Up-regulating Plant Defenses in Populus Increases Phytoremediation Capacity for Carbon Tetrachloride

Phytoremediation is a technology that exploits plants to clean up polluted soil and water, with a potential for enormous benefit to mankind. It is cost-effective and certainly more aesthetically pleasing than aggressive land reforming approaches. Such technology is proving successful, but could benefit from a stronger research base. For example, our landmark results suggest that remediation by poplar could be made more efficient by exploiting the plant’s own natural defense mechanisms. This raises the prospect of bio-engineering plants to enhance defense mechanisms and improve their remediation of ground-water contaminants including organic compounds and heavy metals. Our experimental approach to research plant function is also unusual, relying on non-invasive nuclear imaging of carbon-11, a short-lived positron emitting radioisotope that allows us to trace the fate of newly acquired carbon within intact plants and perform repeat tests on the same plant over time. Such information is not available by using carbon-14 or carbon-13. INTRODUCTION There is a growing concern over continued elevated levels of halocarbon contaminants in groundwater which present human health risks as potential carcinogens. Carbon tetrachloride (CCl4), one of several compounds in this class of contaminants, has been targeted for environmental remediation. Unlike many organic compounds, halocarbons can persist in the environment for decades (Wackett et al., 1989). Use of poplar trees to clean up such groundwater contaminants has proven to be a cost-effective (Schnoor et al., 1995; Kassel et al., 2002; Nyer and Gatliff, 1996; Newman et al., 1998), but we still face the challenge of minimizing foliar emissions of volatile chlorocarbon compounds as they can become atmospheric pollutants capable of depleting the ozone layer (Van der Lelie et al., 2002; Schwitzguebel et al., 2002; Ma and Burken, 2003). However it turns out that chlorocarbons can be metabolized, and the plants need not act only as agents to transfer chlorocarbons from soil to atmosphere. Our research is focused on elucidating the mechanistic pathways through which plants naturally metabolize halocarbon compounds, and on improving these processes. One strategy we have been exploring is to heighten a plant’s natural defense system using treatments of specific compounds known to be biosynthesized as part of the plant’s defense processes. We recently showed that treatment of poplar saplings using jasmonic acid, a potent plant hormone involved in responses to wounding and other environmental cues, will result in a significant rise in sugar intermediates at the expense of starch biosynthesis with Eighth International Symposium on In Situ and On-Site Bioremediation, Baltimore, June 2005. consequential increases in both leaf export of mobile sugar and emission of isoprene (Ferrieri et al., 2005; Babst et al., 2005). This suggests that jasmonate may serve as a cue for redirection of recently fixed carbon. We hypothesize that plant utilization of carbon newly acquired through leaf photosynthesis will have major effects on plant metabolism of CCl4, thereby altering foliar emissions. Through labeling of intact leaves of poplar clones (OP637) with the radioactive isotope carbon-11 (t1⁄2 20.4 m) using C-labeled carbon dioxide gas, we have been able to test this hypothesis owing to the extremely short half-life of the tracer which reflects only those processes involving newly acquired carbon. Further, the short half-life of the tracer affords a unique opportunity to retest same plants over time and treatment. MATERIALS AND METHODS Plant growth — Hardwood cuttings from poplar clone (OP637) were greenhouse grown in hydroponics using Hoagland nutrient solution. Age matched plants at 4 weeks were used in all studies and maintained using metal halide lamps (1000 μmol m s). CCl4 Exposure – Excess CCl4 was introduced into the hydroponics solution providing a saturation concentration of 520 ppm. Leaf Emission – Air flowing at 150 mL m passed through a plastic containment bag tied around the entire foliage of the plant. Volatile emissions were captured on molecular sieve cartridges (Tenex GR carbon-based molecular sieve) placed on the outflow. Samples were collected on 2 h intervals during the lighted period. Gas Chromatography Analysis of Volatile Emissions – The glass cartridges were designed to insert directly into the heated injector of a Hewlett Packard 5280A gas chromatograph equipped with a flame ionization detector. Volatile components were thermally desorbed from the cartridge, and individual components isolated using a 8 ft x 1/8 in o.d. stainless steel column packed with Porapak T (Analabs). Data for both isoprene emission and CCl4 emission was obtained from a single analysis. Gas Chromatography Analysis of Leaf TCA Levels – Frozen leaf tissue was ground up, extracted into 80% aqueous methanol at 80C, and samples analyzed by capillary gas chromatography using a 25 m x 0.32 mm o.d. Carbowax 20M column coupled with a flame ionization detector. Radiotracer Administration – Carbon-11 (t 1⁄2 20.4 m) was produced on a JSW 41 in. cyclotron using the N(p, )C nuclear transformation. The isotope was extracted from the target gas as CO2 was administered directly to an intact source leaf using a lighted leaf chamber (Ferrieri et al., 2005). Positron Autoradiogaphy – In some experiments, 90 min after CO2 was administered to a leaf the plant was transferred to a phosphor plate for contact-exposure, and the image recorded (Fuji BAS model 2500) to show radioactivity distribution throughout the plant due to [C]carbohydrate transport. Fuji Image Gauge software was used to quantify radioactivity partitioning between leaf load zone, apex, and lower stem. Eighth International Symposium on In Situ and On-Site Bioremediation, Baltimore, June 2005. FIGURE 1. Photograph on left shows a poplar clone (OP637) set on the imaging bed. Radiographic images, on the right, show extent of [C]partitioning of a control plant acquired at baseline (time zero) and 24 hours later, and of the CCl4 exposed plant shown in the photograph over the same time period. Separate doses of CO2 were administered to the selected leaf encircled. Metabolite Analysis – In other plants, 30 minutes after tracer administration, the exposed leaf was surgically excised at the petiole and extracted using methods described above.