| The effect of the antibiotic and environmental pollutant, chlortetracycline (CTC), on the growth and viability of maize and pinto bean plants was investigated. An interdisciplinary approach was used that combined biochemical and molecular biology tools, in coordination with liquid chromatography/mass spectrometry (LC/MS) methods. It was first necessary to determine what effect chlortetracycline uptake by the plants would have on overall protein expression levels. Second, any identifiable differences in protein expression levels needed to be characterized. Using sodium dodecylsulfate--polyacrylamide gel electrophoresis (SDS-PAGE), a protein band with a molecular weight in the range of 20-30 kDa was visually observed to increase in the maize plants treated with CTC, relative to the untreated controls. This difference was not observed in pinto beans. Since maize is known to be tolerant to CTC, while pinto beans are intolerant, these findings suggest that the protein induced in response in CTC-treated maize may play a role in CTC tolerance. Further analysis showed that, in correlation with the SDS-PAGE data, glutathione S-transferase (GST) is induced in CTC-treated maize. Using liquid chromatography with ion trap mass spectrometry (LC-IT/MS/MS), it was shown that GSTs isolated from CTC-treated maize were capable of conjugating glutathione (GSH) to CTC in vitro . At the same time, this process could not be observed in GSTs isolated from pinto bean plants. Using bottom-up mass spectrometry techniques, as well as quantitative polymerase chain reaction (QPCR), the GST 27 isoform was identified as a putative candidate specific for CTC detoxification in maize. However, it appears that CTC detoxification in maize has adverse side effects with respect to detoxification of herbicides, which are also xenobiotic stressors of agricultural plants. In vitro analysis of GSTs isolated from CTC-treated maize show a reduced response to detoxification of two common classes of herbicides, chlorotriazine and chloracetanilide, relative to GSTs isolated from the untreated maize. Pinto beans, on the other hand, did not show any observable response to CTC uptake, lacked the ability to detoxify CTC, and show adverse effects in response to CTC. Since CTC is a known calcium chelator, a biolistic assay was developed to determine the effects of CTC on intracellular calcium levels in pinto bean leaves. This novel method showed that CTC is a significant intracellular calcium chelator in pinto beans, and suggests that calcium chelation is one possible mechanism for CTC phytotoxicity in pinto beans. |
