Co-transfection of tobacco protoplasts with plasmids encoding eqFP611 and GFP targeted to peroxisomes. Transfection of N. tabacum wild-type protoplasts with two separate plasmids encoding eqFP611 with a peroxisomal targeting signal 2 (pKAT2-eqFP611) and GFP targeted to the peroxisomal membrane (p35S-N-TAP2(G)pex). Pictures of the same protoplast taken through MitoTracker (A) and FITC (B) filter sets, respectively. Scale bars: 10 μM (C) Plasmid maps. Black arrow, CaMV 35S: cauliflower mosaic virus 35S promoter; grey box, KAT2: N-terminal 297 nucleotides of the KAT2 coding sequence; grey box, TAP: chimeric sequence for tandem affinity purification; red box, eqFP611: eqFP611 coding sequence; green box, GFP(S65T): GFP coding sequence including the S65T modification; grey box, APX: sequence encoding the C-terminal 36 amino acids of cotton ascorbate peroxidase; black box, NOS T: nopaline synthase terminator. H: HindIII, P: PstI, Xb: XbaI, Sm: SmaI, Sa: SacI, E: EcoRI, Xh: XhoI restrictions sites. Vector backbone: pUC19 and pGreenII, respectively.