Effects of Enhanced UV-B Radiation on the Activity and Expression of Alternative Oxidase in Red Kidney Bean Leaves

An increase in ultraviolet （UV） B radiation on the earth＇s surface is a feature of current global climate changes. It has been reported that alternative oxidase （AOX） may have a protective role against oxidative stress induced by environmental stresses, such as UV-B. To better understand the characteristic tolerance of plants to UV-B radiation, the effects of enhanced UV-B radiation on the activity and expression of AOX in red kidney bean （Phaseolus vulgaris） leaves were investigated in the present study. The results show that the total respiration rate and AOX activity in red kidney bean leaves increased significantly during treatment with enhanced UV-B. However, cytochrome oxidase （COX） activity did not change significantly. The H2O2 content was also markedly increased and reached a maximum of 4.45 mmol·L^-1·g^-1 DW （dry weight） at 24 h of UV-B treatment, before dropping rapidly. Both alternative pathway content and alternative pathway activity were increased in the presence of exogenous H2O2. Immunoblotting analysis with anti-AOX monoclonal antibody revealed that expression of the AOX protein increased in red kidney bean leaves under enhanced UV-B radiation, reaching a peak at 72 h. In addition, AOX expression in red kidney bean leaves was induced by exogenous H2O2. These data indicate that the increase in AOX activity in red kidney bean leaves under enhanced UV-B radiation was mainly due to H2O2-induced AOX expression.     

Isolation of a Tomato Protease that May Be Involved in Proteolysis of 1-Aminocyclopropane-l-Carboxylate Synthase

1-aminocyclopropane- 1-carboxylate （ACC） synthase is a principal enzyme that catalyses the committed step in phytohormone ethylene biosynthesis. Previous evidence indicates that the hypervariable C-terminus of ACC synthase is most likely to be processed proteolytically in vivo. However, the protease responsible has not been identified thus far. In the present study, we detected proteolytic activity against ACC synthase （LeACS2） in tomato （Lycopersicon esculentum Mill.） fruit extract based on a newly established in vitro assay system. Purification of the protease through DEAE, gel filtration and MonoQ chromatography resulted in considerable enrichment of a 64-kDa protein species. Subsequent biochemical analysis of the purified tomato protease revealed that the optimal conditions for its proteolytic activity were at pH 8.0 and at 37 ~C. In addition, the protease activity was blocked completely by the metalloprotease inhibitor 1,10-phenanthroline. The present study represents the first report on the isolation of an ACC synthase- processing protease from plant tissues.     

Effects of Enhanced UV-B Radiation on the Activity and Expression of Alternative Oxidase in Red Kidney Bean Leaves

An increase in ultraviolet(UV)B radiation on the earth's surface is a feature of current global climate changes.It hasbeen reported that alternative oxidase(AOX)may have a protective role against oxidative stress induced by environmentalstresses,such as UV-B.To better understand the characteristic tolerance of plants to UV-B radiation,the effects ofenhanced UV-B radiation on the activity and expression of AOX in red kidney bean(Phaseolus vulgaris)leaves wereinvestigated in the present study.The results show that the total respiration rate and AOX activity in red kidney bean leavesincreased significantly during treatment with enhanced UV-B.However,cytochrome oxidase(COX)activity did not changesignificantly.The H_2O_2 content was also markedly increased and reached a maximum of 4.45 mmol.L~(-1)·g~(-1)DW(dry weight)at 24 h of UV-B treatment,before dropping rapidly.Both alternative pathway content and alternative pathway activity wereincreased in the presence of exogenous H_2O_2.Immunoblotting analysis with anti-AOX monoclonal antibody revealed thatexpression of the AOX protein increased in red kidney bean leaves under enhanced UV-B radiation,reaching a peak at 72h.In addition,AOX expression in red kidney bean leaves was induced by exogenous H_2O_2.These data indicate that theincrease in AOX activity in red kidney bean leaves under enhanced UV-B radiation was mainly due to H_2O_2-induced AOXexpression.     

Variations in Growth, Photosynthesis and Defense System Among Four Weed Species Under Increased UV-B Radiation

Weed tolerance of UV-B radiation varies with species, and the radiation could affect weed ecology and management. Variations In growth, photosynthesis and defense system among four important agronomic weeds, Abutllon theophrastl Medlk, Amaranthus retroflexus L., Digitaria sanguinalis （L.） Scop and Chloris virgata Swartz, under Increased UV-B radiation （ambient and increased radiation at 2.7, 5.4 and 10.8 kJ.m^-2.d-1） were studied In the greenhouse experiment. After 2 weeks of radiation, the shoots＇ dry mass decreased with increasing UV-B radiation except for D. sanguinalis. The reduction in biomass was the result of changes in morphology and physiology. Higher levels of UV-B treatment decreased the leaf area, plant height, net photosynthetic rate and chlorophyll contents, while it increased the contents of wax and UV-B absorbing compound in all species, except for A. retroflexus, which did not increase significantly. The activity of superoxide dismutase, catalase, ascorbate peroxide and the content of ascorblc acid changed differently among the weed species as UV-B radiation increased. D. sangulnalls was the most tolerant and A. retroflexus the most sensitive to increased UV-B radiation. The results also show that the two grass species （D. sanguinalis and C. virgata） were more tolerant to UV-B radiation than the two broadleafed species （A. theophrasti and A. retroflexus）. The UV-B absorbing compound and leaf wax played Important roles against UV-B damages in the two grass weeds. The overall results suggest that weed community, competition and management will be altered by continuous ozone depletion.     

Expression of an antisense 1-aminocyclopropane-1-carboxylate oxidase gene stimulates shoot regeneration in Cucumis melo

The role of ethylene in shoot regeneration was investigated using transgenic Cucumis melo plants expressing an antisense 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene. ACC oxidase catalyses the last step of ethylene biosynthesis. Leaf and cotyledon explants from the transgenic plants exhibited low ACC oxidase activity and ethylene production, whereas the regeneration capacity of the tissues was greatly enhanced (3.5- and 2.8-fold, respectively) compared to untransformed control tissues. Addition of ethylene released by 50 or 100 μm 2-chloroethylphosphonic acid dramatically reduced the shoot regeneration rate of the transgenic tissues. The results clearly demonstrate that ethylene plays an important role in C. melo morphogenesis in vitro. Received: 23 April 1997 / Revision received: 9 June 1997 / Accepted: 2 July 1997     

Drought-Stimulated Activity of Plasma Membrane Nicotinamide Adenine Dinucleotide Phosphate Oxidase and Its Catalytic Properties in Rice

The activity of plasma membrane （PM） nicotinamide adenine dinucleotide phosphate （NADPH） oxidase and its catalytic properties in rice was investigated under drought stress conditions. Drought stress led to decreased leaf relative water content （RWC） and, as a result of drought-induced oxidative stress, the activities of antioxidant enzymes increased significantly. More interestingly, the intensity of applied water stress was correlated with increased production of H2O2 and O2^- and elevated activity of PM NADPH oxidase, a key enzyme of reactive oxygen species generation in plants. Histochemical analyses also revealed increased H2O2 and O2^- production in drought-stressed leaves. Application of diphenylene iodonium （DPI）, an inhibitor of PM NADPH oxidase, did not alleviate drought-induced production of H2O2 and O2^-. Catalysis experiments indicated that the rice PM NADPH oxidase was partially fiavin-dependent. The pH and temperature optima for this enzyme were 9.8 and 40 ℃, respectively. In addition, drought stress enhanced the activity under alkaline pH and high temperature conditions. These results suggest that a complex regulatory mechanism, associated with the NADPH oxidase-H2O2 system, is involved in the response of rice to drought stress.     

UV-B-induced Oxidative Damage and Protective Role of Exopolysaccharides in Desert Cyanobacterium Microcoleus vaginatus

UV-B-induced oxidative damage and the protective effect of exopolysaccharides （EPS） in Microcoleus vaginatus, a cyanobacterium isolated from desert crust, were investigated. After being irradiated with UV-B radiation, photosynthetic activity （FvlFm）, cellular total carbohydrates, EPS and sucrose production of irradiated cells decreased, while reducing sugars, reactive oxygen species （ROS） generation, malondialdehyde （MDA） production and DNA strand breaks increased significantly. However, when pretreated with 100 mg/L exogenous EPS, EPS production in the culture medium of UVo B stressed cells decreased significantly; Fv/Fm, cellular total carbohydrates, reducing sugars and sucrose synthase （SS） activity of irradiated cells increased significantly, while ROS generation, MDA production and DNA strand breaks of irradiated cells decreased significantly. The results suggested that EPS exhibited a significant protective effect on DNA strand breaks and lipid peroxidation by effectively eliminating ROS induced by UV-B radiation in M. vaginatus.     

Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 Gene

Loss-of-function ethylene insensitive 2 （EIN2） mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expres- sion during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and Iocules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones.     

UV-B Radiation Suppresses the Growth and Antioxidant Systems of Two Marine Microalgae, Platymonas subcordiformis （Wille） Hazen and Nitzschia closterium （Ehrenb.） W. Sm

This study investigated whether increased solar UV-B radiation (280-315nm) could suppress the growth of marine microalgae through effects on their antioxidant systems. Two marine microalgae species, Platymonas subcordiformis (Wille) Hazen and Nitzschia closterium (Ehrenb.) W. Sm, were exposed to a range of UV-B radiation and both showed reductions in their growth rates, and the chlorophyll a(Chl a) and carotenoid (Car) contents when UV-B radiation dose increased. Superoxide anion radical (O2^ )production and the concentration of hydrogen peroxide (H2O2) and malodiadehyde (MDA) also increased with the increasing of UV-B radiation. Antioxidant systems, non-enzymic components (Car and glutathione content) and enzymic components (superoxide dismutase (SOD) and catalase (CAT) activity), decreased as a result of enhanced UV-B radiation. When the exogenous glutathione (GSH) was added, the effects of UV-B radiation on the growth of the two species were alleviated. These results suggest that enhanced UV-B radiation suppressed the antioxidant systems and caused some active oxygen species to accumulate, which in turns retarded the development of the marine microalgae.     

Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

A chloroplast-localized tomato （Lycopersicon esculentum Mill.） ω-3 fatty acid desaturase gene （LeFADT） was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturasegene family. Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7 was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7 was induced by chilling stress （4 ℃）, but was inhibited by high temperature （45 ℃）, in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18 ： 3 content could maintain a higher maximal photochemical efficiency （Fv/Fm） and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18 ： 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.     

Metabolism of 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

In preclimacteric apple fruits ( Malus × domestica Borkh. cv. Golden Delicious) ethylene production is controlled by the rates of 1-aminocyclopropane-1-carboxylic acid (ACC) synthesis, and by its metabolism to ethylene by the ethylene-forming enzyme and to 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by malonyl CoA-ACC transferase. The onset of the climacteric in ethylene production is associated with an increase in the activity of the ethylene-forming enzyme in the pulp and with a rise in the activity of ACC synthase. Malonyl transferase activity is very high in the skin of immature fruit, decreases sharply before the onset of the climacteric, and remains nearly constant thereafter. More than 40% of the ACC synthesized in the skin and around 5% in the flesh, are diverted to MACC at early climacteric. At the climacteric peak there are substantial gradients in ethylene production between different portions of the tissue, the inner cortical tissues producing up to twice as much as the external tissues. This increased production is associated with, and apparently due to, increased content of ACC synthase. Less than 1% of the synthesized ACC is diverted to MACC in the flesh of climacteric apples. In contrast, the skin contains high activity of malonyl transferase, and correspondingly high levels [1000 nmol (g dry weight)⁻¹] of MACC.     

The quantification of 1-(malonylamino)cyclopropane-1-carboxylic acid in plant tissues by quantitative mass spectrometry

A method for the quantitation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugated form of 1-aminocyclopropane-1-carboxylic acid (ACC), in plants is described. [2,2,3,3-²H₄]MACC has been used as an internal standard for selected ion monitoring/isotope dilution quantitation of MACC in wheat seedlings and in tomato leaves. This method is compared with a widely-used two step indirect assay for MACC, which is based upon hydrolysis of MACC to ACC and conversion of ACC by hypochlorite reagent to ethylene which is subsequently quantified by gas chromatography.     

The Conversion of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid to 1-Aminocyclopropane-1-Carboxylic Acid in Plant Tissues

Since 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the major conjugate of 1-aminocyclopropane-1-carboxylic acid (ACC) in plant tissues, is a poor ethylene producer, it is generally thought that MACC is a biologically inactive end product of ACC. In the present study we have shown that the capability of watercress (Nasturtium officinale R. Br) stem sections and tobacco (Nicotiana tabacum L.) leaf discs to convert exogenously applied MACC to ACC increased with increasing MACC concentrations (0.2-5 millimolar) and duration (4-48 hours) of the treatment. The MACC-induced ethylene production was inhibited by CoCl₂ but not by aminoethoxyvinylglycin, suggesting that the ACC formed is derived from the MACC applied, and not from the methionine pathway. This was further confirmed by the observation that radioactive MACC released radioactive ACC and ethylene. A cell-free extract, which catalyzes the conversion of MACC to ACC, was prepared from watercress stems which were preincubated with 1 millimolar MACC for 24 hours. Neither fresh tissues nor aged tissues incubated without external MACC exhibited enzymic activity, confirming the view that the enzyme is induced by MACC. The enzyme had a K_m of 0.45 millimolar for MACC and showed maximal activity at pH 8.0 in the presence of 1 millimolar MnSO₄. The present study indicates that high MACC levels in the plant tissue can induce to some extent the capability to convert MACC to ACC.     

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10^–3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.     

Changes in Ethylene Production and in 1-Aminocyclopropane-1-carboxylic Acid (ACC) and Malonyl-ACC Contents of Cocklebur Seeds during Their Development

Ethylene production in developing cocklebur (Xanthium pennsyluanicumWallr.) seeds peaked when the dry weight of the seeds beganto increase in the early period of development. The productionthen began to decrease and stopped when the dry weight increasewas completed. The upsurge of ethylene production in the earlydevelopmental period paralleled increases in ACC synthase activityand the 1-aminocyclopropane-1-carboxylic acid (ACC) contentof the seeds, both of which rapidly decreased later. Malonyl-ACC (MACC) accumulated in developing cocklebur seedsduring the early period of development, before the ACC contentand ethylene production increased. Although the ACC synthaseactivity, ACC content and ethylene production showed markeddecreases, the MACC content remained almost unchanged duringthe middle period of seed development, with a pronounced decreaseoccurring in the late period. Exogenous application of MACCdid not promote ethylene production of seeds collected at thelate developmental stage. Aminoethoxyvinylglycine, an inhibitorof ACC synthase, strongly inhibited the ethylene productionof the same lot of seeds. Therefore, the decrease in the MACCcontent in developing cocklebur seeds was not due to reuse ofMACC for ethylene production. (Received May 24, 1984; Accepted August 15, 1984)     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Regulation of ethylene biosynthesis after short-term heat treatment in etiolated pea stems

Incubation of plant tissues at a constant elevated temperature greatly inhibits both basal and wound ethylene production. However, recovery from heat treatment is relatively rapid and is followed by stimulated ethylene production. The present investigation examines the kinetics of ethylene production after short-term heal treatment and the regulation of heat-altered ethylene production. Subapical stem segments of 7-day-old etiolated pea L. cv. Alaska) seedlings were analyzed for ethylene production, 1-aminocyclopropane-l-carboxylic acid (ACC) oxidation, and ACC and l-(malonylamino)cyclopropane-l-carboxylic acid (MACC) content after a 2-min 40°C heat pulse. The short-term heat pulse transiently inhibited ethylene production and ACC oxidation accompanied by a slight ACC accumulation within a 30-min time period. Conjugation to MACC did not appear to play an integral role in heat-regulated ethylene production. It was concluded that the major factor affecting ethylene production after heat treatment is the temporary inactivation of ACC oxidation. The possible roles of ACC synthase, ACC oxidase and lipoxygenase in regulating ethylene production after heat treatment are discussed.