Sharon L. Doty

Enhancing phytoremediation through the use of transgenics and endophytes

In the last decade, there has been an increase in research on improving the ability of plants to remove environmental pollution. Genes from microbes, plants, and animals are being used successfully to enhance the ability of plants to tolerate, remove, and degrade pollutants. Through expression of specific bacterial genes in transgenic plants, the phytotoxic effects of nitroaromatic pollutants were overcome, resulting in increased removal of these chemicals. Overexpression of mammalian genes encoding cytochrome P450s led to increased metabolism and removal of a variety of organic pollutants and herbicides. Genes involved in the uptake or detoxification of metal pollutants were used to enhance phytoremediation of this important class of pollutants. Transgenic plants containing specific bacterial genes converted mercury and selenium to less toxic forms. In addition to these transgenic approaches, the use of microbes that live within plants, termed endophytes, also led to improved tolerance to normally phytotoxic chemicals and increased removal of the pollutants. Bacteria that degraded a herbicide imparted resistance to the herbicide when inoculated into plants. In another study, plants harboring bacteria capable of degrading toluene were more tolerant to normally phytotoxic concentrations of the chemical, and transpired less of it into the atmosphere. This review examines the recent advances in enhancing phytoremediation through transgenic plant research and through the use of symbiotic endophytic microorganisms within plant tissues.

Enhanced phytoremediation of volatile environmental pollutants with transgenic trees.

Small, volatile hydrocarbons, including trichloroethylene, vinyl chloride, carbon tetrachloride, benzene, and chloroform, are common environmental pollutants that pose serious health effects. We have developed transgenic poplar (Populus tremula × Populus alba) plants with greatly increased rates of metabolism and removal of these pollutants through the overexpression of cytochrome P450 2E1, a key enzyme in the metabolism of a variety of halogenated compounds. The transgenic poplar plants exhibited increased removal rates of these pollutants from hydroponic solution. When the plants were exposed to gaseous trichloroethylene, chloroform, and benzene, they also demonstrated superior removal of the pollutants from the air. In view of their large size and extensive root systems, these transgenic poplars may provide the means to effectively remediate sites contaminated with a variety of pollutants at much faster rates and at lower costs than can be achieved with current conventional techniques.

Diazotrophic endophytes of native black cottonwood and willow

Poplar and willow are economically-important, fast-growing tree species with the ability to colonize nutrient-poor environments. To initiate a study on the possible contribution of endophytes to this ability, we isolated bacteria from within surface-sterilized stems of native poplar (Populus trichocarpa) and willow (Salix sitchensis) in a riparian system in western Washington state. Several of the isolates grew well in nitrogen-limited medium. The presence ofnifH, a gene encoding one of the subunits of nitrogenase, was confirmed in several of the isolates including species ofBurkholderia, Rahnella, Sphingomonas, andAcinetobacter. Nitrogenase activity (as measured by the acetylene reduction assay) was also confirmed in some of the isolates. The presence of these diazotrophic microorganisms may help explain the ability of these pioneering tree species to grow under nitrogen limitation.

Characterization of bacterial endophytes of sweet potato plants.

Endophytic bacteria associated with sweet potato plants (Ipomoea batatas (L.) Lam.) were isolated, identified and tested for their ability to fix nitrogen, produce indole acetic acid (IAA), and exhibit stress tolerance. Eleven different strains belonging to the genera, Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas and Phyllobacterium, were identified. Four strains were shown to produce IAA (a plant growth hormone) and one strain showed the ability to grow in nitrogen free medium and had the nitrogenase subunit gene, nifH. To determine if IAA production by the endophytes had any role in protecting the cells against adverse conditions, different stress tests were conducted. The IAA producer grew well in the presence of some antibiotics, UV and cold treatments but the response to pH, osmotic shock, thermal and oxidative treatments was the same for both the IAA producer and the no IAA producer. To determine if IAA produced by the strains was biologically relevant to plants, cuttings of poplar were inoculated with the highest IAA producing strain. The inoculated cuttings produced roots sooner and grew more rapidly than uninoculated cuttings. These studies indicate that endophytes of sweet potato plants are beneficial to plant growth.

Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees

Three endophytic yeast, one isolated from stems of wild cottonwood (Populus trichocarpa), two from stems of hybrid poplar (P. trichocarpaxPopulus deltoides), were characterized by analyzing three ribosomal genes, the small subunit (18S), internal transcribed spacer (ITS), and D1/D2 region of the large subunit (26S). Phenotypic characteristics of the yeast isolates were also obtained using a commercial yeast identification kit and used for assisting the species identification. The isolate from wild cottonwood was identified to be closest to species Rhodotorula graminis. The two isolates from hybrid poplar were identified to be species Rhodotorula mucilaginosa. In addition, the three yeast isolates were observed to be able to produce indole-3-acetic acid (IAA), a phytohormone which can promote plant growth, when incubated with l-tryptophan. To our knowledge, the yeast strains presented in this study were the first endophytic yeast strains isolated from species of Populus.

Phytoremediation of Organic Contaminants: A Review of Phytoremediation Research at the University of Washington

As overwhelmingly positive results have become available regarding the ability of plants to degrade compounds such as trichloroethylene, phytoremediation studies are expanding. Studies to determine the potential for phytoremediation of fully chlorinated compounds, such as carbon tetrachloride and tetrachloroethylene, brominated compounds, such as ethylene dibromide and dibromochloropropane, and nonhalogenated compounds, such as methyl-t-butyl ether (MTBE), are underway. When using phytoremediation, it is important to select not only a plant that is capable of degrading the pollutant in question, but also one that will grow well in that specific environment. In ecologically sensitive areas, such as the Hawaiian Islands, only plants native to the area can be used. One way to supplement the arsenal of plants available for remedial actions is to utilize genetic engineering tools to insert into plants those genes that will enable the plant to metabolize a particular pollutant. Hybrid technologies, such as usin...

Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Recently, plants, poplar trees in particular, have been investigated as a tool to remove TCE from soil and groundwater. The metabolism of TCE in plants is being investigated for two reasons: one, plant uptake and metabolism represent an important aspect of the environmental fate of the contaminant; two, metabolism pattern and metabolite identification will help assess the applicability of phytoremediation. It was previously shown that TCE metabolites in plants are similar to ones that result from cytochrome P450-mediated oxidation in mammals: trichloroethanol, trichloroacetate and dichloroacetate. Our measurements indicate that one of these metabolites, trichloroethanol, is further glycosylated in tobacco and poplar. The glycoside was detected in all tissues (roots, stems and leaves) in comparable levels, and was at least 10 fold more abundant than free trichloroethanol. The glycoside in tobacco was identified as the ss-D-glucoside of trichloroethanol by comparison of the mass spectra and the chromatographic retention time of its acetylation product to that of the synthesized standard. Trichloroethanol and its glucoside did not persist in plant tissue once plants are removed from TCE contaminated water, indicating further metabolism.

Biological nitrogen fixation and biomass accumulation within poplar clones as a result of inoculations with diazotrophic endophyte consortia

Sustainable production of biomass for bioenergy relies on low-input crop production. Inoculation of bioenergy crops with plant growth-promoting endophytes has the potential to reduce fertilizer inputs through the enhancement of biological nitrogen fixation (BNF). Endophytes isolated from native poplar growing in nutrient-poor conditions were selected for a series of glasshouse and field trials designed to test the overall hypothesis that naturally occurring diazotrophic endophytes impart growth promotion of the host plants. Endophyte inoculations contributed to increased biomass over uninoculated control plants. This growth promotion was more pronounced with multi-strain consortia than with single-strain inocula. Biological nitrogen fixation was estimated through (15)N isotope dilution to be 65% nitrogen derived from air (Ndfa). Phenotypic plasticity in biomass allocation and branch production observed as a result of endophyte inoculations may be useful in bioenergy crop breeding and engineering programs.

Latent homology and convergent regulatory evolution underlies the repeated emergence of yeasts

Convergent evolution is common throughout the tree of life, but the molecular mechanisms causing similar phenotypes to appear repeatedly are obscure. Yeasts have arisen in multiple fungal clades, but the genetic causes and consequences of their evolutionary origins are unknown. Here we show that the potential to develop yeast forms arose early in fungal evolution and became dominant independently in multiple clades, most likely via parallel diversification of Zn-cluster transcription factors, a fungal-specific family involved in regulating yeast-filamentous switches. Our results imply that convergent evolution can happen by the repeated deployment of a conserved genetic toolkit for the same function in distinct clades via regulatory evolution. We suggest that this mechanism might be a common source of evolutionary convergence even at large time scales.

Improving phytoremediation through biotechnology.

Sharon Doty received a BS degree in genetics from the University of California, Davis (1989), and a PhD in Microbiology from the University of Washington (1995). She did postdoctoral research in plant biochemistry (UW). Her current position is assistant professor in the College of Forest Resources, UW. Her research program covers three main areas: phytoremediation, the use of plants for removing environmental pollutants; bioenergy, specifically the use of non-food plants; and biological nitrogen fixation in non-legume plants. Phytoremediation of contaminated soils offers an environmentally friendly, cost effective, and carbon neutral approach for the clean up of toxic pollutants in the environment. Plants with abilities to hyperaccummulate heavy metals, uptake volatile organic compounds, and sequester pollutants have been proposed as a solution to the treatment of toxic contamination in situ. However, the use of plant-based technologies has a number of limitations, primarily due to the fact that plants are autotrophs and not ideally suited for the metabolism and breakdown of organic compounds. One of the major limitations with current phytoremediation is the often slow time-scale for remediation to acceptable levels and also toxicity to the plants themselves. To some extent, this can be addressed through interactions with the natural micro flora associated with plants; both endophytic bacteria and rhizosphere bacteria have been shown to have the potential to degrade organic compounds in association with plants.