Yukari Sunohara

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.

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.

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.

Effects of trampling on morphology and ethylene production in asiatic plantain

Abstract The effects of simulated trampling on shoot morphology and ethylene production of a trampling-tolerant perennial forb asiatic plantain were investigated. Trampling increased the number of leaves or inflorescences per plant, the petiole diameter, and the leaf blade length to width ratio but decreased the leaf blade width to petiole diameter ratio and the inflorescence length. Ramets subjected to trampling produced more ethylene than did nontrampled ramets originating from the same root crown. Moreover, an ethylene releaser ethephon decreased the leaf blade width to petiole diameter ratio and increased the leaf blade length to width ratio, in a manner similar to the changes induced by trampling. These results suggested that trampling-induced ethylene might be closely related to some of the adaptive morphological changes in asiatic plantain in response to trampling. Nomenclature: Ethephon, 2-chloroethylphosphonic acid; asiatic plantain, Plantago asiatica L.

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.

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.

Isolation of ambiguine D isonitrile from Hapalosiphon sp. and characterization of its phytotoxic activity

Cyanobacterium Hapalosiphon sp. contains phytotoxic substances. Fractionation of Hapalosiphon sp. crude extract guided by lettuce seedlings bioassay revealed that multiple fractions contained differential plant growth suppression activity. From the most active fraction inhibiting the growth of lettuce, ambiguine D isonitrile was isolated. A physiological study with ambiguine D isonitrile showed that it effectively inhibited mitosis, which resulted in the suppression of root growth. Ambiguine D isonitrile caused increased reactive oxygen species (ROS) generation and was associated with observed lipid peroxidation and cell death. These oxidative processes probably play a key role in the phytotoxic action of ambiguine D isonitrile in plants.

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.

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, fructose‑bisphosphate aldolase was increased; while, phosphotransferase and phosphoglycero mutase were decreased in glycolysis. In addition, phosphoglyceraldehyde‑3‑phosphate 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 glyceraldehyde‑3‑phosphate 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.

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.