Qualitative and quantitative dataset of TROL protein interaction with C3 and C4 ferredoxin: NADP+ oxidoreductases

Last step of electron transport from ferredoxin to NADP+ in photosynthesis light reactions catalyses ferredoxin: NADP+ oxidoreductase (FNR). FNR is present as soluble protein in stroma, but also bound to the protein complexes on the membrane with thylakoid rhodanase-like protein (TROL) and translocon on the inner envelope chloroplast membrane (Tic62), which have identical C terminal FNR binding domain [1,2]. During the electron transport, FNR anchored by TROL protein transfers electrons on NADP+ and forms NADPH which is then used in Calvin cycle as reducing agent. TROL is an integral membrane protein [3] with an inactive rhodanase-like domain (RHO) facing stroma which, as proposed earlier [4], could bind a small ligand leading to releasing or binding of FNR. FNR-TROL protein complex is necessary for optimal photosynthetic electron flow [1]. It has been shown that C4 plant maize FNR isomers have different N-terminal structure which determines binding affinity to protein complexes and different ratios of bound and unbound FNR in bundle sheath and mesophyll cells, depending on preferable photosynthetic electron transport [5]. Mutant Arabidopsis plant that contain maize FNR1 protein showed influence on dynamic association of FNR and change in excitation balance between photosystems which then influenced photo induced electron transport and finally photosynthesis [5]. In order to determine the influence of maize FNR1 on photosynthesis in C3 plants and difference in interaction strength with TROL, we preformed Yeast two hybrid screening, x-alpha-gal assay and β-galactosidase assay.

Photosynthesis FNR TROL Protein interaction Electron transport chain a b s t r a c t Last step of electron transport from ferredoxin to NADPþ in photosynthesis light reactions catalyses ferredoxin: NADP þ oxidoreductase (FNR). FNR is present as soluble protein in stroma, but also bound to the protein complexes on the membrane with thylakoid rhodanase-like protein (TROL) and translocon on the inner envelope chloroplast membrane (Tic62), which have identical C terminal FNR binding domain [1,2]. During the electron transport, FNR anchored by TROL protein transfers electrons on NADPþ and forms NADPH which is then used in Calvin cycle as reducing agent. TROL is an integral membrane protein [3] with an inactive rhodanase-like domain (RHO) facing stroma which, as proposed earlier [4], could bind a small ligand leading to releasing or binding of FNR. FNR-TROL protein complex is necessary for optimal photosynthetic electron flow [1]. It has been shown that C4 plant maize FNR isomers have different N-terminal structure which determines binding affinity to protein complexes and different ratios of bound and unbound FNR in bundle sheath and mesophyll cells, depending on preferable photosynthetic electron transport [5]. Mutant Arabidopsis plant that contain maize FNR1 protein showed influence on dynamic association of FNR and change in excitation balance between photosystems which then influenced photo induced electron transport and finally photosynthesis [5]. In order to determine the influence of maize FNR1 on photosynthesis in C3 plants and difference in interaction strength

Data
The data indicates stronger interaction [4] between maize FNR1 with ITEP region of TROL [5] than one between pea FNR1 and ITEP. Yeast two hybrid interaction screening showed positive interactions between both FNR's with ITEP and IA2 region and negative with module 220 and PEPE (Fig. 1). Vigorous yeast cell growth and intensive blue color development from yeast MEL1 gene activation in X-alpha-gal assay was detected in ZmFNR1FLAGHA-ITEP protein interaction and lighter blue color was developed in interaction PsFNR1 with ITEP and IA2 regions and ZmFNR1FLAGHA with IA2 (Fig. 2). b-galactosidase assay showed that ZmFNR1FLAGHA-ITEP interaction is 10 times stronger than PsFNR1-ITEP interaction (Table 1). Raw data of b-galactosidase assay, Yeast two hybrid screening and X-alpha-gal assay figures can be found in Supplementary materials.

Experimental design, materials, and methods
C-terminal regions of TROL named ITEP (residues I429eP466) and Tic62 named IA2 (residues M263-S444), were introduced in bait vector pBD-Gal4 Cam, as well as additional regions of TROL which don't participate in TROL-FNR interaction -module 220 (residues N180eV220) and PEPE (residues A379-A419) [1,3]. IA2 C-terminal region was used as positive control [2] and empty bait vector as Specifications Table   Subject Biology, Biochemistry Specific subject area Protein interactions Type of data Image Chart How data were acquired Yeast two hybrid, b-galactosidase assay, X-alpha-gal assay, yeast transformation Yeast cells were transformed by modified LieO-Ac method. Cells were grown overnight in YPD medium (Yeast extract 10 g/l (w/v), Peptone 20 g/l (w/v), Glucose 20 g/l (w/v)) on 30 C with constant mixing on rotator. One ml of cells was pelleted in a microfuge for 5 sec. prior to each transformation.
Pellet was resuspended by shaking in 50e100 ml of the remaining medium. Two ml of carrier DNA (10 mg/ml, denatured by boiling) were then added, followed by addition of 1 mg of plasmid construct. Mixture was vortexed and 500 ml of PLATE mixture (90 ml 45% PEG, 10 ml 1 M LieO-Ac, 1 ml 1 M Tris-Cl and 200 ml 0,5 M EDTA) was added. Fifty ml of 1 M DTT was added and the mixture was vortexed again, followed by 6 to 8-h incubation on room temperature. Cells were heat shocked for 10 minutes at 42 C and plated on selective medium plates.
Transformants were selected on selective medium plates lacking Leucine and Tryptophan, grown for three days on 30 C and transferred in same liquid medium overnight, 200 rpm on 30 C. For  protein-protein interaction screening cells were plated on selective medium lacking Leucine, Tryptophan, Histidine and Adenine and grown on 30 C for 3 days. b-galactosidase assay is done according to manufacturer's instructions (Thermo Fischer), same as X-alpha-galactosidase assay (Takara).