Fig 2: Biological networks generated for combination of twelve differential proteins.Fifteen differentially up-regulated proteins including ATP synthase subunit beta, alcohol dehydrogenase, beta-glucosidase, phosphoglycerate kinase, triose phosphate isomerase, RCA, Rubisco, APX2, CDSP3, peroxiredoxin, thioredoxin translation elongation factor, glutamate-ammonia ligase, chaperone and 14-3-3 in cassava autotetraploid genotypes were used to generate a protein-protein interaction network through Pathway Studio analysis. Regulation is marked as an arrow with R, Chemical Reaction as an arrow with C and Binding as an arrow without any marks. The entity table, relation table and reference table data were presented in in Tables S1, S2, S3.

Fig 3: Analysis of ascorbate peroxidase (APX) activity and cytosolic (cAPX) or stromal (sAPX) APX abundances in wild type (WT), fsd1-1, fsd1-2 and GFP-FSD1 lines after methyl viologen (MV) treatment. (A) Specific activity of APX measured spectrophotometrically. (B,D,F,H) Immunoblots of cAPX in wild type (WT; B), fsd1-1 (D), fsd1-2 (F) and GFP-FSD1 line (H), supplemented with respective controls of protein loading as visualized on Stain-free gels. Each immunoblot contains protein extracts from mock (lane C) and 1 µM MV treated (lane MV) plants. (C,E,G,I) Quantification of bands optical density in (B,D,F,H), respectively. Blots for each line were prepared on separate membranes. Raw band optical densities were normalized according to the total density of the respective specific bands on the membrane (mean ± SD, N = 3). Asterisks indicate a statistically significant difference between mock and 1 µM MV treated plants in designated time points as calculated by one-way ANOVA with post hoc Tukey HSD test (asterisk indicates statistical significance at p < 0.05). Uncropped, full original images of the blots and gels are documented in Supplementary Figures 9, 10.

Fig 4: Western blotting of Rubisco, APX and PrxQ.The expression of Rubisco, APX and PrxQ in leaves of cassava NZ199 diploid (a) and autotetraploid (b) genotypes were detected by western blotting using antiRubisco-polyclonal antibody (AS07218), anti-APX antibody (AS08368) and anti-PrxQ antibody (AS05093) from Agrisera, respectively.

Fig 5: Zinc-binding motif is required for the functionality but not for VIR3 binding to tAPX and GAPB.(A) Representative images of WT, vir3-1, vir3-1/VIR3OE, vir3-1/pUBQ::VIR3-FLAG, and vir3-1/pUBQ::VIR3H239Y-FLAG seedlings grown in long days (16-hour light/8-hour dark), without (control) or with exposure to 7 days of constant light leading to the exclusive development of variegation patterns on vir3-1 and vir3-1/pUBQ::VIR3H239Y-FLAG leaves but not in the other genotypes. (B) Representative images of infiltrated N. benthamiana leaves (top) and the corresponding split luciferase (LUC) complementation assays (bottom), expressing permutations of VIR3 or VIR3H239Y with tAPX (top) or GAPB (bottom), and each fused to N- and C-terminal fragments of LUC (NL and CL, respectively). Controls consisted of LUC fusion constructs between tAPX and GAPB. LUC luminescence was analyzed after 2 days. The experiments were repeated at least three times. (C) Co-IP assay displays the in vivo interaction between VIR3 with tAPX and GAPB in vir3-1/pUBQ::VIR3-FLAG and vir3-1/pUBQ::VIR3H239T-FLAG seedlings, using FLAG antibody for Co-IP, and tAPX and GAPB- specific antibodies for immunoblot assays. The lanes between IP and flow are blank.