Visualizing the air-to-leaf transfer and within-leaf movement and distribution of phenanthrene: further studies utilizing two-photon excitation microscopy.

Two-photon excitation microscopy (TPEM) was used to monitor the air-to-leaf transfer and within-leaf movement and distribution of phenanthrene in two plant species (maize and spinach) grown within a contaminated atmosphere. Phenanthrene was visualized within the leaf cuticle, epidermis, mesophyll, and vascular system of living maize and spinach plants. No detectable levels of phenanthrene were observed in the roots or stems of either species, suggesting phenanthrene entered the leaves only from the air. Phenanthrene was observed in both the abaxial and adaxial cuticles of both species. Particulate material (aerosols/dust) contaminated with phenanthrene was located at the surface of the cuticle and became encapsulated within the cuticularwaxes. Overtime, diffuse areas of phenanthrene formed within the adjacent cuticle. However, most of the visualized phenanthrene reaching the leaves arrived via gas-phase transfer. Phenanthrene was found within the wax plugs of stomata of both species and on the external surface of the stomatal pore, but not on the internal surface, or within the sub-stomatal cavity. Phenanthrene diffused through the cuticles of both species in 24-48 h, entering the epidermis to reside predominantly within the cell walls of maize (indicative of apoplastic transport) and the cellular cytoplasm of spinach (indicative of symplastic transport). Phenanthrene accumulated within the spinach cytoplasm where it concentrated into the vacuoles of the epidermal cells. Phenanthrene was not observed to accumulate in the cytoplasm of maize cells. Phenanthrene entered the internal mesophyll of both species, and was found within the mesophyll cell walls, at the surface of the chloroplasts, and within the cellular cytoplasm. Phenanthrene was observed within the xylem of maize following 12 days exposure. The cuticle and epidermis at the edges of spinach leaves had a systematically higher concentration of phenanthrene than the cuticle and epidermal cells at the center of the leaf. These results provide important new information about how such compounds enter, move, and distribute within leaves, and suggest that contemporary views of such processes based on data obtained from traditional analytical methods may need to be revised.