Β-cyanoalanine Synthase Activity Research Articles

β-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified β-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and β-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its β-cyanoalanine synthase activity. Consistent with the β-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the β-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite’s ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Horizontally acquired cysteine synthase genes undergo functional divergence in lepidopteran herbivores

Horizontal gene transfer (HGT) plays an important role in evolutionary processes as organisms adapt to their environments, and now cases of gene duplication after HGT in eukaryotes are emerging at an increasing rate. However, the fate and roles of the duplicated genes over time in eukaryotes remain unclear. Here we conducted a comprehensive analysis of the evolution of cysteine synthase (CYS) in lepidopteran insects. Our results indicate that HGT-derived CYS genes are widespread and have undergone duplication following horizontal transfer in many lepidopteran insects. Moreover, lepidopteran CYS proteins not only have β-cyanoalanine synthase activity but also possess cysteine synthase activity that is involved in sulfur amino acid biosynthesis. Duplicated CYS genes show marked divergence in gene expression patterns and enzymatic properties, suggesting that they probably have undergone subfunctionalization and/or neofunctionalization in Lepidoptera. The gene transfer of CYS genes and subsequent duplication appears to have facilitated the adaptation of lepidopteran insects to different diets and promoted their ecological diversification. Overall, this study provides valuable insights into the ecological and evolutionary contributions of CYS in lepidopteran insects.

Cyanogenic glycosides can function as nitrogen reservoir for flax plants cultured under N-deficient conditions

Soil nitrogen (N) deficiency is a common phenomenon that plagues both naturally growing plants as well as agricultural crops, ultimately affecting their growth and productivity. The aim of our study is to determine the effect of short-term N deprivation on secondary metabolites production in developing and mature leaves of flax (Linum usitatissimum L.). Two weeks under low-N conditions decrease plant growth, N concentration, and soluble proteins content in leaves. Reduction in photosynthesis intensity was also observed. A decrease of cyanogenic glycoside content under N-deficient conditions was most visible in mature leaves. However, the content of linamarin and lotaustralin was about 10-fold higher in younger than in mature leaves, in which play probably protective role due to the possibility of toxic HCN release. Despite the N deficit, flax plants accumulated nitrogen in cyanoglycosides. N from cyanoglycosides can be used to synthesise amino acids; this possibility is supported by the high activity of β-cyanoalanine synthase, especially in N-deficient leaves. On the other hand, the content of different types of phenolic compounds increased in N-deficient plants, especially in young leaves, possibly replacing cyanoglucosides in protective functions. Our results indicated that cyanogenic glycosides could be an important nitrogen source for flax plants grown under temporary N-deficient conditions.

Iron homeostasis regulates maturation of tomato (climacteric) and capsicum (non-climacteric) fruits

Endogenous iron quickly interchanges its oxidation states (Fe III and Fe II), which modulate production of reactive oxygen species (ROS) and nitric oxide (NO), and also serve as co-factor for various enzymes. A sharp increase in Fe(III) content in mature fruits of tomato (climacteric) is a unique observation from the present work. Iron homeostasis in non-climacteric capsicum fruits, however, accompanies a balancing of Fe(III) and Fe(II) levels during turning and ripe stages. Heme oxygenase-1 (HO-1), which mobilizes toxic heme and produces Fe(II), apparently regulates iron homeostasis in tomato. Nearly static HO-1 activity in capsicum indicates that HO-1 probably does not have a major role in iron homeostasis in non-climacteric fruit maturation. Endogenous NO content is much higher during tomato fruit maturation than in capsicum. ROS production is highest in green (unripe) tomato and capsicum fruits and it sharply declines with fruit maturation. Tomato fruits exhibit very high β-cyanoalanine synthase (β-CAS) activity in turning stage and mature fruits, highlighting its crucial role in maintaining a static level of cyanide (CN) generated as a byproduct of ethylene biosynthesis during fruit maturation. In contrast, β-CAS activity is very low in maturing capsicum fruits. Thus, a coordination between iron signal and, NO and ROS accumulation in climacteric and non-climacteric fruits is apparently perceived by hormonal (ethylene) signals getting altered differentially during fruit ripening.

Assimilation of exogenous cyanide cross talk in Oryza sativa L. to the key nodes in nitrogen metabolism.

Exogenous cyanide (CN-) effects on nitrogen (N) uptake, transport, and assimilation in rice seedlings were investigated at the biochemical and molecular levels. Seedlings were treated with either a 2-d or 4-d supply of potassium cyanide (KCN) in the nutrient solution containing nitrate (NO3-) or ammonium (NH4+). Although a KCN-induced increase was recorded in the activity of β-cyanoalanine synthase (β-CAS) in rice tissues of both NH4+-fed and NO3--fed seedlings, the former showed a significantly greater assimilation rate for CN- than the latter. The addition of KCN decreased NO3- uptake and assimilation, whereas a negligible impact was observed in NH4+ treatments. Enzymatic assays showed a marked activities enhancement of glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in NH4+-fed seedlings after KCN exposure. Similarly, the rises occurring in nitrate reductase (NR) and GDH activity in NO3--fed seedlings were also detected after 2-d exposure to KCN, whereas a significant reduction of GS and GOGAT activities was determined. The results suggest that although exogenous KCN at moderate or high concentrations caused repressively effects on biomass growth of both NH4+-fed and NO3--fed rice seedlings, the nontoxic concentration of KCN supplied can serve as a supplemental N source in plant nutrition and N metabolism.

Limited induction of ethylene and cyanide synthesis are observed in quinclorac-resistant barnyardgrass (Echinochloa crus-galli) in Uruguay

AbstractBarnyardgrass [Echinochloa crus-galli(L.) P. Beauv] is the foremost weed in rice (Oryza sativaL.) systems, and its control is crucial to successful rice production. Quinclorac, a synthetic auxin herbicide, has been used effectively to manageE. crus-galli. However, occurrences of quinclorac-resistant genotypes are frequently reported, and its resistance evolution has led to questions about the continued utility of quinclorac for grass control. Identification of the resistance mechanism(s) of resistant genotypes will facilitate development of integrated weed management strategies that sustain quinclorac use for management ofE. crus-galli. We evaluated the responses to quinclorac of two contrasting genotypes: E7 (resistant, R) and LM04 (susceptible, S). Quinclorac induced ethylene and cyanide biosynthesis in the S-genotype. Both genotypes responded similarly to an increasing application of exogenous 1-carboxylic acid aminocyclopropane (ACC) and potassium cyanide, and their growth was inhibited at higher doses. The key mechanism for cyanide (HCN) detoxification in plants, β-cyanoalanine synthase (β-CAS) activity, was evaluated in both genotypes, and no significant difference was observed in the basal activity. However, quinclorac significantly induced β-CAS–like activity in the S-genotype, which is consistent with the increased synthesis of ethylene and cyanide. This work suggests that the resistance to quinclorac of the E7 R-genotype is likely related to an alteration in the auxin signal transduction pathway, causing a lower stimulation of ACC synthase and, therefore, limited synthesis of ethylene and HCN after quinclorac treatment.

Ethylene Biosynthesis Inhibition Combined with Cyanide Degradation Confer Resistance to Quinclorac in Echinochloa crus-galli var. mitis.

Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of β-cyanoalanine synthase (β-CAS). Resistant biotypes exhibited higher β-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities—due to less relative expression of ACS and ACO genes and enhanced β-CAS activity, as well as mutation and increased relative expression of EcCAS gene—can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.

Quinclorac resistance in Echinochloa phyllopogon is associated with reduced ethylene synthesis rather than enhanced cyanide detoxification by β-cyanoalanine synthase.

Multiple herbicide resistant Echinochloa phyllopogon exhibits resistance to the auxin herbicide quinclorac. Previous research observed enhanced activity of the cyanide-detoxifying enzyme β-cyanoalanine synthase (β-CAS) and reduced ethylene production in the resistant line, suggesting β-CAS-mediated cyanide detoxification and insensitivity to quinclorac stimulation as the resistance mechanisms. To investigate the molecular mechanisms of quinclorac resistance, we characterized the β-CAS genes alongside plant transformation studies. The association of β-CAS activity and ethylene production to quinclorac resistance was assayed in the F6 progeny of susceptible and resistant lines of E. phyllopogon. A single nucleotide polymorphism in a β-CAS1 intron deleted aberrantly spliced mRNAs and enhanced β-CAS activity in the resistant line. The enhanced activity, however, was not associated with quinclorac resistance in F6 lines. The results were supported by lack of quinclorac resistance in Arabidopsis thaliana expressing E. phyllopogon β-CAS1 and no difference in quinclorac sensitivity between β-CAS knockout and wild-type rice. Reduced ethylene production co-segregated with quinclorac resistance in F6 lines which were previously characterized to be resistant to other herbicides by an enhanced metabolism. β-CAS does not participate in quinclorac sensitivity in E. phyllopogon. Our results suggest that a mechanism(s) leading to reduced ethylene production is behind the resistance. © 2019 Society of Chemical Industry.

Physiological assessment of non–target site restistance in multiple-resistant junglerice (Echinochloa colona)

AbstractHerbicide-resistant Echinochloa species are among the most problematic weeds in agricultural crops globally. Recurring herbicide selection pressure in the absence of diverse management practices has resulted in greater than 20% of sampled Echinochloa populations from rice (Oryza sativa L.) fields demonstrating multiple resistance to herbicides in Arkansas, USA. We assessed the resistance profile and potential mechanisms of resistance in a multiple herbicide–resistant junglerice [Echinochloa colona (L.) Link] (ECO-R) population. Whole-plant and laboratory bioassays were conducted to identify the potential mechanisms of non–target site resistance in this population. ECO-R was highly resistant to propanil (>37,800 g ha−1) and quinclorac (>17,920 g ha−1) and had elevated tolerance to cyhalofop (R/S = 1.9) and glufosinate (R/S = 1.2) compared to the susceptible standard. The addition of glufosinate (590 g ha−1) to cyhalofop (314 g ha−1), propanil (4,500 g ha−1), or quinclorac (560 g ha−1) controlled ECO-R 100%. However, cyhalofop applied with propanil (48% control) or quinclorac (15% control) was antagonistic. The application of the known metabolic enzyme inhibitors malathion, carbaryl, and piperonyl butoxide increased control of ECO-R with propanil (>75%) but not with other herbicides. Neither absorption nor translocation of [14C]cyhalofop or propanil was different between ECO-R and ECO-S. [14C]Quinclorac absorption was also similar between ECO-R and ECO-S; however, translocation of quinclorac into tissues above the treated leaf of ECO-R was >20% higher than that in ECO-S. The abundance of metabolites was higher (∼10%) in the treated leaves of ECO-R than in ECO-S beginning 48 h after treatment. The activity of β-cyanoalanine synthase, which detoxifies hydrogen cyanide, was not different between ECO-R and ECO-S following quinclorac treatment. Resistance to propanil was due to herbicide detoxification by metabolic enzymes. Resistance to quinclorac was due to a detoxification mechanism yet to be understood. The reduction in sensitivity to cyhalofop and glufosinate might be a secondary effect of the mechanisms conferring high resistance to propanil and quinclorac.

Regulation of Hydrogen Sulfide Metabolism by Nitric Oxide Inhibitors and the Quality of Peaches during Cold Storage.

Both nitric oxide (NO) and hydrogen sulfide (H2S) have been shown to have positive effects on the maintenance of fruit quality during storage; however, the mechanisms by which NO regulates the endogenous H2S metabolism remain unknown. In this experiment, peaches were immersed in solutions of NO, potassium 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO, as an NO scavenger), N-nitro-l-arginine methyl ester (l-NAME, as an inhibitor of nitric oxide synthase (NOS)-like activity), and sodium tungstate (as an inhibitor of nitrate reductase), and the resulting changes in the H2S metabolism of peaches were studied. The results showed that exogenous NO reduced the contents of endogenous H2S, Cys, and sulfite; decreased the activities of l-/d-cysteine desulfhydrase (l-/d-CD), O-acetylserine (thiol)lyase (OAS-TL), and sulfite reductase (SiR); and increased the activity of β-cyanoalanine synthase (β-CAS). Both c-PTIO and sodium tungstate had similar roles in increasing the H2S content by sustaining the activities of l-/d-CDs, OAS-TL, and SiR. l-NAME increased the H2S content, mainly by maintaining the d-CD activity. The results suggest that NO, c-PTIO, l-NAME, and sodium tungstate differently regulate the H2S metabolism of peaches during storage.

Resistance to quinclorac caused by the enhanced ability to detoxify cyanide and its molecular mechanism in Echinochloa crus-galli var. zelayensis

Quinclorac, an auxin-type herbicide, is widely used to control barnyardgrass and some dicotyledon weeds. Echinochloa crus-galli var. zelayensis, a variety of E. crus-galli (L.) Beauv., is widespread in China and some populations have resistance to quinclorac. E. crus-galli var. zelayensis seeds with varying sensitivity to quinclorac were used in the present study. The expression of the ADP/ATP carrier protein (ANT) gene, which plays an important role in the maintenance of cellular energy balance, dramatically rose in the S biotype after exposure to quinclorac, while no change was found in two R biotypes. The activity of β-cyanoalanine synthase (β-CAS), which is the key enzyme for cyanide degradation, was higher in two R biotypes than in the S biotype before and after treatment with quinclorac. One single-nucleotide difference was detected in the EcCAS gene of two R biotypes compared with the S biotype. The nucleotide change, which caused one amino acid substitution, replacing Methionine (Met)-295 with Lysine (Lys)-295 in the two R biotypes, which are same as the rice β-CAS gene at this position. In addition, EcCAS gene expression was higher in the two R biotypes than in the S biotype. In conclusion, β-CAS may play a crucial role in the resistance of E. crus-galli var. zelayensis to quinclorac. EcCAS gene mutation and higher gene expression may enhance the activity of β-CAS to avoid the accumulation of toxic cyanide in resistant populations, thus contributing to the resistance mechanism of E. crus-galli var. zelayensis. to quinclorac.

β-Cyanoalanine Synthases and Their Possible Role in Pierid Host Plant Adaptation.

Cyanide is generated in larvae of the glucosinolate-specialist Pieris rapae (Lepidoptera:Pieridae) upon ingestion of plant material containing phenylalanine-derived glucosinolates as chemical defenses. As these glucosinolates were widespread within ancient Brassicales, the ability to detoxify cyanide may therefore have been essential for the host plant shift of Pierid species from Fabales to Brassicales species giving rise to the Pierinae subfamily. Previous research identified β-cyanoalanine and thiocyanate as products of cyanide detoxification in P. rapae larvae as well as three cDNAs encoding the β-cyanoalanine synthases PrBSAS1-PrBSAS3. Here, we analyzed a total of eight species of four lepidopteran families to test if their cyanide detoxification capacity correlates with their feeding specialization. We detected β-cyanoalanine synthase activity in gut protein extracts of all six species tested, which included Pierid species with glucosinolate-containing host plants, Pierids with other hosts, and other Lepidoptera with varying food specialization. Rhodanese activity was only scarcely detectable with the highest levels appearing in the two glucosinolate-feeding Pierids. We then amplified by polymerase chain reaction (PCR) 14 cDNAs encoding β-cyanoalanine synthases from seven species. Enzyme characterization and phylogenetic analysis indicated that lepidopterans are generally equipped with one PrBSAS2 homolog with high affinity for cyanide. A second β-cyanoalanine synthase which grouped with PrBSAS3 was restricted to Pierid species, while a third variant (i.e., homologs of PrBSAS1), was only present in members of the Pierinae subfamily. These results are in agreement with the hypothesis that the host shift to Brassicales was associated with the requirement for a specialized cyanide detoxification machinery.

A gene horizontally transferred from bacteria protects arthropods from host plant cyanide poisoning.

Cyanogenic glucosides are among the most widespread defense chemicals of plants. Upon plant tissue disruption, these glucosides are hydrolyzed to a reactive hydroxynitrile that releases toxic hydrogen cyanide (HCN). Yet many mite and lepidopteran species can thrive on plants defended by cyanogenic glucosides. The nature of the enzyme known to detoxify HCN to β-cyanoalanine in arthropods has remained enigmatic. Here we identify this enzyme by transcriptome analysis and functional expression. Phylogenetic analysis showed that the gene is a member of the cysteine synthase family horizontally transferred from bacteria to phytophagous mites and Lepidoptera. The recombinant mite enzyme had both β-cyanoalanine synthase and cysteine synthase activity but enzyme kinetics showed that cyanide detoxification activity was strongly favored. Our results therefore suggest that an ancient horizontal transfer of a gene originally involved in sulfur amino acid biosynthesis in bacteria was co-opted by herbivorous arthropods to detoxify plant produced cyanide.DOI: http://dx.doi.org/10.7554/eLife.02365.001.

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 (ΔAtnit4) 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.

The β-cyanoalanine pathway is involved in the response to water deficit in Arabidopsis thaliana

The β-cyanoalanine pathway is primarily responsible for detoxification of excess cyanide produced by plants. Recent evidence suggests that cyanide detoxification via this pathway may be involved in the response and tolerance to water deficit in plants. The aim of this study was to explore this role in Arabidopsis thaliana in greater detail. The first objective was to establish responsiveness of the pathway to the magnitude and duration of water deficit. The second objective was to examine how interruption of single genes (AtCysA1, AtCysC1 and AtNIT4) encoding enzymes of the pathway influenced the ability to metabolize cyanide and withstand water deficit. Arabidopsis plants were exposed to conditions which emulated acute and chronic water deficit, followed by measurement of tissue cyanide concentration, activity of enzymes, and physiological parameters. The results for wild-type Arabidopsis demonstrated a transient increase in cyanide concentration and β-cyanoalanine synthase activity, followed by a decrease in both. The increase in enzyme activity was localized to the tissue in direct proximity to the stress. The knockdown AtCysA1 mutant did not differ from wild-type while AtCysC1 mutants were slightly more sensitive to water deficit. The AtNIT4 mutant was the most sensitive showing decreased growth along with altered chlorophyll content under water deficit as compared to wild-type. Collectively, the results indicated that the pathway is responsive to water deficit although the severity of stress did not alter the nature of the response, implying that the capacity to remove cyanide generated during water deficit may contribute to tolerance to this stress in Arabidopsis.

Quinclorac resistance: a concerted hormonal and enzymatic effort in Echinochloa phyllopogon

Quinclorac (3,7-dichloro-quinoline-carboxylic acid) is a selective herbicide widely used to control annual grasses and certain broadleaf weeds. Echinochloa phyllopogon (Stapf) Koss. is the most noxious grass weed in California rice fields and has evolved resistance to multiple herbicides with different modes of action. A quinclorac-resistant (R) E. phyllopogon biotype found in a Sacramento Valley rice field where quinclorac has never been applied was investigated. Resistant to susceptible (S) GR(50) (herbicide rate for 50% growth reduction) ratios ranged from 6 to 17. The cytochrome P450 inhibitor malathion (200 mg L(-1)) caused R plants to become as quinclorac susceptible as S plants. Quinclorac rapidly (6 HAT) stimulated ethylene formation in S plants, but only marginally in R plants. Malathion pretreatment did not reduce ethylene formation by quinclorac-treated S and R plants. Activity of β-cyanoalanine synthase (β-CAS) in tissue extracts was 2-3-fold greater in R than in S plants, and incubation of shoot extracts with 1 mM malathion reduced β-CAS activity by 40% in both biotypes. Resistance to quinclorac in R E. phyllopogon involved at least two mechanisms: (a) insensitivity along the response pathway whereby quinclorac induces ethylene production; (b) enhanced β-CAS activity, which should enable greater HCN detoxification following quinclorac stimulation of ethylene biosynthesis. This unveils new resistance mechanisms for this multiple-resistant biotype widely spread throughout California rice fields.

A possible new mechanism involved in ferro-cyanide metabolism by plants

Ferro-cyanide is one of the commonly found species at cyanide-contaminated soils and groundwater. Unlike botanical metabolism of KCN via the β-cyanoalanine pathway, processes involved in the plant-mediated assimilation of ferro-cyanide are still unclear. The objective of this study was to investigate a possible mechanism involved in uptake and assimilation of ferro-cyanide by plants. Detached roots of plants were exposed to ferro-cyanide in a closed-dark hydroponic system amended with HgCl(2), AgNO(3), LaCl(3), tetraethylammonium chloride (TEACl), or Na(3)VO(4), respectively, at 25 ± 0.5°C for 24 h. Total CN, free CN(-), and dissolved Fe(2+) were analyzed spectrophotometrically. Activity of β-cyanoalanine synthase involved in cyanide assimilation was also assayed using detached roots of plants in vivo. Dissociation of ferro-cyanide [Fe(II)(CN)(6)](-4) to free CN(-) and Fe(2+) in solution was negligible. The applied inhibitors did not show any significant impact on the uptake of ferro-cyanide by soybean (Glycine max L. cv. JD 1) and hybrid willows (Salix matsudana Koidz × alba L.; p > 0.05), but rice (Oryza sativa L. cv. JY 98) was more susceptible to the inhibitors compared with the controls (p < 0.05). However, TEACl had the most severe effect on the assimilation of ferro-cyanide by soybean, hybrid willows, and maize (Zea mays L. cv. PA 78; p < 0.01), whereas AgNO(3) was the most sensitive inhibitor to rice (p < 0.01). No measurable difference in β-cyanoalanine synthase activity of roots exposed to ferro-cyanide was observed compared with the control without any cyanides (p > 0.05), whereas roots exposed to KCN showed a considerable increase in enzyme activity (p < 0.05). Plants take up Fe(2+) and CN(-) as a whole complex, and in vivo dissociation to free CN(-) is not prerequisite during the botanical assimilation of ferro-cyanide. Ferro-cyanide is likely metabolized by plants directly through an unknown pathway rather than the β-cyanoalanine pathway.

Mitochondrial β-Cyanoalanine Synthase Is Essential for Root Hair Formation in Arabidopsis thaliana

Cyanide is stoichiometrically produced as a coproduct of the ethylene biosynthesis pathway and is detoxified by β-cyanoalanine synthase enzymes. The molecular and phenotypical analysis of T-DNA insertion mutants of the mitochondrial β-cyanoalanine synthase CYS-C1 suggests that discrete accumulation of cyanide is not toxic for the plant and does not alter mitochondrial respiration rates but does act as a strong inhibitor of root hair development. The cys-c1 null allele is defective in root hair formation and accumulates cyanide in root tissues. The root hair defect is phenocopied in wild-type plants by the exogenous addition of cyanide to the growth medium and is reversed by the addition of hydroxocobalamin or by genetic complementation with the CYS-C1 gene. Hydroxocobalamin not only recovers the root phenotype of the mutant but also the formation of reactive oxygen species at the initial step of root hair tip growth. Transcriptional profiling of the cys-c1 mutant reveals that cyanide accumulation acts as a repressive signal for several genes encoding enzymes involved in cell wall rebuilding and the formation of the root hair tip as well as genes involved in ethylene signaling and metabolism. Our results demonstrate that mitochondrial β-cyanoalanine synthase activity is essential to maintain a low level of cyanide for proper root hair development.

Role of ethylene in abnormal shoot growth induced by high concentration of brassinolide in rice seedlings

A high concentration of brassinolide (BL) induced abnormal shoot shape in rice seedlings, that is, the newly developed leaf sheath was shorter than the old sheath, and increased the number of leaves. The involvement of ethylene and cyanide, co-metabolites during ethylene biosynthesis, in abnormal shoot growth was analyzed by comparing the action mechanism of BL with that of quinclorac in barnyard grass seedlings. BL strongly stimulated ethylene production, and the amount of ethylene and the third leaf sheath length in rice seedlings were negatively correlated. Exogenously applied ethephon also induced abnormal growth of rice seedlings. L-α-amino-ethoxyvinyl-glycine (AVG), an ethylene biosynthetic inhibitor, partially reversed abnormal shoot growth. The level of endogenous cyanide was low in rice seedlings, because of the high level of β-cyanoalanine synthase (CAS) activity, and exogenously applied KCN did not induce abnormal shoot growth. The results indicate that BL-induced abnormal shoot growth of rice seedlings was probably mediated by ethylene production.

Mechanism of resistance to quinclorac in smooth crabgrass ( Digitaria ischaemum)

The mechanism of resistance to quinclorac was investigated in a smooth crabgrass biotype [ Digitaria ischaemum (Schreb. ex Schweig) Schreb. ex Muhl] from Tulare County, California. Quinclorac (8.96 kg a.i. ha −1) had no effect ( P = 0.18) on the resistant (R) biotype, but reduced fresh weight of a susceptible (S) biotype by 93%. After treatment with 4.48 kg a.i. quinclorac ha −1, the S biotype produced about three times more ethylene than the R biotype and accumulated cyanide in tissues. Similar amounts of endogenous cyanide resulting from treatment with KCN reproduced quinclorac phytotoxicity. Pre-treatment with the ACC synthase inhibitor AVG reduced quinclorac phytotoxicity by 37% and ethylene production by 89%. These data suggest a target site-based mechanism of resistance involving stimulation of ACC synthesis and accumulation of cyanide. Also, the R biotype had four times more β-cyanoalanine synthase activity than the S biotype, suggesting a higher ability to detoxify cyanide.