Ethylene-promoted ascorbate peroxidase activity protects plants against hydrogen peroxide, ozone and paraquat

Abstract. In experiments where mung beans ( Vigna radiata L.) and peas ( Pisum sativum L.) have been pre-exposed to ethylene and afterwards treated with ozone, it has been shown that such ethylenepretreated plants may become more resistant to ozone. Further experiments with hydrogen peroxide (H₂O₂) and the herbicide paraquat suggest that this increased resistance against ozone depends on the stimulation of ascorbate peroxidase activity which provides cells with increased resistance against the formation of H₂O₂ which is also formed when plants are fumigated with ozone. These results explain why increased production of ethylene can be observed in plants exposed with ozone or other oxidative stress and clearly demonstrate that in plants, as well as animals, peroxidases protect cells against harmful concentrations of hydroperoxides.     

Role of the testa epidermis in the leakage of intracellular substances from imbibing soybean seeds and its implications for seedling survival

The generation of ethylene from 1-aminocyclopropane-1-carboxylic acid (ACC) added to a cell-free preparation from etiolated pea ( Pisum sativum L. cv. Alaska) epicotyls was found not to be due to a specific ACC oxidase or to oxygen radicals. Rather, endogenously produced H₂O and manganese ions are coupled in a reaction sequence which produces ethylene from ACC. In a model system, H₂O and Mn²⁺ converted ACC to ethylene under conditions similar to those in the pea preparation. Ultrafiltration of the pea preparation inhibited ethylene production, but it could be reconstituted either by adding an H₂O₂-generating system to the ultrafiltrate or Mn²⁺ to the retentate. H₂O₂-generating systems could reconstitute ethylene formation in a heat-inactivated cell-free sample while the loss of ability to produce ethylene upon dialysis of the pea preparation correlated with the loss of Mn²⁺ from the sample. Studies using cell-free preparations to investigate ethylene synthesis should take care to exclude the possible involvement of H₂O₂ and Mn^2+.     

Survey of wound-induced ethylene production by excised root segments

Ethylene production was measured from excised 10-mm apical and subapical root segments from 50 cultivars in 19 species of 7 families. Monocotyledonous species tended to have much lower rates of ethylene production than dicotyledonous species. Ethylene production was generally higher in apical root segments than in subapical segments within 1 h of wounding. However, cultivars of Cucumis melo , C. sativus , Helianthus annuus , Hibiscus esculentus , and Zea mays had higher rates of ethylene production from subapical segments. In apical root segments, Phaseolus aureus cv. Berken had the highest ethylene production rate (76.7 ηl g⁻¹ h⁻¹), while Zea mays cv. Silver Queen had the lowest rate (0.6 ηl g⁻¹ h⁻¹). In subapical root segments, Cucumis sativus cv. Armenian had the highest rate (55.7 ηl g⁻¹ h⁻¹), while Zea mays cv. Silver Queen again had the lowest rate (0.6 ηl g⁻¹ h⁻¹). The many different responses in magnitude and kinetics of wound-induced ethylene production among the species, cultivars and tissues should provide interesting and useful systems with which to study wound responses and induced ethylene production.     

Regulation by CO2 of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climacteric fruits

Cheverry, J. L., Sy, M. O., Pouliquen, J. and Marcellin, P. 1988. Regulation by CO₂ of 1-aminocyclopropane-1-carboxylic acid conversion to ethylene in climateric fruits. - Physiol. Plant. 72: 535–540.
A high CO₂ concentration (20%) at 20°C rapidly and strongly inhibited the development of the climacteric ethylene burst in apple ( Malus domestica Borkh. cv. Granny Smith) and avocado ( Persea americana Mill. cv. Fuerte) fruits and did not change 1-aminocyclopropane-l-carboxylic acid (ACC) content. Treatment with 20% CO₂ markedly decreased ACC-dependent ethylene biosynthesis at 20°C in climacteric pericarp tissues. It is suggested, therefore, that high CO₂ levels inhibit conversion of ACC to ethylene.
Synthesis of the ethylene forming enzyme (EFE) was enhanced when intact preclimacteric apples or early climacteric avocados were pretreated for 40 h with 10 μ11^-1 ethylene. When CO₂ (20%) and ethylene were both applied, a reduced stimulatory effect of ethylene on EFE synthesis was observed. A high CO₂ concentration enhanced EFE acivity in excised tissues of apples and avocados incubated with ACC (2 m M ) and cycloheximide (1 m M ) or 2–5-norbornadiene (5 ml 1^-1). In the autocatalytic process, 20% CO₂ antagonized the stimulation of EFE synthesis by ethylene, but promoted EFE activity.     

Gibberellin-induced elongation and osmoregulation in internodes of floating rice

Internodal elongation in floating rice ( Oryza sativa L. cv. Habiganj Aman II) is known to be enhanced by treatment with ethylene or gibberellic acid (GA₃) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH. while GA₃-induced internodal elongation is hardly affected by humidity. We examined the possible involvement of osmoregulation in the stimulation by GA₃ of the elongation of internodes at low RH. Submergence and treatment with ethylene or GA3₃ at 100% RH increased the osmotic potential in internodes of excised stem segments, while GA₃ at 20% RH maintained the osmotic potential at a low level. In internodes of stem segments that had been treated with GA₃ at 20% RH, the activity of invertase and the level of soluble sugars were almost 2- and 1.5-fold higher, respectively, than those in internodes that had been treated with GA₃ at 100% RH. These results indicate that one of the possible mechanisms by which GA₃ promotes elongation of internodes at low RH involves the osmoregulation that is achieved by promotion of the synthesis of invertase.     

Carbon dioxide-independent and -dependent components of light inhibition of the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in oat leaves

Light inhibits while carbon dioxide enhances the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene in oat ( Avena sativa L. cv. Victory) leaf segments. The possibility that the light inhibition is mediated through changes of carbon dioxide has been investigated. The level of CO₂ increases or decreases in the sealed incubation vial in darkness or in light, respectively, which can apparently account for the differences in ACC-dependent ethylene production between the dark and light treatments. However, although the evolution of ethylene from ACC in the dark is reduced upon depletion of CO₂, the difference between light and dark is still very noticeable. Moreover, the production of the ethylene in CO₂-free air in the dark was still higher than in the light, where the concentration of CO₂ was 0.01%. It is proposed that the light effect on the conversion of ACC to ethylene is composed of two distinguishable components: one CO₂-mediated and the other CO₂-independent.     

Cytokinins as inhibitors of root growth

The elongation of roots of wheat ( Triticum aestivum L. cv. Diamant II), flax ( Linum usitatissimum L. cv. Concurrent) and cucumber ( Cucumis sativus L. cv. Favör) seedlings in the dark was strongly inhibited by various native and synthetic cytokinins (kinetin, benzyladenine, isopentenyladenine, zeatin and their corresponding 9-ribosides). An inhibition of 50% was obtained for wheat roots with 3 · 10⁻⁹ M zeatin and for flax roots with 6 · 10⁻⁹ M isopentenyladenine. The ribosides were in all cases less inhibitory. The inhibition was reversed by various types of 'antiauxins' and 'antiethylenes' (such as structural auxin analogues, uncouplers, specific inhibitors of ethylene synthesis, free radical scavengers, inhibitors of ethylene action). These substances as a rule counteract also inhibitions caused by auxins. Auxins and cytokinins stimulate ethylene production synergistically, and the similar inhibitory effects of these two types of hormone can be understood if it is assumed that their effect is at least partly mediated through ethylene. The cytokinins must be considered as possible natural inhibitors and regulators of root growth.     

Survival and recovery of perennial forage grasses under prolonged Mediterranean drought: II. Water status, solute accumulation, abscisic acid concentration and accumulation of dehydrin transcripts in bases of immature leaves

Effects of ozone on spring wheat ( Triticum aestivum L. cv. Satu) were studied in an open-top chamber experiment during two growing seasons (1992–1993) at Jokioinen in south-west Finland. The wheat was exposed to filtered air (CF), non-filtered air (NF), non-filtered air+35 nl l⁻¹ ozone for 8 h d⁻¹ (NF⁺) and ambient air (AA). Each treatment was replicated five times. Two wk after anthesis, after 4 wk of ozone treatment (NF⁺, 45 nl l⁻¹ 1000–1800 hours, seasonal mean) the net CO₂ uptake of wheat flag leaves was decreased by c . 40% relative to CF and NF treatments, both initial and total activity of Rubisco and the quantity of protein-bound SH groups were decreased significantly. Added ozone also significantly accelerated flag leaf senescence recorded as a decrease in chloroplast size. The effect was significant 2 wk after anthesis, and senescence was complete after 4 wk. In the CF and NF treatments senescence was complete 5 wk after anthesis. The significant effect of ozone on the chloroplasts and net CO₂ uptake 2 wk after anthesis did not affect the grain filling rate. However, since the grain filling period was shorter for ozone fumigated plants, kernels were smaller. The decrease in 1000-grain weight explained most of the yield reduction in the plants under NF⁺ treatment. The results indicate that wheat plants are well buffered against substantial decrease in source activity, and that shortened flag leaf duration is the major factor causing ozone-induced yield loss.     

Ethylene formation in plant mitochondria. Dependence on the transport of 1-aminocyclopropane-1-carboxylic acid across the membrane

The mitochondrial fraction isolated from plumular hooks of etiolated pea seedlings ( Pisum sativum L. cv. Kelvedon Wonder) displayed a ten-fold higher rate of ethylene formation from 1-aminocyclopropane-1-carboxylic acid [ACC; 3.2 nmol C₂H₄ (mg protein ⁻¹)h⁻¹], than the tissue from which it was isolated. When the ionophores valinomycin or nigericin were added, a 60- to 70-fold increase in activity in intact mitochondria over the activity in plumular hooks was obtained for ethylene formation under the same conditions, and a 20-fold increase was obtained upon addition of gramicidin. The addition of ionophores did not affect the rate of ethylene formation in submitochondrial particles (55% inside-out as determined by cytochrome oxidase latency) which already exhibited a 2–3-fold higher specific activity than intact mitochondria. Low concentrations of the detergents cholate and deoxycholate increased mitochondrial ethylene formation activity and had no effect on the rate of the reaction in submitochondrial particles. These results support the suggestion that ACC conversion to ethylene is associated with the inner side of the inner mitochondrial membrane and that transport across the intact mitochondrial membrane is rate-limiting in the reaction.     

Ethylene production in habituated and auxin-requiring tobacco callus cultures. Does ethylene play a role in the habituation?

Ethylene production of habituated and auxin-requiring tobacco ( Nicotiana tabacum L. cv. Xanthi) callus cultures were compared. More ethylene was produced by auxinrequiring i.e. auxin-heterotrophic cultures than by habituated ones. Treatment with 2,4-dichlorophenoxyacetic acid increased the ethylene evolution of habituated cultures over the range 10⁻⁷ to 10⁻⁴ M , which suggests that the higher ethylene production of auxin-dependent callus is caused by the 2,4-D in the medium. The IAA levels depended on the age of both types of callus cultures.     

Impact of rising tropospheric ozone on potato: effects on photosynthesis, growth, productivity and yield quality

In view of its importance for human nutrition, the European Commission funded a collaborative research programme (1998–99) to evaluate the impact of future increases in atmospheric ozone (O₃) and carbon dioxide (CO₂) concentrations on yield and tuber quality in potato ( Solanum tuberosum L.). This was the first large-scale open-top chamber project to provide field-based data spanning a wide range of European climatic conditions and ozone concentrations for a widely used cultivar, cv. Bintje. Intensive measurements of physiological and developmental effects were made throughout crop growth to improve the mechanistic understanding of the processes involved. Analysis of the experimental results and modelling work based on the extensive database revealed that the increasing tropospheric O₃ concentrations predicted under future climatic scenarios in Europe are likely to reduce tuber yield in potato. However, season-long exposure to elevated O₃ had both beneficial and detrimental effects on tuber quality. Most of the significant physiological and growth effects occurred during the later stages of crop development, when elevated O₃ reduced photosynthesis and promoted senescence. The observed changes in the quality and nutritional attributes of tubers, such as decreased content of reducing sugars and increased nitrogen concentrations, may be attributable to reduced carbohydrate allocation.     

Chromium-induced inhibition of ethylene evolution in bean (Phaseolus vulgaris) leaves

The influence of chromium concentration on ethylene production in bean plants ( Phaseolus vulgaris L. cv. Contender) was investigated. A Cr ion-induced inhibition of ethylene synthesis from endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) was observed within both leaf discs floated on 2 m M CrO²⁻₄ or Cr³⁺ and leaf discs from plants cultured in nutrient solutions containing 10, 20 or 40 μ M CrO²⁻₄. However, Cr ions supplied either to plants with the nutrient solution or to discs with the incubation medium rather increased the conversion of exogenous ACC to ethylene. Primary leaves of plants exposed to CrO²⁻₄-containing nutrient solutions showed a statistically insignificant decrease of ACC-synthase activity. In the trifoliolate leaves of plants exposed to 10 μ M CrO²⁻₄, in which a significant decrease of ethylene production from endogenous ACC was observed, a substantial increase of ACC synthase was found. These results indicate that Cr ion-induced inhibition of ethylene production is not due to a breakdown of membrane integrity, which is necessary for ethylene forming enzyme activity, but caused by metabolic alterations leading to decreased ACC availability. Chromium ions may act by inhibiting ACC synthase activity or by diverting a metabolic step prior to the ACC synthase catalyzed reaction.     

Lack of ethylene involvement in tulip tepal abscission

The tepals of cut flowers of Tulipa hybrida cv. Golden Apeldoorn and Tulipa kaufmanniana cv. Shakespeare abscise 3–4 days after harvest. The weakening of the abscission zones is accompanied by cell wall breakdown and the separation of 3–4 rows of intact cells at the base of the tepal. During senescence, there is no ethylene climacteric and ethylene production rates remain low, between 0.07 and 0.4 nl g⁻¹ fresh weight h⁻¹. Adding 3–5 μl l⁻¹ ethylene slightly accelerated the weakening of the abscission zones but had no effect on the time of first abscission. Neither 0.5 m M silver thiosulphate nor 5 m M aminoethoxyvinylglycine delayed the time to abscission. It is concluded that tulip tepal fall does not involve primary regulation by ethylene, unlike the majority of other abscission systems that have been studied.     

Scavenging Effects of Dopamine Agonists on Nitric Oxide Radicals

Abstract: It has recently been considered that free radicals are closely involved in the pathogenesis of Parkinson's disease (PD), and the level of nitric oxide radical (^•NO), one of the free radicals, is reported to increase in PD brain. In the present study, we established a direct detection system for ^•NO in an in vitro ^•NO-generating system using 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)- N -methyl-1-propanamine as an ^•NO donor and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) by electron spin resonance (ESR) spectrometry and examined the quenching effects of the dopamine agonists pergolide and bromocriptine on the amount of ^•NO generated. ^•NO appeared to be scavenged by pergolide and, to a lesser extent, by bromocriptine. In the competition assay, the 50% inhibitory concentration values for pergolide and bromocriptine were estimated to be ∼23 and 200 µ M , respectively. It was previously reported that in vivo treatment of pergolide and bromocriptine completely protected against the decrease in levels of striatal dopamine and its metabolites in the 6-hydroxydopamine-injected mouse. Considering these findings, pergolide and probably bromocriptine may also protect against dysfunction of dopaminergic neurons because of its multiple effects; not only does it stimulate the presynaptic autoreceptors, but it also directly scavenges ^•NO radicals and hence protects against ^•NO-related cytotoxicity. This ESR spectrometry method using carboxy-PTIO may be useful for screening other drugs that can quench ^•NO.     

Ethylene reduces gas exchange and growth of lettuce plants under hypobaric and normal atmospheric conditions

Elevated levels of ethylene occur in controlled environment agriculture and in spaceflight environments, leading to adverse plant growth and sterility. The objectives of this research were to characterize the influence of ethylene on carbon dioxide (CO₂) assimilation (C_A), dark period respiration (DPR) and growth of lettuce ( Lactuca sativa L. cv. Buttercrunch) under ambient and low total pressure conditions. Lettuce plants were grown under variable total gas pressures of 25 kPa (hypobaric) and 101 kPa (ambient) pressure. Endogenously produced ethylene accumulated and reduced C_A, DPR and plant growth of ambient and hypobaric plants. There was a negative linear correlation between increasing ethylene concentrations [from 0 to around 1000 nmol mol⁻¹ (ppb)] on C_A, DPR and growth of ambient and hypobaric plants. Declines in C_A and DPR occurred with both exogenous and endogenous ethylene treatments. C_A was more sensitive to increasing ethylene concentration than DPR. There was a direct, negative effect of increasing ethylene concentration reducing gas exchange as well as an indirect ethylene effect on leaf epinasty, which reduced light capture and C_A. While the C_A was comparable, there was a lower DPR in hypobaric than ambient pressure plants – independent of ethylene and under non-limiting CO₂ levels (100 Pa pCO₂, nearly three-fold that in normal air). This research shows that lettuce can be grown under hypobaria (≅25% of normal earth ambient total pressure); however, hypobaria caused no significant reduction of endogenous ethylene production.     

Effects of ethylene biosynthesis in carrot root slices on 6-methoxymellein accumulation and resistance to Botrytis cinerea

The role of ethylene biosynthesis in the resistance response of carrot ( Daucus carota L., cv. Chantenay red-cored) slices to infection by Botrytis cinerea Pers. ex Pers. was investigated using aminoethoxyvinylglycine (AVG) and inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.-), and norbornadiene, an inhibitor of ethylene binding. Carrot slices became susceptible to a normally non-invasive level (10⁵ ml^-1) of spores of B. cinerea after treatment with AVG. ACC partially reversed the susceptibility induced by AVG. The ability of a crude pectic enzyme preparation from B. cinerea to induce resistance to a normally invasive level of B. cinerea spores (10⁶ ml^-1) was prevented by AVG. Accumulation of the carrot phyto-alexin 6-methoxymellein (6-MM) was prevented by norbornadiene, but it had no effect on the resistance response. An event associated with ethylene biosynthesis other than 6-MM accumulation appears to be responsible for the resistance of carrot slices to infection by B. cinerea.     

Quantitative relationships between osmotic potential amd epicotyl growth in Vigna radiata as affected by osmotic stress and cotyledon excision

Ethylene biosynthesis in leaf discs of tobacco ( Nicotiana tabacum L. cv. Xanthi), as measured by the conversion of L-[3,4-¹⁴C]-methionine to ¹⁴C₂H₄, was markedly inhibited by exogenous ethylene. This inhibition was accompanied by a decrease in total (free + conjugated) content of 1-aminocyclopropane-1-carboxylic acid (ACC), most of which appeared in its conjugated inactive form. The autoinhibitory effect of ethylene was reversible and could be relieved by Ag⁺. The Ag⁺-treated leaf discs, with or without ethylene, contained only free ACC at an increased level. The results suggest that in tobacco leaves, the autoinhibition of ethylene production resulted from reduction in the availability of free ACC, through both suppression of ACC formation and increased ACC conjugation.     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Ethylene-induced putrescine accumulation modulates K+ partitioning between roots and shoots in barley seedlings

We investigated the cause and effect relationships among ethylene, polyamines, and K⁺ in barley ( Hordeum vulgare L. cv. Amagi) seedlings. Application of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, to the growth medium caused a decrease in K⁺ concentration in roots and an increase in shoots. Addition of ACC induced putrescine accumulation in roots, while spermidine and spermine levels remained unchanged. Exogenous supply of putrescine led to putrescine accumulation and reduced K⁺ concentration. Application of Co²⁺, an inhibitor of ethylene biosynthesis, together with ACC, inhibited putrescine accumulation with a decrease in K⁺ concentration in roots. ACC-treated roots showed K⁺ uptake capacity equivalent to that of control roots, implying that the majority of K⁺ is translocated to shoots. These results suggest that ethylene regulates K⁺ partitioning between roots and shoots through the level of accumulation of putrescine in barley seedlings.     

Changes in catalase activity and hydrogen peroxide concentration in plants in response to low temperature

Taxicity of oxygen species such as free radicals and H₂O₂ has been invoked to explain a number of degradative processes in plants, most involving photo-oxidation. Since catalase is a major protectant against accumulation and toxicity of H₂O₂, we examined alterations in catalase activity in several plant species ( Pisum sativum L. cv. Greenfeast, Vigna radiata (L.) R. Wilcz, Cucumis sativus L. cv. Heinz Pickling, and Passiflora spp.) during chilling, and compared this change to change in H₂O₂ content. Catalase activity was reduced in a range of chilling sensitive and tolerant species by exposure to low temperature. This reduction in catalase activity correlated better with the onset of visible symptoms than with the treatment itself. Visible injury in turn was dependent on light and temperature differences. Hydrogen peroxide concentrations invariably decreased with low temperatures.
Reduction in catalase activity therefore does not necessarily imply accumulation of H₂O₂ to damaging levels. The absence of a clear inverse relationship between catalase activity and H₂O₂ concentration suggests the continued activity of other reactions that remove H₂O₂ and these may be important in the tolerance of plants to oxidative attack. Loss of catalase activity may result from the inability of damaged peroxisomal membranes to transport catalase precursors into the peroxisome.