Effect of paraquat on ethylene biosynthesis by intact green Phaseolus vulgaris seedlings

Ethylene production by intact green bean ( Phaseolus vulgaris L. cv. Limburgse vroege) seedlings was investigated in white light and in darkness. In white light both endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene production were stimulated. A decrease in the 1-(malonylamino)cyclopropane-1-carboxylic acid (M-ACC) level and a slight increase in the free ACC concentration could be observed in light. The total amount of endogenous ACC was not changed by light. We related the effect of light to the effect of paraquat on ethylene biosynthesis. Paraquat caused a strong increase of endogenous ethylene production in light. However, the conversion of exogenously applied ACC in light was not influenced by the paraquat treatment, although the presence of the herbicide in the chloroplasts was evident through the inhibition of net photosynthesis. In light, paraquat increased the total ACC content. This was due to an enlargement of the free ACC pool. The effects of white light and paraquat on ethylene biosynthesis can be differentiated from one another: white light exerts its influence on the conversion of ACC to ethylene; it also seems to inhibit the malonylation and may act on the formation of ACC itself. Paraquat influences only ACC synthesis.     

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.     

Irradiance-induced alterations of growth and cytokinins in Phaseolus vulgaris seedlings

Light is a major environmental factor affecting plant growth and development. The cytokinins have many similar effects on these processes and may be involved in photomorphogenesis. In order to study the correlation between light and endogenous cytokinins, we have examined growth parameters and endogenous cytokinins in stems, leaves and other organs of Phaseolus vulgaris, cultivated for 10 days under a range of irradiances (25, 110, 350 and 500 µmol m^–2 s^–1). The nucleotides isopentenyladenosine-5-monophosphate and zeatin riboside-5-monophosphate were the dominant cytokinins, whereas both free bases and ribosides were below the detection level (0.5 pmol g^–1). Plants grown at the highest irradiance had in their stems, leaves, petioles and roots significantly higher levels of cytokinins than had plants grown at the lowest irradiance. As expected, increased light influx increased the dry weight of the root, petiole and leaf, and increased the leaf area, with concomitant increases in the cytokinins in these plant parts. However, the stem showed a different and more complex relationship with irradiance. Stem cytokinin levels increased drastically between 350 and 500 µmol m^–2 s^–1, but this was not correlated with any change in stem length; the light inhibition of stem elongation was mainly seen when irradiance was increased to 110 µmol m^–2 s^–1. Taken as a whole, the results are consistent with an effect of irradiance and cytokinins on the processes favouring biomass production.     

Stimulation of ACC-dependent ethylene production in citrus leaf discs by light

The influence of light and darkness incubation on in vivo ethylene forming enzyme (EFE) activity in citrus ( Citrus sinensis L. Osbeck cv. Salustiana) mature leaf discs was studied. Leaf discs incubated in light produced higher amounts of ethylene than in darkness. Transfer of discs from light to the dark resulted in a marked inhibition of EFE activity, whereas transfer of discs from the dark to light enhanced ethylene forming activity considerably. Light did not affect 1-aminocyclopropane-l-carboxylie acid (ACC) uptake. Incubation in a CO₂-eniiched atmosphere enhanced EFE activity both in light and in darkness, but light stimulation of EFE activity was apparently not affected by CO₂. Effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, inhibitor of photosynthetic electron flow) and KCN (inhibitor of cytochrome oxidase) were studied. DCMU at 0.2 m M inhibited EFE activity in light, whereas no effect was detected in the dark. On the other hand 1 m M KCN stimulated EFE activity in the light, and no significant effect was observed in the dark. CoCl₂ at 1 m M inhibited ACC-dependent ethylene production, suggesting that ethylene production from ACC is mediated by EFE in citrus leaf discs both in light and in the dark. Cycloheximide also inhibited EFE activity in the light and no effects were detected in the dark. Therefore protein synthesis in light (perhaps EFE synthesis) could be required for the light stimulation of the in vivo EFE activity.     

Translatable mRNA changes in ethylene induced abscission zones of Phaseolus vulgaris (Red Kidney)

Abstract The wheat germ translation system was programmed with soluble RNA extracted from foliar abscission zones of Phaseolus vulgaris, These extracts were taken at various times after the induction of abscission. A translation product with a molecular weight of 42 kilodalton (kD) was only present after this treatment, though three other species 32, 27 and 17 kD increased substantially. The isozyme of cellulase with a pi of 9.5 could not be conclusively identified amongst the products though the 32 kD protein is probably chitinase. Comparison of the abscission zone translatable RNA with that from adjacent petiole and stem tissues showed the 17 kD protein developed in all these location. The 42, 32 and 27 kD bands were found predominantly in the zone and petiole.     

Difference in light-induced increase in ploidy level and cell size between adaxial and abaxial epidermal pavement cells of Phaseolus vulgaris primary leaves

Changes in nuclear DNA content and cell size of adaxial andabaxial epidermal pavement cells were investigated using brightlight-induced leaf expansion of Phaseolus vulgaris plants. Inprimary leaves of bean plants grown under high (sunlight) ormoderate (ML; photon flux density, 163 µmol m^–2s^–1) light, most adaxial epidermal pavement cells hada nucleus with the 4C amount of DNA, whereas most abaxial pavementcells had a 2C nucleus. In contrast, plants grown under lowintensity white light (LL; 15 µmol m^–2 s^–1)for 13 d, when cell proliferation of epidermal pavement cellshad already finished, had a 2C nuclear DNA content in most adaxialpavement cells. When these LL-grown plants were transferredto ML, the increase in irradiance raised the frequency of 4Cnuclei in adaxial but not in abaxial pavement cells within 4d. On the other hand, the size of abaxial pavement cells increasedby 53% within 4 d of transfer to ML and remained unchanged thereafter,whereas adaxial pavement cells continuously enlarged for 12d. This suggests that the increase in adaxial cell size after4 d is supported by the nuclear DNA doubling. The differentresponses between adaxial and abaxial epidermal cells were notinduced by the different light intensity at both surfaces. Itwas shown that adaxial epidermal cells have a different propertythan abaxial ones. Key words: Cell enlargement, endopolyploidization, epidermal pavement cells, incident light intensity, leaf expansion, nuclear DNA content, Phaseolus vulgaris     

The physiological role of lipoxygenase in ethylene formation from 1-aminocyclopropane-1-carboxylic acid in oat leaves

In order to understand the physiological significance of the in-vitro lipoxygenase (EC 1.13.11.12)-mediated ethylene-forming system (J.F. Bousquet and K.V. Thimann 1984, Proc. Natl. Acad. Sci. USA 81, 1724–1727), its characteristics were compared to those of an in-vivo ethylene-forming system. While oat (Avena sativa L.) leaves, as other plant tissues, preferentially converted only one of the 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) isomers to 1-butene, the lipoxygenase system converted all four AEC isomers to 1-butene with nearly equal efficiencies. While the in-vivo ethylene-forming system of oat leaves was saturable with ACC with a K_m of 16 M, the lipoxygenase system was not saturated with ACC even at 10 mM. In contrast to the in-vivo results, only 10% of the ACC consumed in the lipoxygenase system was converted to ethylene, indicating that the reaction is not specific for ethylene formation. Increased ACC-dependent ethylene production in oat leaves following pretreatment with linoleic acid has been inferred as evidence of the involvement of lipoxygenase in ethylene production. We found that pretreating oat leaves with linoleic acid resulted in increased ACC uptake and thereby increased ethylene production. A similar effect was observed with oleic acid, which is not a substrate of lipoxygenase. Since linoleic acid hydroperoxide can substitute for lipoxygenase and linoleic acid in this system, it is assumed that the alkoxy radicals generated during the decomposion of linoleic acid hydroperoxide are responsible for the degradation of ACC to ethylene. Our results collectively indicate that the reported lipoxygenase system is not the in-vivo ethylene-forming enzyme.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AEC 1-amino-2-ethylcyclopropane-1-carboxylic acid - Epps N-(2-hydroxyethyl)-piperazine-N-3-propanesulfonic acid - LH linoleic acid - LOOH linoleic acid hydroperoxide - pyridoxal-P pyridoxal-phosphate This work was presented at the 12th International Conference on Plant Growth Substances, Heidelberg, FRG, August 1985 (Abstract No. PO 5-52)     

Apical localization of 1-aminocyclopropane-1-carboxylic acid and its conversion to ethylene in etiolated pea seedlings

The biosynthetic basis for the high rates of ethylene production by the apical region of etiolated pea (Pisum sativum L.) seedlings was investigated. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) was quantified in extracts of various regions of seedlings by measuring isotopic dilution of a ²H-labelled internal standard using selected-ion-monitoring gas chromatography/mass spectrometry. The ACC levels in the apical hook and leaves were much higher than in the expanded internodes of the epicotyl. The capacity of excised tissue sections to convert exogenous ACC to ethylene was also much greater in the apical region, reflecting the distribution of soluble protein in the epicotyl.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - FW fresh weight - GC/MS coupled gas chromatography/mass spectrometry - HPLC high-performance liquid chromatography     

Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper

We studied the sequence of several metabolic reactions, representative for oxidative damage and protection, in primary leaves of Phaseolus vulgaris (cv. Limburgse vroege) as a function of root assimilation of a toxic sublethal Cu concentration (630 μ M ). A transient increase of products of membrane peroxidation was observed in the primary leaves during the period of Cu uptake. This rise was mainly due to the oxidizing properties of copper itself and not to a stimulation of the lipoxygenase (EC 1.13.11.12) activity. In our experimental conditions, membrane lipid peroxidation and K⁺-leakage were not directly related; during at least three days after Cu application to the roots, when products of lipid peroxidation were already detected in the leaf, permeability of the cytoplasmic membrane for K⁺ was improved. However, Cu stimulated the capacity of catalase (EC 1.11.1.6) and ascorbate peroxidase (EC 1.11.1.11). These enzymes protect the tissue against oxidative stress since at least the hydrogen peroxide content was significantly reduced. Superoxide dismutase (EC 1.15.1.1) was not involved in this defense mechanism.     

The effect of light on the production of ethylene from 1-aminocyclopropane-1-carboxylic acid by leaves

White light inhibits the conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) in discs of green leaves of tobacco (Nicotiana tabacum L.) and segments of oat (Avena sativa L.) leaves by from 60 to 90%. Etiolated oat leaves do not show this effect. The general nature of the effect is shown by its presence in both a mono- and a dicotyledon. Since the leaves have been grown and pre-incubated in light, yet can produce from 2 to 9 times as much ethylene in the dark as in the light, it follows that the light inhibition is fully reversible. The inhibition by light is about equal to that exerted in the dark by CoCl₂; it can be partly reversed by dithiothreitol and completely by mercaptoethanol. Thus the light is probably acting, via the photosynthetic system, on the SH group(s) of the enzyme system converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

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.     

Changes in properties of phosphoenolpyruvate carboxylase from the CAM plant Sedum praealtum D.C. upon dark/light transition and their stabilization by glycerol

A prenounced decrease in phosphoenolpyruvate earboxylase (PEPC) activity is observed upon dark/light transition in Sedum praealtum D.C., only when glycerol is included in the extraction medium. If glycerol is omitted, the activity extracted in light is initially low, but soon reaches night levels. The stabilization of the light-induced form of the enzyme by glycerol, in crude or desalted extracts, made it possible to study its kinetic properties in comparison to those of the dark form. The behaviour towards substrate (PEP) changes from hyperbolic (dark) to sigmoid (light), S_0.5 is increased and the enzymic activity becomes more sensitive to malate inhibition. Quite different activity/pH profiles are also obtained for the two forms of PEPC.It is inferred that the in vivo regulation of PEPC in CAM is effected by a concerted action of light, malate and pH shifting.     

The effect of CO2 on ethylene evolution and elongation rate in roots of sunflower (Helianthus annuus) seedlings

Both carbon dioxide and ethylene can affect the rate of root elongation. Carbon dioxide can also promote ethylene biosynthesis by enhancing the activity of 1-aminocylopropane-1-carboxylic acid (ACC) oxidase. Since the amount of CO₂ in the soil air, and in the atmosphere surrounding roots held in enclosed containers, is known to vary widely, we investigated the effects of varying CO₂ concentrations on ethylene production by excised and intact sunflower roots (Helianthus annuus L. cv. Dahlgren 131). Seedlings were germinated in an aeroponic system in which the roots hung freely in a chamber and were misted with nutrient solution. This allowed for treatment, manipulation and harvest of undamaged and minimally disturbed roots. While exposure of excised roots to 0.5% CO₂ could produce a small increase in ethylene production (compared to roots in ambient CO₂), CO₂ concentrations of 2% and above always inhibited ethylene evolution. This inhibition of ethylene production by CO₂ was attributed to a reduction in the availability of ACC: however, elevated CO₂ had no effect on ACC oxidase activity. ACC levels in excised roots were depressed by CO₂ at a concentration of 2% (as compared to ambient CO₂), but n-malonyl-ACC (MACC) levels were not affected. Treating intact roots with 2% CO₂ inhibited elongation by over 50%. Maximum inhibition of elongation occurred 1 h after the CO₂ treatment began, but elongation rates returned to untreated values by 6 h. Supplying these same intact roots with 2% CO₂ did not alter ethylene evolution. Thus, in excised sunflower roots 2% CO₂ treatment reduces ethylene evolution by lowering the availability of ACC. Intact seedlings respond differently in that 2% CO₂ does not affect ethylene production in roots. These intact roots also temporarily exhibit a significantly reduced rate of elongation in response to 2% CO₂.