Screening of Mutant Arabidopsis Thaliana and Chlamydomonas Reinhardtii for their Potential Use as Phytosensors in 2,4,6 Trinitrotoluene (TNT) Contaminated Environments

Plant biotechnology is a diverse field that is expanding from agricultural research towards environmental applications. The focus of this project was to exploit vegetative effects, such as photosynthesis and growth in genomic model organisms Arabidopsis thaliana and Chlamydomonas reinhardtii to 2,4,6-trinitrotoluene (TNT) with a goal to develop biomonitoring systems. Plants and algae have evolved with various biochemical pathways that have the potential to be exploited for the use of sensing explosives and chemical warfare agents in soil, water and air. The first part of the project involved characterizing the effects of TNT on germination and early seedling development of wild-type Arabidopsis thaliana. It was determined that 10 mM TNT was the tolerance level for Arabidopsis and was used to screen fast neutron irradiated mutant Arabidopsis to evaluate the phenotypic stress responses in the seedlings. TNT responsive mutant lines (lines 1, 2, 3, and 4) were selected on a basis of a leaf color change from dark green to pale green. The second part of the project was to determine the growth response of wild-type and mutant Chlamydomonas reinhardtii to TNT. Growth response studies of wild-type Chlamydomonas revealed that 3 mg/ml of TNT was the maximum TNT concentration that allowed growth. Insertional mutant lines were screened on 3 mg/ml TNT where one mutant (CL48) was selected on the basis of a color change from green to white. Growth response of CL48 in TNT indicated that this mutant line was hypersensitive to TNT compared with transformation recipient line and wild-type Chlamydomonas. The third part of the project involved using microarray technology to determine the differential gene expression of Chlamydomonas in response to TNT. Approximately 158 responsive genes were differentially expressed. Genes involved in photosynthesis and energy metabolism were up-regulated in the presence of TNT. TNT may cause oxidative stress since many oxidative stress related genes were up-regulated. Among the down-regulated genes, the expression of cell wall-related genes was repressed. Several unidentified genes were also induced or repressed. The overall study promotes future work involving the identification of the genes that are involved in TNT response.