FIGURE 5.

Localization of labeled cryptogein after direct infiltration into tobacco leaves and after aspiration though leaf petiole. (A) Three hours after direct infiltration of biotin-labeled capsicein (Caps), cryptogein (Cry) and its variants into leaf mesophyll of three different leaves, samples from the site of infiltration and the surrounding area were collected, extracted with phosphate buffer with 1% SDS and submitted to 15% SDS-polyacrylamide gel electrophoresis followed by western-blot analysis using streptavidin labeled with horseradish peroxidase and chemiluminescence detection (STV). Except for the mutant K48T all proteins showed higher concentration at the zone of infiltration in comparison with the surrounding zone. (B) Six hours after aspirating of biotin-labeled cryptogein (Cry) and its variants through petiole of detached leaves to three different leaves, samples from two different regions corresponding to the lower and upper part of the leaf were collected. Majority of the proteins exhibited approximately equal distribution in both, lower and upper parts of the leaf, whereas K61T and K48T/K94T mutants showed a little higher concentration in the lower part than in the upper part of the leaf. In addition, a second band with molecular mass of about 16 kDa was detected in all proteins suggesting a possible modification of proteins in plants. (C) Intensities of biotin-labeled cryptogein and its variants 6 h after aspirating through leaf petiole of detached leaves recalculated according to different efficiency of biotinylation reaction (Figure 3). Control (CTRL) represents the samples without any treatment and Cry-B represents 5 ng of biotin-labeled wt cryptogein.