Increased β-Cyanoalanine Nitrilase Activity Improves Cyanide Tolerance and Assimilation in Arabidopsis

Plants naturally produce cyanide （CN） which is maintained at low levels in their cells by a process of rapid assimilation. However, high concentrations of environmental CN associated with activities such as industrial pollution are toxic to plants. There is thus an interest in increasing the CN detoxification capacity of plants as a potential route to phytoremediation. Here, Arabidopsis seedlings overexpressing the Pseudomonas fluorescens β-cyanoalanine nitrilase pinA were compared with wild-type and a β-cyanoalanine nitrilase knockout line （△Atnit4） for growth in the presence of exogenous CN. After incubation with CN, ＋PfpinA seedlings had increased root length, increased fresh weight, and decreased leaf bleaching compared with wild-type, indicating increased CN tolerance. The increased tolerance was achieved without an increase in β-cyanoalanine synthase activity, the other enzyme in the cyanide assimilation pathway, suggesting that nitrilase activity is the limiting factor for cyanide detoxification. Labeling experiments with [^13C] KCN demonstrated that the altered CN tolerance could be explained by differences in flux from CN to Asn caused by altered β-cyanoalanine nitrilase activity. Metabolite profiling after CN treatment provided new insight into downstream metabolism, revealing onward metabolism of Asn by the photorespiratory nitrogen cycle and accumulation of aromatic amino acids.

Quinclorac Resistance in Echinochloa crus-galli from China

Echinochloa crus-galli is a major weed in rice fields in China,and quinclorac has been long used for its control.Over-reliance of quinclorac has resulted in quinclorac resistance in E.crus-galli.Two resistant(R)E.crus-galli populations from Hunan,China were confirmed to be at least 78-fold more resistant to quinclorac than the susceptible(S)population.No difference in foliar uptake of 14C-labelled quinclorac was detected between the R and S plants.However,a higher level of 14C translocation and a lower level of quinclorac metabolism were found in the R plants.Basal and induced expression levels ofβ-cyanoalanine synthase(β-CAS)gene andβ-CAS activity were not significantly different between the R and S plants.However,the induction expression of 1-aminocyclopropane-1-carboxylic acid oxidase(ACO1)gene by quinclorac treatment was evident in the S plants but not in the R plants.Quinclorac resistance in the two resistant E.crus-galli populations was not likely to be related to foliar uptake,translocation or metabolism of quinclorac,nor to cyanide detoxification viaβ-CAS.Thus,target-site based quinclorac signal reception and transduction and regulation of the ethylene synthesis pathway should be the focus for further research.

Cysteine and Cysteine-Related SignalingPathways in Arabidopsis thaliana

Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor moleculeinvolved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its deriva-tive molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine issynthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed byO-acetylserine（thiol）lyase （OASTL）. Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resultingin a complex array of isoforms and subcellular cysteine pools, in recent years, significant progress has been made inArabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the dis-covery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCSwith S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions.Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signalingmolecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essentialrole in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which isessential for root hair development and plant responses to pathogens.

Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine（thiol）lyase C in Arabidopsis thaliana

In non-cyanogenic species, the main source of cyanide derives from ethylene and camalexin biosyntheses. In mitochondria, cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolized by the β-cyanoalanine synthase CYS-C1, catalyzing the conversion of cysteine and cyanide to hydrogen sulfide and β-cyanoalanine. The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine（thiol）lyase mitochondrial isoform, OAS-C, which catalyzes the incorporation of sulfide to O-acetylserine to produce cysteine, thus generating a cyclic pathway in the mitochondria. The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme, showing defects in root hair formation. Genetic complementation with the OAS-Cgene rescues the impairment of root hair elongation, restoring the wild-type phenotype. The mitochondria compromise their capacity to properly detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant. Consequently, we observe an accumulation of sulfide and cyanide and of the alternative oxidase, which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules. Our results allow us to suggest that the significance of OAS-C is related to its role in the proper sulfide and cyanide detoxification in mitochondria.