EFFECT OF ABIOTIC STRESSES AND CYANIDE TREATMENT ON THE CYANIDE ASSIMILATORY PATHWAY IN ARABIDOPSIS THALIANA

AN ABSTRACT OF THE THESIS OF: Aissatou Sidibe, for the Master of Science degree in Plant Biology, presented on November 6, 2008, at Southern Illinois University Carbondale TITLE: EFFECT OF ABIOTIC STRESSES AND CYANIDE TREATMENT ON THE CYANIDE ASSIMILATORY PATHWAY IN ARABIDOPSIS THALIANA MAJOR PROFESSOR: Dr. Stephen D. Ebbs Cyanide HCN is a vey toxic chemical and typically targets the mitochondrion to block the electron transport chain among other effects. Beside its toxicity, cyanide occurs naturally in plants, predominantly from the synthesis of ethylene and from the hydrolysis of cyanogenic glycosides in some plant families. Higher plants biosynthesize cyanide mainly from oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) which is also precursor of ethylene biosynthesis. Ethylene production is known to increase under certain developmental stage such as germination, senescence, abscission and fruit ripening. Stress cyanide is also produced during abiotic and biotic stress in plants because stress elicits ethylene synthesis. Endogenous cyanide as well the cyanide that is acquired from the environment is detoxified by the cyanoalanine synthase (CAS) pathway. CAS (EC 4.4.1.9), which is localized to the mitochondrion is the principal enzyme of cyanide metabolism in higher plants and is responsible for the formation of β-cyanoalanine in the presence of cysteine and cyanide. The β-cyanoalanine formed is turned into asparagine by cyanoalanine hydratase. Given that abiotic and biotic stress elicit ethylene synthesis and that ethylene elicits CAS activity, this relationship suggests that the CAS pathway may provided a means for recycling stress cyanide back into amino acid pools. In addition to potentially poisoning metabolism, the production of cyanide represents a fundamental loss of assimilation carbon and nitrogen. The CAS pathway would provide a mechanism of returning those atoms to primary metabolism, which would be particularly critical when plants are confronted with stress. The most important goal of this research was to provide data on the response of the CAS pathway to selected abiotic stresses as well as to exposure to exogenous cyanide. The experiments were conducted with the model plant Arabidopsis thaliana (Col-0) type and the SALK mutants lines with a t-DNA inserted into the genes AtCysC1 and AtNIT4. The first objective of this research was to determine how AtCysC1 and AtNIT4 gene expression responds to specific abiotic stresses (water deficit, temperature) and cyanide exposure in Arabidopsis thaliana Col 0 wild type. Non-quantitative and Real Time PCR analysis were used to address the issue. The second objective was to determine the effect of abiotic stresses on CAS enzyme activity and cyanide biosynthesis in the wild type and mutants lines. The final objective was to determine if exposure to 100 μM KCN cyanide in hydroponics increased the enzymatic activity of CAS in the Arabidopsis Salk lines and the wild type. A colorimetric assay was used to analyze the cyanide concentration and CAS activity. The Real time PCR results revealed a significant increase in transcript for AtCysC1 of the water deficit, cold and cyanide treatment. On the other hand, the AtNIT4 Real Time PCR showed transcript induction only on the cold treated plants. The tissue analysis results have shown a significantly increase of CAS activity in water deficit and cold treated Arabidopsis wild type shoots. However the cyanide content in the plants treated with abiotic stress and cyanide did not change in comparison to the control. The cyanide treatment did not have an affect on the CAS activity of Arabidopsis wild type and mutants. There was no significant difference between the two mutant\u27s lines in terms of cyanide content and CAS activity and there was a significant difference between the mutants and the wild type in CAS activity. Given the physiological and molecular results of this research, we can say that cyanide assimilation in Arabidopsis thaliana is controlled by a transcriptional and post transcriptional mechanism and involved AtCysC1 and AtNIT4 genes as well as the CAS