1. Volume 10, Issue 1, Pages 168-182 (January 2017)
Thioredoxins Play a Crucial Role in Dynamic Acclimation of Photosynthesis in Fluctuating Light 
Ina Thormählen, Arkadiusz Zupok, Josephin Rescher, Jochen Leger, Stefan Weissenberger, Julia Groysman, Anne Orwat, Gilles Chatel- Innocenti, Emmanuelle Issakidis-Bourguet, Ute Armbruster, Peter Geigenberger 
Molecular Plant 
Volume 10, Issue 1, Pages (January 2017)
DOI: /j.molp
Copyright © 2017 The Author Terms and Conditions

2. Figure 1
Light-Activation Kinetics of NADP-MDH are Strongly Restricted in trxm1m2 and ntrc Mutants.
Light-activation kinetics of NADP-MDH in response to a rapid dark–light transition in leaves of (A–C) trxm1.1, trxm2.1, trxm1.1 m2.1, and (D–F) trxf1, ntrc, trxf1 ntrc Arabidopsis mutants compared with Col-0 wild-type (WT). Dark-adapted plants at the end of the night were exposed to sudden illumination for different time intervals (0, 2, 5, 10, 20, and 30 min) with 160 μmol photons m−2 s−1 light intensity to analyze NADP-MDH activity in the rosettes using different assay conditions: Initial activity without DTT additions in the assay (A and D), maximal activity with 10 mM DTT included in the assay (B and E), and redox-activation state (initial activity/maximal activity × 100) (C and F). The light transition is indicated by black and white bars. Plants used in these experiments were grown for 4 weeks under short-day conditions (8 h/16 h day/night) with 160 μmol photons m−2 s−1 during the day. Results are the mean and the error bars represent SEM (n = 5 biological replicates for A–C and n = 9 biological replicates for D–F). Asterisks indicate the individual time points when the values of the mutants are significantly different to WT (trxm1.1, purple; trxm1.1 m2.1, blue; trxf1, orange; ntrc, red; trxf1 ntrc, yellow; *0.01 < P < 0.05, **0.005 < P < 0.01, ***0.001 < P < 0.005, ****P < 0.001, two-way ANOVA with Tukey post hoc test). Supplemental Table 1 shows significant differences from WT over all time points of the dark–light transition kinetics (main effect analysis).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

3. Figure 2
Adjustment of Chlorophyll Fluorescence Kinetics to Dark–Light Transitions is Slightly Affected in trxm1m2 but Strongly Impaired in ntrc Mutants.
Transient changes in chlorophyll fluorescence parameters in response to rapid dark–light transitions in leaves of (A and B) trxm1.1, trxm2.1, trxm1.1 m2.1 and (C and D) trxf1, ntrc, trxf1 ntrc Arabidopsis mutants compared with Col-0 wild-type (WT). (A and C) Non-photochemical quenching of light energy (NPQ) and (B and D) PSII quantum efficiency (ΦII) were monitored in a time-resolved manner in plants after 30 min dark adaptation starting with a saturating pulse of 2700 μmol photons m−2 s−1 for 0.8 s, followed by illumination with 145 μmol photons m−2 s−1 of actinic light for 15 min and a subsequent recovery phase of further 15 min in darkness (indicated by white and black bars, respectively). Plants used in these experiments were grown for 4 weeks under short-day conditions (8 h/16 h day/night) with 160 μmol photons m−2 s−1 during the day. Results are the mean and the error bars represent SEM (n = 7 biological replicates for A and B and n = 6 biological replicates for C and D). Asterisks indicate where the values of mutants are significantly different to WT (trxm1.1, purple; trxm1.1 m2.1, blue; trxf1, orange; ntrc, red; trxf1 ntrc, yellow; *0.01 < P < 0.05, ***0.001 < P < 0.005, ****P < 0.001, one-way ANOVA with Tukey post hoc test).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

4. Figure 3
Changes of the NADPH/NADP+ Ratio in Response to Dark–Light Transition in trxm1m2 and ntrc Mutants.
Transient kinetics in NADPH/NADP+ ratios in response to a rapid dark–light transition in leaves of (A–C) trxm1.1, trxm2.1, trxm1.1 m2.1 and (D–F) trxf1, ntrc, trxf1 ntrc Arabidopsis mutants compared with Col-0 wild-type (Col-0). Dark-adapted plants at the end of the night were exposed to sudden illumination for different time intervals (0, 2, 5, 10, 20, and 30 min) with 160 μmol photons m−2 s−1 light intensity to analyze the levels of NADPH (A and D), NADP+ (B and E), and NADPH/NADP+ ratios (C and F) in the rosettes. The light transition is indicated by black and white bars. Plants used in these experiments were grown for 4 weeks under short-day conditions (8 h/16 h day/night) with 160 μmol photons m−2 s−1 during the day. Results are the mean and the error bars represent SEM (n = 6 biological replicates). Asterisks indicate the individual time points when the values of mutants are significantly different to WT (ntrc, red; trxf1 ntrc, yellow; *0.01 < P < 0.05, ***0.001 < P < 0.005, ****P < 0.001, two-way ANOVA with Tukey post hoc test). Supplemental Table 1 shows significant differences from WT over all time points of the dark–light transition kinetics (main effect analysis).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

5. Figure 4
Acclimation and Growth of trxm1m2 and ntrc Mutants in Different Light Intensities and in Fluctuating Light.
Col-0 wild-type (Col-0), trxm1.1m2.1, trxm1.2m2.2, ntrc, Col gl1 wild-type (Col gl1), and pgr5 were grown in short days at medium light intensity (ML, 250 μmol photons m−2 s−1) for 18 days after germination (d.a.g.) and then kept at this light intensity or shifted to either LL (90 μmol photons m−2 s−1), HL (900 μmol photons m−2 s−1), or fluctuating LL/HL (FL; 4 min LL, 1 min HL, average light intensity ∼250 μmol photons m−2 s−1).
(A and B) Pictures of 5-week-old Col-0, trxm1.1m2.1, trxm1.2m2.2, and ntrc plants shifted from ML to FL after 18 d.a.g. (A) or grown at 250 μmol photons m−2 s−1 for the entire period (B).
(C) Pictures of Col gl1 and pgr5 as described in (A) and (B).
(D and E) Growth rates relative to the average of the respective wild-type represented as boxplots of trxm1.1m2.1, trxm1.2m2.2, ntrc (D) and pgr5 (E) mutants. The calculated growth rates with number of plants analyzed are depicted in Supplemental Figure 5.
(F–H) Fv/Fm of plants growing in ML (F) and after shift for 2 h, 2 days, 4 days, 6 days, 8 days, and 10 days to HL (G) or FL (H). Error bars represent SEM (n = 20 biological replicates per genotype and condition, except for Col gl1 and pgr5 with n = 12 biological replicates).
Asterisks indicate where the values of mutants are significantly smaller compared with the respective wild-type (trxm1m2 double mutants, blue; ntrc, red; pgr5, pink; *0.01 < P < 0.05, **0.005 < P < 0.01, ***0.001 < P < 0.005, ****P < 0.001, one-way ANOVA with Tukey post hoc test).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

6. Figure 5
Acclimation of Photosynthetic Efficiency in trxm1m2 and ntrc Mutants Growing in Different Light Intensities.
Chl a fluorescence parameters NPQ (A, C, and E) and PSII quantum efficiency (B, D, and F) were analyzed in Col-0 wild-type (Col-0), trxm1.1m2.1, trxm1.2m2.2, and ntrc plants at actinic light intensities similar to growth light. Plants used for these experiments were grown under different light intensities as described in Figure 4, being acclimated to LL (A and B), ML (C and D), and HL (E and F). ΦII, PSII quantum efficiency; NPQ, non-photochemical quenching. Error bars represent SEM (n = 6 biological replicates). Asterisks indicate where the values of mutants are significantly different compared with the respective wild-type (trxm1m2 double mutants, blue; ntrc, red; *0.01 < P < 0.05, **0.005 < P < 0.01, ***0.001 < P < 0.005, ****P < 0.001, one-way ANOVA with Tukey post hoc test).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

7. Figure 6
ntrc Mutant Shows a Decreased Ability to Respond to Rapid Changes in Light Intensity in Fluctuating Light.
(A and B) Representative false color images of FL-grown Col-0 wild-type (Col-0) and ntrc in HL (A) and LL (B) periods of FL (LL, 4 min 90 μmol photons m−2 s−1; HL, 1 min 900 μmol photons m−2 s−1) representing non-photochemical quenching (NPQ), PSII quantum efficiency (ΦII), and PQ pool oxidation state (qL).
(C) NPQ is higher in ntrc in the LL periods of FL compared with Col-0, but not in HL. Asterisks above traces mark were NPQ is significantly increased compared with Col-0 (*0.01 < P < 0.05, **0.005 < P < 0.01, ***0.001 < P < 0.005, ****P < 0.001, Student's t-test).
(D) ΦII is severely decreased throughout the light fluctuations compared with Col-0.
(E) The PQ pool reduction expressed as 1 − qL is increased throughout the light fluctuations compared with Col-0.
In (D) and (E) the values of ntrc are significantly different from Col-0 at all the time points analyzed (P < 0.01, Student's t-test). Error bars represent SEM (n = 6 biological replicates).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions

8. Figure 7
In trxm1m2 Mutants a Compensatory Mechanism Upregulates Photosynthesis in the Low-Light Periods of Fluctuating Light.
(A and B) Representative false color images of FL-grown Col-0 wild-type (Col-0), trxm1.1m2.1, and trxm1.2m2.2 in HL (A) and LL (B) periods of FL (LL, 4 min 90 μmol photons m−2 s−1; HL, 1 min 900 μmol photons m−2 s−1) representing non-photochemical quenching (NPQ), PSII quantum efficiency (ΦII), and the PQ pool oxidation (qL).
(C) NPQ is higher in trxm1m2 mutants in the HL periods of FL compared with Col-0.
(D) ΦII is decreased in the HL periods, but increased in the LL periods compared with Col-0.
(E) The PQ pool reduction expressed as 1 − qL is lower in trxm1m2 in LL periods compared with Col-0.
(F) ΦI is increased in trxm1m2 in LL periods compared with Col-0. Error bars represent SEM (n = 10 biological replicates for fluorescence analyses and n = 5 biological replicates for PSI analysis).
Asterisks above or below traces mark where values for both trxm1m2 lines are significantly increased or decreased compared with Col-0, respectively (*0.01 < P < 0.05, **0.005 < P < 0.01, ***0.001 < P < 0.005, ****P < 0.001, one-way ANOVA with Tukey post hoc test). For fluorescence analyses statistical analysis was performed on single time points; for PSI analysis statistical analysis was performed on all four time points from each LL period.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2017 The Author Terms and Conditions