GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA by AMPARO LIMA (Under the Direction of Scott Arthur Merkle) ABSTRACT Arsenic pollution is an environmental problem affecting the health of millions of people worldwide. Unfortunately, conventional remediation technologies for this toxic pollutant are costly and environmentally destructive. An alternative to conventional remediation methods is phytoremediation, the use of plants to extract pollutants from contaminated soil, water and air. Recent studies demonstrated that increasing the thiol- sinks in transgenic plants by over-expressing the bacterial γ-glutamylcysteine synthetase gene resulted in a higher tolerance and accumulation of arsenic. To further explore the potential of transgenic plants to remove arsenate from polluted soil, we genetically engineered eastern cottonwood (Populus deltoides) trees to over-express γ-ECS and, we also established an in vitro propagation system for another phytoremediation candidate, Salix nigra. Our results show that eastern cottonwood trees over-expressing the γ-ECS gene were able to grow normally on toxic levels of arsenate. We also established an in vitro regeneration system for Salix nigra from immature inflorescence explants. INDEX WORDS: Phytoremediation, arsenate, γ-glutamylcysteine synthetase. GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA by AMPARO LIMA Biologo. Autonomous University of the State of Morelos. Mexico. 1999 A Thesis Submitted to The Graduate Faculty of The University of Georgia in Partial Fulfillment of The Requirements for The Degree MASTER OF SCIENCE ATHENS, GEORGIA 2003 2003 AMPARO LIMA All Rights Reserved GENETIC ENGINEERING OF POPULUS DELTOIDES FOR ARSENIC PHYTOREMEDIATION AND THE ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA by AMPARO LIMA Major Professor: Scott A. Merkle Committee: Jeffrey F.D. Dean C. Joseph Nairn Richard B. Meagher Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia May 2003 iv TABLE OF CONTENTS CHAPTER I INTRODUCTION AND LITERATURE REVIEW …………………………..1 CHAPTER II ENHANCED ARSENIC TOLERANCE OF TRANSGENIC EASTERN COTTONWOOD PLANTS OVEREXPRESSING γ-GLUTAMYLCYSTEINE SYNTHETASE………………………………………..…….…………………33 CHAPTER III ESTABLISHMENT OF AN IN VITRO PROPAGATION SYSTEM FOR SALIX NIGRA……………………………………………………………………….…53 CHAPTER IV CONCLUSIONS………………………………………………………………67 1 CHAPTER I INTRODUCTION AND LITERATURE REVIEW Arsenic Contamination Over the past century, mining, agriculture, manufacturing and urban activities have all contributed to extensive soil and water contamination (Cunningham et al., 1995). High on the list of toxic pollutants affecting the health of millions of people worldwide is arsenic (Nriagu, 1994). Arsenic is a naturally occurring element widely distributed on the earth's crust, mainly existing as arsenic sulfide, metal arsenates or arsenites (Emsley, 1991). Arsenic contamination can be from natural or man-made sources. Natural contamination results from the dissolution of naturally existent minerals/ores or soils and up-flow of geothermal water (Emsley, 1991). Man-made pollution generates from most industrial effluents, copper smelting, pesticides and atmospheric deposition (Nriagu, 1988). In the environment, arsenic combines with oxygen, chlorine, and sulfur to form inorganic arsenic compounds (Nriagu, 1994). These toxic metalloids, classified as “group A” human carcinogens, can cause skin lesions, lung, kidney and liver cancer, and damage to the nervous system (U.S. EPA 1996: www.epa.gov/ogwdw/ars/arsenic.htm1). In the United States, hundreds of superfund sites are listed on the National Priority List as having unacceptably high levels of arsenic (www.epa.gov). The processes currently being used to remediate contaminated soils are physical, chemical and biological (Cunningham et al., 1995). These processes either decontaminate the soil or stabilize the pollutant within. Decontamination reduces the amount of pollutants by 2 removing them. Stabilization does not reduce the quantity of pollutant at a site, but makes use of soil amendments to alter the soil chemistry so as to sequester or absorb the pollutant into the matrix, thereby reducing or eliminating environmental risks (Pignatello, 1989; Merian and Haerdi, 1992). Traditional arsenic remediation methods include oxidation, co-precipitation, filtration, adsorption, ion exchange and reverse osmosis. Unfortunately, managing contaminated soils, sludge, and groundwater is costly and the resultant environmental damage is very high (U.S. Army Toxic and Hazardous materials Agency, 1987). The enormous costs and relative ineffectiveness of traditional remediation methods have prompted the development of alternative remediation methods. Phytoremediation There are several species of plants that can survive on highly polluted sites. Most survive by either avoiding toxic materials or by accumulating and sequestering them in their tissues (Baker and Brooks, 1989; Hedge and Fletcher, 1996; Chaudhry et al., 1998; Khan et al., 1998; Schnoor et al., 1995). Plants that use the latter mechanism are known as hyper-accumulators. The following foliar concentrations have been suggested as a threshold to define hyper-accumulation: 10,000 mg/kg for zinc, 1000 mg/kg for copper and 100 mg/kg for cadmium (Reeves et al., 1995). The ability of some plants to hyper- accumulate, and in some cases degrade, toxic compounds gave rise to an alternative remediation method known as phytoremediation. Phytoremediation uses plants to extract, sequester or detoxify pollutants from soil, water and air (Rashkin, 1996). This innovative technology offers advantages over 3 conventional physical or chemical techniques. It is estimated that phytoremediation costs can be between two- and four-fold less than existing remediation technologies (Meagher and Rugh, 1996). In addition, this approach is an ecologically preferable method because it reclaims soil in situ instead of permanently removing it to a storage site (Salt et al., 1995). Although phytoremediation as a technology is still in its development stages, it has become a rapidly expanding research area because of its promise for the remediation of organic and inorganic pollutants. Organic pollutants include polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), nitroaromatics, and linear halogenated hydrocarbons (Meagher, 2000). In phytoremediation, the main goal is to completely mineralize these compounds into relatively non-toxic constituents, such as carbon dioxide, nitrate, chlorine, and ammonia (Cunningham et al., 1996). Using plants, organic pollutants can be remediated through several biophysical and biochemical processes including absorption, transport and translocation or hyper-accumulation, or transformation and mineralization (Meagher 2000). Inorganic pollutants include toxic metals such as aluminum, arsenic, cadmium, chromium, copper, lead, mercury, nickel, zinc, cesium, strontium and uranium (Salt et al., 1998). Inorganic pollutants are immutable at an elemental level and cannot be degraded or mineralized (Salt et al., 1998); thus, their remediation is difficult to achieve (Meagher and Rugh, 1996). Plant-based phytoremediation strategies for inorganic pollutants rely on plant roots to extract, vascular systems to transport, and leaves to act as sinks to concentrate these pollutants (Dhankher et al., 2002). 4 Phytoremediation strategies for arsenic contaminated soils are not very common, but the few existing studies show great promise for the potential applications of this alternative remediation method. Arsenic Phytoremediation As previously mentioned, certain plant species have the capacity to extract pollutants from soil or water through their normal root uptake of nutrients. The plants then store these compounds in their cells or convert them into less toxic forms (Meagher 2000). To date, there is only one report of a plant with the ability to handle arsenic in this manner. Pteris vittata, a fern indigenous to the southern parts of the U.S., has the capacity to hyper-accumulate arsenic to very high levels (7500 ppm; Ma et al., 2001). Unfortunately, the enzymes responsible for arsenic hyper-accumulation in this plant are not yet available for manipulation into other plant species. Although specific arsenic hyper-accumulation enzymes have not been isolated, increased tolerance and accumulation of arsenic has been reported in plants over-expressing the bacterial enzyme γ-glutamylcysteine synthetase (Dr. Yujing Li, Genetics Department, University of Georgia, personal communication). Gamma-glutamylcysteine synthetase (γ-ECS) forms part of a three-step enzymatic pathway responsible for the synthesis of phytochelatins. In plants, heavy metal detoxification often occurs through the chelation of metal ions by metal-binding ligands (Cobbett, 2000). To date, a number of metal-binding ligands have been recognized and among the most studied are the phytochelatins. They are members of a small class of Cys sulfhydryl residue-rich peptides [γ-glumatylcysteine (γ-EC), glutathione (GSH) and 5 phytochelatins (PC)] that play an important role in the detoxification and sequestration of thiol-reactive heavy metals (Noctor et al., 1998; Zhu et al., 1999b; Xiang et al., 2001). These γ-EC containing peptides are derived from common amino acids in a three-step reaction.