Functional redundancies in cyanide tolerance provided by β-cyanoalanine pathway genes in arabidopsis thaliana

The β-cyanoalanine pathway is the primary mechanism for detoxification and assimilation of excess cyanide in higher plants. Two genes (AtCysA1 and AtCysC1) mediate the first step of the pathway, and a single gene (AtNit4) mediates the second step. The potential functional redundancy between the enzymes encoded by AtCysA1 or AtCysC1 was examined with knockdown lines for each gene to determine the extent to which cyanide tolerance was altered. A knockdown line for AtNit4 was also examined for alterations in cyanide tolerance and to infer whether activity of the sulfurtransferase pathway can compensate to provide cyanide homeostasis and detoxification if the β-cyanoalanine pathway is disrupted. Methodology. Arabidopsis thaliana (Col-0) and single SALK line knockdown mutants for β-cyanoalanine pathway genes were germinated on Murashige and Skoog medium in plates and grown in hydroponics to accumulate sufficient biomass. Plants were exposed to chronic or acute doses of cyanide, and physiological and biochemical measurements, including transpiration, chlorophyll content and fluorescence, and oxidative stress, were taken to assess the response of the lines to cyanide exposure. Pivotal results. There were only modest phenotypic differences between Col-0 and the mutants AtcysA1 and AtcysC1 in response to cyanide exposure, suggesting that these mutant lines were not impaired in their tolerance to cyanide. The Atnit4 mutant was highly sensitive to cyanide exposure, with significant reductions in all measured parameters in response to cyanide exposure. Conclusions. The results suggest that the AtcysA1 and AtcysC1 mutants are functionally redundant in mediating cyanide detoxification in vivo and that disruption of one gene does not compromise cyanide tolerance. The sensitivity of the Atnit4 mutant implies that when this gene associated with the β-cyanoalanine pathway is disrupted, the sulfurtransferase pathway for cyanide detoxification does not compensate to protect the plants from cyanide exposure. Alternately, the increased sensitivity of the Atnit4 line may be due to the toxicity of the β-cyanoalanine intermediate.