to a fine powder. Extraction buffer [50 mM Tris–HCl pH 8.0, 0.15 M NaCl, 0.5% (v/v) NP-40] was immediately added, mixed on a vortex and centrifuged at 16,100gat 4C for 20 min. Protein was quantified using the Bradford reagent (Bio-Rad). Alkylation assays were performed as previously described (Naranjo et al. 2016b) using MM(PEG) 24 from Thermo Scientific. Protein samples were subjected to SDS–PAGE under reducing (NTRC, 2-Cys Prxs, f-type Trxs) or non-reducing (FBPase) conditions using an acrylamide gel concentration of 9.5% (FBPase alkylation assays), 12% (protein levels of NTRC and 2- Cys Prxs) and 14% (alkylation assays of f-type Trxs and 2-Cys Prxs). Resolved proteins were transferred to nitrocellulose membranes and probed with the indicated antibody. Specific antibodies for NTRC (Serrato et al. 2004), 2-Cys Prxs (Pe ´rez-Ruiz et al. 2006) and Trxs f(Naranjo et al. 2016b) were previously raised in our laboratory. The anti-FBPase antibody was kindly provided by Dr. Sahrawy (Estacio ´n Experimental del Zaidı ´n, Granada, Spain). Determination of chlorophylls and measurements of Chl afluorescence Chl levels were measured as previously described (Pe ´rez-Ruiz et al. 2006). Room temperature Chl afluorescence and Chl fluorescence imaging of whole rosettes were performed using a pulse-amplitude modulation fluorometer (IMAGINGPAM M-Series instrument, Walz). Induction–recovery curves were performed using blue (450 nm) actinic light (81 mEm –2 s –1 ) at the intensities specified for each experiment during 6 min. Saturating pulses of blue light (10,000 mEm –2 s –1 ) and 0.6 s duration were applied every 60 s, and recovery in darkness was recorded for another 6 min. The maximum PSII quantum yield, determined as the ratio of variable fluorescence (F v ) to maximal fluoresence (F m ), F v /F m , the parameters Y(II) and Y(NPQ), corresponding to the respective quantum yields of PSII photochemistry and non-regulated basal quenching, were calculated by the ImagingWin v2.46i software according to the equations in Kramer et al. (2004). Measurements of relative linear electron transport rates were based on Chl fluorescence of pre-illuminated plants applying stepwise increasing actinic light intensities up to 146 mEm –2 s –1 . Supplementary Data Supplementary data are available at PCP online. Funding This work was supported by Spanish Ministry of Innovation and Competitiveness (MINECO) [a European Regional Development Fund-co-financed grant (BIO2017-85195-C2-1- P), and a pre-doctoral fellowship to V.O.]. Disclosures The authors have no conflicts of interest to declare. References Alkhalfioui, F., Renard, M. and Montrichard, F. (2007) Unique properties of NADP-thioredoxin reductase C in legumes. J. Exp. Bot. 58: 969–978. 2162 V. Ojeda et al. | The central role of 2-Cys Prxs in redox regulation Downloaded from https://academic.oup.com/pcp/article-abstract/59/10/2155/5053952 by UNIVERSIDAD DE SEVILLA user on 03 June 2019 Arsova, B., Hoja, U., Wimmelbacher, M., Greiner, E., Ustun, S., Melzer, M., et al. (2010) Plastidial thioredoxin z interacts with two fructokinase-like proteins in a thiol-dependent manner: evidence for an essential role in chloroplast development in Arabidopsis and Nicotiana benthamiana. Plant Cell 22: 1498–1515. Broin, M and Rey, P. 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