Figure 5.

Alanine scanning of the five sequence-dependent CPuORFs. (A–E) Effects of Ala substitutions and conservation scores of amino acid residues in the ANAC082 (A), CIPK6 (B), At3g15430 (C), At5g27920 (D) and OTLD1 (E) CPuORFs. The 35S::UTR:RLUC reporter plasmid harboring a WT CPuORF or its mutant with an Ala substitution was co-transfected with the 35S::FLUC internal control plasmid into MM2d protoplasts by electroporation, and the reporter activities were analyzed as in Figure 2. Means ± S.D. of at least three biological replicates are shown. Each graph is representative of at least two separate experiments using independently prepared protoplasts. Single and double asterisks indicate significant differences from the corresponding WT at P < 0.05 and P < 0.01 by t-test, respectively. The amino acid sequence of each CPuORF is indicated below the graph. Amino acid residues analyzed in the Ala scanning mutagenesis are shown in bold. The numbers below the amino acid residues indicate the positions of the residues in each CPuORF peptide. Conservation scores of the amino acid residues of each CPuORF were calculated based on the alignments presented in Supplementary Figure S2, using the Scorecons server (40), and shown in the graph below the numbers indicating the positions of the amino acid residues.