The chloroplast NADPH thioredoxin reductase C, NTRC, controls non-photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis

Original Article The chloroplast NADPH thioredoxin reductase C, NTRC, controls non-photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis Belén Naranjo 1 ,ClaraMignée 2 , Anja Krieger-Liszkay 2 , Dámaso Hornero-Méndez 3 , Lourdes Gallardo-Guerrero 3 , Francisco Javier Cejudo 1 &MarikaLindahl 1 1 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas (CSIC) –Universidad de Sevilla, 410 92 Seville, Spain, 2 Institute for Integrative Biology of the Cell (I2BC), Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA) Saclay, Institut de Biologie et de Technologie de Saclay, Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud, 91191 Gif-sur-Yvette Cedex, France and 3 Departamento de Fitoquímica de los Alimentos, Instituto de la Grasa, CSIC, 41013 Seville, Spain ABSTRACT High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under lightlimiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NTRC). Whereas Arabidopsis thaliana plants lacking NTRC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an ntrc–psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the ntrc mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the ntrc–psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking ntrc alone. These data uncover a new role for NTRC in the control of photosynthetic yield. Key-words: high-light acclimation; oxidative stress; photosynthesis; redox signalling; thioredoxin; thylakoid. INTRODUCTION In plants, not only are reactive oxygen species (ROS) produced through normal aerobic metabolism, but additional ROS are also generated via the photosynthetic electron transport (PET) and oxygen evolution, which take place in the chloroplast (Foyer & Noctor 2009). At elevated light intensities, when the PETrates exceed photosynthetic capacity, the excess light energy absorbed yields increased production of ROS, including superoxide anion radicals, hydrogen peroxide, hydroxyl radicals and singlet oxygen (Foyer & Noctor 2009; Li et al. 2009). Adverse environmental conditions, such as extreme temperatures, high salinity and drought have in common that photosynthesis is slowed down, and this leads to enhanced formation of ROS even under moderate light intensities, as molecular oxygen acts as a sink for PET-derived electrons (Takahashi & Murata 2008; Li et al.2009).Adaptation to high light intensities is essential for plant survival and involves changes of gene expression and enzymatic activities in order to optimize photosynthesis and to avoid toxic ROS levels (Li et al. 2009; Foyer et al.2012;Tikkanenet al. 2012; Spetea et al. 2014; Dietz 2015). Photosystem II (PSII) is intrinsically sensitive to strong irradiance, as excess excitation increases the production of singlet oxygen in the reaction centre, which impairs the adjacent proteins, in particular the D1 protein (Aro et al.1993; Krieger-Liszkay et al. 2008; Tyystjarvi 2013). Damaged inactive D1 protein becomes proteolytically degraded and replaced with a de novo synthesized copy in a costly repair process (Nixon et al. 2010; Tyystjarvi 2013; Jarvi et al.2015).One of the protection mechanisms of plants against excess light involves thermal dissipation of the light energy captured by light-harvesting complex II (LHCII), the light-harvesting antenna of PSII (Szabo et al. 2005). The fate of the light energy absorbed can be inferred from measurements of chlorophyll a fluorescence (Baker 2008), and the loss of chlorophyll fluorescence that is not due to photochemistry is referred to as nonphotochemical quenching (NPQ). The major component of NPQ in plants, qE, requires acidification of the thylakoid lumen, the presence of the PSII PsbS protein (Li et al.2000) and the conversion of the xanthophyll violaxanthin to zeaxanthin, via antheraxanthin, catalysed by violaxanthin deepoxidase (VDE) (Szabo et al. 2005; Jahns et al. 2009; Jahns & Holzwarth 2012; Ruban et al. 2012). VDE is located in the thylakoid lumen and is regulated by pH, the availability of ascorbate and, possibly, by its redox state (Pfundel & Dilley 1993; Bratt et al. 1995; Hall et al. 2010; Simionato et al.2015). The effectiveness of qE in preventing production of singlet Correspondence: M. Lindahl. e-mail: [email protected] © 2015 John Wiley & Sons Ltd804 doi: 10.1111/pce.12652Plant, Cell & Environment (2016) 39,804–822 oxygen and photoinhibition has been confirmed in studies of an Arabidopsis mutant lacking PsbS (npq4) and in transgenic Arabidopsis overexpressing PsbS (oePsbS), which induces very high levels of NPQ (Li et al. 2002; Roach & Krieger-Liszkay 2012). Plants in the field may experience a number of sudden changes in light intensity throughout the day. Therefore, rapid reversibility of NPQ should be advantageous, as efficient use of the incident light to promote photosynthesis under light-limiting conditions is as important as protection of the photosynthetic apparatus against excess excitation. Indeed, the time required for reversal of heat dissipation after each shift from high to low light is predicted to cause large losses in carbon assimilation by crop canopies (Zhu et al. 2004). The activity of zeaxanthin epoxidase (ZE), which converts zeaxanthin back to violaxanthin, is necessary to revert qE (Jahns et al. 2009; Jahns & Holzwarth 2012) and, hence, to down-regulate the protection of plants against excess light in order to allow for a more efficient utilization when light energy is scarce. ZE is thought to be constitutively active, and the possible regulation of this enzyme is not well understood (Jahns et al. 2009). Numerous findings indicate that induction and reversal of the qE component of NPQ may be more complex than previously thought. Heat stress reduces the ΔpH component of the trans-thylakoid proton motive force and affects qE (Zhang et al. 2009). Plants lacking the PGR5 or PGRL1 proteins, which function in the switch between linear electron flow (LEF) and cyclic electron flow (CEF), are unable to induce qE (Munekage et al. 2002; DalCorso et al. 2008; Suorsa et al. 2012). Furthermore, plants devoid of the thylakoid H + /K + antiporter KEA3 display retardation of recovery from NPQ after transfer from high to low light, suggesting a role for KEA3 in this process through direct control of the trans-thylakoid proton gradient (Armbruster et al. 2014). In addition, mutants affected in the thylakoid two-pore K + channel TPK3 have been shown to be deficient in NPQ (Carraretto et al. 2013). Probably, more factors regulating these processes remain to be identified. Redox regulation is a means of regulating enzyme activity that implies reversible post-translational changes in the redox state of functional groups in the amino acid side chains (Buchanan & Balmer 2005). Oxidation of reactive cysteine residues to sulphenic acids or disulphides may be reversed through the action of thioredoxins and glutaredoxins (Meyer et al. 2009). Chloroplasts contain a rich variety of thioredoxins, which receive reducing equivalents from the PET via ferredoxin–thioredoxin reductase (Schurmann & Buchanan 2008) and in this manner catalyse light-dependent reduction of target enzymes. In addition, there is a chloroplast NADPH-dependent thioredoxin reductase, NTRC, which contains a thioredoxin domain fused to the C-terminus (Serrato et al. 2004; Perez-Ruiz et al. 2006). NTRC constitutes a functional homodimer, where the NTR domain of one subunit reduces the active site of the thioredoxin domain in the other subunit (Perez-Ruiz & Cejudo 2009; Bernal-Bayard et al. 2012). NTRC is a very efficient reductant for the thiol-dependent peroxidase 2-Cys peroxiredoxin (2-Cys Prx) (Moon et al. 2006; Perez-Ruiz et al. 2006; Alkhalfioui et al. 2007; Puerto-Galan et al. 2015). Hence, a role in chloroplast oxidative stress tolerance has been proposed for this enzyme. The Arabidopsis thaliana ntrc knockout mutant is pale green and displays retarded growth (Serrato et al. 2004; Perez-Ruiz et al. 2006; Lepisto et al. 2009), while plants overexpressing NTRC are larger than wild-type (wt) plants (Toivola et al. 2013). This phenotype of the ntrc mutant is particularly pronounced under short-day photoperiods (Perez-Ruiz et al. 2006; Lepisto et al. 2009; Lepisto et al.2013).Thentrc mutant was reported to be hypersensitive to several kinds of abiotic stress, such as high salinity and drought (Serrato et al. 2004), prolonged darkness (Perez-Ruiz et al. 2006) and heat (Chae et al.2013).Under these conditions, the already low growth rate of the ntrc mutant is further reduced in comparison with wt plants (Serrato et al. 2004; Perez-Ruiz et al. 2006). The possible implication of chloroplast redox enzymes, for example thioredoxins, as signalling molecules in highlight acclimation and protection against excess excitation was postulated several years ago (Li et al. 2009). Recently, the relevance of thioredoxins in adaptation of plants to fluctuating light conditions has been considered (Nikkanen & Rintamaki 2014). This motivated us to investigate a potential connection between light acclimation processes and NTRC, previously implied in peroxide detoxification and abiotic stress tolerance. MATERIALS AND METHODS Plant material and growth conditions Arabidopsis thaliana plants were grown on soil in a growth chamber FitoClima 700 EDTU from Aralab (Rio de Mouro, Portugal) under a photoperiod of 10h light/14 h darkness and at temperatures of 22°C/20°C during the day/night cycle. The light intensities applied were 50 (low light), 120 (normal light), 600 (moderately high light) or 1000 μmol quanta m 2 s 1 (high light). Temperature and relative humidity (60%) were strictly controlled at all light intensities. Chlorophyll content was measured using the equations in Lichtenthaler (1987) after extraction from leaf discs with 100% methanol overnight. The Arabidopsis mutants used were the ntrc knockout mutant (Serrato et al. 2004; Perez-Ruiz et al. 2006) and the trxx and Δ2cp mutants (Pulido et al. 2010). The psbs mutant was selected from the line SALK_095156, which contains a T-DNA insertion in the second exon of the gene At1g44575 encoding the PSII PsbS subunit. The ntrc–psbs mutant was obtained by manually crossing the psbs mutant with the ntrc mutant. PCR analyses of these insertion lines were performed using the oligonucleotides in Supporting Information Tables S1 and S2. Measurements of chlorophyll fluorescence and of the photosystem I P700 redox state Room temperature chlorophyll afluorescence was measured using a pulse-amplitude modulation fluorometer (DUALPAM-100, Walz, Effeltrich, Germany). The maximum quantum NTRC controls photosynthetic electron transport 805 ©2015JohnWiley&SonsLtd,Plant, Cell and Environment,39,804–822 yield of PSII was assayed after incubation of plants in the dark for 30 min by calculating the ratio of the variable fluorescence, F v ,tomaximalfluorescence, F m (F v /F m ). Induction–recovery curves were performed using red (635nm) actinic light at the intensities specified for each experiment during 8 min. Saturating pulses of red light at 10 000μmol quanta m 2 s 1 intensity and 0.6s duration were applied every 60 s, and recovery in darkness was recorded for up to 12min. The parameters Y(II), Y(NPQ) and Y(NO), corresponding to the respective quantum yields of PSII photochemistry, NPQ and non-regulated basal quenching (NO), were calculated by the DUAL-PAM-100 software according to the equations in Krameret al. (2004b). Measurements of relative linear electron transport rates were based on chlorophyll fluorescence of preilluminated plants applying stepwise increasing actinic light intensities up to 2000 μmol quantam 2 s 1 . For measurement of post-illumination fluorescence induction, dark-adapted leaves were subjected to one saturating pulse and illuminated with actinic light at 126 μmol quanta m 2 s 1 intensity, whereafter fluorescence was recorded for 5 min in darkness. Chlorophyll fluorescence imaging of whole rosettes was performed using an IMAGING-PAM M-Series instrument (Walz). The redox state of photosystem I (PSI) P700 was monitored by following the changes in absorbance of 30 min dark-adapted plants at 830 versus 875 nm using the DUALPAM-100. To probe the maximum extent of P700 oxidation, leaves were illuminated with far red light superimposed on the saturating pulse of red light. Electron paramagnetic resonance for measurement of peroxide-derived hydroxyl radicals in vivo Spin trapping assays with N-(4-pyridylmethylene)-tert- butylamine N,N′-dioxide (4-POBN) were carried out using leaves from plants grown at 120 μmol quanta m 2 s 1 essentially as in Michelet and Krieger-Liszkay (2012). Pieces of 15–20 mg of leaf per sample were infiltrated with a solution containing 50mM4-POBN, 4% ethanol, 2.5 μMFe-ethyl- enediaminetetraacetic acid (EDTA) and 20 mMphosphate buffer (pH7.0). Infiltrated leaves were incubated in 250 μLof the same solution for 1 h under the light conditions indicated. Electron paramagnetic resonance (EPR) spectra of the 4-POBN/α-hydroxyethyl adducts were recorded at room temperature in a standard quartz flat cell using an e-scan X-band spectrometer (Bruker, Rheinstetten, Germany). The following parameters were used: microwave frequency, 9.73 GHz; modulation frequency, 80kHz; modulation amplitude, 1 G; microwave power, 6.3 mW; and number of scans, 2. Signals were normalized to leaf weight. Cloning, expression and purification For the expression of proteins, the coding sequences for VDE (At1g08550) and ZE (At5g67030), without chloroplast and luminal transit peptides, were amplified by PCR using genespecific oligonucleotides, which included restriction sites for cloning into the pQE-30 vector (Qiagen Sciences, Germantown, MD, USA). The sequences of these oligonucleotides were as follows: VDE, forward 5′-GA- GGATCCGTTGATGCACTTAAA-3′(BamH1) and reverse 5′-AACTGCAGCCTGACCTTCCTGAT-3′(PstI), and ZE, forward 5′-AAGGATCCGCGGCGACG-3′(BamH1) and reverse 5′-GCAAGCTTAGCTGTCTGAAGTAA-3′ (HindIII). Proteins were expressed in Escherichia coli XL1-Blue cells from Stratagene (Agilent Technologies, Santa Clara, CA, USA). The recombinant proteins were purified by nickel-affinity chromatography using the Ni-NTA Agarose resin (Qiagen). Purified recombinant rice NTRC was prepared as in Serrato et al. (2004), but using nickel-affinity chromatography. Electrophoresis and protein gel blot analysis Total leaf proteins or thylakoid proteins were extracted as described by Lepisto et al. (2009), the chlorophyll concentrations were measured in 80% acetone as in Lichtenthaler (1987), and the protein concentrations were determined according to Markwell et al. (1978). Proteins were resolved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels at 12% or 10% acrylamide concentration. Antibodies against purified recombinant VDE were raised in rabbit and used at 1:2500 dilution. Antibodies against PsbS, ZE and the ATP synthase γsubunit (AtpC) were from Agrisera (Vännäs, Sweden) and used at dilutions of 1:2000, 1:3000 and 1:5000, respectively. The antibody against NTRC (Serrato et al. 2004) was used at dilution 1:1000. Signals were visualized with enhanced chemiluminescence (ECL) using the Immobilon ™ Western Chemiluminescent horseradish peroxidase (HRP) substrate reagent from Millipore (Billerica, MA, USA). Alkylation of cysteine thiols was performed using MM(PEG) 24 from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Determination of ATP hydrolysis and ΔpH in vitro using 9-aminoacridine fluorescence Thylakoids isolated from wt plants were diluted to a chlorophyll concentration of 5 μgmL 1 . The thylakoid suspension was incubated in the presence of 2.5 μM9-aminoacridine and with the additions of 100μMNADPH and 5 μMNTRC, 100 μMNADPH alone or 5 mMDTT for 5 min. The fluorescence at 420–550 nm was recorded using the DUALPAM-100 fluorometer. ATP (50 μM)andNH 4 Cl (20 mM)were added at the indicated time points. For maximal fluorescence quenching, thylakoids were illuminated with red light at 500 μmol quanta m 2 s 1 intensity in the presence of 100 μM methyl viologen. Measurement of ΔpH in vivo using the electrochromic shift assay Spectroscopic measurements of electrochromic shift (ECS) based on absorbance changes at 520 nm were performed 806 B. Naranjo et al. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment,39,804–822 essentially as in Takizawa et al.(2007)usingaJTS-10Joliot- type spectrometer (BioLogic, Claix, France). ECS signals and dark relaxation were measured after 15 min illumination of plants with actinic light at intensities between 12 and 940 μmol quanta m 2 s 1 . The slowly reversible component ECS inv is proportional to the ΔpH. For each leaf, the maximum signal was obtained using a saturating laser flash. The flashinduced signal was used for normalization. Analysis of xanthophylls Pigments were extracted by grinding 0.5 g of leaves in liquid nitrogen and resuspending the leaf powder in 10mL 90% acetone. Quantitative analysis of the carotenoids involved in the xanthophyll cycle (violaxanthin, antheraxanthin and zeaxanthin) was performed using high-performance liquid chromatography (HPLC) as described by Hornero-Mendez et al. (2000). All operations were carried out under dimmed light to prevent isomerization and photodegradation of pigments. RESULTS The Arabidopsis ntrc mutant tolerates high light intensities In order to address the possible involvement of NTRC in long-term high-light acclimation, we examined the growth of Arabidopsis ntrc andwtplantsatdifferentlightintensities. Elevated light intensities did not further compromise the growth of the ntrc plants (Fig. 1a). The rosettes of the mutant plants maintained nearly their proportions to those of wt plants even at higher light intensities (Fig. 1b). The ntrc rosettes weighed 12 ± 2.7 and 8.6 ± 1.9% of wt rosettes at 600 Figure 1. Tolerance of Arabidopsis wild-type (wt) and ntrc knockout mutant plants to different irradiances. Plants were grown under a photoperiod of 10h light/14 h darkness at 50, 120, 600 or 1000 μmol quanta m 2 s 1 intensity. (a) wt and ntrc mutant plants grown for 5 weeks at the respective light intensities. (b) Fresh weight of rosettes from 5-week-old plants. Each data point is the mean of 10–20 plants, and standard deviations (SDs) are presented as error bars. (c) Content of total chlorophylls aand bdetermined in leaf discs from leaves of 5-week-old plants. Each data point is the mean of discs from 10 plants ± SDs. (d) Five-week-old plants grown at 120 μmol quanta m 2 s 1 light intensity were transferred to an intensity of 800 μmol quanta m 2 s 1 . Time 0 corresponds to dawn of the last day in normal light. Each data point is the mean of the F v /F m values measured from 12 plants (three leaves from each), and SDs are presented as error bars. (e) Relative content of peroxide-derived hydroxyl radicals based on electron paramagnetic resonance (EPR) signals from wt and ntrc mutant leaves in the dark, D; growth light, GL (120 μmolquantam 2 s 1 ); and high light, HL (500 μmol quanta m 2 s 1 ). The presence of hydroxyl radicals was shown by indirect spin trapping with N-(4-pyridylmethylene)-tert-butylamine N,N′- dioxide (4-POBN)/ethanol in the presence of Fe-ethylenediaminetetraacetic acid (EDTA). The signal from the ntrc mutant is expressed as a percentage of the corresponding wt control sample ± SE. Darkness, n= 5; growth light, n= 8; high light, n= 4. Significantly different values according to Student’st-test are marked with different letters (P<0.05). NTRC controls photosynthetic electron transport 807 ©2015JohnWiley&SonsLtd,Plant, Cell and Environment,39,804–822 and 1000 μmol quanta m 2 s 1 , respectively, as compared with 10 ± 3.8% at 120 μmol quanta m 2 s 1 . Regarding the amount of total chlorophyll per leaf area, the mutant plants hadabout60%ofthevaluesofwtplantsatalllightintensities examined (Fig. 1c). To test short-term high-light tolerance, plants grown at a light intensity of 120 μmol quanta m 2 s 1 were transferred to 800 μmol quanta m 2 s 1 , and the integrity of PSII was assayed as variable fluorescence normalized to maximal fluorescence (F v /F m ). Before the transfer to high light, the ntrc plants had an F v /F m ratio of about 0.78, which is slightly lower than the typical 0.83 of wt plants (Fig. 1d). After 3h of illumination at 800 μmol quantam 2 s 1 , these values had decreased to 0.74 and 0.76, respectively. The results indicate that the ntrc mutant does not suffer increased damage to PSII (Fig. 1d). Assays were also performed using detached leaves in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis and, therefore, impedes PSII repair. These experiments confirmed that there is hardly any difference in sensitivity to photoinhibition between mutant and wt plants (Supporting Information Fig. S1). Under high-light conditions, the increase in formation of superoxide anion radicals leads to enhanced production of hydrogen peroxide and hydroxyl radicals (Foyer & Noctor 2009; Li et al. 2009). Taking into account that NTRC is an important source of electrons for one of the most abundant chloroplast peroxidases, the 2-Cys Prx (Moon et al. 2006; Perez-Ruiz et al. 2006; Alkhalfioui et al. 2007), we examined the content of superoxide, peroxides and hydroxyl radicals in plants at their growth light intensity (120 μmol quanta m 2 s 1 ) and after exposure to high light (500 μmolquantam 2 s 1 )or darkness by an indirect spin trapping assay (Heyno et al. 2009). To this end, leaves were immersed in 4-POBN/ethanol in the presence of Fe-EDTA, as a catalyst for the Haber–Weiss reaction, while illuminated and EPR spectra of the 4-POBN/αhydroxyethyl adducts were recorded (Supporting Information Fig. S1). Notably, the ntrc plants had 40% higher content of peroxide-derived hydroxyl radicals than the corresponding wt controls at growth light intensity, but not under strong illumination (Fig. 1e). These results on ROS content in the ntrc mutant are in agreement with a previous report (Lepisto et al. 2013). This would speak against a function of NTRC in peroxide detoxification at high irradiances. The absence of NADPH thioredoxin reductase C causes drastically enhanced non-photochemical quenching at lower light intensities To analyse the function of NTRC in plant adaptation to changes of light intensity, we measured the ability of the ntrc mutant to adapt to a sudden increase in light intensity. Chlorophyll fluorescence was monitored during illumination with actinic light followed by darkness (Fig. 2a). The intensity of the actinic light in this experiment was about twice the growth light of these plants. The saturating light pulseinduced peaks of maximal fluorescence during illumination (F′ m) were smaller than the maximal fluorescence in the dark-adapted state (F m ) for both wt and ntrc mutant plants (Fig. 2a). This shows that the mutant is capable of responding adequately to an increase in light intensity by inducing NPQ. During the subsequent dark period, fluorescence maxima were recovered in both wt and mutant plants with similar kinetics (Fig. 2a). At actinic light intensities lower than the growth light intensity, NPQ is normally not observed, except shortly after the onset of light due to transient acidification of the thylakoid lumen (Kalituho et al. 2007). This was confirmed in the fluorescence curves recorded for the wt plants (Fig. 2b). In contrast, the ntrc mutant displayed extensive NPQ lasting during the entire illumination period, as revealed by the dramatic reduction of the F′ mvalues (Fig. 2b). This response is abnormal and distinguishes the plants lacking NTRC from wt plants. In order to compare quantitatively the photosynthetic performance of these plants during induction and recovery, the respective quantum yields of NPQ, PSII photochemistry and non-regulated, or basal, energy dissipation (NO) was analysed according to Kramer et al. (2004b). At 75 μmol quanta m 2 s 1 actinic light, the fraction of total energy dissipated through NPQ in the ntrc mutant plants was as high as 0.5 under steady-state photosynthesis, whereas the corresponding value for wt plants was close to 0.05 (Fig. 2c). Accordingly, the effective quantum yield of PSII in the light was more than four times lower in the mutant as compared with wt plants, while NO did not differ (Fig. 2c). Chlorophyll fluorescence imaging showed that the high-NPQ phenotype is particularly pronounced in the young expanding leaves of the ntrc plants and that there is variegation within single leaves (Fig. 2d). Because some aspects of the ntrc mutant phenotype depend on the day length during growth (Perez-Ruiz et al. 2006; Lepisto et al. 2009; Lepisto et al. 2013) and these experiments were performed using plants grown under short-day conditions (10 h light and 14 h darkness), we also measured chlorophyll fluorescence of plants grown under long-day (16 h light/8 h dark) photoperiod. The ntrc mutant plants grown under long photoperiods also displayed exceptionally high and persisting NPQ at low light intensities (Supporting Information Fig. S2). Photosystem I in the ntrc mutant suffers donor-side limitations in the light, but not lack of acceptors Once it is established that the lack of NTRC strongly influences the levels of NPQ and PSII activity, we proceeded to investigate the PSI activity. It should be noted that various biosynthetic processes, such as starch synthesis, are deficient in the ntrc mutant (Michalska et al. 2009; Toivola et al. 2013), and therefore, a shortage of electron acceptors for PSI might be expected. Furthermore, poor consumption of ATP associated with deficient biosynthesis might lead to activation of NPQ. To test the possibility that the role of NTRC in starch synthesis could be related to the high NPQ in the ntrc mutant, we focused on adenosine diphosphate (ADP) glucose pyrophosphorylase (AGPase), a key enzyme in this process, which was previously 808 B. Naranjo et al. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment,39,804–822 reported to be reduced and activated by NTRC (Michalska et al. 2009). An Arabidopsis mutant lacking the regulatory small subunit of the AGPase, aps1, is devoid of leaf starch and grows poorly, except under continuous-light conditions (Ventriglia et al. 2008). However, aps1 shows a pattern of NPQ identical to wt plants (Supporting Information Fig. S3), indicating that the role of NTRC in starch synthesis is not relevant for the increased energy dissipation. Measurements of the PSI activity in wt and ntrc plants based on P700 absorbance changes showed that the redox state of P700 during illumination is affected in plants lacking NTRC (Supporting Information Fig. S4). Analysis of the respective quantum yields of PSI activity, Y(I); donor-side limitations, Y (ND); and acceptor-side limitations, Y(NA), revealed that the ntrc mutant has a low effective PSI quantum yield under growth light intensity (Fig. 3). This turned out to be the result of limitations on the donor side of PSI in the electron transport chain during illumination (Fig. 3). In contrast, wt and ntrc plants were indistinguishable with respect to acceptor-side limitations (Fig. 3). Thus, the absence of NTRC leads to a deficiency affecting specifically the supply of electrons to PSI in the light. However, the demand for electrons from PSI appears not to be altered. The influence of NADPH thioredoxin reductase C on non-photochemical quenching is unrelated to its function as a reductant for the 2-Cys peroxiredoxin NADPH thioredoxin reductase C is known to be an important electron donor to the chloroplast 2-Cys Prx (Perez-Ruiz et al. 2006; Pulido et al. 2010), and because ROS are involved in Figure 2. Chlorophyll fluorescence in wild-type (wt) and ntrc mutant plants. Fluorescence was measured with a pulse-amplitude modulation fluorometer using attached leaves of 5-week-old plants grown at 120 μmolquantam 2 s 1 light intensity. Following 30min dark incubation and determination of F 0 and F m , the actinic light was turned on, and saturating pulses were applied every 60 s. After 8 min illumination, measurements were continued for another 10 min in the dark. White and black bars below graphs indicate periods of illumination with actinic light and darkness, respectively. (a) Induction and recovery curves with 278 μmol quanta m 2 s 1 intensity actinic light. (b) Induction and recovery curves applying 75 μmol quanta m 2 s 1 intensity actinic light. (c) Quantum yields of NPQ, Y(NPQ); photosystem II (PSII) photochemistry, Y(II); and non-regulated energy dissipation, Y(NO), based on experiments such as in (b). Y(NPQ), open circles; Y(II), closed squares; Y(NO), closed triangles. Each data point is the mean of the values from four plants and standard deviations (SDs) are indicated by error bars. (d) False-colour images representing F v /F m in wt and ntrc mutant plants and the respective Y(NPQ) and Y(II) after 3 min of 80 μmol quanta m 2 s 1 intensity illumination. Images of ntrc plants have been enlarged to facilitate viewing. NTRC controls photosynthetic electron transport 809 ©2015JohnWiley&SonsLtd,Plant, Cell and Environment,39,804–822 the regulation of various responses to excess light (Li et al. 2009), we addressed the relevance of this function for the control of NPQ. A severe knockdown mutant with minimal levels of 2-Cys Prx, the Δ2cp mutant (Pulido et al. 2010), was examined for its capacity to induce NPQ as a function of light intensity and compared with ntrc and wt plants. In addition, mutant plants devoid of the chloroplast thioredoxin Trx x (Pulido et al. 2010) were included in these experiments, as Trx xhas been proposed as an alternative electron donor for 2-Cys Prx (Collin et al. 2003). Plants were grown at four different light intensities, and fluorescence induction–recovery curves were recorded using three actinic light intensities for each set of plants. The yields of NPQ for the Δ2cp and trxx mutants were similar to those of wt plants (Fig. 4 and Supporting Information Fig. S5), except the Δ2cp plants grown at high light intensities, which had less NPQ under lower actinic light. In contrast, the yields of NPQ for the ntrc mutant were higher under all conditions tested (Fig. 4 and Supporting Information Fig. S5). This indicates that the effect that NTRC exerts on NPQ is not related to reduction of 2-Cys Prx. To compare the photosynthetic performance of these plants, the relative rates of linear PET were analysed under gradually increasing intensities of actinic light up to 2000 μmol quanta m 2 s 1 (Fig. 5). wt plants adapted to higher light intensities had higher linear PET rates, but the electron transport for the ntrc mutant remained extremely slow at all intensities examined. The electron transport rates of plants deficient in Trx xand 2-Cys Prx were comparable with those of the wt (Fig. 5). The yields of NPQ in these experiments were always higher for ntrc plants, particularly at lower light intensities (Supporting Information Fig. S6). The absence of NADPH thioredoxin reductase C leads to alterations of the xanthophyll cycle In order to establish the reason why plants lacking NTRC have higher levels of NPQ, different factors contributing to NPQ were analysed. The presence of the xanthophyll zeaxanthin is a prerequisite for formation of the qE component of NPQ, while violaxanthin is inactive in this process (Jahns et al. 2009; Jahns & Holzwarth 2012). Therefore, we determined the levels of the xanthophyll cycle pigments violaxanthin, antheraxanthin and zeaxanthin in wt and ntrc mutant plants. Samples were collected at the end of the night and following the onset of light. As expected, wt leaves displayed only a minor increase in the content of antheraxanthin and zeaxanthin 20min after dawn, which reverted after 40 min of illumination (Fig. 6a). Then, as the light was turned off and after an additional 90min of darkness, the original violaxanthin levels were nearly completely recovered in wt leaves. In contrast, the ntrc mutant showed 10-fold higher levels of zeaxanthin throughout the 40min of illumination (Fig. 6a). This implies that the high NPQ of the ntrc plants at normal light intensities is a direct consequence of excess zeaxanthin and antheraxanthin. Because the relative content of xanthophyll cycle enzymes could affect the composition of xanthophylls, the levels of both VDE and ZE were analysed. However, VDE, the enzyme responsible for the conversion of violaxanthin to zeaxanthin, and ZE, which catalyses the conversion of zeaxanthin back to violaxanthin, were equally abundant in ntrc mutant and wt Figure 3. Activity of photosystem I (PSI) in wild-type (wt) and ntrc mutant plants. The redox state of the photosystem I (PSI) reaction centre P700 was monitored through the changes in absorbance at 830 versus 875 nm. Five-week-old plants grown at 120 μmol quanta m 2 s 1 light intensity were kept in the dark for 30 min prior to the measurements. Following the initial determination of maximal oxidation of P700, the actinic light at an intensity of 126 μmol quanta m 2 s 1 was turned on, and saturating pulses were applied every 20 s. After 5 min, the actinic light was switched off, and measurements were continued for another 5 min. The quantum yields of PSI photochemistry, Y(I); donor-side limitations Y(ND); and acceptor-side limitations Y(NA) are based on saturating pulseanalyses. White and black bars below graphs indicate periods of illumination with actinic light and darkness, respectively. Each data point is the mean of the values from six plants, and standard deviations (SDs) are shown as error bars. 810 B. Naranjo et al. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment,39,804–822 plants (Fig. 6b). Hence, there is no evidence for differences in the content of xanthophyll cycle enzymes that could contribute to explaining the observed changes in xanthophyll composition. The qE component of NPQ depends on ΔpH and, hence, is sensitive to nigericin (Ruban et al. 2012). Treatment with nigericin in vivo reduced markedly the NPQ in ntrc plants (Fig. 7a). To test the short-term effect of thiol reduction in vivo in the ntrc mutant, detached leaves were treated with DTT, a known inhibitor of VDE (Yamamoto & Kamite 1972), before fluorescence was recorded. Indeed, the yield of NPQ in the ntrc mutant leaves treated with DTT decreased prominently (Fig. 7b). The titration showed that incubation with 2 mM DTT could reduce the yield of NPQ to wt levels (Fig. 7c). Concomitantly, the effective quantum yield of PSII in the light increased significantly in the mutant leaves upon incubation with DTT (Fig. 7d). Taken together, the effects of nigericin and DTT and the elevated zeaxanthin levels indicate that the energy-dependent quenching qE is permanently activated under low-light conditions in ntrc plants. The redox state of the xanthophyll cycle enzymes violaxanthin de-epoxidase and zeaxanthin epoxidase in vivo It is conceivable that NTRC could modulate the activities of VDE and/or ZE through reduction of regulatory disulphides. VDE is known to be inhibited by disulphide reduction in vitro (Yamamoto & Kamite 1972; Hall et al. 2010; Simionato Figure 4. Non-photochemical quenching (NPQ) in wild-type (wt) and ntrc,Δ2cp and trxx mutant plants grown under different irradiances. Chlorophyll fluorescence was measured using attached leaves of 5-week-old plants grown at 50, 120 and 600 μmol quanta m 2 s 1 . Plants were kept in the dark for 30 min prior to measurements. Three different actinic light intensities of 18, 75 and 278 μmol quanta m 2 s 1 were used, and saturating pulses were applied every 60 s. Quantum yields of NPQ, Y(NPQ), were calculated from saturating pulse analyses. White and black bars below graphs indicate periods of illumination with actinic light and darkness, respectively. Each data point is the mean of the Y(NPQ) from four plants, and standard deviations (SDs) are indicated by error bars. NTRC controls photosynthetic electron transport 811 ©2015JohnWiley&SonsLtd,Plant, Cell and Environment,39,804–822 et al. 2015), although the physiological meaning of this remains unknown. The Arabidopsis VDE contains 12 cysteines in the sequence of the mature protein, and, yet, a single substitution of cysteine-72 to tyrosine is sufficient to abolish activity (Niyogi et al. 1998). ZE contains nine cysteine residues, five of which are conserved in orthologues from monocotyledons and four are also conserved in green algae (Supporting Information Fig. S7). Thiol-based redox regulation of ZE has not been previously reported, although ZE was found to be inhibited in vivo by photooxidative stress under high-light conditions (Reinhold et al. 2008). Thus, to test whether VDE is a redox-regulated enzyme, the redox state of VDE in vivo in the ntrc mutant and wt plants was examined (Fig. 8a), taking advantage of the fact that reduced VDE forms migrates slower on SDS-PAGE (Supporting Information Fig. S8). While the most oxidized form of VDE is somewhat more abundant in the ntrc mutant in the dark, there is no significant difference between the mutant and wt VDE redox states in the light (Fig. 8a). This would rule out a possible role for NTRC in reductive inactivation of VDE in the light. Purified recombinant ZE was found to be prone to the formation of intermolecular disulphides under non-reducing conditions, and purified NTRC is able to catalyse the reduction of these disulphides in vitro (Supporting Information Fig. S9). In contrast, analysis of the redox state of ZE in vivo using alkylation with methyl-polyethylene glycol 24 - maleimide, MM(PEG), showed no changes in the wt and the ntrc mutant (Fig. 8b). This indicates that ZE is not a target for redox regulation under normal-light conditions. Another potential target for redox control through NTRC is the γsubunit of the thylakoid ATP synthase, which is known to mediate the activation of ATP synthesis (Hisabori et al. 2013). A possible deficiency in the reduction of the γsubunit would lead to a build-up of the trans-thylakoid proton gradient and, thus, enhanced NPQ. In the dark, this subunit is present in the oxidized form in both the wt and the ntrc mutant (Fig. 8c). After only 1 min of illumination, the γsubunit is fully reduced in the wt, while a minor amount remains oxidized in the ntrc mutant plants in the light (Fig. 8c). The trans-thylakoid ΔpH is larger in plants lacking NADPH thioredoxin reductase C under low light intensities Given that low pH of the thylakoid lumen is a key factor for formation of qE and an active ATP synthase is a prerequisite for consumption of protons, it is relevant to know whether NTRC is capable of activating this enzyme. Activity of the Figure 5. Linear photosynthetic electron transport in wild-type (wt) and ntrc,Δ2cp and trxx mutant plants grown under different irradiances. Chlorophyll fluorescence was measured with a pulse-amplitude modulation fluorometer using attached leaves of plants grown at 50, 120, 600 and 1000 μmol quanta m 2 s 1 for 5 weeks. Relative electron transport rates of photosystem II (PSII), ETR(II), were determined during stepwise increasing photosynthetically active radiation (PAR). Each data point is the mean of the ETR(II) from eight plants, and standard deviations (SDs) are presented as error bars. 812 B. Naranjo et al. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment,39,804–822 and stable levels of NPQ observed. However, a mutant lacking the regulatory small subunit of the AGPase, aps1 (Ventriglia et al. 2008), which is devoid of leaf starch, shows a pattern of NPQ identical to wt plants. Moreover, our determinations of PSI activity based on P700 absorbance show that the ntrc mutant does not suffer more limitations at the acceptor side of PSI than the wt. In contrast, whereas the PSI donor-side limitations are gradually relieved after a dark–light transition in the wt, these limitations persist in plants lacking NTRC during the entire period of illumination. These results speak against the idea that limited activation of chloroplast biosynthetic processes would contribute to the high-qE phenotype of ntrc plants. Wild-type plants transferred from darkness to weak or moderate light display a brief initial peak of NPQ that relaxes within less than 2 min, as protons accumulating in the thylakoid lumen are consumed through ATP synthesis. This phenomenon has been suggested to reflect the lightinduced activation of the ATP synthase (Kalituho et al. 2007). An attractive hypothesis would be that NADPH, the substrate for NTRC, might regulate ATP synthesis through NTRC-mediated reduction of the ATP synthase γsubunit, which is well known to be redox regulated (Hisabori et al. 2013). Indeed, plants with a defective chloroplast ATP synthase have high constitutive NPQ at low light intensities (Dal Bosco et al. 2004). The ATPase was activated by NTRC and NADPH in vitro, indicating a direct effect of NTRC on the activation of the ATP synthase. Light-induced reduction of the ATP synthase γsubunit in vivo proved to be somewhat less efficient in the ntrc mutant than in wt plants. However, it is uncertain whether this alone would account for the increase in ΔpH observed in the ntrc mutant, and it is obvious that there are other enzymes more efficient than NTRC in catalysing this reaction in vivo. The distribution of photosynthetic electron flow between LEF and CEF is crucial for balancing the synthesis of ATP and NADPH and for acclimation of plants to fluctuating light conditions (Kramer et al. 2004a; Foyer et al.2012; Allahverdiyeva et al. 2015). Interestingly, measurements of constitutive high qE under moderate-light conditions have been applied previously to screen for high-CEF (hcef) mutants, which have higher rates of proton translocation and larger proton motive force (Livingston et al.2010).Thethioredoxin Trx m4 has recently been shown in vivo to inhibit CEF dependent on the NDH (Courteille et al. 2013). However, mutants lacking Trx m4 did not show enhanced NPQ or lower effective PSII quantum yield in the light (Courteille et al. 2013). The ntrc mutant displays elevated and stable post-illumination chlorophyll fluorescence indicative of an enhanced NDH activity and possible high CEF. Nevertheless, the limitation of electron donation to PSI in the ntrc mutant in comparison with wt plants indicates that there is not more CEF in the mutant. Hence, this mechanism cannot contribute to the larger ΔpH found in the ntrc plants. The precise mechanism of action of NTRC in down-regulation of qE remains to be determined and might involve one or several means to limit influx and to enhance efflux of protons from the thylakoid lumen, such as inhibition of NDH and the PGR5/PGRL1 pathways or activation of the KEA3 antiporter. Another possibility is that reduction of the ATP synthase γsubunit might not be sufficient to fully activate this enzyme and that the simultaneous reduction of other subunits, perhaps catalysed by NTRC, would be required, as the α,β,δand εsubunits are also potential thioredoxin targets (Lindahl & Kieselbach 2009). In summary, our results show that plants lacking NTRC accumulate more protons in the thylakoid lumen at low and moderate light intensities, which results in rapid synthesis of zeaxanthin and high levels of dissipation of the light energy absorbed by LHCII. This, in turn, leads to a low effective quantum yield of PSII and, hence, to shortage of electron donors for PSI. A further implication of this enhanced dissipation and slow electron transport is that less energy is available for biosynthesis. The ntrc mutant was previously reported to have lower rates of carbon dioxide fixation, particularly at lower light intensities (Perez-Ruiz et al.2006).Hence,thelow starch content in ntrc plants (Michalska et al. 2009; Lepisto et al. 2013; Toivola et al. 2013) may also reflect the modest production of photosynthates. Indeed, this view affords an explanation to several aspects of the ntrc mutant phenotype. Plants that are continuously starved for light energy should be undersized and grow slowly (Serrato et al. 2004; Perez-Ruiz et al. 2006; Lepisto et al. 2009). Furthermore, longer days should promote growth of such plants by adding to the quantity of total energy absorbed, and this is also true for plants lacking NTRC (Perez-Ruiz et al. 2006; Lepisto et al. 2009, 2013). Here, we show that more electrons can be forced through the LEF pathway in plants lacking both NTRC and PsbS than in plants lacking NTRC alone. This rescues part of the ntrc phenotype with respect to growth and pigmentation. In conclusion, NTRC is required for down-regulation of qE-mediated energy dissipation and stimulation of LEF, which is essential for plant growth, particularly during early leaf development. NTRC is not involved in long-term or short-term high-light tolerance, nor is it implied in peroxide detoxification at high light intensities. On the contrary, plants lacking NTRC are overprotected against light even at low and moderate irradiances, leading to a state of permanent starvation for light energy. ACKNOWLEDGEMENTS This work was supported by a European Regional Development Fund-cofinanced grant (BIO2013-43556-P) from the Spanish Ministry of Economy and Competitiveness (MINECO). B.N. was a recipient of a pre-doctoral fellowship from MINECO. J.M. Romero, Instituto de Bioquímica Vegetal y Fotosíntesis, Sevilla (Spain), is acknowledged for sharing the aps1 mutant. We would like to thank P. 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Non-photochemical quenching (NPQ) in the aps1 mutant in comparison towith wild -type and ntrc mutant plants. Figure S4. Redox state of the photosystem I P700 in wild -type and ntrc mutant plants. Figure S5. Non-photochemical quenching (NPQ) in wild -type and ntrc,Δ2cp and trx x mutant plants grown at high light intensity. Figure S6. Non-photochemical quenching (NPQ) as a function of light intensity in wild -type and ntrc, Δ2cp and trx x mutant plants grown at different light intensities. Figure S7. Domain architectures and positions of cysteines in the Arabidopsis thaliana VDE and ZE. Figure S8. Migration of reduced and oxidised VDE in SDSPAGE. NTRC controls photosynthetic electron transport 821 ©2015JohnWiley&SonsLtd,Plant, Cell and Environment,39,804–822 Figure S9. NTRC reduces intermolecular disulphides of ZE in vitro. Figure S10. Isolation of a psbs knockout mutant and ntrc-psbs double knockout mutants. Table S1. DNA sequences of specific oligonucleotides used as primers for genotyping ntrc, psbs and ntrc-psbs knockout mutants. Table S2. Primer pairs of specific oligonucleotides used for genotyping ntrc, psbs and ntrc-psbs knockout mutants, and their templates. 822 B. Naranjo et al. © 2015 John Wiley & Sons Ltd, Plant, Cell and Environment,39,804–822