Comparison of herbicide specificity of CYP81A cytochrome P450s from rice and a multiple-herbicide resistant weed, Echinochloa phyllopogon.

BACKGROUND: CYP81A cytochrome P450s (CYP81As) play a key role in herbicide detoxification in Poaceae plants. Crop CYP81As confer natural tolerance to multiple herbicides, whereas CYP81As in weeds disrupt herbicide action. Identifying differences in CYP81A herbicide specificity between crops and weeds could provide valuable information for controlling weeds. In this study, we quantitatively compared herbicide specificity between CYP81A6 from rice (Oryza sativa) and CYP81A12 and CYP81A21 from a weed, Echinochloa phyllopogon, using transgenic Escherichia coli and Arabidopsis. RESULTS: All three CYP81As metabolized the five tested herbicides and formed similar metabolites, with the highest relative activities of 400 to 580% toward bentazone compared to their activity on bensulfuron-methyl (defined as 100%). However, they showed differing activity toward propyrisulfuron. The relative activities of Echinochloa phyllopogon CYP81A12 (12.2%) and CYP81A21 (34.4%) toward propyrisulfuron were lower than that of rice CYP81A6 (98.5%). Additionally, rice CYP81A6 produced O-demethylated propyrisulfuron and hydroxylated products, whereas Echinochloa phyllopogon CYP81As produced only hydroxylated products. Arabidopsis expressing CYP81A12 and CYP81A21 exhibited lower levels of resistance against propyrisulfuron compared to that in Arabidopsis expressing CYP81A6. Homology modeling and in silico docking revealed that bensulfuron-methyl docked well into the active centers of all three CYP81As, whereas propyrisulfuron docked into rice CYP81A6 but not into Echinochloa phyllopogon CYP81As. CONCLUSION: The differing herbicide specificity displayed by rice CYP81A6 and Echinochloa phyllopogon CYP81A12 and CYP81A21 will help design inhibitors (synergists) of weed CYP81As, as well as develop novel herbicide ingredients that are selectively metabolized by crop CYP81As, to overcome the problem of herbicide resistance. © 2022 Society of Chemical Industry.

A promiscuous fatty acid ω-hydroxylase CYP94A90 is likely to be involved in biosynthesis of a floral nitro compound in loquat (Eriobotrya japonica).

Nitro groups are often associated with synthetically manufactured compounds such as medicines and explosives, and rarely with natural products. Loquat emits a nitro compound, (2-nitroethyl)benzene, as a flower scent. The nitro compound exhibits fungistatic activity and is biosynthesised from l-phenylalanine via (E/Z)-phenylacetaldoxime. Although aldoxime-producing CYP79s have been intensively studied, it is unclear what enzymes form nitro groups from aldoximes either in plants or in other organisms. Here, we report the identification of two cytochrome P450s that are likely to be involved in (2-nitroethyl)benzene biosynthesis in loquat through differential gene expression analysis using RNA-seq and functional identification using yeast and tobacco. CYP79D80 and CYP94A90 catalysed the formation of (E/Z)-phenylacetaldoxime from l-phenylalanine and (2-nitroethyl)benzene from the aldoxime, respectively. Expression profiles of CYP79D80 and CYP94A90 were correlated with the emission of (2-nitroethyl)benzene from loquat flowers. CYP94A90 also functioned as a fatty acid ω-hydroxylase as do other CYP94A fatty acid ω-hydroxylases. The CYP94As tested from other plants were all found to catalyse the formation of (2-nitroethyl)benzene from (E/Z)-phenylacetaldoxime. CYP79D80 and CYP94A90 are likely to operate in concert to biosynthesise (2-nitroethyl)benzene in loquat. CYP94A90 and other CYP94As are 'promiscuous fatty acid ω-hydroxylases', catalysing the formation of nitro groups from aldoximes, and are widely distributed in dicot plants.

Investigation of clomazone-tolerance mechanism in a long-grain cultivar of rice.

BACKGROUND: Clomazone is a potent herbicide for controlling weeds that have evolved resistance to other herbicides due to its unique mode of action. Clomazone is used in rice cultivation, but is limited to long-grain cultivars because other cultivars are highly sensitive to it. In this study, we investigated the mechanism of clomazone tolerance in a long-grain cultivar. RESULTS: The long-grain cultivar Kasalath tolerated approximately five-fold higher doses of clomazone compared to two short-grain cultivars, Nipponbare and Koshihikari. While Arabidopsis thaliana transformed with a rice cytochrome P450, CYP81A6, showed resistance to clomazone, the cyp81a6 knockout Kasalath was unchanged in its clomazone sensitivity. The inheritance of clomazone sensitivity in the F1 and F2 of Kasalath and Nipponbare indicated the involvement of multiple loci for clomazone tolerance. Four chromosome segment substitution lines of Nipponbare/Kasalath and Koshihikari/Kasalath exhibited partial tolerance to clomazone. The overlapping DNA region among the four lines is on chromosome 5 within 11.5 Mb. CONCLUSION: Multiple loci are involved in clomazone tolerance in Kasalath, one of which is located on chromosome 5. This information will help develop short-grain cultivars tolerant to clomazone. © 2021 Society of Chemical Industry.

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

BACKGROUND: Multiple herbicide resistant Echinochloa phyllopogon exhibits resistance to the auxin herbicide quinclorac. Previous research observed enhanced activity of the cyanide-detoxifying enzyme ß-cyanoalanine synthase (ß-CAS) and reduced ethylene production in the resistant line, suggesting ß-CAS-mediated cyanide detoxification and insensitivity to quinclorac stimulation as the resistance mechanisms. To investigate the molecular mechanisms of quinclorac resistance, we characterized the ß-CAS genes alongside plant transformation studies. The association of ß-CAS activity and ethylene production to quinclorac resistance was assayed in the F6 progeny of susceptible and resistant lines of E. phyllopogon. RESULTS: A single nucleotide polymorphism in a ß-CAS1 intron deleted aberrantly spliced mRNAs and enhanced ß-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 ß-CAS1 and no difference in quinclorac sensitivity between ß-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. CONCLUSION: ß-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.

Role of CYP81A cytochrome P450s in clomazone metabolism in Echinochloa phyllopogon.

Clomazone is a herbicide used in the cultivation of numerous crops due to its unique site of action and effectiveness on weeds. The differences in clomazone susceptibility among plants have been attributed to the differences in their complex clomazone metabolic pathways that are not fully understood. We previously identified two CYP81A cytochrome P450 monooxygenases that metabolize five chemically unrelated herbicides in multiple-herbicide resistant Echinochloa phyllopogon. Since the resistant E. phyllopogon have decreased clomazone susceptibility, involvement of these P450s in clomazone resistance was suggested. In this study, we revealed that each P450 gene endowed Arabidopsis thaliana (Arabidopsis) with clomazone resistance. Consistent with this, clomazone resistance co-segregated with resistance to other herbicides in F6 progenies of crosses between susceptible and resistant E. phyllopogon, suggesting that the P450s are involved in differential clomazone susceptibility in E. phyllopogon. Arabidopsis transformations of the other seven CYP81As of E. phyllopogon found that two more genes, CYP81A15 and CYP81A24, decreased Arabidopsis susceptibility to clomazone. Differences in substrate preference between clomazone and a herbicide that inhibits acetolactate synthase were suggested among the four CYP81A P450s. This study provides insights into clomazone metabolism in plants.

Molecular Responses of Maize Shoot to a Plant Derived Smoke Solution.

Plant-derived smoke has effects on plant growth. To find the molecular mechanism of plant-derived smoke on maize, a gel-free/label-free proteomic technique was used. The length of root and shoot were increased in maize by plant-derived smoke. Proteomic analysis revealed that 2000 ppm plant-derived smoke changed the abundance of 69 proteins in 4-days old maize shoot. Proteins in cytoplasm, chloroplast, and cell membrane were altered by plant-derived smoke. Catalytic, signaling, and nucleotide binding proteins were changed. Proteins related to sucrose synthase, nucleotides, signaling, and glutathione were significantly increased; however, cell wall, lipids, photosynthetic, and amino acid degradations related proteins were decreased. Based on proteomic and immunoblot analyses, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) was decreased; however, RuBisCO activase was not changed by plant-derived smoke in maize shoot. Ascorbate peroxidase was not affected; however, peroxiredoxin was decreased by plant-derived smoke. Furthermore, the results from enzyme-activity and mRNA-expression analyses confirmed regulation of ascorbate peroxidase and the peroxiredoxinin reactive oxygen scavenging system. These results suggest that increases in sucrose synthase, nucleotides, signaling, and glutathione related proteins combined with regulation of reactive oxygen species and their scavenging system in response to plant-derived smoke may improve maize growth.

CYP81A P450s are involved in concomitant cross-resistance to acetolactate synthase and acetyl-CoA carboxylase herbicides in Echinochloa phyllopogon.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.

Proteomic analysis of the effect of plant-derived smoke on soybean during recovery from flooding stress.

Flooding negatively affects the growth of soybean, whereas the plant-derived smoke enhances seedling growth of crops. To clarify the mechanism underlying the recovery from flooding stress, proteomic analysis was performed based on morphological results. Growth of soybean seedlings was inhibited under flooding stress, but it recovered after water removal following treatment with plant-derived smoke. Sucrose/starch metabolism and glycolysis were suppressed in smoke-treated flooded soybean compared to flooded soybean. The protein abundance and gene expression of O-fucosyltransferase family proteins related to the cell wall were higher in smoke-treated flooded soybean than in flooded soybean. Protein abundance and gene expression of peptidyl-prolyl cis-trans isomerase and Bowman-Birk proteinase isoinhibitor D-II were lower in smoke-treated flooded soybean than in flooded soybean. Taken together, these results suggest that plant-derived smoke enhances soybean growth during recovery from flooding stress through the balance of sucrose/starch metabolism and glycolysis. Furthermore, the accumulation of cell-wall related protein might be an important factor contributing to recovery of soybean from flooding stress. BIOLOGICAL SIGNIFICANCE: Flooding negatively affects the growth of soybean, whereas the plant-derived smoke enhances the seedling growth of crops. To clarify the mechanism underlying the recovery from flooding stress, proteomic analysis of soybean with different treatments including normal conditions, flooding stress, and flooding stress in the presence of plant-derived smoke was performed in this study. Growth of soybean seedlings was inhibited under flooding stress, however, it recovered with plant-derived smoke treatment during recovery from flooding stress. Sucrose/starch metabolism and glycolysis were suppressed in smoke-treated flooded soybean compared to flooded soybean, which suggests altered sucrose/starch metabolism and glycolysis contribute to soybean growth recovery from flood stress. Furthermore, the protein abundance and gene expression of O-fucosyltransferase family proteins related to the cell wall was higher in smoke-treated flooded soybean than in flooded soybean, which might be an important factor contributing to the recovery of soybean from flooding stress.

Proteomic analysis of the promotive effect of plant-derived smoke on plant growth of chickpea.

Plant-derived smoke plays a key role in seed germination and plant growth. To investigate the effect of plant-derived smoke on chickpea, a gel-free/label-free proteomic technique was used. Germination percentage, root/shoot length, and fresh biomass were increased in chickpea treated with 2000 ppm plant-derived smoke within 6 days. On treatment with 2000 ppm plant-derived smoke for 6 days, the abundance of 90 proteins including glycolysis-related proteins significantly changed in chickpea root. Proteins related to signaling and transport were increased; however, protein metabolism, cell, and cell wall were decreased. The sucrose synthase for starch degradation was increased and total soluble sugar was induced. The proteins for nitrate pathway were increased and nitrate content was improved. On the other hand, although secondary metabolism related proteins were decreased, flavonoid contents were increased. Based on proteomic and immuno-blot analyses, proteins related to redox homeostasis were decreased and increased in root and shoot, respectively. Furthermore, fructose­bisphosphate aldolase was increased; while, phosphotransferase and phosphoglycero mutase were decreased in glycolysis. In addition, phosphoglyceraldehyde­3­phosphate dehydrogenase and glutamine synthetase related genes were up-regulated. These results suggest that plant-derived smoke improves early stage of growth in chickpea with the balance of many cascades such as glycolysis, redox homeostasis, and secondary metabolism. BIOLOGICAL SIGNIFICANCE: The current study examined the effects of plant-derived smoke on root of chickpea seedlings using a gel-free/label-free proteomic technique. Based on functional categorization of results from proteomics, proteins related to glycolysis, signaling, transport, protein metabolism, cell wall, and cell were predominantly changed in chickpea. The proteins related to carbohydrate and nitrate pathways were increased, while, those of secondary metabolism were decreased. Physiological analysis indicated that flavonoid, total soluble sugar, and nitrate content were increased in root of chickpea treated with plant-derived smoke for 6 days. Moreover, accumulated protein abundance of glyceraldehyde­3­phosphate dehydrogenase and fructose-bisphosphate aldolase was in agreement with immuno-blot results, which suggests that glycolysis process might be enhanced in root of chickpea in response to plant-derived smoke.

Involvement of H2O2 in fluazifop-P-butyl-induced cell death in bristly starbur seedlings.

In order to understand the action mechanism of fluazifop-P-butyl (FB) in bristly starbur (Acanthospermum hispidum D.C.), a susceptible plant, the role of active oxygen species (ROS) in herbicide-induced cell death in shoots was investigated. FB-induced phytotoxicity was not reduced by the antioxidants, 1,4-diazabicyclooctane (dabaco), sodium azide, l-tryptophan, d-tryptophan, hydroquinone and dimethyl pyridine N-oxide (DMPO). The activities of superoxide dismutase (SOD) and catalase (CAT), in bristly starbur seedlings were significantly increased by FB at 12 HAT and 24 HAT, while ascorbate peroxidase (APX) and glutathione reductase (GR) activities increased only at 12 HAT. The contents of H2O2 in FB-treated bristly starbur seedlings were significantly higher to that of control between 8 and 24 HAT. According to the analysis of potassium iodide - starch or 3,3-diaminobenzidine, the accumulation of hydrogen peroxide was observed in the apical growing point, stem, petiole and veins of FB-treated bristly starbur seedlings at 24 HAT. The cell viability of bristly starbur seedlings treated by 10µM FB decreased at 18 HAT. These results suggested that FB-induced cell death in bristly starbur shoots may be caused by ROS (O2- and H2O2) generation and lipid peroxidation.

Improved tolerance to transplanting injury and chilling stress in rice seedlings treated with orysastrobin.

In addition to their fungicidal activity, strobilurin-type fungicides are reported to show enhancing effects on crop growth and yield. Previous studies suggested that the fungicide has a mitigating effect on abiotic stresses. However, there are few reports about growth enhancement through abiotic stress alleviation by strobilurin-type fungicides, but the mechanism of action of the growth enhancement is still not clear. The present study revealed that orysastrobin enhanced rice seedling growth after root cutting injury and chilling stress. We also found that orysastrobin decreased the transpiration rate and increased ascorbate peroxidase and glutathione reductase activities. This stress alleviation was eliminated by the application of naproxen, a putative abscisic acid biosynthesis inhibitor. These results suggested that orysastrobin improved tolerance against transplanting injury and chilling stress in rice seedlings by inducing water-retaining activity through the suppression of transpiration, and also by inducing reactive oxygen scavenging activity thus inhibiting reactive oxygen species accumulation.

Screening and identification of phytotoxic volatile compounds in medicinal plants and characterizations of a selected compound, eucarvone.

Screening and identification of phytotoxic volatile compounds were performed using 71 medicinal plant species to find new natural compounds, and the characterization of the promising compound was investigated to understand the mode of action. The volatile compounds from Asarum sieboldii Miq. showed the strongest inhibitory effect on the hypocotyl growth of lettuce seedlings (Lactuca sativa L.cv. Great Lakes 366), followed by those from Schizonepeta tenuifolia Briquet and Zanthoxylum piperitum (L.) DC.. Gas chromatography-mass spectrometry (GC/MS) identified four volatile compounds, α-pinene (2,6,6-trimethylbicyclo[3.1.1]hept-2-ene), ß-pinene (6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane), 3-carene (3,7,7-trimethylbicyclo[4.1.0]hept-3-ene), and eucarvone (2,6,6-trimethy-2,4-cycloheptadien-1-one), from A. sieboldii, and three volatile compounds, limonene (1-methyl-4-(1-methylethenyl)-cyclohexene), menthone (5-methyl-2-(propan-2-yl)cyclohexan-1-one), and pulegone (5-methyl-2-propan-2-ylidenecyclohexan-1-one), from S. tenuifolia. Among these volatile compounds, eucarvone, menthone, and pulegone exhibited strong inhibitory effects on both the root and shoot growth of lettuce seedlings. Eucarvone-induced growth inhibition was species-selective. Cell death, the generation of reactive oxygen species (ROS), and lipid peroxidation were induced in susceptible finger millet seedlings by eucarvone treatment, whereas this compound (≤158 µM) did not cause the increase of lipid peroxidation and ROS production in tolerant maize. The results of the present study show that eucarvone can have strong phytotoxic activity, which may be due to ROS overproduction and subsequent oxidative damage in finger millet seedlings.

Allelochemical L-DOPA induces quinoprotein adducts and inhibits NADH dehydrogenase activity and root growth of cucumber.

Allelochemical L-DOPA (l-3,4-dihydroxyphenylalanine) inhibits growth of several plant species. However, its mode of action is not well clarified in plants. The present studies were conducted to explore the action mechanism of L-DOPA in cucumber roots. The results revealed that L-DOPA suppressed the root growth of cucumber and induced quinoprotein and melanin formation in the roots. Moreover, L-DOPA not only decreased mitochondrial viability and NADH dehydrogenase (complex I) activity but also increased quinoprotein formation in vitro in isolated mitochondria from cucumber roots. Strong correlations were observed between quinoprotein formation and root growth inhibition, quinoprotein formation and NADH dehydrogenase activity, after L-DOPA treatment. The results suggest that quinoprotein formation and mitochondrial impairment might be involved in growth-inhibition mechanism of L-DOPA in cucumber roots.

Phytotoxic action mechanism of hapalocyclamide in lettuce seedlings.

Hapalocyclamide (cyclo-thiazole-L-alanine-oxazole-D-alanine-D-thiazoline-d-phenylalanine), a hexapeptide phytotoxic compound, was isolated from the terrestrial cyanobacterium Hapalosiphon sp. The phytotoxic action of the compound was investigated in lettuce (Lactuca sativa L. cv. Great Lakes no. 366) by determining its effects on several physiological processes. Hapalocyclamide effectively inhibited mitosis process in root tips, which resulted in the suppression of primary root growth of lettuce. The compound also induced overproduction of reactive oxygen species (ROS) and loss of cell viability in root cells. Moreover, hapalocyclamide-induced lipid peroxidation in both roots and shoots. Therefore, the primary action of hapalocyclamide to suppress lettuce growth might be caused by ROS overproduction, which induces major oxidative damage to membrane lipids, resulting in cell death and growth inhibition.

Characterization of calnexin in soybean roots and hypocotyls under osmotic stress.

Calnexin is an endoplasmic reticulum-localized molecular chaperone protein which is involved in folding and quality control of proteins. To evaluate the expression of calnexin in soybean seedlings under osmotic stress, immunoblot analysis was performed using a total membrane protein fraction. Calnexin constantly accumulated at an early growth stage of soybean under normal growth conditions. Expression of this protein decreased in 14-day-old soybean roots when treated with 10% polyethylene glycol for 2 days. Other abiotic stresses such as drought, salinity, cold as well as abscisic acid treatment, similarly reduced accumulation of calnexin and this reduction was correlated with reduction in root length in soybean seedlings under abiotic stresses. When compared between soybean and rice, calnexin expression was not changed in rice under abiotic stresses. Using Flag-tagged calnexin, a 70 kDa heat shock cognate protein was identified as an interacting protein. These results suggest that osmotic or other abiotic stresses highly reduce accumulation of the calnexin protein in developing soybean roots. It is also suggested that calnexin interacts with a 70 kDa heat shock cognate protein and probably functions as molecular chaperone in soybean.

Mutation of alpha-tubulin genes in trifluralin-resistant water foxtail (Alopecurus aequalis).

BACKGROUND: Trifluralin-resistant biotypes of water foxtail (Alopecurus aequalis) have been identified in wheat fields from northern Kyushu, Japan. Water foxtail is a winter-annual grassy weed, causing substantial crop losses. This study reports on mutation in α-tubulin (TUA) genes from water foxtail, the site of action of trifluralin. RESULTS: Two trifluralin-sensitive (S) Chikugo and Ukiha biotypes and four trifluralin-resistant (R) Asakura-1, Asakura-2, Tamana and Tosu biotypes of water foxtail were used for herbicide resistance analysis. R biotypes showed 5.7-30.7-fold trifluralin resistance compared with the S biotypes. No differences in the uptake and translocation of (14)C-trifluralin were observed between Chikugo (S) biotype and Asakura-1 (R) biotype. Most of the (14)C detected in the plant material was in the root tissue, and no substantial increases were noted in shoot tissues. Comparative TUA sequence analysis revealed two independent single amino acid changes: change of Val into Phe at position 202 in TUA1 and change of Leu into Met at position 125 in TUA3 in Asakura-1 biotype. In the Tamana (R) biotype, two amino acid changes of Leu to Phe at position 136 and Val to Phe at position 202 were observed in the predicted amino acid sequence of TUA1, compared with Chikugo (S) biotype. CONCLUSION: The results provide preliminary molecular explanation for the resistance of water foxtail to trifluralin, a phenomenon that has arisen as a result of repeated exposure to this class of herbicide. This is the first report of α-tubulin mutation in water foxtail and for any Alopecurus species reported in the literature.

Quinclorac-induced cell death is accompanied by generation of reactive oxygen species in maize root tissue.

The importance of reactive oxygen species for herbicide quinclorac (3,7-dichloro-8-quinolinecarboxylic acid)-induced cell death in roots was investigated. This was in order to understand its mode of action in grass species grown in the dark. Under these dark conditions, quinclorac suppressed the shoot and root growth of maize (Zea mays L. cv. Honey Bantam) in a concentration-dependent manner (50microM), although the inhibition level was less than that observed under growth conditions in the light. Analysis of cell viability using Evans blue or fluorescein diacetate-propidium iodide (FDA-PI) staining showed that the maize root cells significantly lost their viability after 14h root treatment with 10microM quinclorac, but not 10microM 2,4-dichlorophenoxyacetic acid (2,4-D). Determination of reactive oxygen species (ROS) in maize roots using a superoxide anion (O2-)-specific indicator, dihydroethidium (DHE), indicated that 50microM quinclorac induced a high level of O2- production in maize roots after 14h root treatment than that of either the control (non-treated) or with 50microM 2,4-D. Moreover, either cell death or ethane evolution, an indicator of lipid peroxide formation, in maize root segments was significantly enhanced by 50microM quinclorac, but not by 50microM 2,4-D. On the other hand, the 50microM 2,4-D treatment induced much higher ethylene and cyanide production in the root segments than with the 50microM quinclorac. These results suggest that quinclorac-induced cell death in maize roots may be caused by ROS and lipid peroxidation, but not by ethylene and its biosynthetic pathway-related substances including cyanide, which have been thought to be the causative factor of quinclorac-induced phytotoxicity in susceptible grass weeds such as Echinochloa, Digitaria, and Setaria.