Regulation of photosynthetic electron flow on dark to light transition by ferredoxin:NADP(H) oxidoreductase interactions

  1. Manuela Kramer
  2. Melvin Rodriguez-Heredia
  3. Francesco Saccon
  4. Laura Mosebach
  5. Manuel Twachtmann
  6. Anja Krieger-Liszkay
  7. Chris Duffy
  8. Robert J Knell
  9. Giovanni Finazzi
  10. Guy Thomas Hanke  Is a corresponding author
  1. School of Biochemistry and Chemistry, Queen Mary University of London, United Kingdom
  2. Department of Plant Physiology, Faculty of Biology and Chemistry, University of Osnabrück, Germany
  3. Institute of Plant Biology and Biotechnology, University of Münster, Germany
  4. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, France
  5. Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, Centre National de la Recherche Scientifique (CNRS), Commissariat a` l’Energie Atomique et aux Energies Alternatives (CEA), Université Grenoble Alpes, Institut National Recherche Agronomique (INRA), Institut de Recherche en Sciences et Technologies pour le Vivant (iRTSV), CEA Grenoble, France
7 figures, 4 tables and 3 additional files

Figures

Figure 1 with 2 supplements
FNR is not detected in the chloroplast stroma of higher plants.

(A) Representative micrograph showing immunogold labelling of FNR in sections of chloroplasts from Wt Arabidopsis detected by IGL-TEM. White arrows indicate example gold particles. (B) Immunogold …

Figure 1—figure supplement 1
Specificity of antisera used for Immunogold labelling and blue native PAGE western blots.

Mature Arabidopsis leaf protein extract was subjected to SDS-PAGE before immunoblotting and detection of (A) FNR, (B) cytochrome f (Cyt f), and (C) TROL. The gel was loaded with 20 µg protein for …

Figure 1—figure supplement 2
Detecting protein localisation in sub-chloroplast compartments.

(A) Representative micrograph of a wt chloroplast, with acceptable uranyl acetate staining for detection of sub-chloroplast localisation of proteins. Chloroplast was immunolabelled to detect FNR. (B)…

Arabidopsis plants with variable FNR:membrane tether interactions.

Leaf extracts of Arabidopsis wt, fnr1, and fnr1 mutants transformed to express genes for the maize FNR proteins ZmFNR1, ZmFNR2, and ZmFNR3 in the fnr1 background were separated into soluble and …

Figure 3 with 1 supplement
Tether interactions determine FNR sub-chloroplast location.

Density of immunogold labelled FNR in different sub-chloroplast compartments of the indicated genotypes. Values are averages of three biological replicates, with combined label and area for the …

Figure 3—figure supplement 1
Comparison of areas of different chloroplast sub-compartments between genotypes.

Areas of chloroplast sub-compartments were defined as displayed in Figure 1—figure supplement 2, before calculating the percentage area within the chloroplast of each indicated sub-compartment for …

Figure 4 with 1 supplement
Light-dependent NADP(H) reduction and oxidation kinetics are influenced by FNR location and binding partners.

(A) Traces show NADPH fluorescence of dark adapted Arabidopsis chloroplasts measured over a short light exposure from 10 to 40 s. Traces are averages of three to five independent chloroplast …

Figure 4—figure supplement 1
Abundance of the major photosynthetic complexes in the genotypes used in this study.

Arabidopsis plants of the indicated genotypes were grown in 12 hr light conditions at 150 µE/12 hr dark and mature leaf protein extract was subjected to SDS-PAGE before immunoblotting and detection …

Figure 5 with 2 supplements
Impact of FNR location on photosynthetic electron transport in Arabidopsis following dark adaptation or light acclimation.

(A) ECS measurements after a 20 s high light pulse on dark adapted leaves (grey background) and light acclimated leaves (5 min 150 µE m−2 s−1, actinic light). The relaxation kinetics of ECS were …

Figure 5—figure supplement 1
DCMU inhibition of PSII for measurement of cyclic electron flow in Arabidopsis.

Electrochromic band shift (ECS) assay to compare generation of ΔpH without (stimulation of both photosystems) or with 20 µM DCMU (inhibition of photosystem II). (A) ECS measurements on leaves from …

Figure 5—figure supplement 2
Raw data from ECS and PSI responses to high light treatment.

(A) Representative normalised ECS traces following high light exposure to either red light (filled circles) or far red light (empty circles) after dark adaptation (left) and light acclimation at 150 …

Figure 6 with 1 supplement
FNR sub-chloroplast distribution changes in response to light.

Density of immunogold labelled FNR in different sub-chloroplast compartments of Wt Arabidopsis either dark incubated (left panel, same data as in Figure 3) or light incubated (right panel), prior to …

Figure 6—figure supplement 1
Two models describing potential mechanisms that could explain the impact of FNR interactions on photosynthetic electron transport during the dark to light transition.

Models are based on data presented in this work that indicate (i) FNR is overwhelmingly membrane bound irrespective of interaction with the Tic62/TROL tethers (Figures 1 and 3); (ii) Strong …

Appendix 1—figure 1
Examination of the two phases detected in fluorescent measurement of light-dependent NADP+ reduction.

Traces show NADPH fluorescence of dark adapted Arabidopsis chloroplasts measured over a short light exposure from 10 to 40 s. Traces are averages of five separate chloroplast preparations (wt), or …

Tables

Table 1
Mixed effects model investigating changes in FNR density between total chloroplast sub-compartments of three individuals each of WT and fnr1 Arabidopsis.

Analysis of data presented in Figure 1. Fixed effects taking either label density in the stroma as the intercept or label density in the margins/lamellae as the intercept. Linear mixed model fit by …

Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017). The model is a mixed effects model with random intercepts. The square root of response is the response variable, tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum sqMean sqNum DFDen DFF valuePr (>F)
Sub-compartment14.2317.11532295.587.45650.000693***
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
individual(Intercept)0.18960.4354
Residual0.95420.9769
Number of obs: 306, groups: individual, 6
Fixed effects when fnr1 stroma is set as the intercept
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)0.91650.27914.26863.2830.0276*
Grana−0.36070.1714295.5794−2.1050.0361*
Margin/lamellae2.28840.1714295.579413.355<2−16***
Genotype comparison WT:fnr10.12300.41285.03750.2980.7776
WT grana: fnr1 grana0.62860.2845295.57942.2100.0279*
WT margin/lamellae: fnr1 margin/lamellae−0.46610.2845295.5794−1.6380.1025
Fixed effects when fnr1 margin/lamellae is set as the intercept
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)0.91650.27914.268611.4820.000228***
Grana−2.64910.1714295.5794−15.460<2−16***
Stroma−2.28840.1714295.5794−13.355<2−16***
Genotype comparison WT:fnr1−0.34300.41285.0375−0.8310.443524
WT grana: fnr1 grana1.09470.2845295.57943.8480.000146***
WT stroma: fnr1 stroma−0.46610.2845295.57941.6380.102459
Fixed effects when Wt stroma is set as the intercept
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.03950.30415.85763.4180.0147*
Grana0.26790.2271295.57941.1800.2391
Margin/lamellae1.82230.2271295.57948.0242.43−14***
Genotype comparison WT:fnr1−0.12300.41285.0375−0.2980.7776
Wt grana: fnr1 grana−0.62860.2845295.5794−2.2100.0279*
Wt margin/lamellae: fnr1 margin/lamellae0.46610.2845295.57941.6380.1025
Fixed effects when Wt margin/lamellae is set as the intercept
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)2.86180.30415.85769.4119.41−05***
Grana−1.55440.2271295.5794−6.8444.44−11***
Stroma−1.82230.2271295.5794−8.0242.43−14***
Genotype comparison WT:fnr10.3430.41285.03750.8310.443524
Wt grana: fnr1 grana−1.09470.2845295.5794−3.8480.000146***
Wt stroma: fnr1 stroma−0.46610.2845295.5794−1.6380.102459
Table 2
Mixed effects model investigating changes in FNR density between total chloroplast sub-compartments of three individuals each from Arabidopsis genotypes WT, fnr1 mutant and expressing ZmFNR1; ZmFNR2 and ZmFNR3 in the fnr1 background.

Analysis of data presented in Figure 3. Fixed effects taking either label density in the stroma as the intercept or label density in the margins/lamellae as the intercept. Linear mixed model fit by …

Wt
Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017).
The model is a mixed effects model with random intercepts. The square root of response is the response variable,
tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum SqMean SqNum DFDen DFF valuePr (>F)
Sub-compartment4.9911.66373636.1520.0003089***
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
Individual(Intercept)0.210970.4593
Residual0.046020.2145
Number of obs: 12, groups: individual, 3
Fixed effects when stroma is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.17430.29272.64754.0120.034961*
Grana0.31040.175261.7720.126711
Lamellae1.47480.175268.420.000153***
Margin1.37360.175267.8420.000227***
Fixed effects when lamellae is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)2.64910.29272.64759.0510.004601**
Grana−1.16440.17526−6.6480.00056***
Margin−0.10120.17526−0.5780.584522
Stroma−1.47480.17526−8.420.000153***
fnr1
Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017).
The model is a mixed effects model with random intercepts. The square root of response is the response variable,
tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum SqMean SqNum DFDen DFF valuePr (>F)
Sub-compartment5.65161.88393626.2040.000759***
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
Individual(Intercept)0.075210.2742
Residual0.071890.2681
Number of obs: 12, groups: individual, 3
Fixed effects when stroma is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.21170.22144.48365.4720.003875**
Grana−0.15840.21896−0.7240.496563
Lamellae0.93530.218964.2720.005251**
Margin1.51610.218966.9250.000449***
Fixed effects when lamellae is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)2.14690.22144.48369.6950.000356***
Grana−1.09370.21896−4.9960.002463**
Margin0.58080.218962.6530.037882*
Stroma−0.93530.21896−4.2720.005251**
fnr1:ZmFNR1
Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017).
The model is a mixed effects model with random intercepts. The square root of response is the response variable,
tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum SqMean SqNum DFDen DFF valuePr (>F)
Sub-compartment4.52421.508136.0123.5580.001009**
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
Individual(Intercept)0.00059840.02446
Residual0.0640170.25302
Number of obs: 12, groups: individual, 3
Fixed effects when stroma is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.17540.14687.99798.0094.34−05***
Relevel grana0.23180.20666.011.1220.304712
Relevel lamellae1.37740.20666.016.6680.000547***
Relevel margin1.28490.20666.016.220.000793***
Fixed effects when lamellae is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)2.552850.146767.997917.3951.22−07***
Relevel grana−1.145670.206596.01002−5.5460.001444**
Relevel margin−0.092520.206596.01002−0.4480.669949
Relevel stroma−1.377440.206596.01002−6.6680.000547***
fnr1:ZmFNR2
Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017).
The model is a mixed effects model with random intercepts. The square root of response is the response variable,
tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum SqMean SqNum DFDen DFF valuePr (>F)
Sub-compartment5.44141.81383622.8490.001106**
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
Individual(Intercept)0.027410.1656
Residual0.079380.2817
Number of obs: 12, groups: individual, 3
Fixed effects when stroma is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.18860.18876.67966.30.000488***
Grana0.48640.2362.1140.078885.
Lamellae1.82980.2367.9540.00021***
Margin0.92390.2364.0160.006989**
Fixed effects when lamellae is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)3.01840.18876.679615.9981.42−06***
Grana−1.34340.236−5.840.00111**
Margin−0.90590.236−3.9380.00764**
Stroma−1.82980.236−7.9540.00021***
fnr1:ZmFNR3
Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017).
The model is a mixed effects model with random intercepts. The square root of response is the response variable,
tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum SqMean SqNum DFDen DFF valuePr (>F)
Sub-compartment9.00463.00153825.4160.0001923***
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
Individual(Intercept)00
Residual0.11810.3436
Number of obs: 12, groups: individual, 3
Fixed effects when stroma is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.067940.1984185.3830.00066***
Grana0.062840.2805980.2240.828395
Lamellae1.994470.2805987.1080.000101***
Margin1.443430.2805985.1440.00088***
Fixed effects when lamellae is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)3.06240.1984815.4353.09−07***
Grana−1.93160.28068−6.8840.000127***
Margin−0.5510.28068−1.9640.085144.
Stroma−1.99450.28068−7.1080.000101***
Table 3
Mixed effects model investigating changes in FNR density between different the chloroplast sub-compartments of WT Arabidopsis following dark adaptation or light adaptation of leaves.

Analysis performed using the data in Figure 6. Fixed effects taking either label density in the stroma as the intercept or label density in the margins/lamellae as the intercept. Linear mixed model …

Deletion test carried out using Satterthwaite’s method with the R package lmerTest (Kuznetsova, Brockhoff & Christensen 2017). The model is a mixed effects model with random intercepts. The square root of response is the response variable, tissue is the fixed effect, and individual the random effect.
Fixed effect deletedSum SqMean SqNum DFDen DFF valuePr (>F)
Sub-compartment0.301840.100613122.36130.1227
Model summary:
Random effects:
GroupsNameVarianceStd. Dev.
Individual(Intercept)0.10730.3276
Residual0.042610.2064
Number of obs: 24, groups: individual, 6
Fixed effects when dark adapted stroma is set as the intercept::
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.17430.22356.30675.2530.00164**
Grana0.31040.1685121.8420.09033.
Lamellae1.47480.1685128.751.48−06***
Margins1.37360.1685128.153.11−06***
Light:dark comparison0.41570.31616.30671.3150.23427
Light:dark grana0.14210.2384120.5960.5622
Light:dark lamellae0.14820.2384120.6220.54562
Light:dark margins0.59630.2384122.5020.02782*
Fixed effects when dark adapted lamellae is set as the intercept::
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)2.649130.2235426.30673811.8511.52−05***
Grana−1.164370.16854312.000001−6.9081.63−05***
Margins−0.1011750.16854312.000001−0.60.5595
Stroma−1.4748070.16854312.000001−8.751.48−06***
Light:dark comparison0.5639880.3161366.3067381.7840.1223
Light:dark grana−0.0061640.23835612.000001−0.0260.9798
Light:dark margins0.448080.23835612.0000011.880.0846.
Light:dark stroma−0.1482410.23835612.000001−0.6220.5456
Fixed effects when light acclimated stroma is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)1.59010.22356.30677.1130.00031***
Grana0.45250.1685122.6850.01986*
Lamellae1.6230.1685129.635.37−07***
Margins1.970.16851211.6886.48−08***
Dark:light comparison−0.41570.31616.3067−1.3150.23427
Dark:light grana−0.14210.238412−0.5960.5622
Dark:light lamellae−0.14820.238412−0.6220.54562
Dark:light margins−0.59630.238412−2.5020.02782*
Fixed effects when light acclimated lamellae is set as the intercept:
EstimateStd. ErrorDFt valuePr (>|t|)
(Intercept)3.2131180.2235426.30673814.3744.68−06***
Grana−1.1705350.16854312.000001−6.9451.55−05***
Margins0.3469050.16854312.0000012.0580.062.
Stroma−1.6230480.16854312.000001−9.635.37−07***
Dark:light comparison−0.5639880.3161366.306738−1.7840.1223
Dark:light grana0.0061640.23835612.0000010.0260.9798
Dark:light margins−0.448080.23835612.000001−1.880.0846.
Dark:light stroma0.1482410.23835612.0000010.6220.5456
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Arabidopsis thaliana)FNR1;
FNR2
GenBankAT5G66190; AT1G20020
Gene (Zea mays)FNR1;
FNR2;
FNR3;
TROL
GenBankBAA88236; BAA88237; ACF85815;
ACF79627.1
Strain, strain background (Escherichia coli)BL21(DE3)Thermo FisherEC0114Competent cells
Genetic reagent (A. thaliana)Columbia;
Columbia fnr1;
Columbia ZmFNR1;
Columbia ZmFNR3
Arabidopsis Biological Resource Center (ABRC)
See Twachtmann et al., 2012 for over-expression line generation
wt;
AT1G20020 T-DNA insertion mutant;
Line expressing BAA88236 under control of the AT1G20020 promoter;
Line expressing ACF85815 under control of the AT1G20020 promoter
See Hanke et al., 2008 for mutant isolation;
AntibodyAnti-maize FNR2 (Rabbit polyclonal)This paper
(Okutani et al., 2005)
BAA88237IG-TEM (1:200), WB (1:50,000). Raised in rabbit using recombinant mature purified maize FNR2 protein
AntibodyAnti-maize Cyt f (Rabbit polyclonal)This paper
(Okutani et al., 2005)
IG-TEM (1:200), WB (1:5000)
Raised in rabbit using Cyt f protein purified from maize leaves
AntibodyAnti-maize TROL (guinea pig polyclonal)This paperWB (1:10,000) Raised in guinea pig using TROL protein purified as described in the Materials and methods
Recombinant DNA reagentpCold His-TROL (plasmid)Takara-Bio;
This paper
ACF79627.1Cloned as described in the Materials and methods
Recombinant DNA reagentpSAt-FNR1pro-Zm-FNR2 (plasmid)From Twachtmann et al., 2012AT1G20020;BAA88237Plasmid for expressing maize FNR2 under control of the Arabidopsis FNR1 promoter
Sequence-based reagentTROL -FEurofins genomics (Ebersberg, Germany)PCR primersGTCGACGAGGATCGACAAAA
Sequence-based reagentTROL -REurofins genomics (Ebersberg, Germany)PCR primersGAATTCCTAGACCCGGTTTCTT
Peptide, recombinant proteinMaize TROLEurofins genomics (Ebersberg, Germany)ACF79627.1Cloned and purified as described in the Materials and methods

Additional files

Supplementary file 1

Summary of published information about Arabidopsis and maize FNR iso-proteins.

Taken from Hanke et al., 2005, Okutani et al., 2005, Lintala et al., 2009, Lintala et al., 2007, and Twachtmann et al., 2012. Kinetic parameters are for the reverse direction to photosynthesis (NADPH-dependent reduction of Fd).

https://cdn.elifesciences.org/articles/56088/elife-56088-supp1-v2.docx
Supplementary file 2

Additional statistical analysis.

(a) Table of mixed effects model investigating changes in FNR density between different chloroplast sub-compartments in WT Arabidopsis. Analysis of data presented in Figure 1—figure supplement 2. Fixed effects taking either label density in the stroma as the intercept or label density in the margins/lamellae as the intercept. Linear mixed model fit by REML. Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1. (b) Table of mixed effects model investigating changes in cytochrome f density between different chloroplast sub-compartments in WT Arabidopsis. Analysis of data presented in Figure 1—figure supplement 2. Fixed effects taking either label density in the stroma as the intercept or label density in the margins/lamellae as the intercept. Linear mixed model fit by REML. Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1. (c) Table of fitting parameters and errors in comparison of light-dependent NADP+ reduction by different genotypes. Analysis performed using the data in Figure 4. Fits were calculated from experiments on individual chloroplast preparations, and then the parameters, and the fitting errors averaged. (d) Table of statistical analysis on the contribution of the fast phase to total amplitude of light-dependent fluorescence change in the chloroplast assay of NADP+ reduction. Analysis performed using the data averaged in Figure 4 and in (c). (e) Table of fitting parameters and errors in comparison of dark NADPH oxidation by different genotypes. Analysis performed using the data in Figure 4. Fits were calculated from experiments on individual chloroplast preparations, and then the parameters, and the fitting errors averaged. (f) Table of Pm values and statistical analysis of plants used for PAM analysis of the high light response. Analysis performed using the data in Figure 5, with example traces given in Figure 5—figure supplement 2. Pm determination of dark adapted leaves in order to calculate PSI parameters in response to high light treatment of Wt, fnr1, and fnr1 plants expressing either ZmFNR1, ZmFNR2, or ZmFNR3 Arabidopsis plants (see Figure 5). n = 5–7 replicates. Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘’ 1.

https://cdn.elifesciences.org/articles/56088/elife-56088-supp2-v2.docx
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