Distribution of DsRed, phl p 1 and M12 accumulation in vacuum-infiltrated tobacco leaves.

Four leaves of various ages from eight-week-old N. tabacum plants were vacuum-infiltrated with Agrobacterium containing a plasmid encoding one of three model proteins: DsRed, phl p 1 or M12. After incubation (5 d, 25°C, 16/8 h light/dark cycle, 7 klux), the leaf discs were cut using a cork borer (22-mm inner diameter) according to the schema shown in Figure 1A. Figures 1B–D show the protein accumulation distribution for each of the three proteins with respect to the leaf age and position within the leaf. To simplify the evaluation and allow for a graphical illustration of the results, missing values between the data points were added by linear interpolation using Origin 8.1. For each of the three replicates, the four boxes designated L1 to L4 represent the four different leaf ages, with L4 being the oldest and L1 being the youngest. The notations 1–3 (y-axis) and −2−2 (x-axis) are the positional coordinates of the leaf discs (see Figure 1A). The level of rpa was related to the TSP to control for differences in leaf weight and extraction efficiency between individual leaf discs. The results obtained from each plant were normalized, with the highest rpa value set at 100%. The color scale represents the normalized rpa percentage.