Dysfunction of Chloroplast Protease Activity Mitigates pgr5 Phenotype in the Green Algae Chlamydomonas reinhardtii

Researchers have described protection mechanisms against the photoinhibition of photosystems under strong-light stress. Cyclic Electron Flow (CEF) mitigates electron acceptor-side limitation, and thus contributes to Photosystem I (PSI) protection. Chloroplast protease removes damaged protein to assist with protein turn over, which contributes to the quality control of Photosystem II (PSII). The PGR5 protein is involved in PGR5-dependent CEF. The FTSH protein is a chloroplast protease which effectively degrades the damaged PSII reaction center subunit, D1 protein. To investigate how the PSI photoinhibition phenotype in pgr5 would be affected by adding the ftsh mutation, we generated double-mutant pgr5ftsh via crossing, and its phenotype was characterized in the green algae Chlamydomonas reinhardtii. The cells underwent high-light incubation as well as low-light incubation after high-light incubation. The time course of Fv/Fm values in pgr5ftsh showed the same phenotype with ftsh1-1. The amplitude of light-induced P700 photo-oxidation absorbance change was measured. The amplitude was maintained at a low value in the control and pgr5ftsh during high-light incubation, but was continuously decreased in pgr5. During the low-light incubation after high-light incubation, amplitude was more rapidly recovered in pgr5ftsh than pgr5. We concluded that the PSI photoinhibition by the pgr5 mutation is mitigated by an additional ftsh1-1 mutation, in which plastoquinone pool would be less reduced due to damaged PSII accumulation.


Introduction
Under light, P680 in Photosystem II (PSII) and P700 in Photosystem I (PSI) are oxidized and a photosynthesis reaction takes place.PSII oxidizes water molecules in the Mn 4 CaO 5 cluster, and the electrons from the water molecules are transferred via electron transfer pathway [1].Most of the cofactors are ligated in D1/D2 heterodimer, and reduce plastoquinone to generate plastoquinol in the thylakoid membrane.The Cytochrome b 6 f complex (b 6 f ) transfers electrons from plastoquinol to plastocyanin, coupled with proton translocation from the stroma to the lumen.The b 6 f reduces plastoquinone, consuming the stromal proton, and oxidizes plastoquinol, releasing the proton to the lumen [2].The oxidized P700 (P700 + ) receives an electron from plastocyanin, and the electron transfer reaction within PSI eventually reduces ferredoxin (Fd) in the stroma.The Ferredoxin NADP(+) reductase receives an electron from Fd and synthesizes NADPH.This electron transfer coupled with proton translocation forms a proton gradient (∆pH) across the thylakoid membrane that is essential for ATP synthesis by chloroplast ATP synthase [3].∆pH also induces photoprotection mechanisms such as non-photochemical chlorophyll fluorescence quenching (NPQ) [4].Throughout this overall electron transfer reaction, linear electron flow (LEF) will generate oxygen, ATP, and NADPH.
Under strong light, both photosystems are potentially photoinhibited and need to be protected [5].Reactive oxygen species (ROS) are generated under strong-light conditions Plants 2024, 13, 606 3 of 13

Growth Check
We crossed pgr5 (mt + ) and ftsh1-1 (mt − ) to generate the pgr5ftsh double mutant (Figure S1).All experiments included the pgr5pgrl1 double mutant to consider the situation for abolishing CEF.To evaluate the qualitative cellular growth, we performed a spot test under continuous light (Figure 1).The cell suspensions were spotted on TAP or TP medium and exposed to different light intensities (0 to 200 µmol photons•m −2 •s −1 ), with the LED having a 620 nm primary peak and a 453 nm secondary peak (Figure S2).The control and the PGR5-complemented strain (pgr5 + PGR5) grew normally, indicating that the light source set up did not inhibit C. reinhardtii cellular growth.A previous report [32] showed that pgr5 can grow photoautotrophically in up to 200 µmol photons•m −2 •s −1 of continuous light, while its growth is significantly lower under fluctuating light conditions.However, we found that the photoautotrophic growth of pgr5 (TP medium in Figure 1) was severely impaired even under 50 µmol photons•m −2 •s −1 with our light source.These results indicate that our light source more effectively inhibited photosynthesis activity in pgr5 than that in the previous report without fluctuating conditions.
Plants 2024, 13, x FOR PEER REVIEW 3 of

Growth Check
We crossed pgr5 (mt + ) and ftsh1-1 (mt − ) to generate the pgr5ftsh double mutant (Figu S1).All experiments included the pgr5pgrl1 double mutant to consider the situation f abolishing CEF.To evaluate the qualitative cellular growth, we performed a spot test u der continuous light (Figure 1).The cell suspensions were spotted on TAP or TP mediu and exposed to different light intensities (0 to 200 μmol photons•m −2 •s −1 ), with the LE having a 620 nm primary peak and a 453 nm secondary peak (Figure S2).The control an the PGR5-complemented strain (pgr5 + PGR5) grew normally, indicating that the lig source set up did not inhibit C. reinhardtii cellular growth.A previous report [32] show that pgr5 can grow photoautotrophically in up to 200 μmol photons•m −2 •s −1 of continuo light, while its growth is significantly lower under fluctuating light conditions.Howeve we found that the photoautotrophic growth of pgr5 (TP medium in Figure 1) was severe impaired even under 50 μmol photons•m −2 •s −1 with our light source.These results indica that our light source more effectively inhibited photosynthesis activity in pgr5 than that the previous report without fluctuating conditions.

Time Course of Fv/Fm
To roughly evaluate photoinhibition using this light source treatment, we monitor the time course of the Fv/Fm values after strong-light treatment.The Chlamydomon cells were grown under low light (10 μmol photons•m −2 •s −1 ), and then, exposed to hig light (250 μmol photons•m −2 •s −1 ) for 6 h, low light for 6 h, or high light for 2 h followi low light for 5 h (Figure 2).The Fv/Fm values were decreased in all strains after 2 h high-light incubation.Following an additional 4 h of high-light treatment, the Fv/Fm v ues remained 0.5 in the control, pgr5pgrl1, and pgr5-complemented strain (pgr5 + PGR However, the Fv/Fm values decreased in pgr5, ftsh1-1, and pgr5ftsh with this trend bein slightly milder in pgr5.To confirm the effect on photosystem protein accumulation durin

Time Course of Fv/Fm
To roughly evaluate photoinhibition using this light source treatment, we monitored the time course of the Fv/Fm values after strong-light treatment.The Chlamydomonas cells were grown under low light (10 µmol photons•m −2 •s −1 ), and then, exposed to high light (250 µmol photons•m −2 •s −1 ) for 6 h, low light for 6 h, or high light for 2 h following low light for 5 h (Figure 2).The Fv/Fm values were decreased in all strains after 2 h of high-light incubation.Following an additional 4 h of high-light treatment, the Fv/Fm values remained 0.5 in the control, pgr5pgrl1, and pgr5-complemented strain (pgr5 + PGR5).However, the Fv/Fm values decreased in pgr5, ftsh1-1, and pgr5ftsh with this trend being slightly milder in pgr5.To confirm the effect on photosystem protein accumulation during highlight treatment, we analyzed photosynthesis protein accumulation via immunoblotting Plants 2024, 13, 606 4 of 13 (Figure S3).By adding chloramphenicol, which blocks chloroplast translation, we evaluated the stability of the chloroplast-encoded protein (Figure S3 upper).We confirmed that the D1 protein level decreased in the presence of chloramphenicol during high-light exposure, and this effect became milder with an ftsh1-1 background, which is consistent with previous reports [12,13].However, we did not observe substantial changes in PSI reaction center protein accumulation.We observed an increment in the FTSH signal during high-light incubation, which is consistent with a previous report [35].These results confirmed the effect of the ftsh1-1 back-ground and that the accumulation levels of the PSI core protein was not affected under the high light condition.To investigate the recovery process after high-light treatment, we incubated the cells for up to 5 h under low light after 2 h of high-light treatment.The Fv/Fm values were slightly recovered in pgr5, pgr5pgrl1, ftsh1-1, and pgr5ftsh, while we confirmed complete recovery after 1 h of incubation in the control and pgr5 + PGR5.These results indicate that our high-light treatment was able to induce photoinhibition, and following low-light treatment was able to recover the photosystem.Of note, Fv/Fm was decreased after 2 h of high-light incubation and remained around 0.5 in pgr5pgrl1, and pgr5 followed this trend.This suggests complete CEF abolishment would potentially mitigate PSII photoinhibition.pgr5ftsh and ftsh1-1 showed continuous Fv/Fm value decrement during high light, indicating that PSII was already photoinhibited, and the additional pgr5 mutation did not rescue the phenotype for the Fv/Fm value in pgr5ftsh.
Plants 2024, 13, x FOR PEER REVIEW 4 of 13 high-light treatment, we analyzed photosynthesis protein accumulation via immunoblotting (Figure S3).By adding chloramphenicol, which blocks chloroplast translation, we evaluated the stability of the chloroplast-encoded protein (Figure S3 upper).We confirmed that the D1 protein level decreased in the presence of chloramphenicol during high-light exposure, and this effect became milder with an ftsh1-1 background, which is consistent with previous reports [12,13].However, we did not observe substantial changes in PSI reaction center protein accumulation.We observed an increment in the FTSH signal during high-light incubation, which is consistent with a previous report [35].These results confirmed the effect of the ftsh1-1 back-ground and that the accumulation levels of the PSI core protein was not affected under the high light condition.To investigate the recovery process after high-light treatment, we incubated the cells for up to 5 h under low light after 2 h of high-light treatment.The Fv/Fm values were slightly recovered in pgr5, pgr5pgrl1, ftsh1-1, and pgr5ftsh, while we confirmed complete recovery after 1 h of incubation in the control and pgr5 + PGR5.These results indicate that our high-light treatment was able to induce photoinhibition, and following low-light treatment was able to recover the photosystem.Of note, Fv/Fm was decreased after 2 h of high-light incubation and remained around 0.5 in pgr5pgrl1, and pgr5 followed this trend.This suggests complete CEF abolishment would potentially mitigate PSII photoinhibition.pgr5ftsh and ftsh1-1 showed continuous Fv/Fm value decrement during high light, indicating that PSII was already photoinhibited, and the additional pgr5 mutation did not rescue the phenotype for the Fv/Fm value in pgr5ftsh.Plants 2024, 13, 606 5 of 13

Time Course of Light-Induced Absorbance Changes at 705 nm
To evaluate the electron transfer activity of PSI, we measured light induced absorbance changes at 705 nm which correlate with P700 photooxidation [36][37][38].The absorbance at 705 nm decreased in response to continuous actinic light illumination due to the accumulation of P700 + .This absorbance decrement can be recovered through the cessation of actinic light illumination because of the reduction in P700 + resulting from electron transfer from plastocyanin, CEF, and back-reactions [38].Because far-red light illumination barely induces an absorbance changes at 705 nm in C. reinhardtii, we illuminated the cells with red LEDs (represented as an orange ring, in the Materials and Method Section with a single peak at 630 nm).However, to evaluate light-induced PSI photo-oxidation activity, as 630 nm excites both PSI and PSII, we added DCMU to block PSII's contribution, as well as DBMIB to eliminate the effect of electrons from the Cyt b 6 f complex on PSI [38].DCMU and DBMIB were added to the sample just before taking the measurement.We observed that the 705 nm absorbance changes almost reached a plateau after 5 s of illumination; thus, we roughly estimated the effect on light-induced P700 + accumulation based on the amplitude just before the cessation of actinic light.
We found that the amplitude of absorbance changes during actinic light illumination was decreased in all strains after 2 h of high-light (250 µmol photons•m −2 •s −1 ) treatment (Figure 3).Particularly, pgr5pgrl1 showed no absorbance changes at 705 nm, and this did not change after 4 h, indicating that our light source setup effectively induced PSI photoinhibition without fluctuation.The pgr5 strain also showed a strong reduction in the absorbance amplitude, which reduced further following 4 h of high-light treatment, indicating that it has a weaker phenotype compared with pgr5pgrl1.We barely observed any absorbance change at 705 nm after 6 h of high-light treatment.On the other hand, the control, pgr5 + PGR5, ftsh1-1, and pgr5ftsh maintained their absorbance amplitudes even after 4 and 6 h of high-light treatment.This means that PSI function can be maintained and/or resist further PSI photoinhibition in pgr5ftsh, unlike the pgr5 single mutant.
We also measured the light-induced absorbance changes in the cells during lowlight (10 µmol photons•m −2 •s −1 ) treatment after high-light (250 µmol photons•m −2 •s −1 ) treatment (Figure 4).We reproduced the decrease in the light-induced absorbance change amplitude after 2 h of high-light treatment in all strains, and a particularly strong reduction was observed in pgr5pgrl1 and pgr5.After 2 h of low-light treatment following 2 h of high-light treatment, the amplitude of the absorbance changes returned to the same level as before the high-light treatment in the control, pgr5+PGR5, and pgr5ftsh.We found that the amplitude was recovered, but the level was not the same as that before the high-light treatment in pgr5 and ftsh1-1.After 4 h of low-light treatment, the amplitudes of pgr5 and ftsh1-1 returned to the same levels as before the high-light treatment.These results indicate that the PSI photoinhibition phenotype of pgr5 was less observable in pgr5ftsh.However, the amplitude of pgr5pgrl1 did not recover at all, even after 4 h of low light-incubation.That indicates that, in the case of the complete abolishment of CEF, our low-light treatment was not able to recover its PSI photoinhibition, and the 2-h high-light treatment inflicted irreversible damage on PSI.We also measured the light-induced absorbance changes in the cells during low-light (10 μmol photons•m −2 •s −1 ) treatment after high-light (250 μmol photons•m −2 •s −1 ) treatment (Figure 4).We reproduced the decrease in the light-induced absorbance change amplitude after 2 h of high-light treatment in all strains, and a particularly strong reduction was observed in pgr5pgrl1 and pgr5.After 2 h of low-light treatment following 2 h of high-light treatment, the amplitude of the absorbance changes returned to the same level as before the high-light treatment in the control, pgr5+PGR5, and pgr5ftsh.We found that the ftsh1-1 returned to the same levels as before the high-light treatment.These results indicate that the PSI photoinhibition phenotype of pgr5 was less observable in pgr5ftsh.However, the amplitude of pgr5pgrl1 did not recover at all, even after 4 h of low light-incubation.That indicates that, in the case of the complete abolishment of CEF, our low-light treatment was not able to recover its PSI photoinhibition, and the 2-h high-light treatment inflicted irreversible damage on PSI.

Discussion
Under high-light treatment, in which CEF is activated, the electrons from PSI will be reinjected to the plastoquinone pool by CEF, and the electron-transfer rate within the b 6 f complex will be elevated through photosynthetic control.These mechanisms prevent an over-reduction in the plastoquinone pool under high light.An over-reduction in the PQ pool leads the formation of singlet oxygen, and then, plastoquinol scavenges singlet oxygen to protect D1 degradation [39][40][41].Other PQ derivatives also work as potential ROS scavengers [42,43].In the case of ftsh1-1, damaged PSII subunits, such as D1, were accumulated; additionally, we observed lower Fv/Fm during the high-light treatment, and the Fv/Fm-recovery rate became slower due to the lower rate of removal of damaged D1, which is consistent with a previous report (Figure 2) [12].PSII deactivation did not affect P700 photo-oxidation kinetics when we poised the sample with DCMU and DBMIB, which was confirmed by the result of the light-induced absorbance changes at 705 nm; this possibility was also suggested in a previous report (Figure 3) [38].The light-induced absorbance changes were not completely recovered after 2 h of low-light incubation in ftsh1-1, suggesting that damaged-PSII accumulation could also propagate PSI damage by reactive oxygen species (Figure 4).In the case of pgr5, PSI is photoinhibited, as confirmed in Figure 3, and the b 6 f complex electron-transfer rate is also inhibited according to Buchert, 2020 [33].In this situation, the plastoquinone pool would be relatively more reduced compared to ftsh1-1 but more oxidized compared to control; thus, the efficiency of ROS scavenging would be lower than in the control and less damaged-D1 accumulation would occur during high light.When we consider the low-light treatment after high-light treatment, the damaged-PSI-recovery rate is slower than in the control and is similar to that in ftsh1-1 (Figure 4); this result in similar to the Fv/Fm-recovery rate for pgr5 and ftsh1-1 during low-light incubation after high-light treatment.These consideration are consistent with the trend of Fv/Fm decrement during high-light treatment (pgr5 > ftsh1-1) and similar to the trend during low-light treatment after high-light treatment (Figure 2).
When we completely abolish CEF, such as in pgr5pgrl1, the plastoquinone pool is more oxidized than in pgr5, while the electron-transfer rate of the b 6 f complex is also slowed down.As shown in Figures 3 and 4, the light-induced absorbance change was completely lost after 2 h of high-light treatment and did not recover, even after 4 h of low-light treatment.Therefore, the plastoquinone redox state is more reduced compared to pgr5 after high-light treatment, which would enable it to more effectively scavenge single oxygen to protect D1 degradation.During low-light treatment after high light-treatment, electron transfer activity will not recover completely or will recover at the slowest rate because PSI activity is already impaired with high-light treatment.These considerations are consistent with our observation of Fv/Fm under high-light treatment and following low-light incubation, in which the reduced Fv/Fm value was maintained during highlight treatment and the slowest Fv/Fm-recovery rate occurred during low-light incubation (Figure 2).From another perspective, the complete abolishment of CEF would not generate less ∆pH to drive the synthesis of ATP molecules, therefore, its repair efficiency would be slowed down, resulting in the slowest Fv/Fm-recovery rate during low light after high-light treatment.
In the double-mutant pgr5ftsh, we observed that the time course of Fv/Fm during high light and during low light after high light exhibited the same trend as ftsh1-1, and the pgr5 phenotype was masked (Figure 2).This indicates the pgr5ftsh double mutant accumulates damaged PSII in the same way as the ftsh1-1 single mutant, as confirmed via immunoblotting (Figure S3).The light-induced P700 photo-oxidation kinetics also exhibited the same trend with ftsh1-1 during both high light and low light after high light (Figures 3 and 4).The pgr5 mutation decreases electron reinjection into the plastoquinone pool, as well as the b 6 f complex electron-transfer rate; thus, PSI photoinhibition should occur as observed in the single mutant (Figures 3 and 4).In the case of the pgr5ftsh double mutant, damaged-PSII accumulation would enable less electron transfer from PSII to the plastoquinone pool, which could lead to the plastoquinone pool exhibiting less over-reduction than pgr5 (because Plants 2024, 13, 606 9 of 13 pgr5 does not accumulate damaged PSII).Lower over-reduction of the plastoquinone pool will generate less singlet oxygen [39], which would cause less damage to PSI.In addition, the electron-transfer rate from plastoquinone pool to PSI would be slowed down due to lowered b 6 f electron-transfer rate in pgr5's genetic background.Therefore, mitigated PSI photoinhibition was observed (Figure 5).Another possibility is that FTSH is also involved in damaged-PSI subunit removal.In this scenario, oxidized PSI subunits (which might be enhanced by the pgr5 mutation) would be recognized by FTSH and removed.Unlike PSII, oxidized PSI subunits might be more stable because they have been constitutively detected in field-grown spinach.In addition, slower PSI-synthesis rate and the removal of PSI subunits would lead to irreversible/hard-to-recover PSI photoinhibition.However, intensive biochemical experiments are required to show that FTSH targets PSI as a substrate, which is impossible to confirm from the present data.The limitation of PSI photoinhibition in pgr5ftsh was found to be an experimentally observable phenotype within a period of several hours period.In fact, the cellular growth of pgr5ftsh was impaired in the photoautotrophic condition and strongly reduced in the photomixotrophic condition under the higher-light condition (more than 100 µmol photons•m −2 •s −1 ) (Figure 1).
Plants 2024, 13, x FOR PEER REVIEW 9 of 13 occur as observed in the single mutant (Figures 3 and 4).In the case of the pgr5ftsh double mutant, damaged-PSII accumulation would enable less electron transfer from PSII to the plastoquinone pool, which could lead to the plastoquinone pool exhibiting less over-reduction than pgr5 (because pgr5 does not accumulate damaged PSII).Lower over-reduction of the plastoquinone pool will generate less singlet oxygen [39], which would cause less damage to PSI.In addition, the electron-transfer rate from plastoquinone pool to PSI would be slowed down due to lowered b6f electron-transfer rate in pgr5's genetic background.Therefore, mitigated PSI photoinhibition was observed (Figure 5).Another possibility is that FTSH is also involved in damaged-PSI subunit removal.In this scenario, oxidized PSI subunits (which might be enhanced by the pgr5 mutation) would be recognized by FTSH and removed.Unlike PSII, oxidized PSI subunits might be more stable because they have been constitutively detected in field-grown spinach.In addition, slower PSI-synthesis rate and the removal of PSI subunits would lead to irreversible/hard-to-recover PSI photoinhibition.However, intensive biochemical experiments are required to show that FTSH targets PSI as a substrate, which is impossible to confirm from the present data.The limitation of PSI photoinhibition in pgr5ftsh was found to be an experimentally observable phenotype within a period of several hours period.In fact, the cellular growth of pgr5ftsh was impaired in the photoautotrophic condition and strongly reduced in the photomixotrophic condition under the higher-light condition (more than 100 μmol photons•m −2 •s −1 ) (Figure 1).

Strains, Growth, Crossing, Spot Test and Immunoblotting
The Chlamydomonas reinhardtii cells were grown in Tris-acetate phosphate (TAP), Trisphosphate (TP) medium, or minimum medium (MM) under low (10 µmol photons•m −2 •s −1 ) or high light intensity (250 µmol photons m −2 s −1 ).Please refer to the Section 4.2 for the equipment setup.The pgr5 (mt + ) [32] and ftsh1-1 (mt − ) strains [12] were crossed according to Harris [44].To determine ftsh1-1's genetic background, the 333 bp DNA fragment was prepared via PCR with L1 and R2 primers, including the fts1-1 mutation site which is not digested, using EagI resriction enzyme in ftsh1-1 (Figure S1a,b).The PCR product was digested with EagI and 207 bp and 126 bp fragments were generated from the wild-type sequence of FTSH1's genetic background, not from ftsh1-1's background (Figure S1c).We determined pgr5's genetic background in the photomixotrophic growth condition under ambient light supplemented with 20 µg paromomycin•mL −1 as well as genotyping with two independent PCRs using L2 + R2 and L3 + R3, of which the genomic DNA was deleted and replaced with an antibiotic resistance marker cassette in pgr5's background.Finally, we obtained the pgr5ftsh double mutant (Figure S1d).The pgr5-complemented strain, pgr5 + PGR5, was from Johnson et al. [32].The pgr5pgrl1 double mutant was from Steinbeck et al. [45].Cells grown in liquid TAP medium under low light were suspended in TP medium and incubated for 12 h under low light.To perform the high-light treatment, the cell concentration was adjusted with fresh TP medium at 20 µg chlorophyll•mL −1 and incubated for several hours with shaking under high light.To maintain uniform brightness on the surface of the cell culture, cell suspensions were placed in a low-form beaker, and the depth of the cell suspension in the beaker was no more than 5 mm.To perform spot test, the cell concentration was adjusted with fresh TP medium at 25 ng chlorophyll•µL −1 , and then, 4 µL of suspension was spotted on a TAP or TP medium plate.SDS-PAGE followed by immunoblotting for whole-cell polypeptides were performed according to a previous article [46].The signals from enhanced chemiluminescence were detected using ChemiDoc (BioRad, Hercules, CA, USA).

Light Source
For the measurement and spot test for the high-light treatment and the following low-light treatment, pink and/or yellow PLANTFLECs (NK system, Osaka, Japan) were used in the following combinations.For the high-light treatment and spot test, three pink PLANTFLECs and three yellow PLANTFLECs were placed alternately, and a 620 nm primary emission peak and a 453 nm secondary emission peak were prepared (Figure S2).The light intensities for the spot test were adjusted by changing the distance between the light source and plates or by applying several sheets of white papers.For the following low-light treatment, a yellow PLANTFLEC was used (according to the instrument manual, the yellow PLANTFLEC has a 600 nm primary emission peak and a 453 nm secondary emission peak).The emission spectrum of the light source was recorded with a light analyzer LA-105 (NK system, Japan).

Chlorophyll Fluorescence Measurement and Absorbance Changes in Cells at 705 nm
Cells were suspended with liquid TP medium at 20 µg chlorophyhll•mL −1 before measurement.Chlorophyll fluorescence measurement was performed with a DUAL-PAM-100 (Walz, Effeltrich, Germany).For the measurement of light-induced absorbance changes at 705 nm, the cell suspension was mixed with the same volume of TP medium containing 20% (w/v) Ficol, and DCMU (1 µM) and DBMIB (1 µM) were added just before measurement.The samples were placed for 30 s in the darkness in a sample cuvette holder and illuminated with an orange actinic light at an intensity of 82 µmol photons m −2 s −1 for 5 s, followed by darkness for 5 s.The absorbance changes were recorded with a JTS-10 (BioLogic, Seyssinet-Pariset, France) using the following sequence program: 3(10msD)5s2(10ms)10msI{200µs,35,5s,J200µsD15µsI}20µsJ200µsD{2ms,35,5s,D}50msD.

Figure 1 .
Figure 1.Spot test on solid medium.Corresponding 100 ng chlorophyll cell suspensions were sp ted on TAP or TP medium and incubated under different light intensities emitted from the lig source shown in Figure S2.A representative solid plate is shown here.Cells prepared from the thi biologically replicated batch were spotted on solid medium and incubated for 5 days at 25 °C und each light intensity.

Figure 1 .
Figure 1.Spot test on solid medium.Corresponding 100 ng chlorophyll cell suspensions were spotted on TAP or TP medium and incubated under different light intensities emitted from the light source shown in Figure S2.A representative solid plate is shown here.Cells prepared from the third biologically replicated batch were spotted on solid medium and incubated for 5 days at 25 • C under each light intensity.

Figure 2 .
Figure 2. Time course of Fv/Fm under light treatment.Cells were incubated under high light for 6 h (upper left) and low light for 6 h (upper right), and underwent 2 h of high-light treatment

Figure 2 .
Figure 2. Time course of Fv/Fm under light treatment.Cells were incubated under high light for 6 h (upper left) and low light for 6 h (upper right), and underwent 2 h of high-light treatment following 5 h of low-light treatment (lower left).The Fv/Fm value was evaluated every 2 h in the only high-light and only low-light treatment, and every hour in the high-light and treatment followed by recovery.The mean values with standard error bars from three biological replicates were plotted, and each point was connected with a straight line.The color codes are as follows: control (gray), pgr5 (magenta), pgr5pgrl1 (cyan), pgr5 + PGR5 (green), ftsh1-1 (yellow), and pgr5ftsh (red).

Figure 3 .
Figure 3. Light-induced absorbance changes at 705 nm from the cells incubated under high light.Cells were sampled from the cell culture incubated under high light every 2 h, and the light-induced absorbance changes at 705 nm were measured.The color codes of the traces are as follows: before high light (black); 2 h (magenta), 4 h (cyan), and 6 h (green) after high-light treatment.The strain names are shown at the bottom right in each panel.The cells were poised with 1 μM DCMU and 1 μM DBMIB just before measurement.The actinic light was switched on and off at points indicated by downward and upward arrows, respectively.The mean values with standard error bars from three biological replicates were plotted and connected with straight lines.

Figure 3 .
Figure 3. Light-induced absorbance changes at 705 nm from the cells incubated under high light.Cells were sampled from the cell culture incubated under high light every 2 h, and the light-induced absorbance changes at 705 nm were measured.The color codes of the traces are as follows: before high light (black); 2 h (magenta), 4 h (cyan), and 6 h (green) after high-light treatment.The strain names are shown at the bottom right in each panel.The cells were poised with 1 µM DCMU and 1 µM DBMIB just before measurement.The actinic light was switched on and off at points indicated by downward and upward arrows, respectively.The mean values with standard error bars from three biological replicates were plotted and connected with straight lines.

Figure 4 .
Figure 4. Light-induced absorbance changes at 705 nm from the cells incubated under high light and followed by low light.Cells was sampled from the cell culture incubated under high light followed by low light every 2 h, and the light-induced absorbance changes at 705 nm were measured.The color codes of the traces are as follows: before high-light treatment (black); 2 h after high light (magenta); and 2 h (cyan) and 4 h (green) after low-light treatment.The strain names are shown at the bottom right in each panel.The cells were poised with 1 μM DCMU and 1 μM DBMIB just before measurement.The actinic light was switched on and off at points indicated by downward and

Figure 4 .
Figure 4. Light-induced absorbance changes at 705 nm from the cells incubated under high light and followed by low light.Cells was sampled from the cell culture incubated under high light followed by low light every 2 h, and the light-induced absorbance changes at 705 nm were measured.The color codes of the traces are as follows: before high-light treatment (black); 2 h after high light (magenta); and 2 h (cyan) and 4 h (green) after low-light treatment.The strain names are shown at the bottom right in each panel.The cells were poised with 1 µM DCMU and 1 µM DBMIB just before measurement.The actinic light was switched on and off at points indicated by downward and upward arrows, respectively.The mean values with standard error bars from three biological replicates were plotted and connected with straight lines.

Figure 5 .
Figure 5. Possible hypothetical model for electron flow in pgr5ftsh double mutant.The pgr5 mutation blocks CEF, resulting in lower electron reinjection to the plastoquinone pool (PQ pool) and lowered b6f electron transfer rate.The ftsh mutation leads to the accumulation of damaged PSII and additional mutation to pgr5 mutation background results in relatively less overreduction of the PQ pool.This situation leads lower electron transfer from PSII to PQ pool.A mitigated PSI photoinhibition phenotype was observed in the pgr5ftsh mutant.

Figure 5 .
Figure 5. Possible hypothetical model for electron flow in pgr5ftsh double mutant.The pgr5 mutation blocks CEF, resulting in lower electron reinjection to the plastoquinone pool (PQ pool) and lowered b 6 f electron transfer rate.The ftsh mutation leads to the accumulation of damaged PSII and additional mutation to pgr5 mutation background results in relatively less overreduction of the PQ pool.This situation leads lower electron transfer from PSII to PQ pool.A mitigated PSI photoinhibition phenotype was observed in the pgr5ftsh mutant.