Yuichi Takeuchi

Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco

Aluminum (Al) inhibits plant growth partly by causing oxidative damage that is promoted by reactive oxygen species and can be prevented by improving antioxidant capacity. Ascorbic acid (AsA), the most abundant antioxidant in plants, is regenerated by the action of monodehydroascorbate reductase (MDAR) and dehydroascorbate reductase (DHAR). We investigated the role of MDAR and DHAR in AsA regeneration during Al stress using transgenic tobacco (Nicotiana tabacum) plants overexpressing Arabidopsis cytosolic MDAR (MDAR-OX) or DHAR (DHAR-OX). DHAR-OX plants showed better root growth than wild-type (SR-1) plants after exposure to Al for 2xa0weeks, but MDAR-OX plants did not. There was no difference in Al distribution and accumulation in the root tips among SR-1, DHAR-OX, and MDAR-OX plants after Al treatment for 24xa0h. However, DHAR-OX plants showed lower hydrogen peroxide content, less lipid peroxidation and lower level of oxidative DNA damage than SR-1 plants, whereas MDAR-OX plants showed the same extent of damage as SR-1 plants. Compared with SR-1 plants, DHAR-OX plants consistently maintained a higher AsA level both with and without Al exposure, while MDAR-OX plants maintained a higher AsA level only without Al exposure. Also, DHAR-OX plants maintained higher APX activity under Al stress. The higher AsA level and APX activity in DHAR-OX plants contributed to their higher antioxidant capacity and higher tolerance to Al stress. These findings show that the overexpression of DHAR, but not of MDAR, confers Al tolerance, and that maintenance of a high AsA level is important to Al tolerance.

A methyl viologen-resistant mutant of Arabidopsis, which is allelic to ozone-sensitive rcd1, is tolerant to supplemental ultraviolet-B irradiation.

To better understand the role of active oxygen species (AOS) in acquired resistance to increased levels of ultraviolet (UV)-B irradiation in plants, we isolated an Arabidopsis mutant that is resistant to methyl viologen, and its sensitivity to UV-B was investigated. A complementation test revealed that the obtained mutant was allelic to the ozone-sensitive radical-induced cell death1-1 (rcd1-1). Therefore, this mutant was named rcd1-2. rcd1-2 was recessive and nearly 4-fold more resistant to methyl viologen than wild type. It exhibited a higher tolerance to short-term UV-B supplementation treatments than the wild type: UV-B-induced formation of cyclobutane pyrimidine dimers was reduced by one-half after 24 h of exposure; the decrease in quantum yield of photosystem II was also diminished by 40% after 12 h of treatment. Furthermore, rcd1-2 was tolerant to freezing. Steady-state mRNA levels of plastidic Cu/Zn superoxide dismutase and stromal ascorbate peroxidase were higher in rcd1-2 than in wild type, and the mRNA level of the latter enzyme was enhanced by UV-B exposure more effectively in rcd1-2. UV-B-absorbing compounds were more accumulated in rcd1-2 than in wild type after UV-B exposure for 24 h. These findings suggest that rcd1-2 methyl viologen resistance is due to the enhanced activities of the AOS-scavenging enzymes in chloroplasts and that the acquired tolerance to the short-term UV-B exposure results from a higher accumulation of sunscreen pigments. rcd1 appears to be a mutant that constitutively shows stress responses, leading to accumulation of more pigments and AOS-scavenging enzymes without any stresses.

Adaptive alterations in the activities of scavengers of active oxygen in cucumber cotyledons irradiated with UV-B

Summary UV-B (290–320 nm) irradiation considerably reduced the cotyledon size of cucumber ( Cucumis sativus L.) seedlings at 20 °C, and the extent of growth inhibition was reduced at 25 °C. At both temperatures, levels of endogenous scavengers and activities of active oxygen-scavenging enzymes were affected by UV-B irradiation. In particular, ascorbate peroxidase activity increased considerably, suggesting that active oxygen species might participate in the growth inhibition induced by UV-B irradiation. However, since no positive correlation was detected between the dependence of growth inhibition on temperature and the capacity to scavenge active species of oxygen, other mechanisms must be involved in the changes in the responses to UV-B that are related to temperature.

Peroxidation of lipids and growth inhibition induced by UV-B irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumis sativus L.) were cultured in vitro under UV radiation at different wavelengths, obtained by passage of light through cut-off filters with different transmittance properties. Growth and the synthesis of chlorophyll (Chl) in cotyledons were inhibited and malondialdehyde was accumulated upon irradiation at wavelengths below 320 nm. Exogenous application of scavengers of free radicals reversed the growth inhibition induced by UV-B. Measurement of the fluorescence of Chl a suggested that electron transfer in photosystems was affected by UV-B irradiation. On the basis of these results, the involvement is postulated of active species of oxygen in damages to thylakoid membranes and the growth inhibition that are induced by UV-B irradiation.

Effects of Hypergravity on the Elongation Growth in Radish and Cucumber Hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasaharaet al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+20g) but not under acropetal hypergravity (H-13g), as compared to growth under the control conditions (C+1g and C-1g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.

Ethylene evolution from tobacco leaves irradiated with UV-B

Abstract. Seedlings of Nicotiana tabacum L. (cv. Petit Havana SR1) were grown in the presence or absence of ultraviolet-B (UV-B, 290–320xa0nm) irradiation. The evolution of ethylene from the leaves, the content of 1-aminocyclopropane-1-carboxylic acid (ACC), an endogenous precursor of ethylene, and the activity of ACC synthase, a rate-limiting step in the production of ethylene, were increased by UV-B irradiation. The time course of these increases was parallel with the emergence of damage that was estimated by measuring the chlorophyll (Chl) content and the leakage of ions from leaf cells. Treatment of leaves with aminoethoxy-vinyl-glycine (AVG), a specific inhibitor of ACC synthase, reduced the extent of damage caused by UV-B. These results suggest that ethylene acts on certain processes to cause damage in tobacco leaves irradiated with UV-B.

Effects of ultraviolet-B irradiation on the cuticular wax of cucumber (Cucumis sativus) cotyledons

Cucumber seedlings were grown under three doses of supplemental ultraviolet-B (UV-B) irradiation to examine the effects on the surface structure of the cotyledons. Medium and high doses of irradiation induced glazing (formation of translucent, glossy layers) on the adaxial surfaces of cotyledons, especially those exposed to a high dose of UV-B. Observation with a scanning electron microscope revealed that the adaxial surfaces of cotyledons exposed to a medium dose of UV-B and controls became rough in appearance, but unevenness of the surface was not apparent in cotyledons irradiated with a high dose of UV-B. UV-B irradiation affected the types and amounts of alkanes and primary alcohols, the main components of cucumber cuticular wax. Based on cotyledon area, the amounts of these components were significantly higher in cotyledons irradiated with a medium dose of UV-B than in controls. This effect could be a consequence of small cotyledon area and constant wax production in the cotyledons irradiated with a medium dose of UV-B. The distribution patterns of homologs within the alkane and primary alcohol fractions shifted during growth to longer alkyl chain length in the control cotyledons. UV-B irradiation repressed these changes, suggesting that UV-B acts on cuticular wax biosynthetic pathways.

Accumulation of Pathogenesis-Related Proteins in Tobacco Leaves Irradiated with UV-B

Nicotiana tabacum L. (cv. Petit Havana SR1) were grown under ultraviolet-B (UV-B, 290–320 nm) irradiation, and soluble proteins were extracted from the leaves. Two-dimensional electrophoresis revealed that a minimum of 12 polypeptides were induced by UV-B. Polypeptides which were so abundant as to be detectable by Coomassie brilliant blue staining were then subjected to N-terminal amino acid sequence analyses. Two of the polypeptides were identified as a 23 kDa protein of PS II and 6 as a pathogenesis-related protein 5 (PR-5). Immunoblotting demonstrated that other PR proteins, PR-1 and PR-3 were also induced by UV-B. Salicylic acid (SA), which is an important component of signal transduction that leads to the expression of PR proteins and exhibition of acquired resistance to pathogens, increased in response to exposure to UV-B. In addition, the activity of phenylalanine ammonialyase, which catalyzes the synthesis from phenylalanine of trans-cinnamic acid, the endogenous precursor of SA, was transiently increased by UV-B irradiation. These results suggest that UV-B activates the signal transduction pathway, which is a common step in pathogen infection.

Polysaccharides in primary cell walls of rice cells in suspension culture

Abstract Extracellular polysaccharides in the medium of rice cells in suspension culture were composed of arabinogalactan and arabinoxylan. The amount of cellulose in the cell walls of the suspension-cultured cells was less than 15% (w/w), and non-cellulosic matrix polysaccharides were similar to those reported in graminaceous plants, with the exception that β 1-3, 1-4 glucans that have been widely reported to be major hemicellulosic components were not found.

Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254xa0nm) or UV-B+UV-A (280–360xa0nm; maximum energy at 312xa0nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2′-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2′-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270xa0nm or 290xa0nm increased the 8-OHdG level considerably, while UV at wavelengths above 310xa0nm had only small effects. In situ detection of H2O2 and quantification of H2O2 in plant extracts revealed that H2O2 accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.