BIOINDICATION WITH TRANSGENIC PLANTS

Prior to 1998, transgenic plants were primarily generated to increase the quantity and improve the quality of food, and for cleaning the environment in a process known as phytoremediation. However during that time, scientists from Ukraine and Switzerland also used transgenic plants as biomonitors to assess the quality of environmental surroundings.  Among the first laboratory tests used for biomonitoring were the Ames test and a variety of gain and loss of function tests using E. coli-based systems. However, these methods are insufficient for environmental studies of higher eukaryotes. Other previously available analytical methods and laboratory tests were not designed to mimic environmental conditions for the level of pollutant exposure and type of biological interactions occurring in the field. Therefore, alternative methods of field biomonitoring were developed that allowed tests using organisms involved in absorbing and integrating doses of toxicants from polluted water and soils. Animals are difficult to use as model systems in environmental studies, especially in the analysis of chronic exposure effects, because of their unsettled lifestyle. Two major exceptions are zebrafish and land snails. The transgenic zebrafish assay, which uses a non-active lacI transgene as a target gene, could potentially be applied to evaluate water quality.(1) Land snails can also be considered as an attractive system for soil toxicity assessment. They live on limited territories and therefore are constantly exposed to local contaminants. However, toxic concentrations of heavy metals need to be relatively high in order to be sensed by these animals.

Classical Plant Biosensors

Several studies have reported on plants used as biosensors of genetic toxicity in environmental pollutants. Most of the systems commonly used to study mutations in plants were based on the detection of chromosomal aberrations in Allium cepa, Tradescantia,or Vicia faba plants. Among the plant systems that are applied to environmental studies, specific mention should be made of tobacco plants heterozygous for the Sulfur (Su) nuclear gene that affects the chlorophyll content of leaves. These plants have been used to study the mutagenicity of different chemicals and could also be used for environmental mutagenesis studies, although the type of the alterations that produce the phenotype is still unknown at the molecular level.

Biomonitoring Of Radioactive Pollution With Classical Tests

One of the first attempts to monitor the environmental effects of chronic radiation was carried out in Japan using the stamen-hair mutation assay, TrSHM, of Tradescantia; increased mutation frequencies were correlated with wind direction and nuclear power plant operation periods.(2) The Allium cepa chromosome aberration test was also used for estimating mutagenicity of ionizing radiation. The inhibition of root development in onion bulbs and the increased incidences of chromosomal aberrations in root cells after exposure to radiation were documented.

Recently, we have developed a new microsatellite-based assay for monitoring radiation-induced germline mutation in plants. Thirteen microsatellite loci of two initially genetically-identical populations of wheat (Triticum aestivum, L.) grown in either heavily contaminated or clean control soil have been profiled. A marked 6.5-fold increase in germline mutation rate in the parental generation was found among the offspring of exposed plants.(3) Of special practical importance is the fact that we could show a statistically significant difference in the germline mutation rate with a relatively small sample size. It should be pointed out that detection of the same increase in mutation rate by standard genetic techniques would only have been possible by using nearly a million plants.

Transgenic Plants As Pollution Biosensors

"Plant recombination" assay

We have conducted a number of field and laboratory environmental monitoring experiments using this transgenic Arabidopsis. Plants were grown in seven experimental soil plots in the Chernobyl exclusion zone (zone 1), as well as in different areas of the three other contaminated zones (zones 2 - 4). A dose-dependent increase in HR events to 8.4-fold over the control level was detected in plant populations at pollution levels up to 300 Ci/km2 in the open field and up to 11.0-fold at pollution levels up to 1000 Ci/km2 in the laboratory experiment.(4) "Plant mutation" assay.  The level of precise repair of DNA breaks is measured by homologous recombination events in plants. However, HR does not show the frequency with which repair mistakes lead to mutations. Somatic mutation events are of a particular importance in plants, since they can potentially be passed on to subsequent generations.  A new method of measuring somatic mutation events in plants has been developed. A termination codon introduced into the ?-glucuronidase (uidA) gene completely prevented the translation of active protein; transgenic Arabidopsis plants carrying these inactivated uidA genes were generated. We could observe the spontaneous restoration of uidA activity (as blue spots on plants) due to the reversion of the stop codons to the original codons.

Both types of the transgenic systems ("recombination" and "mutation") were applied to study the genotoxicity of heavy metal ions. Plants sown on media contaminated by the salts of heavy metals, Cd, Pb, Ni, Zn, Cu, and As2O3, exhibited a pronounced uptake-dependent increase in the frequencies of both somatic intrachromosomal homologous recombination and point mutation events. Test plants were also sown in soils collected from sites exhibiting different levels of contamination with Pb, Cd, Zn, and other elements. A four- to sevenfold increase in the frequency of HR and a five- to tenfold induction of point mutations in plants grown in contaminated soils compared to those grown in clean control soil were noted.(5)

The transgenic recombination lines have also been used to evaluate potentially mutagenic exposure to various levels of UV-B radiation.  Experiments using specialized sun-simulators revealed that elevated UV-B increases the frequency of somatic HR in a dose dependent manner.(6) In addition, the system permitted measurement of germline recombination either as a result of an inherited late somatic event or as a meiotic recombination event. Elevated levels of UV-B increased the appearance of plants totally stained blue-that is, plants in which the restoration of the marker gene was inherited by a factor of between two and five.

Outlook

Though several biological systems for evaluating the influence of environmental pollution have been developed, there is no easy test system based on a higher eukaryotic organism yet available. We have introduced a new transgenic plant approach that is fast, sensitive, and in which mutagen-induced HR and point mutation events can be visualized. Since this system allows rapid data collection (around four weeks) and does not require sophisticated equipment and specific knowledge for the detection and scoring of recombination events, it can be broadly used for environmental studies. Soil and water contaminated with metals and organic toxicants pose a major environmental and human health problem that is still in need of an effective and affordable technological solution. We have generated transgenic plants that are able to "sense" the presence of inorganic and, possibly, organic pollutants.  Moreover, these transgenic plants are likely to be useful as phytoremediation devices for removing pollutants from soil and water, accumulating them in their biomass, and detoxifying or vaporizing them. Our plants potentially can be used in remediation quality control tests to evaluate the mutagenicity of contaminated soils before and after remediation. Thus, transgenic plants are beginning to have value as biosensors as well as for the efficient cleanup and post-remediation control of contaminated soil and water. In order to be an effective "alarm system," the test organism needs to provide a warning of a possible hazard before ecologically significant damage can occur. Plants at the base of the food chain are sensitive to toxicants sooner than those at higher trophic levels, thereby reducing the lag period between exposure and significant impact.

Sources

1. Amanuma K, et al. 2000. Transgenic zebrafish for detecting mutations caused by compounds in aquatic environments. Nature Biotechnology 18: 62-65.

2. Ichikawa S. 1981. In situ monitoring with Tradescantia around nuclear power plants. Environmental Health Perspectives 37: 145-164.

3. Kovalchuk O, et al. 2000. Wheat DNA mutation rate after Chernobyl. Nature 407: 583-584.

4. Kovalchuk I, Kovalchuk O, Arkhipov A, and Hohn B. 1998. Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident. Nature Biotechnology 16:1054-1057.

5. Kovalchuk O, Titov V, Hohn B, Kovalchuk I. 2001. A sensitive transgenic plant system to detect toxic inorganic compounds in the environment. Nature Biotechnology 19: 568-72.

6. Ries G, et al. 2000. Elevated UV-B radiation reduces genome stability in plants. Nature 406: 98-101.

Igor Kovalchuk and Olga Kovalchuk

Department of Biological Sciences

University of Lethbridge

mailto:igor.kovalchuk@uleth.ca