-like2/2-Cys redox cascade supports oxidative thiol modulation in chloroplasts

Keisuke Yoshidaa,1, Ayaka Haraa, Kazunori Sugiuraa, Yuki Fukayaa, and Toru Hisaboria,1

aLaboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Midori-ku, 226-8503 Yokohama, Japan

Edited by Bob B. Buchanan, University of California, Berkeley, CA, and approved July 18, 2018 (received for review May 14, 2018) Thiol-based redox regulation is central to adjusting chloroplast characteristics, such as the redox potential and protein surface functions under varying light conditions. A redox cascade via the charge (6–9). Recent studies have revealed the functional diversity ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway of Trx subtypes, as exemplified by their different target selectivities has been well recognized to mediate the light-responsive reduc- (10, 11). tive control of target proteins; however, the molecular basis for In previous work, we studied the diurnal redox behaviors of reoxidizing its targets in the dark remains unidentified. Here, we Trx-targeted proteins in spinach (12) and Arabidopsis (13). Our report a mechanism of oxidative thiol modulation in chloroplasts. results clearly showed that these proteins are sensitively switched We biochemically characterized a chloroplast stroma-localized from oxidized to reduced forms in response to the increase in Arabidopsis atypical Trx from , designated as Trx-like2 (TrxL2). TrxL2 light intensity at dawn, which is indicative of the dynamics of had redox-active properties with an unusually less negative redox chloroplast redox regulation in vivo. These studies also high- potential. By an affinity chromatography-based method, TrxL2 was lighted evidence for another intriguing aspect of chloroplast re- shown to interact with a range of chloroplast redox-regulated pro- teins. The direct discrimination of thiol status indicated that TrxL2 dox regulation; Trx-targeted proteins are reoxidized along with can efficiently oxidize, but not reduce, these proteins. A notable the decrease in light intensity at dusk. This finding raises a exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able fundamental question: What triggers the oxidizing reaction? In to reduce 2CP with high efficiency. We achieved a complete in contrast to the FTR/Trx pathway that is responsible for the re- vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing duction process, the molecular basis for oxidative thiol modula- redox-regulated proteins and draining reducing power to hydro- tion has been unidentified. Therefore, this issue can be regarded

gen peroxide (H2O2). We further addressed the physiological rel- as a critical gap in the current understanding of chloroplast evance of this system by analyzing protein-oxidation dynamics. redox regulation. In Arabidopsis plants, a decreased level of 2CP led to the impair- In this study, we characterized an atypical Trx from Arabi- ment of the reoxidation of redox-regulated proteins during dopsis, designated as Trx-like2 (TrxL2). A combined set of bio- light–dark transitions. A delayed response of protein reoxidation chemical data indicates that (i) TrxL2 acts as the efficient was concomitant with the prolonged accumulation of reducing oxidation factor of several redox-regulated proteins, and (ii) power in TrxL2. These results suggest an in vivo function of the TrxL2 drains reducing power to 2-Cys peroxiredoxin (2CP) and TrxL2/2CP redox cascade for driving oxidative thiol modulation ultimately to (H2O2). On the basis of protein- in chloroplasts. oxidation dynamics in Arabidopsis plants, we further provide physiological insights into oxidative thiol modulation relying on redox regulation | oxidation | chloroplast | TrxL2 | 2-Cys peroxiredoxin the TrxL2/2CP redox cascade.

lant chloroplasts have evolved multiple adaptive strategies Significance Pfor allowing efficient photosynthesis under continuously changing light conditions. Thiol-based redox regulation is a To ensure efficient photosynthetic carbon gain, plant chloro- posttranslational mechanism that plays a major role in the light- plasts have to adjust their own physiology toward changes in responsive control of chloroplast functions. In this regulatory light environments. Specific chloroplast proteins are reversibly system, incident light energy is converted into reducing power by activated–inactivated during light–dark cycles by switching photochemical reactions in thylakoid membrane, which is then the reduction–oxidation states of their Cys residues, which is signaled to redox-regulated target proteins in chloroplasts. The termed redox regulation. A long-standing issue in plant bi- target proteins contain a redox-active Cys pair that forms the ology is the manner in which redox-regulated proteins are disulfide bond in the oxidized state. In most cases, target proteins reoxidized upon the interruption of light exposure. In this are activated upon the reductive cleavage of this bond. Canonical study, we identified the thioredoxin-like2 (TrxL2)/2-Cys per- examples of targets are represented by specific enzymes in the – – oxiredoxin (2CP) redox cascade as a molecular basis for oxi- Calvin Benson cycle (1 3); therefore, chloroplast redox regulation dative thiol modulation in chloroplasts. This finding dissects makes it possible to turn on the CO2 fixation process in concert with the “dark side” of chloroplast redox regulation, providing the excitation of photosynthetic electron transport and, thereby, the an insight into how plants rest their photosynthetic activity light perception. at night. It has been firmly established by earlier pioneering studies that the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) Author contributions: K.Y. and T.H. designed research; K.Y., A.H., K.S., and Y.F. performed pathway serves to transfer reducing power from the electron research; K.Y., K.S., and T.H. analyzed data; and K.Y. and T.H. wrote the paper. transport chain to the target proteins (1, 2). The soluble [4Fe-4S] The authors declare no conflict of interest. protein FTR receives reducing power from photosynthetically This article is a PNAS Direct Submission. reduced ferredoxin and supplies it to Trx (4, 5). Trx is a small Published under the PNAS license. ubiquitous protein with a conserved WCGPC motif in its active 1To whom correspondence may be addressed. Email: [email protected] or site. The reduced form of Trx provides reducing power to target [email protected]. proteins via a dithiol–disulfide exchange reaction, thereby modu- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lating the enzymatic activity of targets. Chloroplasts harbor five 1073/pnas.1808284115/-/DCSupplemental. subtypes of Trx (f, m, x, y,andz), which have distinct molecular Published online August 13, 2018.

E8296–E8304 | PNAS | vol. 115 | no. 35 www.pnas.org/cgi/doi/10.1073/pnas.1808284115 Downloaded by guest on September 28, 2021 Results TrxL2 Has a Cross-Talk with Various Redox-Regulated Proteins. We TrxL2 Is Localized to Chloroplast Stroma. Genomic and phylogenic explored TrxL2-interacting proteins in chloroplasts. To this end, studies have identified multiple Trx-like proteins in plants (14); we applied an affinity chromatography-based screening method however, their biochemical or physiological characteristics re- (18) using TrxL2 monocysteinic mutants (TrxL2CS) as bait. main largely unclear. In this study, we noted one type of Trx-like Chloroplast soluble proteins extracted from spinach leaves were proteins possessing a putative active site motif of WCRKC (SI loaded onto a TrxL2CS-immobilized affinity chromatography Appendix, Fig. S1). This atypical Trx motif is marked by two column. Proteins associated with TrxL2CS via the mixed-disulfide bond were eluted by DTT and then identified using mass spec- positively charged hydrophilic amino acids (Arg and Lys) be- trometry (SI Appendix, Fig. S5A). Some proteins, including tween the Cys residues. This protein was previously termed Rubisco activase (RCA) and 2CP, were identified as possible WCRKC Trx (15) or TrxL2 (14); we adopted the latter name in TrxL2-interacting partners. Further analyses of protein elu- this study. tion patterns by immunoblotting indicated that fructose-1,6- In Arabidopsis, TrxL2 is encoded by two genes (At5g06690 for bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase TrxL2.1 and At5g04260 for TrxL2.2) (SI Appendix, Fig. S1). Both (SBPase), NADP-malate dehydrogenase (NADP-MDH), ADP- isoforms have been previously shown to be targeted to chloro- glucose pyrophosphorylase (AGPase), and peroxiredoxin Q plast stroma by in vitro protein import assays (15). We in- (PrxQ) were also bound to TrxL2CS (SI Appendix, Fig. S5B). It vestigated whether TrxL2 is physically associated with the should be noticed that most of these are already known to be thylakoid membrane. Intact chloroplasts were isolated from Trx-targeted proteins that exhibit dynamic redox shifts in re- Arabidopsis leaves and then fractionated into stroma and thyla- sponse to changes in light intensity (12, 13, 19–21). koid membrane. Immunoblotting analyses using antibodies against TrxL2.1 and TrxL2.2 indicated that both TrxL2 isoforms TrxL2 Fails to Reduce FBPase, SBPase, and RCA. The capture of are exclusively localized to chloroplast stroma (SI Appendix, several Trx-targeted proteins by TrxL2CS (described above) Fig. S2). raised the possibility that TrxL2 has an overlapping function with Trx as a reductive regulatory factor. We thus examined whether TrxL2 Is a Redox-Mediator Protein with an Unusually Less Negative TrxL2 can reduce the proteins captured by TrxL2CS. For use as Redox Potential. To gain biochemical clues into TrxL2 functions, targets, three recombinant proteins involved in the CO2 fixation we prepared recombinant proteins of two TrxL2 isoforms from (FBPase, SBPase, and RCA) were prepared. In agreement with Arabidopsis. Redox state discrimination using the thiol-modifying earlier studies (10, 11, 22), these three proteins were specifically reagent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate (AMS) (for FBPase) or favorably (for SBPase and RCA) shifted from indicated that both TrxL2 isoforms form an intramolecular oxidized to reduced forms by f-type Trx (Trx-f) (Fig. 2A). By disulfide bond in the WCRKC motif, which is cleaved upon re- contrast, FBPase, SBPase, or RCA was not efficiently reduced duction (SI Appendix, Fig. S3A). TrxL2.2 partially formed a by TrxL2. homodimer linked by the intermolecular disulfide bond under oxidative conditions. This might be caused by its additional Cys TrxL2 Reduces 2CP with High Efficiency. We further addressed the residue (Cys82), which is not conserved in TrxL2.1 and other reduction ability of TrxL2 using other target candidate proteins. plant orthologs (SI Appendix, Fig. S1). We also observed that Two different types of Prx (2CP and PrxQ) serve in the antiox- idant defense system by detoxifying H O , a byproduct of pho- TrxL2 has a DTT-dependent insulin reduction activity (SI Ap- 2 2 tosynthesis (23). Unlike other Trx-targeted proteins subject to pendix, Fig. S3B). These results suggest that, as in the case with redox-dependent activity control, these Prxs require reducing Trx, TrxL2 can perform a dithiol–disulfide exchange reaction. power for their catalytic processes directly. We next examined the midpoint redox potential (E )of m Arabidopsis has two isoforms of 2CP, 2CPA and 2CPB, with TrxL2. By fitting the reduction level under several redox buffers almost identical amino acid sequences; the former was used in to the Nernst equation, the Em of TrxL2.1 and TrxL2.2 at − ± − ± this study. The oxidized form of 2CP exists as a homodimer pH 7.5 was determined to be 258 2 mV and 245 2 mV, linked by the intermolecular disulfide bond. Similar to Trxs, respectively (SI Appendix, Fig. S4). The Em of Arabidopsis chlo- TrxL2 converted 2CPA from oxidized to reduced forms (Fig. 2B, roplast redox-mediator proteins, including FTR catalytic subunit Upper), indicating the ability of TrxL2 to transfer reducing power (FTR-C) (16), 10 Trx isoforms (11, 16), and NADPH-Trx re- to 2CP. TrxL2 could reduce PrxQ as well (Fig. 2B, Lower). We ductase C (NTRC) (17), are summarized in Fig. 1. It became then compared the efficiencies of TrxL2 and Trx-f in reducing evident that the Em of TrxL2 is substantially higher (less nega- these Prxs in more detail. The redox shift pattern of 2CPA was tive) than those of other redox-mediator proteins. analyzed with changing the concentration of DTT, a source of reducing power (Fig. 2C and SI Appendix, Fig. S6A) or the re- action time (Fig. 2D and SI Appendix, Fig. S6B). In both cases, Reducing Oxidizing TrxL2 showed much higher efficiency in reducing 2CPA than that of Trx-f. By contrast, such dominant reducing function of NTRC TrxL2 was not observed toward PrxQ (Fig. 2 E and F and SI Appendix, Fig. S6 C and D). TrxL2.1 We have previously shown that Trx-f has a comparatively high FTR-C Trxs TrxL2.2 2CP-reducing activity among five Trx subtypes (17). Therefore,

the present finding has important implications for quite efficient PLANT BIOLOGY coupling of TrxL2 to 2CP. We further monitored the H2O2- -380 -360 -340 -320 -300 -280 -260 -240 -220 detoxifying activity of 2CP. Under the conditions tested here, the activity of 2CP was low in the absence of TrxL2, but largely Midpoint redox potential at pH 7.5 (mV) promoted by the addition of TrxL2 (SI Appendix, Fig. S6E).

Fig. 1. An overview of midpoint redox potentials (Em) of FTR-C, 10 Trx isoforms, NTRC (including NTR domain and Trx domain), and two TrxL2 TrxL2 May Utilize Reducing Power from and NADPH Pools, but Its Efficiency Is Low. isoforms from Arabidopsis. Each Em was determined at pH 7.5. The data for For the next important issue, we TrxL2 are shown in SI Appendix, Fig. S4. The data for other proteins can be attempted to identify the reducing power donor of TrxL2. A found in our earlier studies (11, 16, 17). poplar TrxL2 ortholog has been reported to receive reducing

Yoshida et al. PNAS | vol. 115 | no. 35 | E8297 Downloaded by guest on September 28, 2021 Control 1 1 1 1 m m f ACf BD+ Trx-f1 + Trx-f1 + TrxL2.1 + TrxL2.1 + TrxL2.2 + TrxL2.2

100 100 + DTT / TrxL2.1 + DTT / Trx- + DTT / TrxL2.2 Control + DTT / TrxL2.2 Control + DTT + DTT / Trx- + DTT / TrxL2.1 + DTT + DTT / Trx- + DTT / Trx- 80 80 100 50 75 Ox 37 60 60 50 Red FBPase Ox 37 40 40 25 2CPA Red 20 20 20 25 20 15 2CPA reduction level (%) 0 0 0 10 100 1000 0102030 15 DTT (M) Time (min) 10 E F

10 50 100 100 37 80 80 50 Red 25 60 60 SBPase 37 Ox 20 Red 40 40 75 PrxQ 15 Ox 50 Red 20 20 RCA Ox 37 PrxQ reduction level (%) 0 0 (kDa) 10 10 100 0102030 (kDa) 0 DTT (M) Time (min)

Fig. 2. Biochemical characterization of target-reducing activity of Trx and TrxL2. (A) Redox shift visualization of FBPase, SBPase, and RCA. (B) Redox shift visualization of 2CPA and PrxQ. (A and B) Each target protein (oxidized form; 2 μM) was incubated with Trx-f1, Trx-m1, TrxL2.1, or TrxL2.2 (1 μM) in the presence of DTT (0.5 mM for FBPase, SBPase, RCA, and 2CPA; 0.05 mM for PrxQ) for 30 min. Free thiols were labeled with AMS (for FBPase, SBPase, RCA, and PrxQ) or N-ethylmaleimide (for 2CPA), and proteins were subjected to nonreducing SDS/PAGE. (C–F) Comparison of Prx-reducing activity of Trx-f1 and TrxL2. Reduction level of 2CPA (C and D) or PrxQ (E and F) was calculated as the ratio of the reduced form to the total, and plotted against the DTT concentration (C and E) or the reaction time (D and F). Raw images of the SDS/PAGE and immunoblotting are shown in SI Appendix, Fig. S6 A–D. Each value represents the mean ± SD (n = 3). Ox, oxidized form; Red, reduced form.

power from reduced glutathione (GSH) (24). In agreement with time-dependent changes in their redox states were analyzed (Fig. this report, we detected a GSH-dependent shift from oxidized to 3A). In the absence of TrxL2, RCA maintained its reduced state reduced forms in TrxL2, but its redox change was limited to only for 15 min. By contrast, RCA was gradually oxidized in the a slight level (SI Appendix, Fig. S7 A and B). We investigated presence of TrxL2. Notably, this oxidation response was con- whether (Grx) can promote TrxL2 reduction in the comitant with TrxL2 reduction. These results strongly suggest presence of GSH. Two types of chloroplast Grx, GrxC5 and that TrxL2 can receive reducing power directly from RCA. GrxS12, were prepared (25), both of which exhibited GSH- We performed similar experiments using other redox- dependent insulin reduction activity (SI Appendix, Fig. S7C). regulated proteins (FBPase and SBPase) as oxidation targets. However, neither GrxC5 nor GrxS12 facilitated TrxL2 reduction A whole set of the resulting SDS/PAGE profiles and the quan- significantly (SI Appendix, Fig. S7 A and B). We also addressed titative data on the redox changes are described in SI Appendix, NTRC, a notable redox-mediator protein that uses NADPH as a Fig. S8 and Fig. 3 B–D, respectively. RCA, FBPase, and SBPase source of reducing power (26). TrxL2.1, but not TrxL2.2, was were commonly oxidized in a TrxL2-dependent manner, al- able to be partially reduced in an NTRC-dependent manner (SI though their extents and kinetics were not uniform. For com- Appendix, Fig. S7 A and B). We further assessed the possibility parison, we investigated whether Trxs can oxidize these proteins. SI that FTR acts as the transmitter of reducing power to TrxL2 ( Trx-f oxidized each target, but its efficiency was lower than that Appendix, Fig. S7 D and E). As expected, FTR could reduce Trx- of TrxL2 in all cases (Fig. 3 B–D and SI Appendix, Fig. S8). SI f (SI Appendix, Fig. S7F) and other all subtypes of chloroplast Appendix, Fig. S9 shows the target reduction levels upon in- Trx (16). In contrast, FTR failed to reduce TrxL2 (SI Appendix, m x y z Fig. S7 D and E). Taken together, it was shown that TrxL2 has cubation with other Trx subtypes ( , , , and ) for 15 min. the potential to use reducing power from GSH and NADPH These Trxs were also less effective in oxidizing each of the tar- pools, but its efficiency is low. It is, therefore, considered that gets. All of these results let us conclude that TrxL2 is an efficient TrxL2 needs other sources of reducing power to achieve oxidation factor of several redox-regulated proteins. adequate activity. The TrxL2/2CP Redox Cascade Oxidizes Target Proteins and Drains TrxL2 Efficiently Oxidizes RCA, FBPase, and SBPase. A remarkably Reducing Power to H2O2. The estimated in vivo amounts of ± ± less negative redox potential (Fig. 1 and SI Appendix, Fig. S4) TrxL2.1 and TrxL2.2 were 1.4 0.1 and 2.4 0.8 nmol/g leaf and a physical interaction with several redox-regulated protein protein, respectively (SI Appendix, Fig. S10), which are roughly (SI Appendix, Fig. S5) led us to wonder whether TrxL2 may have 10-fold lower than that of FBPase (11). Furthermore, a variety of an ability to oxidize redox-regulated proteins; we next addressed redox-regulated proteins coexist in chloroplasts. It is thus un- this possibility. RCA was first tested as an example of oxidation likely that TrxL2 can function as the final acceptor of reducing targets. RCA was precedently reduced using DTT and Trx-f. power. Namely, TrxL2 must continue to drain reducing power to Thereafter, DTT and Trx-f were removed by gel filtration. RCA ensure an efficient oxidation reaction. Strong candidates for the in the highly reduced state was incubated with an equimolar acceptor are 2CP and its downstream molecule H2O2 (Fig. 2 and concentration of TrxL2 in the oxidized state (2 μM each), and SI Appendix, Fig. S6).

E8298 | www.pnas.org/cgi/doi/10.1073/pnas.1808284115 Yoshida et al. Downloaded by guest on September 28, 2021 On the basis of this hypothesis, we attempted to reconstitute a A redox cascade from redox-regulated proteins to H2O2 (SI Ap- Control+ TrxL2.1 + TrxL2.2 pendix, Fig. S11 for the raw data on the SDS/PAGE and im- munoblotting images, and Fig. 4 for the quantitative data on the redox changes). In these experiments, each redox-regulated 5 min 0 min 15 min 2 min 5 mn 15 min 2 min 15 min 75 protein (RCA, FBPase, or SBPase), TrxL2, and 2CPA were in- cubated at a molar ratio of 5:1:2 (in micromoles). An excess of 50 RCA Red RCA Ox H2O2 (100 μM) was added to the reaction. The protein bands of 37 RCA, FBPase, and SBPase overlapped with that of oxidized 2CPA in the SDS/PAGE, making it difficult to discriminate their redox 25 state precisely; therefore, they were detected by immunoblotting. 20 TrxL2.2 Red TrxL2.2 Ox RCA was largely maintained in the reduced form even in the TrxL2.1 Red presence of 2CPA and H2O2 (Fig. 4A, Left and SI Appendix, Fig. 15 TrxL2.1 Ox S11A), indicating that the redox state of RCA is hardly affected by 2CPA or H2O2 directly. Likewise, TrxL2 by itself could oxi- dize RCA only slightly, possibly due to the limited molar amount 10 of TrxL2 compared with that of RCA (one to five). However, (kDa) when 2CPA and H2O2 were incubated together, TrxL2 exerted a drastically elevated activity to oxidize RCA. Under these con- Control + Trx-f1 Trx-f1 ditions, TrxL2 was present in a nearly completely oxidized form + TrxL2.1 TrxL2.1 B + TrxL2.2 TrxL2.2 (Fig. 4A, Right and SI Appendix, Fig. S11A). These results suggest that 2CPA and H2O2 robustly withdrew reducing power from RCA TrxL2, conferring strong oxidative force on TrxL2. Taken to- gether, we have attained an in vitro reconstitution of the TrxL2/ 100 100 2CP redox cascade for oxidizing RCA and draining reducing 80 80 power to H2O2. The TrxL2/2CP redox cascade was capable of

60 60 oxidizing FBPase and SBPase as well, albeit to a lesser extent (Fig. 4 B and C and SI Appendix, Fig. S11 B and C). 40 40

20 20 The TrxL2/2CP Redox Cascade Functions in Vivo. We have shown the biochemical basis for oxidative thiol modulation in chloroplasts.

RCA reduction level (%) 0 0

1 or TrxL2 reduction level (%) Another important issue is the physiological relevance of this 0 5 10 15 f 0 5 10 15 system; we addressed this using Arabidopsis mutant plants. We

Time (min) Trx- Time (min) C obtained T-DNA insertion mutants in the 2CPA and 2CPB genes FBPase (2cpa and 2cpb, respectively) and their double mutant (2cpa 2cpb). These plants were cultivated under long-day conditions 100 100 (16-h day/8-h night) (SI Appendix, Fig. S12A). RT-PCR analysis indicated that both of the 2CPA and 2CPB transcripts were 80 80 largely lowered to an almost undetectable level in the corre- 60 60 sponding mutants (SI Appendix, Fig. S12B). Immunoblotting – – 40 40 analysis indicated that 2CP protein was lowered to 25 30%, 50 55%, and 10–15% of the wild-type level in 2cpa, 2cpb, and 2cpa 20 20 2cpb, respectively (SI Appendix, Fig. S12C). By contrast, there 0 0 was no clear difference in the protein levels of Trxs, TrxL2, 1 or TrxL2 reduction level (%) FBPase reduction level (%) 0 5 10 15 f 051015 NTRC, and PrxQ between wild-type and mutant plants. The D Time (min) Trx- Time (min) aboveground biomass, chlorophyll content, and chlorophyll a/b SBPase ratio were also unchanged in all of the mutants (SI Appendix, Fig. S12 D and E). Some proteins involved in photosynthetic elec- tron transport, including the light-harvesting complex protein 100 100 LHCA1, PSII core D1 protein, and cytochrome b6, normally 80 80 accumulated in mutants as in the wild type (SI Appendix, Fig. 60 60 S12C). We further evaluated photosynthetic electron transport efficiency (SI Appendix, Fig. S13). The results suggested that all 40 40 mutants could perform photosynthetic electron transport com- 20 20 parable to that of wild type. From all of these data, we conclude that the lack of 2CP does not result in drastic phenotypic changes 0 0 1 or TrxL2 reduction level (%) SBPase reduction level (%) f in Arabidopsis, at least under the growth conditions tested in 0 5 10 15 051015 this study. Time (min) Trx- Time (min) Using these plants, we studied protein-oxidation dynamics PLANT BIOLOGY Fig. 3. Biochemical characterization of target-oxidizing activity of TrxL2. (A)An during light–dark transitions. Plants were dark adapted for 12 h, − − example of redox shift visualization. RCA (after reduction treatment; 2 μM) irradiated with high light (HL) (700 μmol photons m 2·s 1) for was incubated with TrxL2 (oxidized form; 2 μM) for the indicated time. Free 30 min, and then returned to the dark. The redox state of Trx- thiols were labeled with AMS, and proteins were subjected to nonreducing targeted proteins, Trxs, and TrxL2 was sequentially determined SDS/PAGE. (B–D) Comparison of target-oxidizing activity of Trx-f1andTrxL2. under this experimental scheme. Photosynthetic electron trans- Reduction level of RCA (B), FBPase (C), or SBPase (D) was calculated as the ratio of the reduced form to the total, and plotted against the reaction time. Data port parameters were almost identical among plants during the on the reduction level of Trx-f1 or TrxL2 are also shown. Raw images of the analysis (SI Appendix, Fig. S14). SDS/PAGE are shown in SI Appendix, Fig. S8. Each value represents the Fig. 5A illustrates the redox behavior of the ATP synthase mean ± SD (n = 3). Ox, oxidized form; Red, reduced form. CF1-γ subunit. CF1-γ was fully reduced upon HL and gradually

Yoshida et al. PNAS | vol. 115 | no. 35 | E8299 Downloaded by guest on September 28, 2021 + 2CPA / H2O2 our earlier observations (11, 13, 17), Trxs were partially reduced + TrxL2.1 TrxL2.1 upon HL. When plants were transferred back to the dark, the + TrxL2.2 TrxL2.2 redox state of Trxs rapidly returned to the original dark-adapted + TrxL2.1 / 2CPA / H2O2 TrxL2.1 (+ 2CPA / H2O2) + TrxL2.2 / 2CPA / H O TrxL2.2 (+ 2CPA / H O ) A 2 2 2 2 level. In particular, Trx-f1, Trx-m2, and Trx-y2 recovered their RCA redox states within 2 min of dark incubation. There was no clear change in the redox behaviors of Trxs between wild-type and 100 100 mutant plants. These results suggest that reducing power transfer via Trxs is dependent on the light-driven electron transport chain 80 80 and is unaffected by the presence or absence of 2CP. 60 60 Two TrxL2 isoforms showed different redox behaviors by HL/ dark treatments (Fig. 6 E and F). TrxL2.1 was entirely converted 40 40 from oxidized to reduced forms upon HL (Fig. 6E). Unlike Trxs, TrxL2.1 was maintained in a fully reduced state even 10 min 20 20 after the onset of the dark period. In the wild type, TrxL2.1 then 0 began to be reoxidized, reaching a completely oxidized state

RCA reduction level (%) 0 TrxL2 reduction level (%) 051015 0 5 10 15 after 30 min in the dark. TrxL2.1 reoxidation was slightly delayed Time (min) Time (min) in 2cpa and 2cpb single mutants. More remarkably, dark- B responsive TrxL2.1 reoxidation was nearly completely blocked FBPase in 2cpa 2cpb double mutant; namely, the lack of 2CP resulted in the accumulation of massive reducing power in TrxL2.1. These 100 100 results provide direct evidence of in vivo cross-talk between TrxL2.1 and 2CP. On the other hand, TrxL2.2 did not shift the 80 80 redox state during light–dark transitions (Fig. 6F). The protein 60 60 signal detected here corresponded to AMS-unlabeled TrxL2.2 (SI Appendix, Fig. S15), indicating that TrxL2.2 existed consti- 40 40 tutively in the disulfide bond-forming oxidized state irrespective 20 20 of light conditions. This result was common to wild-type and mutant plants. It is thus likely that TrxL2.2 was strongly oxidized

0 TrxL2 reduction level (%) 0 by as yet unidentified factors other than 2CP. FBPase reduction level (%) 051015 0 5 10 15 We finally characterized the redox change of 2CP itself in the Time (min) Time (min) wild-type plant (Fig. 6G). In the dark-adapted state, almost all C 2CP was present in the oxidized form. Approximately 20% SBPase of 2CP was converted to the reduced form upon HL. Notably, this 2CP redox state was maintained even after dark treatment 100 100 for 30 min, indicating that 2CP sustained to be supplied with reducing power. All of the data on protein redox dynamics are in 80 80 line with the idea that (i) redox-regulated proteins are oxidized 60 60 by TrxL2 and (ii) 2CP continuously transfers reducing power from TrxL2 to H2O2. We therefore conclude that the TrxL2/2CP 40 40 redox cascade is a functional system for oxidative thiol modu- 20 20 lation in vivo.

0 0 Discussion TrxL2 reduction level (%) SBPase reduction level (%) 051015 051015Thiol-based redox regulation allows chloroplast proteins to Time (min) Time (min) flexibly and appropriately tune their enzymatic functions in re- sponse to changes in light environments. The FTR/Trx redox Fig. 4. Reconstitution of the TrxL2/2CP redox cascade in vitro for oxidizing cascade is well recognized to support the light-dependent re- target protein and draining reducing power to H2O2. Reduction level of RCA ductive response of target proteins (1, 2). By contrast, the (A), FBPase (B), or SBPase (C) was calculated as the ratio of the reduced form mechanism underlying their reoxidation process, which is ob- to the total and plotted against the reaction time. Data on the reduction – level of TrxL2 are also shown. Raw images of the SDS/PAGE and immuno- served during light dark transitions, has been a matter of debate blotting are shown in SI Appendix, Fig. S11. Each value represents the for a long time. In this study, we clarified a redox cascade that mean ± SD (n = 3). enables oxidative thiol modulation in chloroplasts (Fig. 7).

The TrxL2/2CP Redox Cascade Can Oxidize Various Redox-Regulated reoxidized during the subsequent dark incubation. In the wild Proteins in Chloroplasts. In the course of biochemical character- type, CF1-γ was nearly completely reoxidized 30 min after the ization of Arabidopsis TrxL2, we noticed that this protein has the onset of the dark period. By contrast, approximately half of the potential as an oxidation factor for redox-regulated proteins. CF1-γ remained in the reduced form in 2cpa 2cpb at the same Namely, TrxL2 showed a quite less negative redox potential (Fig. time point. Other Trx-targeted proteins, including RCA, 1 and SI Appendix, Fig. S4) and a protein–protein interaction FBPase, and SBPase, also showed the dynamic redox shift from with various redox-regulated proteins via the mixed-disulfide reduced to oxidized forms during light–dark transitions (Fig. 5 bond (SI Appendix, Fig. S5). This potential activity of TrxL2 B–D). Similar to the case of CF1-γ, the reoxidation of these was experimentally demonstrated to be true; TrxL2 could oxidize proteins was partially impaired in mutant plants. The extent of RCA, FBPase, and SBPase efficiently (Fig. 3 and SI Appendix, such impairments was correlated to the accumulation level of Figs. S8 and S9). It should be mentioned that Trx-f (and some of 2CP (SI Appendix, Fig. S12C). These results clearly indicate that other Trx subtypes) was also able to oxidize these proteins to 2CP is a key factor in oxidative thiol modulation in chloroplasts. some extent (Fig. 3 and SI Appendix, Figs. S8 and S9). This is to Fig. 6 A–D shows the redox behavior of four Trx isoforms in be expected, given that Trx-f is a preferred partner for the redox chloroplasts (Trx-f1, Trx-m2, Trx-x, and Trx-y2). In accord with regulation of these three proteins (10, 11, 22) and the redox

E8300 | www.pnas.org/cgi/doi/10.1073/pnas.1808284115 Yoshida et al. Downloaded by guest on September 28, 2021 ABCD

CF1- RCA FBPase SBPase Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark- Dark- adapted Dark- adapted Dark- adapted 02510 15 30 adapted 02510 15 30 02510 15 30 02510 15 30 50 Red 50 Red 50 Red Red WT 37 WT Ox WT WT Ox R.I. Ox 37 Ox (kDa) (kDa) 37 (kDa) (kDa) Red Red Red Red 2cpa 2cpa Ox 2cpa 2cpa Ox R.I. Ox Ox

Red 2cpb Red 2cpb Ox 2cpb Red 2cpb Red Ox R.I. Ox Ox

Red 2cpa Red 2cpa Ox 2cpa Red 2cpa Red 2cpb Ox 2cpb R.I. 2cpb Ox 2cpb Ox

100 100 100 100 80 80 80 80 60 60 60 60 40 WT 40 40 40 2cpa 20 2cpb 20 20 20 2cpa 2cpb 0 0 0 Reduction level (%) Reduction level (%) Reduction level (%) 0 Reduction level (%) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30

Dark- Dark after HL (min) Dark- Dark after HL (min) Dark- Dark after HL (min) Dark- Dark after HL (min) adapted adapted adapted adapted

Fig. 5. Redox dynamics in vivo. Arabidopsis wild-type (WT) and mutant (2cpa, 2cpb, and 2cpa 2cpb) plants were dark adapted for 12 h, irradiated by HL −2 −1 (700 μmol photons m ·s ) for 30 min, and transferred back to the dark. During this period, the redox state of ATP synthase CF1-γ subunit (A), RCA (B), FBPase (C), and SBPase (D) was sequentially determined. Reduction level was calculated as the ratio of the reduced form to the total. Each value represents the mean ± SD from three biological replicates. Ox, oxidized form; Red, reduced form; R.I., redox-insensitive form of RCA.

potential of Trx-f is comparable to those of FBPase and SBPase How Does the TrxL2/2CP Redox Cascade Function in Vivo? It is of (27, 28). However, the molecular functions of TrxL2 are crucially great importance to understand the in vivo dynamics of oxidative different from those of Trxs in the following aspects: First, thiol modulation in chloroplasts. In living plants, the protein TrxL2 shows higher protein-oxidizing efficiency (Fig. 3 and SI redox state is determined by the balance of reduction and oxi- Appendix, Fig. S8); second, TrxL2 has little or no activity to re- dation rates. The TrxL2/2CP redox cascade certainly works even duce redox-regulated proteins (Fig. 2A); and third, TrxL2 fails to under light conditions, as evidenced by the fact that TrxL2.1 and receive reducing power from FTR (SI Appendix, Fig. S7 D–F). 2CP were reduced upon HL (Fig. 6 E and G). Nevertheless, redox-regulated proteins, including CF -γ, RCA, FBPase, and TrxL2 is thus a redox-mediator protein specialized for oxidizing 1 SBPase existed in almost fully reduced forms under identical redox-regulated proteins. conditions (Fig. 5). These results suggest that the protein- Another biochemical feature of TrxL2 is underlined by the reducing rate by the FTR/Trx pathway overwhelmed the oxi- high catalytic rate in transferring reducing power to 2CP (Fig. 2 dizing rate (Fig. 7A). When light is interrupted, the TrxL2/2CP E and D and SI Appendix, Fig. S6 A and B). Owing to its high redox cascade becomes dominant and thereby triggers the affinity to H2O2 (e.g., micromolar range of Km value) (23, 29), apparent oxidative thiol modulation (Fig. 7B); this scenario is 2CP can easily consume reducing power supplied by TrxL2, thus well supported by the dataset of redox dynamics in vivo (Figs. 5 allowing TrxL2 to continuously oxidize redox-regulated proteins. and 6). TrxL2 is widely conserved in land plants (SI Appendix, Indeed, the in vitro reconstitution experiment demonstrated that Fig. S1), but not in cyanobacteria and some algae. Therefore, the protein-oxidizing activity of TrxL2 is strongly dependent on the TrxL2/2CP pathway is thought to be an elaborate system 2CP and H2O2 (Fig. 4 and SI Appendix, Fig. S11). The involve- acquired during adaptation to terrestrial environments, allow- ment of 2CP in protein oxidation has already been proposed (30, ing chloroplasts to turn off the photosynthetic metabolism 31). According to these studies, the atypical Cys His-rich Trx at night. (ACHT) serves as the oxidative thiol modulator and the trans- A delayed response of protein reoxidation (Fig. 5) and a mitter of reducing power to 2CP. However, its oxidation target prolonged accumulation of reducing power in TrxL2.1 (Fig. 6E), reported so far is limited to AGPase (31). By contrast, TrxL2 was which were observed in 2CP-less mutants, underpin the signifi- shown to extensively oxidize RCA, FBPase, and SBPase (Fig. 4 cant impact of the TrxL2/2CP pathway on oxidative thiol mod- and SI Appendix, Fig. S11). Besides, NADP-MDH and AGPase ulation. However, the data on these mutants also provide implications of a more complicated protein-oxidizing network may be additional oxidation targets of TrxL2 (SI Appendix, Fig. in chloroplasts. Considering that protein reoxidation occurred S5), although they are still candidates for further investigation. PLANT BIOLOGY even in 2cpa 2cpb double mutants (Fig. 5), other redox- On the basis of these data, we can consider the TrxL2/2CP mediator proteins and Prxs may redundantly serve in the redox cascade to be a previously unreported system for oxi- protein-oxidizing pathway (Fig. 7B). According to the in vitro dizing a range of redox-regulated proteins in chloroplasts. study, Trxs possessed partial activity to oxidize some redox- Future studies are needed to understand the molecular regulated proteins (Fig. 3 and SI Appendix,Figs.S8andS9). mechanisms underlying the high protein-oxidizing ability of However, Trxs were immediately reoxidized in the dark (Fig. 6 TrxL2. In this regard, the structural information on a poplar A–D), indicating that they mainly function in transmitting the TrxL2 ortholog, which has been recently reported (32), is light-dependent reductive signal to redox-regulated proteins, helpful to attain this goal. as is classically accepted (1). Alternatively, it is highly possible

Yoshida et al. PNAS | vol. 115 | no. 35 | E8301 Downloaded by guest on September 28, 2021 ABCD Trx-f1 Trx-m2Trx-x Trx-y2

Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark- adapted Dark- Dark- adapted adapted Dark- adapted 02510 15 30 02510 15 30 02510 15 30 02510 15 30 Red2 Red2 WT 15 Red1/Ox2 WT 15 Red WT Red WT Red1/Ox2 Ox1 Ox Ox 10 Ox1 (kDa) (kDa) 10 (kDa) (kDa) Red2 Red2 2cpa Red1/Ox2 2cpa Red 2cpa Red 2cpa Red1/Ox2 Ox1 Ox Ox Ox1

Red2 Red2 2cpb Red1/Ox2 2cpb Red 2cpb Red 2cpb Red1/Ox2 Ox1 Ox Ox Ox1 2cpa Red2 2cpa 2cpa 2cpa Red2 Red1/Ox2 Red Red Red1/Ox2 2cpb Ox1 2cpb Ox 2cpb Ox 2cpb Ox1

50 80 40 100 100 60 30 20 80 80 40 index 10 index 20 Oxidation 0 60 60 Oxidation 0 50 40 40 40 40 30 30 20 20 20 20 index 10 index 10 Reduction 0 0 Reduction Reduction level (%) 0 Reduction level (%) 0 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark- Dark- Dark- Dark- adapted adapted adapted adapted EF G TrxL2.1 TrxL2.2 2CP

Dark after HL (min) Dark after HL (min) Dark after HL (min) Dark- Dark- adapted adapted 02510 15 30 02510 15 30 Dark- adapted 02510 15 30 * 20 50 WT 15 Red WT Ox Ox Ox (kDa) 15 37 (kDa) * WT Red Ox 2cpa 2cpa 25 Ox Red 20 2cpb * Red 2cpb Ox Ox 75 50 * 2cpa Red 2cpa Ox (kDa) 2cpb Ox 2cpb 100 100 100 80 80 80 60 60 60 40 40 40 20 0

20 20 Reduction level (%) 0 5 10 15 20 25 30 0 0 Reduction level (%) Reduction level (%) 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Dark- Dark after HL (min) adapted Dark after HL (min) Dark- Dark- Dark after HL (min) adapted adapted

Fig. 6. Redox dynamics in vivo. Arabidopsis wild-type (WT) and mutant (2cpa, 2cpb, and 2cpa 2cpb) plants were dark adapted for 12 h, irradiated by HL (700 − − μmol photons m 2·s 1) for 30 min, and transferred back to the dark. During this period, the redox state of Trx-f1(A), Trx-m2(B), Trx-x (C), Trx-y2(D), TrxL2.1 (E), TrxL2.2 (F), and 2CP (G) was sequentially determined. (A and D) Oxidation index was calculated as the ratio of the Ox1 form to the total. Reduction index was calculated as the ratio of the Red2 form to the total. (B, C, E, and G) Reduction level was calculated as the ratio of the reduced form to the total. (E) Asterisk indicates a possible nonspecific band. (G) As a loading control, Rubisco large subunit was stained with CBB. Each value represents the mean ± SD from three biological replicates. Ox, oxidized form; Red, reduced form.

that some Trx-like proteins with high redox potentials (such as Materials and Methods ACHT) (33) participate in the protein-oxidizing network. Preparation of Expression Plasmids. Total RNA was isolated from Arabidopsis Given that TrxL2.2 was stably present in the oxidized form thaliana as described previously (36) and used as a template for RT-PCR. irrespective of light conditions or plant genotypes (Fig. 6F), Gene fragments encoding the predicted mature protein region of TrxL2.1 77 210 56 192 some unidentified factors are also likely to reside in TrxL2.2 (At5g06690.1; Met -Val ), TrxL2.2 (At5g04260; Ala -Thr ), RCA (At2g39730.1; Ala59-Phe474), GrxC5 (At4g28730; Ser64-Ser174), and GrxS12 downstream and constitutively oxidize this protein. Furthermore, 69 206 NTRC may indirectly affect the protein-oxidizing rate by modu- (At2g20270; Ser -Thr ) were cloned into the pET-23c expression vector (Novagen). Plasmids for TrxL2.1 and TrxL2.2 were designed to express N- lating the redox state of the 2CP pool (34, 35). Our data dem- terminal His-tagged fusion proteins. The primers and restriction enzymes onstrate that the dark side of chloroplast redox regulation is used for expression plasmid construction are listed in SI Appendix, Table S1. supported by the TrxL2/2CP redox cascade, but also open the The sequences of expression plasmids were confirmed to be correct by DNA door for exploring its overall regulatory network and functional sequencing (3730xl DNA Analyzer; Applied Biosystems). Other expression coordination. plasmids were prepared in our earlier studies (11, 16, 17).

E8302 | www.pnas.org/cgi/doi/10.1073/pnas.1808284115 Yoshida et al. Downloaded by guest on September 28, 2021 A Light-side reaction Screening for TrxL2-Interacting Proteins. The method of Trx affinity chro- H H matography (18) was applied to screen for TrxL2-interacting proteins. Light S S TrxL2 monocysteinic mutants (TrxL2.1CS and TrxL2.2CS) were used as baits. Chloroplast soluble proteins were obtained by collecting intact chloroplasts FTR/Trx Red Target from spinach leaves and disrupting these by osmotic stress. The subsequent pathway procedures were similar to those described previously (37).

SS Reducing Power Transfer Reactions. Proteins were reacted under the indicated ETC · Ox Target conditions in medium containing 50 mM Tris HCl (pH 7.5) and 50 mM NaCl at 25 °C. All reactions were conducted under aerobic conditions. The redox state of proteins was determined as described above. For protein-oxidizing B Dark-side reaction experiments, target proteins (RCA, FBPase, or SBPase; 50 μM each) were incubated with 25 μM Trx-f1 and 5 mM DTT for 30 min before the assay. The reduced target proteins were then collected by gel filtration using a H H GSH NADPH S S Superdex 200 10/300 GL column (GE Healthcare). NTRC Red Target H2O2 H2O2-Detoxifying Activity Measurement. H2O2-detoxifying activity was mea- sured according to ref. 38. with modifications. For monitoring 2CP activity, TrxL2 2CP 2CPA (2 μM) was incubated with 0.5 mM DTT, 1 μM TrxL2, and 0.1 mM H O SS 2 2 in a medium containing 50 mM Tris·HCl (pH 7.5) and 50 mM NaCl for the (Trx) (Other Prxs) 2H O Ox Target 2 indicated time at 25 °C. The concentration of H2O2 was then determined by (ACHT) the ferrous oxidation of xylenol orange assay.

Fig. 7. Simplified working model of chloroplast redox regulation. (A) Estimation of TrxL2 Amount in Vivo. The in vivo amount of TrxL2 was esti- “ ” Classically recognized light-side reaction. In response to light-dependent mated by an immunoblotting-based method, as previously described (11). excitation of electron transport chain (ETC), redox-regulated proteins are Briefly, the regression line of signal intensity was generated on a dilution “ ” reduced via the FTR/Trx pathway. (B) Newly emerging dark-side reaction. series of the recombinant protein, which was used as a reference for cal- Redox-regulated proteins are oxidized via the TrxL2/2CP pathway. Several culating the amount in Arabidopsis leaf extracts. other factors may also be involved in this process directly or indirectly. See the main text for details. Ox, oxidized form; Red, reduced form. Plant Materials. A. thaliana wild-type plant (Col-0) and homozygous T-DNA insertion mutants in 2CPA (At3g11630) and 2CPB (At5g06290) genes (2cpa, 133 113 Salk_118663C; 2cpb, Salk_017213C) were used in this study. The 2cpa 2cpb Point mutations in TrxL2CS (Cys to Ser in TrxL2.1; Cys to Ser in TrxL2.2) were introduced using the PrimeSTAR Mutagenesis Basal Kit (Takara) double mutant was obtained by crossing each single mutant and screening according to the manufacturer’s instructions. The primers used for site- from the F2 generation. Genotyping was performed by genomic PCR using the primers listed in SI Appendix, Table S1. Plants were grown in soil in a directed mutagenesis are listed in SI Appendix, Table S1. controlled growth chamber (60 μmol photons m−2·s−1, 22 °C, 16-h day/8-h night) for 4 wk. Protein Expression and Purification. Each expression plasmid was transformed into Escherichia coli strain BL21 (DE3) (for TrxL2.1, TrxL2.2, GrxC5, and Isolation and Subfractionation of Arabidopsis Chloroplasts. For the isolation of GrxS12) or Rosetta (DE3) pLysS (for RCA). Transformed cells were cultured at intact chloroplasts, Arabidopsis wild-type plants were dark incubated over- 37 °C. The expression was induced by the addition of 0.5 mM isopropyl-1- night to promote starch breakdown. Approximately 25 g of leaves were thio-β-D-galactopyranoside followed by overnight culture at 21 °C. Cells were homogenized in a blender with 600 mL of grinding solution containing disrupted by sonication. After centrifugation (125,000 × g for 40 min), the 50 mM Hepes-NaOH (pH 7.6), 330 mM sorbitol, 2 mM EDTA, 1 mM MnCl , resulting supernatant was used to purify the protein. His-tagged TrxL2.1 and 2 1 mM MgCl , 5 mM sodium ascorbate, and 0.05% (wt/vol) BSA. The ho- TrxL2.2 proteins were purified using a Ni-nitrilotriacetic acid affinity column 2 mogenate was filtered by four layers of gauze and centrifuged at 500 × g for as described previously (13). RCA, GrxC5, and GrxS12 proteins were purified 6 min. The resulting precipitates were suspended in wash solution contain- by a combination of anion-exchange chromatography, hydrophobic-interaction ing 50 mM Hepes-NaOH (pH 7.6), 330 mM sorbitol, 2 mM EDTA, 1 mM chromatography, and size-exclusion chromatography, as described previously MnCl2, and 1 mM MgCl2. Intact chloroplasts were isolated by centrifugation (16). Other recombinant proteins were prepared as described previously (11, with a discontinuous gradient of 40% (vol/vol) and 70% (vol/vol) Percoll in 16, 17). The protein concentration was determined with a BCA protein wash solution at 3,200 × g for 20 min. Intact chloroplasts were then washed assay (Pierce). twice by centrifugation with wash solution at 1,300 × g for 2 min. Intact chloroplasts were osmotically ruptured in solution containing 20 mM Tricine- Discrimination of Protein Redox State Using Thiol-Modifying Reagents. The NaOH (pH 8.0), 5 mM MgCl2, and the cOmplete, EDTA-free protease in- redox state of proteins was determined by discriminating the thiol status with hibitor mixture (Roche). Ruptured chloroplasts were centrifuged at 20,000 × the use of thiol-modifying reagents AMS or N-ethylmaleimide. AMS has a g for 20 min. The resulting supernatants and precipitates were collected as molecular mass of 536.44 and thereby lowers protein mobility on SDS/PAGE, stroma and thylakoid membrane fractions, respectively. All procedures were allowing the determination of protein redox state from an observable band conducted at 4 °C. shift. After the in vitro reaction, proteins were precipitated with 10% (wt/vol) trichloroacetic acid and then washed with ice-cold acetone. Precipitated RT-PCR Analysis. Total RNA was extracted from Arabidopsis wild-type and proteins were labeled with the thiol-modifying reagents described in each mutant plants as described above. The transcript abundance of 2CPA and figure. Proteins were subjected to nonreducing SDS/PAGE and stained with 2CPB genes was analyzed using primer pairs specific to each gene (SI Ap- Coomassie Brilliant Blue R-250 (CBB) unless otherwise specified. pendix, Table S1). Actin 2 (ACT2; At3g18780) was used as the control gene. PLANT BIOLOGY Insulin Reduction Activity Measurement. Insulin reduction activity was mea- Immunoblotting Analysis. Total leaf protein was extracted as described pre- sured as described previously (11). For TrxL2.1 and TrxL2.2, 0.5 mM DTT was viously (39). Proteins were separated by SDS/PAGE and transferred to a PVDF added as the reductant. For GrxC5 and GrxS12, 2 mM GSH was added as membrane. Antibodies against TrxL2.1 and TrxL2.2 were prepared using the reductant. each recombinant protein as the antigen. Other antibodies used in this study were prepared previously (11, 13, 17) or commercially available (Agrisera).

Em Determination. The Em of TrxL2.1 and TrxL2.2 was determined under the same conditions as those of our previous assays (11, 16, 17). The ratio of Chlorophyll Content and a/b Ratio. Chlorophyll content and a/b ratio were

reduced DTT (DTTred) to oxidized DTT (DTTox) used for the titration ranged determined after extraction with 80% (vol/vol) acetone according to the from 1 × 10−5 to 5 × 10−2. Assays were performed at pH 7.5. method described in ref. 40.

Yoshida et al. PNAS | vol. 115 | no. 35 | E8303 Downloaded by guest on September 28, 2021 Photosynthetic Electron Transport Measurement. Chlorophyll fluorescence and teins was determined by a method based on thiol modification with AMS and absorbance change at 830 nm were measured simultaneously using a Dual- immunoblotting, as described previously (13). PAM-100 (Walz) with the intact leaves. A saturating pulse of red light −2 −1 (800 ms, >5,000 μmol photons m ·s , 635 nm) was applied to calculate ACKNOWLEDGMENTS. We thank Karl-Josef Dietz, Takushi Hachiya, Kei several parameters. The quantum yields of PSII [Y (II), Y (NO), and Y (NPQ)] Odawara, and Eriko Uchikoshi for discussions; the Biomaterials Analysis and PSI [Y (I), Y (ND), and Y (NA)] were calculated as described in ref. 41. Division, Tokyo Institute of Technology for supporting DNA sequencing Relative electron transport rates of PSII (ETR II) and PSI (ETR I) were calcu- analysis; and the Suzukakedai Materials Analysis Division, Tokyo Institute lated as Y(II) × light intensity and Y (I) × light intensity, respectively. of Technology for supporting mass spectrometry analysis. This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Determination of Protein Redox State in Vivo. Plants were directly frozen using Grants 26840090 (to K.Y.) and 16H06556 (to T.H.) and by Dynamic Alliance liquid nitrogen under the indicated conditions, and the redox state of pro- for Open Innovation Bridging Human, Environment and Materials.

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