TRANSCRIPTIONAL RESPONSES OF SOYBEAN (GLYCINE MAX) AND THALE CRESS (ARABIDOPSIS THALIANA) PLANTS EXPOSED TO DIFFERENT CLASSES OF ENVIRONMENTAL CONTAMINANTS

Abstract

Plants are exposed to various environmental contaminants through irrigation with reclamation water and land application of municipal biosolids. Plants have been shown to take up contaminants from soil and groundwater, and to some extent, metabolize them in their tissues. These mechanisms have potential important implications for the environment and human health. First, as plants constitute the basis of the terrestrial food chain, accumulation of toxic chemicals or their metabolites inside plant tissues may lead to contamination of animals and humans. Second, the recognition of the capability of plants to take up and metabolize contaminants has led to the development of a plant-based remediation technology, referred to as phytoremediation. Phytoremediation is defined as the use of higher plants for the removal of environmental contaminants from soil and groundwater. Although phytoremediation is conceptually attractive as a green, environmental-friendly technology, the metabolism of xenobiotic compounds by plants is often slow and incomplete, possibly resulting in the accumulation of toxic pollutants and/or their metabolites inside plant tissues. Without further detoxification, phytoremediation may result in pollution transfer, potentially threatening the food chain, and eventually humans. Gaining further knowledge about the fate of environmental contaminants inside plant tissues is therefore of paramount importance for conducting environmental risk assessment and enhancing the efficiency of phytoremediation applications. It's an attractive concept today to cultivate plants on contaminated lands, in order to combine the benefits of phytoremediation with plant-based biofuel production. Unlike conventional plant bioenergy production, plant biomass grown on marginal contaminated soil will not compete with land for food production. However, the effect of contaminants on the plant biomass and bioenergy feedstock yield have received little attention. Molecular biology techniques, such as high-throughput gene expression analysis, constitute powerful tools to understand the molecular bases of the plant metabolism and response to environmental contaminants. The objective of this thesis is to understand the physiological and transcriptional responses of two model plants, thale cress (Arabidopsis thaliana) and soybean (Glycine max), exposed to various classes of contaminants, including silver nanoparticles (AgNPs), pharmaceuticals (zanamivir - ZAN and oseltamivir phosphate - OSP), explosives (2,4,6-trinitrotoluene - TNT), and polychlorinated biphenyls (PCBs). Detection of the contaminants inside plants tissues was performed using advance analytical methods, including inductively-coupled plasma - mass spectrometry (ICP-MS), gas-chromatography - mass spectrometry (GC-MS), and liquid-chromatography (LC-MS). The effects of contaminants on plants were assessed by recording various plant metrics, including biomass, root and shoot length, and soybean production. The transcriptional response of plants to exposure to selected contaminants (AgNPs, OSP, and ZAN) was investigated using whole-genome expression microarrays and reverse-transcription real-time (quantitative) PCR (RT-qPCR). In the first experimental phase of this research, the effects of AgNPs and soluble silver (Ag+) on A. thaliana plants were investigated. AgNPs are widely used nanomaterials, which have raised environmental concerns because of their toxicity to most living organisms, including plants. Exposure of hydroponic A. thaliana plants for 14 days to 20-nm AgNPs resulted in a slight increase of the biomass at low concentrations (1.0 and 2.5 mg / L) and a significant decrease of the biomass at higher concentrations (5.0 to 100 mg / L). Exposure to Ag+ for 14 days resulted in a significant reduction of the biomass after 14 days at concentration at and above 5.0 mg / L. Genome-wide expression microarrays revealed that exposure of A. thaliana to AgNPs and Ag+ at the concentration of 5 mg / L for 14 days resulted in differential expression of many genes involved in the plant response to stress and to biotic and abiotic stimuli. Although distinct gene expression patterns developed upon exposure to AgNPs and Ag+, a significant overlap of differentially expressed genes was observed between the two treatments, suggesting that AgNP-induced stress originated partly from silver toxicity and partly from nanoparticle-specific effects. In the second experimental phase of this research, the effects of the antiviral drugs, OSP and ZAN, on A. thaliana were investigated using an approach similar as the one described above. OSP and ZAN are pharmaceutical drugs that currently constitute the last line of defense against influenza infection. These drugs have been widely detected in wastewater effluents, especially during the influenza season, and they have the potential to contaminate agricultural plants through irrigation and land application of biosolids. Exposure of A. thaliana to OSP showed a significant decrease in the plants biomass at the concentrations of 20 and 100 mg / L, although no significant effect on the biomass was recorded upon exposure to ZAN (up to 100 mg / L), suggesting low acute toxicity of these compounds on plants. On the other hand, Arabidopsis exposure to OSP and ZAN at 20 mg / L resulted in significant transcriptional changes, including up- and down-regulation of many genes involved in the plant response to oxidative stresses and response to stimuli. Comparison with an Arabidopsis gene expression database (Genevestigator), revealed that many genes significantly up- and down-regulated by exposure to OSP and/or ZAN were similarly affected by exposure to biotic and abiotic stresses, toxic chemicals, and hormonal stimuli, suggesting that OSP and ZAN have negative chronic effects on plant health. The third experimental phase of this thesis focuses on the effects of two important persistent pollutants, TNT and PCBs, on the growth of soybean plants, with the objective of assessing the potential of using energy crops for the combined benefit of land remediation and biofuel (biodiesel) production. Explosives, such as TNT, are common toxic contaminants frequently observed at explosive manufacturing sites and military training ranges. PCBs are ubiquitous and toxic contaminants that are found in virtually every compartment of the environment. Short-term growth inhibition tests conducted with TNT and selected PCBs (e.g., 2,4'-dichlorobiphenyl - 2,4'-DCB) showed that these compounds exerted no or mild observable effects on plant growth even when applied at very high concentrations (i.e., 100 to 250 mg / kg soil, respectively). Analysis of TNT and 2,4'-DCB in exposed plant tissues showed average concentrations of 30 - 40 ng/g of TNT and 9,000 to 17,000 ng/g of 2,4'-DCB, which is consistent with biotransformation of TNT inside plant tissues. On the other hand, long-term exposure experiments show that exposure to TNT significantly affected soybean growth and production of bean in TNT-exposed plants (25 - 50 mg / kg soil). Exposure to TNT resulted in a significant decrease of the biomass of harvested beans after 120 days, which may have important consequences on the yield of biodiesel obtained from plants grown on contaminated land. Soybean were then exposed to 2,4'-DCB and its major transformation products, 4-OH-2,4'-DCB). Although high concentrations of the parent PCB (100 and 200 mg / kg) resulted in significant decrease of the biomass, high concentrations of the OH-metabolite resulted in increase of the plant biomass. Future research work will include the determination of the molecular bases of the effects - both positive and negative - of TNT, PCBs, and OH-PCBs on soybean plants and beans.