Environ. Sci. Technol. 2004, 38, 4195-4199
A Novel Analytical Approach for Visualizing and Tracking Organic Chemicals in Plants EDWARD WILD, JOHN DENT, JONATHAN L. BARBER, GARETH O. THOMAS, AND KEVIN C. JONES* Departments of Environmental and Biological Sciences, Lancaster University, Lancaster, LA1 4YQ, United Kingdom
Vegetation plays a key role in the environmental fate of many organic chemicals, from pesticides applied to plants, to the air-vegetation exchange and global cycling of atmospheric organic contaminants. Our ability to locate such compounds in plants has traditionally relied on inferences being made from destructive chemical extraction techniques or methods with potential artifacts. Here, for the first time, two-photon excitation microscopy (TPEM) is coupled with plant autofluorescence to visualize and track trace levels of an organic contaminant in living plant tissue, without any form of sample modification or manipulation. Anthracene-a polynuclear aromatic hydrocarbon (PAH)-was selected for study in living maize (Zea mays) leaves. Anthracene was tracked over 96 h, where amounts as low as ∼0.1-10 pg were visible, as it moved through the epicuticular wax and plant cuticle, and was observed reaching the cytoplasm of the epidermal cells. By this stage, anthracene was identifiable in five separate locations within the leaf: (1) as a thin (∼5 µm) diffuse layer, in the upper surface of the epicuticular wax; (2) as thick (∼28 µm) diffuse bands extending from the epicuticular wax through the cuticle, to the cell walls of the epidermal cells; (3) on the external surface of epidermal cell walls; (4) on the internal surface of epidermal cell walls; and (5) within the cytoplasm of the epidermal cells. This technique provides a powerful nonintrusive tool for visualizing and tracking the movement, storage locations, and degradation of organic chemicals within vegetation using only plant and compound autofluorescence. Many other applications are envisaged for TPEM, in visualizing organic chemicals within different matrixes.
Materials and Methods
Introduction Organic chemicals come into intimate contact with vegetation. One obvious example is herbicides; improving their efficacy relies on understanding the mechanisms by which they penetrate, persist, and become activated within the plant (1). Another example relates to persistent organic pollutants (POPs)-such as polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)-which may be emitted to the atmosphere in some regions of the world but may undergo air-vegetation exchange (hopping) to reach locations far from their source (2, 3). For such chemicals, * Corresponding author e-mail:
[email protected]; phone: (44)-1524-593972; fax: (44)-1524-593985. 10.1021/es049915u CCC: $27.50 Published on Web 06/17/2004
vegetation is a key component in their global cycling and environmental fate (4, 5). Herbicide application or atmospheric POP deposition introduces chemicals onto the leaf surface of plants-the cuticle. The cuticle is a hydrophobic lipid structure, typically 0.1-10 µm thick (6, 7), which can be divided into five generalized compartments-the epicuticular wax, the cuticle proper, the cuticle layer, the pectinous layer, and the cell wall. Diffusive movement through these layers is controlled by compound and plant properties, environmental conditions, and time. Diffusion into the leaf controls how much compound is available to be emitted to the atmosphere, or remains to be ingested and enter food chains (8), to be deposited to soil surfaces following foliar shedding (9), or to be metabolized. However, fundamental research questions remain, which hamper our ability to predict and model airvegetation exchange and the role of vegetation in storing and processing these chemicals (5). Examples include ambiguities over the precise storage location(s) within plants (10, 11) and their role in enhancing or retarding dynamic short-term air-surface exchange (12, 13); the role of stomatal versus cuticular uptake (14, 15); and uncertainties over whether plants attain equilibrium with air during their lifetime (8, 16). Addressing these questions has been held back by limitations in the analytical techniques available. To date, methodologies have relied on extracting native or 14Clabeled compounds from intact leaves, isolated cuticles, and reconstructed waxes (10, 11). Time course experiments of plant uptake from contaminated atmospheres and clearance from clean ones have allowed inferences to be made about storage locations within leaves (16), while degradation through photolysis and metabolism tends to be studied in cell cultures (17). However, these approaches are all constrained by current analytical methods and may be unrealistic with respect to intact plant/field conditions. They do not show precisely where within leaves chemicals are located and how quickly they get there, and there is obviously ambiguity over parent/metabolite compound identity when 14C-labeled compounds are used. Confocal laser scanning microscopy has been proposed as a method for visualizing plant cuticles (18). Here, for the first time, two-photon excitation microscopy (TPEM)-a similar but less destructive technique-is used to visualize the locations of an organic chemical in three-dimensional images of whole living leaves, without the need for sample manipulation or modification, through the novel use of both the plant and the compound autofluorescence. TPEM is most commonly used for imaging isolated organelles, living cells, or intact tissues from both plant or animal matrixes, using specific fluorescent dies, markers, or GFP tags (19-23).
2004 American Chemical Society
Chemicals show excitation and fluorescence emission at specific wavelengths, making their identification both rapid and unambiguous. Anthracene was selected as a model compound because it fluoresces at a wavelength below that of autofluorescence from the chosen plant species. It also represents certain properties of POPs, notably their hydrophobicity, lipophilicity, and semivolatility. Replicate time course experiments were conducted, tracking anthracene over 0-96 h using 28 different leaves and 98 locations upon these leaves. The results presented represent average behavior shown by the compound at different locations upon different leaves at set time periods. All images shown were selected to represent average compound behavior at the allocated time. VOL. 38, NO. 15, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
4195
Anthracene was obtained from Aldrich Chemical Co. with a purity of 99.9%. Seeds of Zea mays were obtained from the Maize Genetics Cooperation Stock Center; all plants analyzed were between 21 and 25 days old. Plants were grown in Levington Compost original compost mix, M3, under a 14 h photoperiod, and were illuminated by 400 W sodium solar lighting. Plants were watered four times a day for 5 min periods, using a capillary matting dripper system. The greenhouse temperature was maintained at 25 °C. Anthracene was applied in an acetone solution as a homogeneous layer (
