New Arabidopsis thaliana cytochrome c partners: a look into the elusive role of cytochrome c in programmed cell death in plants

Abstract

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.

Fig. 1.

PCD hallmarks in A. thaliana MM2d cell cultures treated with 35 mm H₂O₂. A, Cell viability measured using trypan blue dye exclusion assay. Untreated and H₂O₂-treated cells are represented by solid and dashed lines, respectively. Data is the result of three independent experiments, each including 500 cells. B, Effect of H₂O₂ treatment on chlorophyll concentration. One milliliter of H₂O₂-treated cell cultures was collected at the indicated times and the chlorophyll amount was calculated according to MacKinney's protocol (29). C, Effect of H₂O₂ treatment on protein concentration. Similar to (B), but the protein amount was determined using the Bradford assay (28). D, Bright-field microscope image showing cells treated with H₂O₂ for 6 h. Cells were stained with trypan blue dye. E, Similar to (D), but after 24 h of H₂O₂ treatment. F, Bright-field microscope image for 35 mm H₂O₂ treated cells, stained with trypan blue dye indicating a cell wall (CW) and cell shrinkage (CS). G–J, Changes in the cellular morphology of H₂O₂-treated cells analyzed by DAPI nuclear staining, chlorophyll fluorescence and bright-field images. Cells were observed under fluorescence (Left Panels G and I) and bright-field (Right panels H and J) microscopy. Upper (G and H) and lower (I and J) panels correspond to 0 and 24 h of H₂O₂ treatment, respectively. Apoptotic nuclei are stained in blue dye and, having undergone shrinkage, lack red fluorescence.

Fig. 2.

BiFC assays in A. thaliana protoplasts. A. thaliana protoplasts were transfected with pSPYCE/pSPYNE vectors, as described in Sheen (34), to corroborate the in vivo interaction of Cc and its potential targets in BiFC. Images were captured 24 h following transient transfection and after 6 h of treatment with 35 mm H₂O₂. Reconstruction of eYFP leads to the obtainment of green fluorescence signal emission, indicative of interaction between Cc and its partners. Protoplasts transfected with chromatin-remodeling complex element SWI3B, a protein unable to interact with Cc, were used as a negative control. The nucleus was stained in blue using DAPI dye. Scale bar is 10 μm.

Fig. 3.

SPR Measurements. A, Sensograms recorded for the binding of plant Cc with GLY2. Three replicate injections were performed for each protein concentration. In each sensogram, the signals from the control surface were subtracted. B, Similar to (A), but for the plant Cc-NRP1 complex. C, Similar to (A), but for the plant Cc-TCL interaction.

Fig. 4.

Principal functions ascribed to novel Cc protein partners. Diagram showing the principal functions of novel plant Cc protein partners identified in vitro with proteomics and corroborated in vivo with BiFC. All targets have been grouped into seven functional categories.