NTRC Effects on Non-Photochemical Quenching Depends on PGR5

Non-photochemical quenching (NPQ) protects plants from the detrimental effects of excess light. NPQ is rapidly induced by the trans-thylakoid proton gradient during photosynthesis, which in turn requires PGR5/PGRL1-dependent cyclic electron flow (CEF). Thus, Arabidopsis thaliana plants lacking either protein cannot induce transient NPQ and die under fluctuating light conditions. Conversely, the NADPH-dependent thioredoxin reductase C (NTRC) is required for efficient energy utilization and plant growth, and in its absence, transient and steady-state NPQ is drastically increased. How NTRC influences NPQ and functionally interacts with CEF is unclear. Therefore, we generated the A. thaliana line pgr5 ntrc, and found that the inactivation of PGR5 suppresses the high transient and steady-state NPQ and impaired growth phenotypes observed in the ntrc mutant under short-day conditions. This implies that NTRC negatively influences PGR5 activity and, accordingly, the lack of NTRC is associated with decreased levels of PGR5, possibly pointing to a mechanism to restrict upregulation of PGR5 activity in the absence of NTRC. When exposed to high light intensities, pgr5 ntrc plants display extremely impaired photosynthesis and growth, indicating additive effects of lack of both proteins. Taken together, these findings suggest that the interplay between NTRC and PGR5 is relevant for photoprotection and that NTRC might regulate PGR5 activity.


Introduction
Plants are sessile organisms and need to cope with continuous changes in light intensity by adjusting energy utilization to metabolic requirements and the amount of light available, which in turn necessitates the constant regulation of photosynthesis. During the photosynthetic process, light energy is utilized to extract electrons from water, which are transferred through the thylakoid membrane of the chloroplasts during linear electron flow (LEF) to ultimately reduce NADP + to NADPH. LEF involves photosystem II (PSII), the cytochrome (cyt) b 6 f complex and photosystem I (PSI). During this process, a transmembrane proton gradient is generated, which is required for ATP synthesis, but also provides protection against damage caused by excess light. The resulting acidification of the thylakoid lumen downregulates the activity of the cyt b 6 f complex and triggers the thermal dissipation of the excess energy at PSII, a phenomenon known as non-photochemical quenching (NPQ) [1]. Both effects decrease levels of LEF, and consequently protect the two photosystems against overreduction and photodamage.
In addition to LEF, alternative thylakoid electron pathways exist, including cyclic electron flow (CEF) around PSI, which effectively returns electrons derived from PSI to the plastoquinone (PQ) pool and the cyt b 6 f complex. Like LEF, CEF contributes to the formation of the proton gradient across the thylakoid membrane, but without the net The Arabidopsis mutants pgr5 (pgr5-1, S130G point mutation), pgrl1ab (SAIL_443E10/ SALK_059233), ntrc (SALK_012208), cgl160 (cgl160-1, SALK_057229) and trx m4 (SALK_023810) were previously described ( [9,10,18,34,39], respectively). The double mutants pgr5 ntrc and pgr5 cgl160 were obtained by manually crossing the respective parental mutant lines and subjecting the progeny to PCR analysis of genomic DNA. Oligonucleotide sequences used for PCR genotyping are listed in Table S1. The pgr5 allele was analyzed by amplifying and sequencing the genomic region spanning the region coding for the S130G point mutation in At2g05620/PGR5 [9].

Chlorophyll a Fluorescence Measurements
In vivo chlorophyll a fluorescence was monitored with the Imaging PAM chlorophyll fluorimeter (Imaging PAM, M-Series; Walz, Effeltrich, Germany). Plants were dark-adapted for 30 min and induction-recovery curves were plotted after exposure to actinic light at 80 or 390 µmol photons m −2 s −1 for 10 min, followed by 8 min of darkness. Saturating pulses (0.8 s duration) were applied every 60 s to determine the different parameters, which were calculated by the Imaging PAM software according to the equations in [40].

Alkylation Assays
To determine the in vivo protein redox state, alkylation assays were performed as previously described by Naranjo et al. (2016) using NEM (N-ethylmaleimide, ThermoFisher Scientific). Leaves were ground in liquid nitrogen and 10% (v/v) trichloroacetic acid (TCA) was immediately added to quench thiol oxidation. Samples were incubated on ice for 20 min and then centrifuged at 16,200× g and 4 • C for 10 min. The pellets were washed with acetone, resuspended in alkylation buffer (2% (w/v) SDS, 50 mM TRIS-HCl pH 7.8, 2.5% (w/v) glycerol, and 4 M urea) with 10 mM NEM, and incubated for protein thiol alkylation for 20 min at room temperature. Alkylated samples were subjected to SDS-PAGE and probed using PGRL1 antibody as described above.

Growth and Photosynthetic Performance of the pgr5 ntrc Double Mutant under Different Light Conditions
To compensate for the deficit of electron acceptors caused by the pgr5 mutation, several approaches have been successfully employed. These include adding methyl viologen (MV) [9] or overexpressing Physcomitrella patens flavodiiron protein genes in the pgr5 mutant [38]. In addition, decreasing LEF and lowering electron donation to PSI complement the pgr5 phenotype, as it has been shown using DCMU [37], by introducing a defect in the oxygen-evolving complex (in the ∆5 pgr5 mutant) [13], and by downregulating the activity of the cyt b 6 f complex (in the pgr5 pgr1 mutant) [38]. Therefore, we hypothesized that decreasing LEF by increasing NPQ in a pgr5 ntrc double mutant would suppress the lethal phenotype of pgr5 under fluctuating light-owing to the absence of NTRC [32]. However, the pgr5 ntrc double mutant, like the pgr5 single mutant, was incapable of surviving under fluctuating light ( Figure 1A), indicating that the ntrc mutation does not suppress the effects of pgr5 in this regard. In marked contrast, the pgr5 mutation largely suppresses the growth defect associated with the ntrc mutation under short-day lighting conditions ( Figure 1A,B).
Plants devoid of NTRC show a stunted and pale phenotype, and are hypersensitive to diverse abiotic stresses [18], but not to high light stress [32]. In addition, it has been shown that, in the ntrc mutant, NPQ is enhanced and the effective quantum yield of photosystem II (Φ II ) is diminished, which protects ntrc against high light but also impairs its growth under low light or short-day conditions ( [32] and Figures 1 and 2). Interestingly, the introduction of the pgr5 mutation into the ntrc background decreased steady-state NPQ to WT-like levels under all the conditions analyzed-including high light (Figures 2A and S1A). However, this effect was not always associated with the recovery of Φ II and growth (Figures 1, 2B and S1B), implying that the WT steady-state NPQ level is not sufficient to cope with light stress in the absence of NTRC and PGR5. For instance, under short-day conditions Φ II and growth rate of pgr5 ntrc were very similar to those of WT plants, whereas under long-day conditions, the pgr5 ntrc mutant failed to reach WT-like Φ II values, and its growth rate resembled that of the ntrc single mutant (Figures 1 and 2B). Under high light, the combination of pgr5 with ntrc was virtually lethal ( Figures 1A and 2B). These results suggest a role for NTRC in photoprotection beyond negatively influencing NPQ induction-possibly via ROS scavenging through the reduction of 2-Cys Prxs-that is usually masked by the high level of NPQ.

NTRC-Dependent NPQ Induction in the Absence of PGR5
In previous studies, the increase in steady-state NPQ in the absence of NTRC was attributed to a lower lumenal proton efflux due to impaired Calvin-Benson cycle and ATP synthase activities, both of which amplify the proton gradient across the thylakoid membrane [27,32,33]. However, here we clearly show that the additional steady-state NPQ induced by the absence of NTRC requires PGR5. Antioxidants 2021, 10, x FOR PEER REVIEW 5 of 13 Plants devoid of NTRC show a stunted and pale phenotype, and are hypersensitive to diverse abiotic stresses [18], but not to high light stress [32]. In addition, it has been day conditions ΦII and growth rate of pgr5 ntrc were very similar to those of WT plants, whereas under long-day conditions, the pgr5 ntrc mutant failed to reach WT-like ΦII values, and its growth rate resembled that of the ntrc single mutant (Figures 1 and 2B). Under high light, the combination of pgr5 with ntrc was virtually lethal (Figures 1A and 2B). These results suggest a role for NTRC in photoprotection beyond negatively influencing NPQ induction-possibly via ROS scavenging through the reduction of 2-Cys Prxs-that is usually masked by the high level of NPQ.

NTRC-Dependent NPQ Induction in the Absence of PGR5
In previous studies, the increase in steady-state NPQ in the absence of NTRC was attributed to a lower lumenal proton efflux due to impaired Calvin-Benson cycle and ATP synthase activities, both of which amplify the proton gradient across the thylakoid membrane [27,32,33]. However, here we clearly show that the additional steady-state NPQ induced by the absence of NTRC requires PGR5.
In addition to steady-state NPQ, we also measured the kinetics of NPQ induction upon a dark-to-light transition, which serves as a proxy for CEF activity [9,10]. The transient induction of NPQ in ntrc is more than doubled compared to WT ( Figure 3A) and its relaxation results in the elevated steady-state NPQ levels described above (see Figure 2A). In addition to steady-state NPQ, we also measured the kinetics of NPQ induction upon a dark-to-light transition, which serves as a proxy for CEF activity [9,10]. The transient induction of NPQ in ntrc is more than doubled compared to WT ( Figure 3A) and its relaxation results in the elevated steady-state NPQ levels described above (see Figure 2A). In the pgr5 mutant, NPQ induction is much weaker than in WT and this has been ascribed to the lack of PGR5/PGRL1-dependent CEF. The transient induction and relaxation of NPQ seen in the pgr5 ntrc mutant is very similar to that of the pgr5 single mutant ( Figure 3A), suggesting that the elevated transient NPQ found in the ntrc mutant itself depends on a functional PGR5 protein. As a control, we used the cgl160 mutant, which is impaired in chloroplast ATPase activity and therefore displays an impaired proton motive force (pmf) and an elevated NPQ [34]. Although the increase in transient NPQ induction seen in cgl160 is very similar to that of ntrc, the combination of the cgl160 mutation with pgr5 reduced the transient NPQ induction to WT but not pgr5 levels, and almost no relaxation of NPQ was seen at later time points. These results indicate that the enhancement of both steady-state and transient NPQ in ntrc depends on PGR5, while this is not the case in the cgl160 mutant. Consequently, owing to the loss of the functional PGR5 protein, pgr5 ntrc lines behave like pgr5 with respect to the expression of NPQ. Moreover, in the absence of PGR5, the cgl160 mutant with a defective chloroplast ATPase still displays transient and steady-state NPQ values that are much higher than those in the pgr5 single mutant, implying that this residual elevated NPQ is specific to the cpATPase defect, and is not related to PGR5/PGRL1-dependent CEF. Indirectly, this also implies that the elevated (steady-state and transient) NPQ observed in plants that lack NTRC is not caused by impaired cpATPase function, but can be explained by the altered activity of PGR5 alone. lines behave like pgr5 with respect to the expression of NPQ. Moreover, in the absence of PGR5, the cgl160 mutant with a defective chloroplast ATPase still displays transient and steady-state NPQ values that are much higher than those in the pgr5 single mutant, implying that this residual elevated NPQ is specific to the cpATPase defect, and is not related to PGR5/PGRL1-dependent CEF. Indirectly, this also implies that the elevated (steadystate and transient) NPQ observed in plants that lack NTRC is not caused by impaired cpATPase function, but can be explained by the altered activity of PGR5 alone. In the pgr5 cgl160 mutant ( Figure S2), the enhanced level of transient NPQ relative to WT was associated with ΦII values that were lower than in WT ( Figure 3A,B), indicating that the pgr5 mutation can restore this component of photosynthesis in the ntrc background, but not in the cgl160 background. In line with this, pgr5 cgl160 plants remain smaller than either of the two parental lines pgr5 and cgl160 under all conditions analysed ( Figure S3). Interestingly, even though pgr5 cgl160 plants are capable of inducing NPQ to levels that are higher than in WT, at least under short-day conditions, these plants are unable to grow under fluctuating light (Figures 3A and S3). These data suggest that the high level of NPQ during the high light period is not decisive for survival under fluctuating light, but possibly its rapid recovery during the low light period is. Moreover, a deficit in ATP synthesis resulting from the absence of the cpATPase assembly factor CGL160 possibly aggravates the stromal overreduction of the pgr5 mutant, increasing the deficit of electron acceptors and the photodamage of P700. In the pgr5 cgl160 mutant ( Figure S2), the enhanced level of transient NPQ relative to WT was associated with Φ II values that were lower than in WT ( Figure 3A,B), indicating that the pgr5 mutation can restore this component of photosynthesis in the ntrc background, but not in the cgl160 background. In line with this, pgr5 cgl160 plants remain smaller than either of the two parental lines pgr5 and cgl160 under all conditions analysed ( Figure S3). Interestingly, even though pgr5 cgl160 plants are capable of inducing NPQ to levels that are higher than in WT, at least under short-day conditions, these plants are unable to grow under fluctuating light (Figures 3A and S3). These data suggest that the high level of NPQ during the high light period is not decisive for survival under fluctuating light, but possibly its rapid recovery during the low light period is. Moreover, a deficit in ATP synthesis resulting from the absence of the cpATPase assembly factor CGL160 possibly aggravates the stromal overreduction of the pgr5 mutant, increasing the deficit of electron acceptors and the photodamage of P700.

PGRL1 Dimer Formation and Protein Content in the Absence of NTRC
Our results indicate that elevated NPQ in ntrc depends on PGR5, which implies that in WT plants the NTRC protein has a negative effect on PGR5 activity. This raises the question of how this effect occurs. A physical interaction between NTRC and PGR5, but not PGRL1, has already been reported [43], although neither of the latter proteins appears as an NTRC interactor in pull-down experiments performed in a more recent study [24]. However, PGRL1 is much more likely to be the target of the redox regulation of PGR5/PGRL1-dependent CEF, since it contains six conserved cysteines (Cys), while PGR5 has only one. Indeed, it has recently been shown that the single Cys in PGR5 is dispensable for CEF in Chlamydomonas [44], while in vitro [11] and in vivo experiments [45,46] have confirmed the redox regulation of PGRL1 by Trxs. Taken together, these considerations are compatible with the direct redox regulation of PGRL1 by NTRC to decrease CEF activityand consequently NPQ. Therefore, we performed in vivo alkylation assays using NEM to analyze the redox dependence of complex formation by PGRL1 in WT and mutant plants after a dark-to-light transition. In fact, PGRL1 was reduced after the dark-tolight transition, with the active monomeric form (m) being more abundant in the light, while the dimer (d, inactive form) was more abundant in the dark and required PGR5 ( Figure 4A), corroborating previous results [11,45]. However, we did not observe any significant difference between the dimer/monomer ratios of PGRL1 in ntrc and WT plants, either in the dark or after a 30-s exposure to light ( Figure 4A), such that an altered PGRL1 dimer/monomer ratio can most likely be excluded as the cause for altered CEF activity in the mutant. Nevertheless, it is necessary to test more light conditions with different exposure times and intensities to definitively rule out this possibility. It has also been shown that Trx m4 forms a complex with PGRL1 and inactivates it [46]. Indeed, phenotypic effects of trx m4 [46,47], but not of other thioredoxin mutants like trx f1f2 [48], can be suppressed by pgr5. Multiple sources of evidence support the notion that chloroplast Fd-FTR-Trxs and NTRC act in a concerted fashion, with 2-Cys Prxs serving as connecting links between the two redox systems [27]. Therefore, another possibility It has also been shown that Trx m4 forms a complex with PGRL1 and inactivates it [46]. Indeed, phenotypic effects of trx m4 [46,47], but not of other thioredoxin mutants like trx f1f2 [48], can be suppressed by pgr5. Multiple sources of evidence support the notion that chloroplast Fd-FTR-Trxs and NTRC act in a concerted fashion, with 2-Cys Prxs serving as connecting links between the two redox systems [27]. Therefore, another possibility is that NTRC influences PGR5/PGRL1-dependent CEF through Trx m4, with NTRC being required for the formation of the PGRL1-Trx m4 complex. However, we could not unambiguously clarify this, since, in our hands, a band (denoted by "c") with the size of the described PGRL1-Trx m4 complex (40 kDa) was not only present in ntrc ( Figure 4A) plants, but also in the trx m4 mutant used as a negative control ( Figure S4). Moreover, the intensity of the "c" band changed only a little, if at all, among genotypes and conditions, arguing against a regulatory role of the represented complex in PGRL1 activity.
Interestingly, some photosynthetic-related proteins such as PsaA and PsbO accumulated less in pgr5 (about 75% and 60% of WT level, respectively) and ntrc (both around 40% of WT) mutants, and both were recovered in the pgr5 ntrc double mutant (90% and 66% of WT, respectively) ( Figure S5). Moreover, the decrease in the NTRC and PGR5 proteins was especially pronounced in the pgr5 and ntrc backgrounds, respectively ( Figures 4B and S5B). While the steady-state level of PGRL1 fell to 73% of WT in the ntrc mutant, amounts of PGR5 dropped to 25% ( Figure 4B) [43]. At first sight, this seems to be at variance with the increased activity of PGR5 that we propose in the absence of NTRC. However, if PGR5 activity is no longer inhibited in the absence of NTRC, then lowering its abundance at the protein level might be necessary to avoid further harmful effects. Accordingly, the enhanced NPQ seen in the ntrc mutant might be the result of the enhanced activity of the remaining 25% of PGR5 proteins relative to WT (and PGRL1 to 70% of WT). Intriguingly, NTRC protein levels were also decreased in the pgr5 mutant to about 50% ( Figure 4B). Since the accumulation of PGR5 and PGRL1 is interdependent [10,11], it is not possible to unambiguously assign each drop in abundance of a given protein specifically to another one in this three-protein set, but there is clearly a two-way type of interdependence of protein levels between PGR5/PGRL1 and NTRC.

Conclusions
We have previously reported that NTRC influences NPQ, probably through the activation of chloroplast metabolism and enhanced thylakoid proton consumption [27,32]. However, by generating and analysing the pgr5 ntrc double mutant, we have demonstrated here that the high NPQ observed in the absence of NTRC is mediated by PGR5 and must be a consequence of the deregulation of its activity. In line with this, PGR5 levels are reduced in the ntrc mutant, probably to compensate for this deregulation. Therefore, we propose that in WT plants, NTRC controls PGR5/PGRL1-dependent CEF activity. Further studies will be needed to clarify the mechanism, which could involve the direct regulation of PGR5, PGRL1 or a third player, either directly by NTRC, or indirectly through other thioredoxins (such as Trx m4) and 2-Cys Prxs. In addition, the pgr5 ntrc mutant uncovered the role of NTRC beyond controlling NPQ induction in the protection of plants against high light stress, possibly through ROS scavenging via 2-Cys Prxs.