Mutation in Arabidopsis β-cyanoalanine synthase overcomes NADPH oxidase action in response to pathogens

Plant responses to pathogens comprise a complex process, implying a plethora of signals and reactions. Among them, endogenous production of hydrogen cyanide (HCN) has been shown to induce resistance in Arabidopsis to the hemibiotrophic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. β-cyanoalanine synthase (CAS-C1) is responsible for the detoxification of HCN in Arabidopsis mitochondria. Here, we show that green fluorescent protein-tagged CAS-C1 is transiently reduced in leaves infected with an avirulent strain of Pst during early interactions and increased in leaves infected with a virulent strain of Pst, supporting previous transcriptional data. Genetic crosses show that mutation in CAS-C1 in Arabidopsis resembles the action of the NADPH oxidase RbohD independently of reactive oxygen species production and that the accumulation of salicylic acid is required for HCN-stimulated resistance to Pst. Finally, we show that the cas-c1 mutation acts on the salicylic acid-dependent response to pathogens by mechanisms other than protein ubiquitination or the increase of monomerization and entry to the nucleus of NPR1, the central regulator of the salicylic acid-mediated response. Considering these results, we propose new mechanisms for modulation of the immune response by HCN.

TISSUE DISTRIBUTION AND BIOCHEMICAL CHARACTERIZATION OF BETA-CYANOALANINE SYNTHASE FROM COMMON BEAN SEEDS (PHASEOLUS VULGARIS)

Cyanide is produced throughout a plant's life cycle, its production increases during certain developmental stages such as seed germination, seedling elongation, fruit ripening and senescence. Beta-cyanoalanine synthase is the most important cyanide metabolizing enzyme found in plants. This work is aimed at studying the tissue distribution, partial purification and some physicochemical properties of β-cyanoalanine synthase from bean seeds (Phaseolus vulgaris). β-cyanolalanine synthase was isolated and partially purified using a combination of ammonium sulphate precipitation, desalting on Sephadex G-10 and gel filtration chromatography on Sephacryl S-200 column. The biochemical characteristics of the enzyme were investigated. Results obtained from this work showed that β-cyanolalanine synthase is more concentrated in the seeds (20.53 nmol/HS/mg) when compared 2 to the cotyledons (10.08 nmol/HS/mg) and the seed coats (5.82 nmol/HS/mg). The partially purified 2 2 enzyme showed a specific activity of 26.77 nmol/HS/mg and an apparent molecular weight of about 2 60,000Da, K values for cyanide and L-cysteine of 0.741 mM and 1.724 mM respectively. The V max m value obtained for cyanide was 25.00 nmol/H S/ml/minwhile that of L-cysteine was 2 o666.67nmol/H S/ml/min. The enzyme showed an optimum temperature of 40C and optimum pH of 2 10.0.Studies on the effect of chloride salt indicated that NaCl and MnCl had strong inhibitory effect 2 on the enzyme; NHCl had slight negative effect while KCl and ZnCl activated the enzyme dose 4 2 dependently.This study showed the presence of β-cyanoalanine synthase in bean seeds which is believed to function in the detoxification of cyanide produced in its tissues especially during germination.

Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.