During photosynthesis, electron transport is necessary for carbon assimilation and must be regulated to minimise free radical damage. There is a longstanding controversy over the role of a critical enzyme in this process (ferredoxin:NADP(H) oxidoreductase, or FNR), and in particular its location within chloroplasts. Here we use immunogold labelling to prove that FNR previously assigned as soluble is in fact membrane associated. We combined this technique with a genetic approach in the model plant Arabidopsis, to show that the distribution of this enzyme between different membrane regions depends on its interaction with specific tether proteins. We further demonstrate a correlation between this distribution and the activity of different photosynthetic electron transport pathways. This supports a role for FNR location in regulating photosynthetic electron flow.

Arabidopsis thaliana plants were all Columbia ecotype. They included wild type (labelled Wt), a mutant of the gene for FNR1 (labelled D1), and 3 genotypes expressing maize isoforms of the FNR enzyme in the D1 background (labelled ZmFNR1, ZmFNR2 and ZmFNR3). All transformed plants were screened at the level of western blotting to confirm expression of heterologous FNR proteins prior to analysis. Plants were grown under a light / dark cycle of 12 h / 12 h with moderate light of 150 µmol photons m^-2 s^-1 at 22°C / 18 °C on soil. Samples were extracted from mature Arabidopsis leaves in the presence of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM EDTA, 20 µg/mL and 0.1 mg/mL polyvinyl-polypyrrolidone. Supernatant and pellet fractions were made from these extracts by centrifugation at 11,000 g 4°C for 5 minutes, and the membrane pellet was resuspended with an equal volume of buffer containing 0.1 % Triton X‑100 to solubilise proteins prior to analysis.

Transmission electron microscopy

The transmission electron micrsocopy was performed on a Jeol JEM-1230 microscope (Jeol, Peabody, MA) equipped with a Morada CCD camera and iTEM Olympus software at 80.00 kV.

Immunogold labelling of leaf sections was carried out as follows. The first fully unfolded leaves of Arabidopsis Wt, fnr1, and plants expressing ZmFNR genes in the fnr1 background were sampled for immunogold labelling to ensure consistency in developmental stage, and kept in the dark until the end of the fixation step. The leaves were harvested at the end of the dark period and cut into 1 mm strips with a sharp razor. The strips were transferred to a 3% paraformaldehyde/0.125 M phosphate buffered saline (PBS) in a syringe, creating an underpressure with the plunger to ensure full penetration of the tissue and removing any air from the parenchyma which would interfere with thin-sectioning later on. The fixation step lasted 5 minutes. For light adaptation, leaves were sampled 2 hr into a light period from under growth lights and maintained at 150 µE until fixation, which was also performed under light.

Leaves were embedded into LR White resin (Agar Scientific, Stansted) by sequential incubation in 70% EtOH 30 min, 90% EtOH 30 min, 100%EtOH 30 min, 100% EtOH 30 min, 50% EtOH/50% LR White 60 min, 100% LR White 60 min, 100% LR White overnight. Embedded strips were transferred to gelatine capsules, filled to the top with LR White resin and covered with a piece of wax. Hardening of the resin took place in an oven at 60° for 2.5 h. The capsule was removed and the resin block, cleaned from the wax and subsequently used for thin-sectioning.

After cutting the blocks on a Reichert-Jung ultramicrotome (Leica, Nussloch, Germany) into 70 nm thin-sections, these were transferred onto EM nickel grids. These were then immunogold labelled by the following sequential incubations in a covered wet chamber: 50 μL 1.25 M phosphate-buffered saline (PBS) 2 min, 20 μL 5% H₂O₂ 5 min, 50 μL PBS / 50 mM glycine 3 x 3 min, 5% BSA in PBS 10 min, 1:200 anti ZmFNR2 or anti Cyt f in 1% BSA in PBS 30 min, 50 μL 1% BSA in PBS 3 x 6 min, 10 μL 1:200 gold particle conjugated anti rabbit IgG in 1% BSA in PBS 30 min, 100 μL PBS 8 x 2 min, 50 μL 1% glutaraldehyde 5 min, 100 μL H₂O 8 x 2 min, 20 μL 4% uranyl acetate 4 min, 100 mL H₂O 3 x 20 min. Following this, the grids were air-dried in a dust-free container and ready to use in transmission electron microscopy.

The areas of interest on the electron micrographs were defined by printing at high resolution and manually colouring in magenta (margins), blue (lamellae) and green (grana core). See text for full explanation of chloroplast sub-compartment definitions in this work. Areas of chloroplasts with poor membrane resolution were not included in analysis. To account for antibody size, an area of 10 nm on either side of both margins and lamellae were included in the area. These data are presented to allow those interested to re-analyse the data if wished.

The genotype is denoted by the first 2 letters (Wt, D1, Z1, Z2 or Z3). The 3rd letter denotes the individual, all other letters relate to grids on which the sample was images taken in a series. Many images were not of a standard high enough for analysis, hence the gaps in numbers. In some case lamellae and margins were counted as a single sub-chloroplast domain, which is indicated in the name.

The chloroplast images have been marked to indicate the appressed membranes of the grana core (coloured green), all stromal lamellae membranes not adjacent to an appressed membrane (coloured blue), and all stromal facing membranes adjacent to appressed membranes (coloured red). The marks cover an area of approximately 10 nm on either side of the membrane, as a conservative estimate of antibody labelling distance.