Cyclic electron transport around photosystem I contributes to photosynthetic induction with Thioredoxin f

In response to light, plants efficiently induce photosynthesis. Light activation of thiol 37 enzymes by the thioredoxin (Trx) systems and cyclic electron transport by the PROTON 38 GRADIENT REGULATION 5 (PGR5)-dependent pathway contribute substantially to 39 regulation of photosynthesis. Arabidopsis thaliana mutants lacking f -type Trxs ( trx f1f2 ) 40 show delayed activation of carbon assimilation due to impaired photoreduction of 41 Calvin-Benson cycle enzymes. To further study regulatory mechanisms that contribute to 42 efficiency during the induction of photosynthesis, we analyzed the contributions of PSI 43 donor- and acceptor-side regulation in the trx f1f2 mutant background. The cytochrome 44 b 6 f complex is involved in PSI donor-side regulation, whereas PGR5-dependent PSI 45 cyclic electron transport is required for both donor and acceptor functions. Introduction of 46 the pgr1 mutation, which is conditionally defective in cytochrome b 6 f complex activity, 47 into the trx f1f2 mutant background did not further affect the induction of photosynthesis, 48 but the combined deficiency of Trx f and PGR5 severely impaired photosynthesis and 49 suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 mutant, the 50 acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI 51 hardly increased during the induction of photosynthesis. We also compared the 52 photoreduction of thiol enzymes between the trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 53 mutation did not result in further impaired photoreduction of Calvin-Benson cycle 54 enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that 55 acceptor-side limitations in the pgr5 trx f1f2 mutant suppress photosynthesis initiation, 56 suggesting that PGR5 is required for efficient photosynthesis induction. ECS ST. is the light-dark difference in the ECS the magnitude of the pmf formed in the light. ECS ST signal was produced by a single turnover light pulse using dark-adapted leaves. B, Proton conductivity of the ( g H+ ). Measurements were performed under the induction of

(www.plantphysiol.org) are: Yuki Okegawa (okegawa@cc.kyoto-su.ac.jp) and Ken 24 Motohashi (motohas@cc.kyoto-su.ac.jp). suppressed plant growth under long-day conditions. In the pgr5 trx f1f2 mutant, the 50 acceptor-side of PSI was almost completely reduced, and quantum yields of PSII and PSI 51 hardly increased during the induction of photosynthesis. We also compared the 52 photoreduction of thiol enzymes between the trx f1f2 and pgr5 trxf1f2 mutants. The pgr5 53 mutation did not result in further impaired photoreduction of Calvin-Benson cycle 54 enzymes or ATP synthase in the trx f1f2 mutant background. These results indicated that 55 Introduction 59 Photosynthesis consists of a series of electron transport reactions in the thylakoid 60 membrane and carbon fixation reactions in the stroma. In the thylakoid reactions, 61 electrons excised from water in Photosystem II (PSII) are transferred to NADP + through 62 the cytochrome b 6 f complex and Photosystem I (PSI), resulting in the production of 63 NADPH. Electron transport is coupled with the translocation of protons across the 64 thylakoid membrane from the stroma to the lumen. The resulting proton motive force 65 (pmf) is utilized in ATP synthesis. NADPH and ATP are used to fix inorganic carbon in 66 the Calvin-Benson cycle. In addition to this linear electron transport from water to 67 NADP + , PSI cyclic electron transport contributes to the supply of ATP for carbon 68 fixation. PSI cyclic electron transport consists of two partially redundant pathways, 69 namely the PROTON GRADIENT REGULATION 5 (PGR5)-dependent and NADH 70 dehydrogenase-like (NDH) complex-dependent pathways (Munekage et al., 2002(Munekage et al., , 200471 DalCorso et al., 2008). In Arabidopsis thaliana, the PGR5-dependent pathway is the main 72 route for PSI cyclic electron transport and contributes to the generation of pmf across the 73 thylakoid membrane and the resulting ATP synthesis (Munekage et al., 2002;DalCorso 74 et al., 2008;Wang et al., 2015). 75 To optimize photosynthetic reactions, chloroplasts have various regulatory 76 mechanisms (Tikhonov, 2015). The downregulation of the cytochrome b 6 f complex, 77 termed photosynthetic control, is a fundamental mechanism involved in the regulation of 78 photosynthesis (Tikhonov, 2013). To avoid acceptor-side limitation of PSI, electron 79 transport via the cytochrome b 6 f complex is slowed through acidification of the thylakoid 80 lumen (Stiehl and Witt, 1969). The Arabidopsis pgr1 mutant, which has an amino acid 81 alteration in the Rieske subunit of the cytochrome b 6 f complex, has a decreased electron 82 essential for plant growth and photoprotection, as its deficiency causes growth defects 107 (Wang et al., 2013;Okegawa and Motohashi, 2015). Trx f is another major Trx; it 108 accounts for approximately 22% of all Trx proteins in the stroma. Initial biochemical 109 analyses in vitro have shown that Trx f plays a central role in the redox regulation of 110 enzymes in the Calvin-Benson cycle, such as fructose-1,6-bisphosphatase (FBPase) and 111 sedoheptulose-1,7-bisphosphatase (SBPase) (Schurmann and Buchanan, 2008;112 Geigenberger and Fernie, 2014;Yoshida et al., 2015). However, an Arabidopsis Trx 113 f-deficient mutant (termed trx f1f2), which lacks both Trx f1 and Trx f2, does not show 114 any growth differences compared with the wild type under long-day conditions (Yoshida 115 et al., 2015;Naranjo et al., 2016). In contrast, Naranjo et al. (2016) reported that the trx 116 f1f2 mutant does display growth inhibition under short-day conditions. They suggested 117 that Trx f is dispensable for plant growth but is required for the efficient induction of 118 photosynthesis. 119 Here, we characterized the trx f1f2 double mutant, the pgr1 trx f1f2 triple mutant (in 120 which the cytochrome b 6 f complex is hypersensitive to luminal acidification), and the 121 pgr5 trx f1f2 triple mutant (in which PGR5-dependent PSI cyclic electron transport is also 122 disturbed). In the trx f1f2 mutant, a delay in the activation of Calvin-Benson cycle 123 enzymes during the induction of photosynthesis caused low activity of ATP synthase. 124 Furthermore, the pgr5 trx f1f2 triple mutant exhibited severe growth defects, suggesting 125 that PGR5-dependent PSI cyclic electron transport is indispensable in the trx f1f2 mutant 126 background. We propose that PGR5-dependent PSI cyclic electron transport also 127 contributes to efficient photosynthetic induction. 128

Results 129
The pgr5 trx f1f2 triple mutant exhibited severe growth defects under long-day 130 conditions 131 To examine the effect of the pgr1 and pgr5 mutations on photosynthesis in the trx f1f2 132 mutant background, the triple mutants pgr1 trx f1f2 and pgr5 trx f1f2 were generated by 133 crossing. Whereas the pgr1 trx f1f2 plants were indistinguishable from the wild-type 134 plants under long-day conditions, growth was severely affected in the pgr5 trx f1f2 135 mutant ( Figure 1A and Supplemental Figure S1A). There was no difference in the fresh 136 weight of 3-week-old plants among the wild-type, pgr1, pgr5, trx f1f2, and pgr1 trx f1f2 137 plants ( Figure 1B). In contrast, the fresh weight of the pgr5 trx f1f2 plants was less than 138 half of that of the wild-type plants ( Figure 1B). Furthermore, the chlorophyll content in 139 the pgr5 trx f1f2 leaves was approximately 58% of that in the wild-type leaves ( Figure  140 1C). These results indicated that the combination of the pgr5 and trx f1f2 mutations led to 141 severe growth defects. Interestingly, the growth retardation in the pgr5 trx f1f2 plants was 142 less evident when they were grown under continuous light conditions (Supplemental 143 Figure S1B). The fresh weight of the pgr5 trx f1f2 plants was approximately 74% of that 144 of the wild-type plants (Supplemental Figure S1C), and its chlorophyll content was 145 almost the same as that of the pgr5 mutant (Supplemental Figure S1D). Since Trx f was 146 proposed to be a requirement for the effective induction of photosynthesis (Naranjo et al., 147 2016), continuous light conditions may be better for the pgr5 trx f1f2 plants. 148 To examine the influence of both the pgr5 and trx f1f2 mutations on the stability of 149 photosynthesis-related proteins, western blot analysis was performed ( Figures 1D and E). 150 As reported previously (Yoshida et al., 2015), in the trx f1f2 mutant, the protein levels of 151 other Trx isoforms did not change ( Figure 1D). In the pgr5 trxf1f2 mutant, the 152 accumulation of Calvin-Benson cycle enzymes and subunits of the photosynthetic 153 complexes were comparable to that in the wild type ( Figures 1D and E). 154

155
The quantum yield of PSII was severely impaired in the pgr5 trx f1f2 mutant 156 To characterize photosynthetic activity in the mutants, their chlorophyll fluorescence 157 parameters were analyzed using a Mini PAM II fluorometer. The maximum quantum 158 yield of PSII (Fv/Fm) was lower in the pgr5 trx f1f2 mutant than in the other genotypes 159 ( Figure 2A). To assess the functionality of PSII in the pgr5 trx f1f2 mutant under growth 160 light conditions, qPd was measured according to Ruban and Murchie (2012). qPd 161 represents the redox state of the Q A site of PSII in the dark and is used to monitor the level 162 of photoinhibition caused by both donor-and acceptor-side limitations of PSII (Wilson 163 and Ruban, 2019). The qPd level less than 0.98 indicates plants to be photoinhibited 164 (Ruban and Murchie, 2012). In the pgr5 trx f1f2 mutant, qPd was lower than 0.98 after 165 actinic light (AL) illumination (0.855 ± 0.061; Figure 2B), indicating that PSII of the 166 pgr5 trx f1f2 mutant was photoinhibited even under constant low-light conditions, 167 although the accumulation of PSII subunits was not affected ( Figure 1E). The 168 light-intensity dependence of the effective quantum yield of PSII [Y(II)] and 169 non-photochemical quenching chlorophyll fluorescence (NPQ) were also measured. As 170 reported previously (Munekage et al., 2001;Munekage et al., 2002), Y(II) was lower in 171 both the pgr1 and pgr5 mutants than in the wild type at high light intensities ( Figure 2C). 172 The trx f1f2 mutant showed substantially decreased Y(II) at low light intensities but a 173 similar Y(II) level to that in the wild type at light intensities higher than 100 µmol 174 photons m −2 s −1 ( Figure 2C). Moreover, a similar trend was also observed in the pgr1 175 trxf1f2 mutant; here, Y(II) was lower than that in the pgr1 mutant at low light intensities, 176 whereas it was identical to that in the pgr1 mutant at light intensities higher than 200 177 µmol photons m −2 s −1 . In contrast, Y(II) remained lower in the pgr5 trx f1f2 mutant under 178 all light intensities, compared to that in the pgr5 and trxf1f2 mutants ( Figure 2C). 179 The NPQ level mainly reflects the size of thermal dissipation in plants. The 180 ∆pH-dependent component of NPQ (qE) was induced in the wild type at light intensities 181 higher than 50 µmol photons m −2 s −1 ( Figure 2D). In the pgr1 and pgr5 single mutants, the 182 decreased ΔpH caused a low NPQ level. In contrast, the trx f1f2 mutant showed higher 183 NPQ than the wild type. In the pgr1 trx f1f2 mutant, the NPQ was slightly higher at low 184 light intensities than that in the pgr1 mutant ( Figure 2D). Unexpectedly, the pgr5 trx f1f2 185 mutant induced a higher NPQ, especially at low light intensities, compared to that in the 186 wild type, and the level was almost identical to that in the trx f1f2 mutant ( Figure 2D). 187 In the analysis of light-intensity dependence, the AL intensity was increased in a 188 step-wise manner at every 2 min after applying a saturating pulse (SP). Since Trx f has 189 been suggested to function in the activation of photosynthesis (Naranjo et al., 2016), 190 photosynthesis may not have been activated in the trx f1f2 mutant background at low light 191 intensities. To evaluate this possibility, Y(II) and NPQ were assessed during the 192 induction of photosynthesis at a low light intensity of 75 µmol photons m −2 s −1 (Figures 193 2E and F). Y(II) reached the steady-state level within 5 min after the onset of AL in the 194 wild type. In the trx f1f2 mutant, however, it took more than 10 min for Y(II) to reach a 195 steady-state level, but the final value was almost identical to that in the wild type ( Figure  196 2E). The pgr5 trx f1f2 mutant showed markedly lower Y(II) than the pgr5 mutant and the 197 Y(II) did not increase at all even 20 min after the onset of AL ( Figure 2E). This result was 198 consistent with the growth defect of the pgr5 trx f1 f2 mutant ( Figures 1A and B). 199 Consistent with the result of the light intensity-dependence analysis ( Figure 2D), the 200 pgr5 trxf1f2 mutant induced a higher NPQ than the wild type ( Figure 2F). The qE 201 component of NPQ is characterized by its relatively fast relaxation kinetics on a 202 physiological time scale of seconds to several minutes (Horton et al., 1996). The majority 203 of NPQ induced in the pgr5 trx f1f2 mutant was relaxed within several minutes in the dark 204 ( Figure 2F). These results indicated that NPQ in the pgr5 trx f1f2 mutant was largely 205 dependent on the qE component, suggesting that the restoration of NPQ in the pgr5 trx 206 f1f2 mutant is attributed to a concomitant restoration of ΔpH. 207 We also measured linear electron transport to NADP + in ruptured chloroplasts 208 ( Figure 2G). Fd and NADP + were added exogenously as electron acceptors to ruptured 209 chloroplasts. The pgr1 mutant showed lower Y(II) than the wild type at a light intensity of 210 167 µmol photons m −2 s −1 owing to its hypersensitivity to low luminal pH (Munekage et 211 al., 2001;Jahns et al., 2002), whereas the pgr1 trx f1f2 mutant had the same Y(II) as the 212 pgr1 mutant ( Figure 2G). In contrast, there was no difference in the Y(II) values among 213 the wild type and pgr5, trx f1f2, and pgr5 trx f1f2 mutants ( Figure 2G). These results 214 indicated that the PSII and PSI activities were not affected in the trx f1f2 mutant 215 background, suggesting that the markedly decreased Y(II) in the pgr5 trx f1f2 mutant was 216 caused by acceptor-side, but not donor-side, limitations of PSI. 217

218
The acceptor-side of PSI was highly reduced in the pgr5 trx f1f2 mutant even at low 219 light intensity 220 The pgr5 trxf1f2 mutant was suggested to be limited on the acceptor-side of PSI. 221 Therefore, we next simultaneously measured the chlorophyll fluorescence and absorption 222 changes in P700 using a Dual-PAM-100 system ( Figure 3). Plants were dark-adapted for 223 30 min and then illuminated with AL (75 µmol photons m −2 s −1 ) for 5 min. The Y(I) 224 parameter is defined by the fraction of P700 that is reduced and not limited by the 225 acceptor side (Klughammer and Schreiber, 2008) and is often used to estimate the 226 effective quantum yield of PSI. In the wild type, Y(I) and Y(II) rapidly increased after a 227 shift from dark to light ( Figures 3A and B). As reported previously (Naranjo et al., 2016), 228 in the trx f1f2 mutant, the initial increases in Y(I) and Y(II) were markedly delayed 229 compared with those in the wild type, and high NPQ was maintained over time (Figures 230 3A,B,and E). This was accompanied by a delay in the relaxation of the acceptor-side 231 limitation of PSI monitored based on Y(NA) ( Figure 3C). Most likely, this phenotype 232 was due to delayed activation of Calvin-Benson cycle enzymes followed by a shortage of 233 electron acceptors in the trx f1f2 mutant. Y(ND), the PSI donor-side limitation in electron 234 transport, is used to estimate the operation of photosynthetic control. In the pgr1 mutant, 235 the transient peak of Y(ND) formed within 60 s of AL onset was higher than that in the 236 wild type owing to enhanced photosynthetic control ( Figure 3D). In contrast, the trx f1f2 237 mutant did not form this peak; instead, a gradual rise in Y(ND) was observed, peaking at 238 3 min after the onset of AL. This suggested that the thylakoid lumen became acidic during 239 this period. The introduction of the pgr1 mutation into the trx f1f2 mutant background did 240 not substantially affect its PSII or PSI photochemistry, though NPQ was partially induced 241 even in the pgr1 mutant background ( Figure 3E). PGR5-dependent PSI cyclic electron 242 transport is required to protect the stroma from overreduction (Munekage et al., 2002;243 DalCorso et al., 2008). In the pgr5 mutant, the P700 + level was drastically reduced at high 244 light intensities (Supplemental Figure S2). Even under the low light intensity used in this 245 study, the relaxation of Y(NA) was markedly delayed in the pgr5 mutant ( Figure 3C). 246 The increase in Y(I) was also delayed, but it reached the wild-type level within 3 min of 247 AL onset ( Figure 3A). In contrast, the increases in Y(I) and Y(II) were severely 248 suppressed in the pgr5 trx f1f2 mutant during the induction of photosynthesis (Figures 3A  249 and B). The high level of Y(NA) was not relaxed at all during the 5 min of illumination 250 ( Figure 3C). Consequently, Y(ND) was close to zero, indicating that the acceptor-side of 251 PSI was largely reduced in the pgr5 trx f1f2 mutant, even under low light conditions. 252 These results indicated that a combination of pgr5 and trx f1f2 mutations synergistically 253 disturbed the initiation of photosynthesis. 254 The high level of Y(NA) in the pgr5 trx f1f2 mutant was not relaxed within 5 min of 255 AL onset ( Figure 3C). To determine whether this acceptor-side limitation could be 256 observed during steady-state photosynthesis, PSI and PSII photosynthetic parameters 257 were measured without dark adaptation (Supplemental Figure S3). In contrast to that in 258 the induction phase of photosynthesis, no difference in the photosynthetic parameters was 259 observed between the wild type and trx f1f2 mutant (  The trx f1f2 mutant exhibited the induction of higher NPQ than the wild type ( Figure 2D  271 and Figure 3E). Furthermore, NPQ was also higher in the pgr1 trxf1f2 and pgr5 trx f1f2 272 mutants than in the pgr1 and pgr5 single mutants, respectively ( Figure 2D and Figure  273 3E). To investigate the reason for this increase in NPQ in the trx f1f2 mutant background, 274 the electrochromic shift (ECS) was analyzed using a Dual PAM system. The ECS signal 275 represents an absorbance change at 515 nm owing to photosynthetic pigments, which is 276 affected by the electric field formed across the thylakoid membrane. ECS t is the 277 light-dark difference in the ECS signal, and represents the magnitude of the pmf formed 278 in the light. ECS t was standardized against ECS ST , which is the ECS signal induced by a 279 single turnover light pulse using dark-adapted leaves. The g H + parameter is determined by 280 monitoring the decay kinetics of the ECS signal in the dark and is considered to mainly 281 represent the proton conductivity of ATP synthase (Takizawa et al., 2008), although the 282 careful inspection is needed for the mutants including pgr1 and pgr5 (Yamamoto and 283 Shikanai, 2020). During the induction of photosynthesis, in the wild type, high-level pmf 284 was transiently formed, decreasing to the steady-state level within 3 min of AL onset 285 ( Figure 4A). Conversely, g H + increased during the induction of photosynthesis, mainly 286 reflecting the activation of chloroplast ATP synthase ( Figure 4B). This process 287 corresponded with the induction and relaxation of NPQ ( Figure 3E). The increase in g H + 288 was suppressed in the trx f1f2 mutant compared to that in the wild type, resulting in 289 delayed relaxation of the transiently induced pmf ( Figures 4A and B). The high-NPQ 290 phenotype observed in the trx f1f2 mutant is probably explained by the suppression of 291 ATP synthase activity. In the pgr1 mutant background, the trx f1f2 defects also enhanced 292 pmf and lowered g H + ( Figures 4A and B), consequently inducing higher NPQ than that in 293 the pgr1 mutant ( Figure 3E). In contrast, the pgr5 mutation further decreased the g H + level 294 in the trx f1f2 mutant background during the period 150-270 s after the onset of AL 295 ( Figure 4B). The pgr5 trx f1f2 triple mutant did not exhibit the induction of transient NPQ 296 ( Figure 3E), and the level of pmf after 270 s of AL onset was similar to the wild-type level 297 and lower than the level observed in the trx f1f2 mutant ( Figure 4A). This is probably 298 because of the very low level of linear electron transport ( Figures 3A and B). Despite the 299 constantly low pmf level, the pgr5 trx f1f2 mutant induced a moderate NPQ level ( Figure  300 3E). A similar trend was also observed when the light-intensity dependence of these 301 parameters was analyzed (Supplemental Figure S4). The pgr5 trx f1f2 mutant showed low 302 pmf similar to that in the pgr5 mutant but induced a higher NPQ than the pgr5 mutant 303 (Supplemental Figure S2E). 304 305

Photoactivation of FBPase and SBPase was delayed in the trx f1f2 mutant 306
In the trx f1f2 mutant, the g H + was lower than that in the wild type ( Figure 4B). ATP 307 synthase in chloroplasts is light-activated via reduction of the CF 1 -ɤ subunit (Nalin and 308 Mccarty, 1984). Trx f contributes to the light-dependent reduction of thiol enzymes, 309 including ATP synthase and Calvin-Benson cycle enzymes (Schwarz et al., 1997). The 310 lower g H + might have reflected the impaired light-dependent reduction of ATP synthase. 311 To investigate this possibility, we examined the redox state of several thiol enzymes 312 during the induction of photosynthesis and under constant low light conditions (80 µmol 313 photons m −2 s −1 ). The thiol enzyme levels in the trx f1f2 mutant background were not 314 affected ( Figures 1D and E). The light-induced state changes in the CF 1 -ɤ subunit were 315 determined by labeling the free thiols with the thiol-reactive 316 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) reagent. In the wild type, 317 CF 1 -ɤ was rapidly reduced upon illumination from zero to the steady-state level over 300 318 s ( Figure 5A). This occurred in all the genotypes ( Figure 5A), suggesting that the lower 319 level of g H + in the trx f1f2 mutant background was not caused by the suppressed activation 320 of ATP synthase due to the impaired reduction of CF 1 -ɤ. As reported previously (Naranjo 321 et al., 2016), however, the light-dependent reduction of FBPase was delayed and the final 322 reduction level was lower in the trx f1f2 mutant background ( Figure 5B). In the wild type, 323 FBPase was gradually reduced within 30 s of the onset of illumination, whereas 300 s was 324 required to start reducing FBPase in the trx f1f2 mutant background ( Figure 5B). SBPase 325 also needs to be photoreduced to be active. Compared with that of FBPase, the reduction 326 of SBPase was slower even in the wild type ( Figure 5C). As observed for FBPase, the 327 reduction of SBPase was delayed in the trx f1f2 mutant background, but the final 328 reduction level was almost identical to that in the wild type ( Figure 5C Figure S4). 370 These results suggest that NDH activity is not affected in the trx f1f2 mutant background 371 and that the phenotype of the pgr5 trx f1f2 mutant is not due to a lack of NDH 372 complex-dependent PSI cyclic electron transport, unlike that with the crr2-2 pgr5 mutant. 373 A study on the high cyclic electron flow 1 (hcef1) mutant, defective in FBPase, 374 showed that the loss of FBPase activity leads to enhancement of NDH 375 complex-dependent PSI cyclic electron transport (Livingston et al., 2010). In the hcef1 376 mutant, the levels of NDH subunits were enhanced and NDH activity was stimulated. 377 Although FBPase was less active in the trx f1f2 mutant than in the wild type ( Figure 5B), 378 the level of the NDH subunit (PnsB1) did not change ( Figure 1E). These results suggest 379 the distinctly different responses of the NDH complex to the hcef1 and trx f1f2 mutations. 380 Since the protein level of FBPase did not decrease in the trx f1f2 mutant ( Figure 1D), 381 other Trx systems could partially compensate in terms of the activation of FBPase. In 382 fact, in the trx f1f2 mutant, the photoreduction rate of FBPase reached approximately 40% 383 of that in the wild type during steady-state photosynthesis ( Figure 5B). Trx m has also 384 been suggested to contribute to the activation of FBPase in vivo (Okegawa and 385 Motohashi, 2015). 386 Unexpectedly, the pgr5 trxf1f2 mutant induced a higher NPQ than the pgr5 mutant 387 ( Figures 2D, F and Figure 3E). The trx f1f2 mutant also exhibited higher NPQ than the 388 wild type, especially during the induction of photosynthesis ( Figure 2F and Figure 3E). In 389 the trx f1f2 mutant, the activation of Calvin-Benson cycle enzymes was delayed during 390 the induction of photosynthesis, though ATP synthase was activated at the same time as 391 that in the wild type (  Figure S4). However, the pgr5 trx f1f2 mutant had a lower pmf level 399 than the wild type, which was similar to that in the pgr5 mutant, (Figure 4 and 400 Supplemental Figure S4), probably due to markedly decreased electron transport activity 401 ( Figures 3A and B). It is still unclear why the pgr5 trx f1f2 mutant induced a higher NPQ 402 than the pgr5 mutant despite the low pmf. To explain this NPQ phenotype, we might have 403 to consider the larger contribution of ∆pH to pmf. In the pgr5 mutant, the contribution of 404 ∆pH to pmf was reported to be larger than that in the wild type (Shikanai and Yamamoto,  Figures S1B-E). In the pgr5 trx f1f2 mutant, the thiol enzymes were 432 activated during steady state photosynthesis, although the reduction level of FBPase was 433 as low as that in the trx f1f2 mutant ( Figure 5). Therefore, continuous light would be 434 suitable for the growth of the pgr5 trx f1f2 plants. However, since PGR5-dependent PSI 435 cyclic electron transport is more important to protect PSI under high light conditions 436 (Munekage et al., 2002), the pgr5 trx f1f2 mutant may show more severe growth defects 437 when grown at higher light intensities, even under continuous light conditions. 438 The ntrc trx f1 double mutant also exhibits severe growth defects (Thormahlen et al., 439 2015;Nikkanen et al., 2016). In this mutant, the light activation of FBPase and 440 ADP-glucose pyrophosphorylase was almost completely suppressed and the 441 NADPH/NADP + ratio was increased (Thormahlen et al., 2015). NTRC has been 442 suggested to contribute to photosynthetic metabolism, especially under low light 443 conditions (Carrillo et al., 2016). As NTRC uses NADPH as an electron donor, NTRC 444 deficiency may enhance the reduction state of the stroma in the trx f1 mutant background. 445 In fact, photoinhibition of PSI was observed in the ntrc trxf1 mutant (Thormahlen et al., 446 2015). Furthermore, compared to that in the wild type, the ntrc trx f1 mutant showed 447 increased activation of NADP-malate dehydrogenase (MDH), which reflects the stromal 448 redox state (Foyer et al., 1992). This indicated that the acceptor-side of PSI was limited in 449 the ntrc trx f1 mutant. Together with the results in the pgr5 trx f1f2 mutant, these results 450 suggest that the acceptor-side limitation of PSI leads to the impaired activation of 451 photosynthesis, resulting in plant growth defects. In the pgr5 single mutant, the induction 452 of photosynthesis was only delayed compared to that in the wild type, at least under the 453 low light conditions used in this study. However, in the trx f1f2 mutant background, the 454 pgr5 mutation markedly suppressed photosynthetic activity. We propose that 455 PGR5-dependent PSI cyclic electron transport is required to induce photosynthesis 456 effectively by preventing overreduction of the stroma. Since PSI cyclic electron transport 457 has been proposed to be regulated by the availability of electron acceptors from PSI 458 (Breyton et al., 2006;Okegawa et al., 2008)

Plant materials and growth conditions 464
Arabidopsis thaliana ecotype Columbia 0 was used as the wild type. Leaves (30 mg fresh weight) were harvested from 3-week-old seedlings grown on MS 478 plates and immediately powdered by grinding in liquid nitrogen. Chlorophyll was 479 extracted in 80% acetone (v/v) and collected by centrifugation at 15,000 × g for 5 min at 480 4°C. The residue was re-extracted with 80% acetone and centrifuged once again (15,000 481 × g, 5 min, 4°C). The chlorophyll content was determined via spectrophotometry, as 482 described previously (Porra et al., 1989). 483 484

In vitro assay of linear electron transport activity 510
Measurement of linear electron transport activity was performed using isolated 511 chloroplasts, as described previously (Munekage et al., 2002). Intact chloroplasts (20 µg 512 ml -1 ) were osmotically ruptured in 50 mM HEPES/NaOH, pH 7.6, containing 7 mM 513 The redox change of P700 was assessed by monitoring the absorbance changes to 544 transmitted light at 830 and 875 nm. Pm (the level of the P700 signal of maximum 545 oxidizable P700) was determined by the application of an SP in the presence of far-red 546 light (720 nm). The maximal level of oxidized P700 during AL illumination (Pm') was 547 determined by SP application. The P700 signal P was recorded immediately before an SP. 548 Y(I) was calculated as (Pm' − P) / Pm. Y(NA) was calculated as (Pm − Pm') / Pm. Y(ND) 549 was calculated as P / Pm. Three complementary quantum yields were defined as follows: 550 Y(I) + Y(NA) + Y(ND) = 1 (Klughammer and Schreiber, 1994). The relative level of 551 reduced P700 was calculated as 1 − Y(ND). The value can vary between 0 (P700 fully 552 oxidized) and 1 (P700 fully reduced) in a given state. 553

ECS analysis 555
The ECS measurements were carried out using the Walz Dual-PAM 100 equipped with a 556 P515/535 module. Each measurement was carried out in ambient air, using 4-5-week-old 557 plants grown under long-day conditions that had been dark-adapted for 30 min. It 558 consisted of 5 min of red AL at 75 μmol photons m −2 s −1 ; a 1 s dark pulse at each different 559 time point was used to record ECS t . This represented the size of the light-induced pmf and 560 was estimated from the total amplitude of the rapid decay of the ECS signal during the 561 dark pulse, as described previously (Wang et al., 2015). The ECS t levels were normalized 562 against a 515-nm absorbance change induced by a single turnover flash (ECS ST ), as 563 measured in dark-adapted leaves before recording. This normalization allowed us to 564 consider possible changes in leaf thickness and chloroplast density between leaves 565 (Takizawa et al., 2008). 566 567

In vivo photoreduction of thiol enzymes 568
Photoreduction of Trx target enzymes in seedlings was determined using the free 569 thiol-specific-modifying reagent, AMS (Thermo Fisher Scientific, USA) as described 570 previously (Okegawa and Motohashi, 2015). Seedlings were dark-adapted for 8 h and 571 exposed to light (80 μmol photons m −2 s −1 ) for up to 60 min. Samples were collected at 572 the indicated time points and detected by western blot analysis. The reduction level of the 573 proteins was quantified using Multi Gauge 3.1 software (Fujifilm, Japan) and presented 574 as the ratio of reduced protein to total protein. 575 576

Statistical analysis 577
Calculations were performed on more than three independent biological replicates (see 578 figure legends). Tukey multiple comparison test was used to determine significant 579 differences among the materials tested (P<0.05). 580 581 www.plantphysiol.org on September 18, 2020 -Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

Accession Numbers 582
Sequence data from this article can be found in the Arabidopsis Genome Initiative or 583 GenBank/EMBL databases under the following accession numbers: Trx f1 (At3g02730), 584 Trx f2 (At5g16400), PGR1 (At4g03280), and PGR5 (At2g05620). 585 586 SUPPLEMENTAL DATA 587 Supplemental Figure S1. Visible phenotypes of the wild type (WT) and pgr1, pgr5, trx 588 f1f2, pgr1 trx f1f2, and pgr5 trx f1f2 mutants. 589 Supplemental Figure S2. Light intensity dependence of PSI and PSII photosynthetic 590 parameters in the wild type (WT) and pgr1, pgr5, trx f1f2, pgr1 trx f1f2, and pgr5 trx f1f2 591 mutants. 592 Supplemental Figure S3. Simultaneous analysis of PSI and PSII photosynthetic 593 parameters during steady-state photosynthesis. 594 SBPase (C) were detected by western blot analysis. The reduction levels of thiol enzymes 663 were indicated as a percentage of the total protein that was reduced. Each value represents 664 the mean ± SD of three independent replicates. Red, reduced; Ox, oxidized. 665 666 A B C D E Figure 1. Visible phenotypes of the wild type (WT) and pgr1, pgr5, trx f1f2, pgr1 trx f1f2, and pgr5 trx f1f2 mutants grown for 3 weeks under long-day conditions. A, Photographs of the plants.
Bars indicate 10 mm. B, Fresh weights of seedlings. Each value is shown as the mean ± standard deviation (SD) of 10 independent replicates. Columns with the same letters are not significantly different between genotypes (Tukey-Kramer test, P < 0.05). C, Chlorophyll content of seedlings, per unit fresh weight. Each value is the mean ± SD of three independent replicates. Columns with the same letters are not significantly different between genotypes (Tukey-Kramer test, P < 0.05). D and E, Western blot analysis. Chloroplasts were fractionated into the stromal fractions (D) and thylakoid membranes (E). For the wild type, dilution series of proteins corresponding to 1.0 (100%), 0.5, 0.25, and 0.125 µg chlorophyll were loaded. Other mutants contained proteins corresponding to 1.0 µg chlorophyll in each lane. A CF 1 -ɤ B FBPase C SBPase Figure 5. Photoreduction of thiol enzymes in the wild type (WT) and pgr1, pgr5, trx f1f2, pgr1 trx f1f2, and pgr5 trx f1f2 mutants. Seedlings were illuminated at a light intensity of 80 µmol photons m −2 s −1 (µE) after a dark period of 8 h and collected at the indicated time points. The extracted proteins were modified with the thiol-reactive 4-acetamido-4'-maleimidylstilbene-2,2'disulfonic acid (AMS) and subjected to non-reducing SDS-PAGE. The redox states of ATP synthase CF 1 -γ (A), FBPase (B), and SBPase (C) were detected by western blot analysis. The reduction levels of thiol enzymes were indicated as a percentage of the total protein that was reduced. Each value represents the mean ± SD of three independent replicates. Red, reduced; Ox, oxidized.