Ethylene production by plant cell cultures: the effect of auxins, abscisic Acid, and kinetin on ethylene production in suspension cultures of rose and ruta cells

Cell suspension cultures of Ruta graveolens (rue) and Rosa sp. produce ethylene. Both cultures grow at a high rate in hormone-free media. The rose cells are undifferentiated while the Ruta cells differentiate and form shoots after extended culture in hormone-free medium. Addition of 2,4-dichlorophenoxyacetic acid stimulated ethylene production in Ruta cells but not in rose cells. Abscisic acid (ABA) inhibited growth and ethylene production in rose, but only ethylene production in Ruta cells. Addition of kinetin reversed the inhibition by abscisic acid in the rose cells but not in the Ruta cells. The results suggested a distinct physiological difference between the two cultures. The Ruta cells responded to the growth regulators in a manner similar to whole plants.     

Gravitropism in Higher Plant Shoots : IV. Further Studies on Participation of Ethylene

Ethylene at 1.0 and 10.0 cubic centimeters per cubic meter decreased the rate of gravitropic bending in stems of cocklebur (Xanthium strumarium L.) and tomato (Lycopersicon esculentum Mill), but 0.1 cubic centimeter per cubic meter ethylene had little effect. Treating cocklebur plants with 1.0 millimolar aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) delayed stem bending compared with controls, but adding 0.1 cubic centimeter per cubic meter ethylene in the surrounding atmosphere (or applying 0.1% ethephon solution) partially restored the rate of bending of AVG-treated plants. Ethylene increases in bending stems, and AVG inhibits this. Virtually all newly synthesized ethylene appeared in bottom halves of horizontal stems, where ethylene concentrations were as much as 100 times those in upright stems or in top halves of horizontal stems. This was especially true when horizontal stems were physically restrained from bending. Ethylene might promote cell elongation in bottom tissues of a horizontal stem or indicate other factors there (e.g. a large amount of `functioning' auxin). Or top and bottom tissues may become differentially sensitive to ethylene. Auxin applied to one side of a vertical stem caused extreme bending away from that side; gibberellic acid, kinetin, and abscisic acid were without effect. Acidic ethephon solutions applied to one side of young seedlings of cocklebur, tomato, sunflower (Helianthus annuus L.), and soybean (Glycine max [L.] Merr.) caused bending away from that side, but neutral ethephon solutions did not cause bending. Buffered or unbuffered acid (HCl) caused similar bending. Neutral ethephon solutions produced typical ethylene symptoms (i.e. epinasty, inhibition of stem elongation). HCl or acidic ethephon applied to the top of horizontal stems caused downward bending, but these substances applied to the bottom of such stems inhibited growth and upward bending—an unexpected result.     

Interpreting Plant Responses to Clinostating: I. MECHANICAL STRESSES AND ETHYLENE

The severe epinasty and other symptoms developed by clinostated leafy plants could be responses to gravity compensation and/or the mechanical stresses of leaf flopping. Epinasty in cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) is delayed by inhibitors of ethylene synthesis and action (aminoethoxyvinylglycine and Ag⁺), confirming the role of ethylene in clinostat epinasty. To test the possibility that clinostat mechanical stresses (leaf flopping) cause ethylene production and, thus, epinasty, vertical plants were stressed with constant, gentle, horizontal, or vertical shaking or with a quick, back-and-forth rotation (twisting). Clinostat leaf flopping was closely approximated but with a minimum of gravity compensation, by turning plants so their stems were horizontal, rotating them quickly about the stem axis, and then returning them to the vertical, repeating the treatment every four minutes (clinostat rotation time). None of these mechanical stresses produced significant epinasties, but vigorous hand-shaking (120 seconds per day) generated minor epinasties, as did Ag⁺ applied daily (concentrations high enough to cause leaf browning). Plants gently inverted every 20 minutes developed epinasty at about the same rate and to about the same extent as clinostated plants, but plants inverted every 20 minutes and immediately returned to the upright position did not become epinastic. It is concluded that clinostat epinasty is probably caused by disturbances in the gravity perception mechanism, rather than by leaf flopping.     

Mechanism of Auxin-induced Ethylene Production

Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.     

Interrelationships between Ethylene Production,Gall Formation,and Root-knot Nematode Development in Tomato Plants Infected with Meloidogyne javanica

Ethylene production was determined in excised tomato (Lycopersicon esculentum) root cultures of Meloidogyne javanica susceptible and resistant cultivars infected with M. javanica. Uninfected cultivars produced very low amounts of ethylene. Relatively high amounts of ethylene were produced by the infected susceptible cultivars. Peak production of 1.6 n moles * g root⁻¹ * h¹⁻ occurred between 9 and 16 days after inoculation (DAI). The period of high ethylene production coincided with that of rapid increase in gall weight. Low amounts of ethylene were also released by the infected resistant cultivar between 9 and 12 DAI, which follows the hypersensitivity reaction. Ethylene production in infected intact plants during the period of rapid gall growth was twice as much as in uninfected plants during the same time. Exposing excised root cultures to 0.5 or l0 ppm ethylene accelerated the rate of increase in gall weight of M. javanica infected roots. In contrast, overall root growth was inhibited by these treatments, compared to infected roots which were not exposed to ethylene.     

Kinetics and Modeling of Lactic Acid Production by Lactobacillus plantarum

An unstructured model was developed to describe bacterial growth, substrate utilization, and lactic acid production by Lactobacillus plantarum in cucumber juice. Significant lactic acid production occurred during growth, as well as stationary phases. The percentage of acid produced after growth ceased was a function of the medium composition. Up to 51% of the lactic acid was produced after growth ceased when NaCl was not present in the medium, whereas not more than 18% of the total lactic acid was produced after the growth ceased in presence of NaCl, probably because of an increase in the cell death rate. An equation relating the specific death rate and NaCl concentration was developed. With the kinetic model proposed by R. Luedeking and E. L. Piret (J. Biochem. Microbiol. Technol. Eng. 1:393-412, 1958) for lactic acid production rate, the growth-associated and non-growth-associated coefficients were determined as 51.9 (±4.2) mmol/g of cells and 7.2 (±0.9) mmol/g of cells h^-1 respectively. The model was demonstrated for batch growth of L. plantarum in cucumber juice. Mathematical simulations were used to predict the influence of variations in death rate, proton concentration when growth ceased, and buffer capacity of the juice on the overall fermentation process.     

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera

Asexual embryogenesis in Daucus carota L. `Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 μl/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.     

Effect of glyphosate on indole-3-acetic acid metabolism in tolerant and susceptible plants

A comparison study was conducted on the effect of glyphosate (N-[phosphonomethyl]glycine) on indole-3-[2-¹⁴C]acetic acid (IAA) metabolism, ethylene production, and growth of 7-day-old seedlings of different plants. The plants tested were American germander (Teucrium canadense L.), soybean (Glycine max L. Merr.), pea (Pisum sativum L. cv. Alaska and Little marvel), mungbean (Vigna radiata L.), and buckwheat (Fagopyrum esculentum Moench). A spray with 2 mM glyphosate affected IAA metabolism to a varied degree. The induced increase of IAA metabolism was greater in buckwheat, Alaska pea, and mungbean than soybean, Little marvel pea, and American germander. The increased IAA metabolism was correlated with the inhibition of growth and with the decrease of ethylene production. The natural rate of IAA metabolism was markedly different among the plant species and cultivars tested and appeared to be related to the sensitivity of the plants to glyphosate. American germander and Little marvel pea with high rates of IAA metabolism were more tolerant to glyphosate than buckwheat and Alaska pea, which had low rates of IAA metabolism. Plants with a high natural rate of IAA metabolism were probably less dependent on IAA and thus less susceptible to glyphosate.     

Ethylene Production by Tobacco (Nicotiana tabacum) Callus

Tobacco callus cultures grown on defined agar-solidified media produced ethylene in differing amounts, which were related to cultural treatment and age of the callus. There was a close correlation between the rate of ethylene production and growth. In darkness, maximal rates occurred in the third week of growth with ethylene production in the range of 750 nl (callus piece)^?1 d^?1 (fr. wt. = 1.5 g), and in the light, maximal rates occurred in the first week of growth, 200 nl (callus piece)^?1 d^?1 (fr. wt. = 200 mg). Growth was also correlated with ethylene production when the latter was altered by exposure of the callus to inhibitors of ethylene synthesis, L-canaline, benzyl isothiocyanate, and 3,5-diiodo-4-hydroxy-benzoic acid. No correlation was found following treatment with AgNO₃, a presumptive inhibitor of ethylene action. The inhibition of growth and ethylene production by L-canaline was partially reversed by gassing the cultures with ethylene (1 μl/1). A mercuric perchlorate sink had no significant effect on growth. A possible relationship between ethylene evolution and growth is discussed.     

Effect of Inhibitors and Stimulators of Ethylene Production on Gall Development in Meloidogyne javanica-Infected Tomato Roots

Excised tomato roots infected with Meloidogyne javanica produced ethylene at 3-6 times the rate of noninfected roots. This increase in ethylene production started 5 days after inoculation. Gall growth and ethylene production in infected roots were accelerated by 1-aminocyclopropane-1-carboxylic acid (ACC), indole acetic acid (IAA), and ethrel known as ethylene production stimulators. When inhibitors of ethylene production, like aminoethoxyvinylglycine (AVG) or aminoxyacetic acid (AOA), or inhibitors of ethylene action like silver thiosulfate (STS), were applied, gall growth and ethylene production were inhibited. Enhanced expansion of parenchymatous cells was observed in sections from nematode-induced galls and ethylene-treated roots. Lignification of xylem elements and fibers in the vascular cylinder was markedly inhibited in the gall, compared with noninfected root tissue. Because ethylene is known to induce cell expansion and to inhibit lignification, it is suggested that this plant hormone plays a major role in the development of M. javanica-induced galls. Ethylene affects gall size by enhancing parenchymatous tissue development and allows expansion of giant cells and the nematode body by reducing tissue lignification.     

Does Water Deficit Stress Promote Ethylene Synthesis by Intact Plants?

The effect of plant water deficit on ethylene production by intact plants was tested in three species, beans (Phaseolus vulgaris L.), cotton (Gossypium hirsutum L.) and miniature rose (Rosa hybrida L., cv Bluesette). Compressed air was passed through glass, plant-containing cuvettes, ethylene collected on chilled columns, and subsequently assayed by gas chromatography. The usual result was that low water potential did not promote ethylene production. When plants were subjected to cessation of irrigation, ethylene production decreased on a per plant or dry weight basis of calculation. No significant promotion of ethylene production above control levels was detected when water deficit-treated bean or cotton plants were rewatered. The one exception to this was for cotton subjected to a range of water deficits, plants subjected to deficits of −1.4 to −1.6 MPa exhibited a transient increase of ethylene production of 40 to 50% above control levels at 24 or 48 hours. Ethylene was collected from intact leaves while plants developed a water deficit stress of −2.9 megapascals after rewatering, and no significant promotion of ethylene production was detected. The shoots of fruited, flowering cotton plants produced less ethylene when subjected to cessation of irrigation. In contrast, the ability of bench drying of detached leaves to increase ethylene production several-fold was verified for both beans and cotton. The data indicate that detached leaves react differently to rapid drying than intact plants react to drying of the soil with regard to ethylene production. This result suggests the need for additional attention to ethylene as a complicating factor in experiments employing excised plant parts and the need to verify the relevance of shock stresses in model systems.     

Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.     

Bacteriocin Production with Lactobacillus amylovorus DCE 471 Is Improved and Stabilized by Fed-Batch Fermentation

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.     

Ethylene Production by Meloidogyne spp.-Infected Plants

Gall size and rates of ethylene production by various hosts infected with Meloidogyne javanica and by excised tomato root cultures infected with M. javanica or M. hapla were measured. Infection with M. javanica increased the rate of ethylene production in dicotyledonous plants (cabbage, pea, carrot, cucumber, carnation, and tomato), but not in infected monocotyledonous plants (corn, wheat, and onion). Nematode infection induced large galls on roots of dicotyledonous, but not monocotyledonous, plants. Excised tomato roots in culture infected with M. javanica produced ethylene at high rates and formed large galls, whereas roots infected with M. hapla produced ethylene at low rates and induced smaller galls.     

The influence of ethylene on proliferation and growth of rose shoot cultures

Ethylene accumulation in four different rose in vitro culture containers was evaluated. Multiplication rate was the highest, and axes most elongated, in the two containers where ethylene accumulation was limited. Pulse treatments of ethylene at various concentrations enhanced proliferation depending on concentration (5 ppm generally was the most favourable) and time of application, while reducing elongation of the shoots. An ethylene trap in the flask atmospheres of the cultures reduced rose shoot proliferation rate but increased elongation of the axes. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) and cobalt chloride (CoCl₂), increased multiplication rate by providing a higher number of axes of a suitable size for subculture. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) had a beneficial effect on multiplication rate, although reducing longitudinal growth of the axes.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - BA benzyladenine - GA₃ gibberellic acid - IBA indolyl-3-butyric acid     

Ethylene is differentially regulated during sugar beet germination and affects early root growth in a dose-dependent manner

Main conclusion

By integrating molecular, biochemical, and physiological data, ethylene biosynthesis in sugar beet was shown to be differentially regulated, affecting root elongation in a concentration-dependent manner. There is a close relation between ethylene production and seedling growth of sugar beet (Beta vulgaris L.), yet the exact function of ethylene during this early developmental stage is still unclear. While ethylene is mostly considered to be a root growth inhibitor, we found that external 1-aminocyclopropane-1-carboxylic acid (ACC) regulates root growth in sugar beet in a concentration-dependent manner: low concentrations stimulate root growth while high concentrations inhibit root growth. These results reveal that ethylene action during root elongation is strongly concentration dependent. Furthermore our detailed study of ethylene biosynthesis kinetics revealed a very strict gene regulation pattern of ACC synthase (ACS) and ACC oxidase (ACO), in which ACS is the rate liming step during sugar beet seedling development.     

Methionine-induced Ethylene Production by Penicillium digitatum

Shake cultures, in contrast to static cultures of Penicillium digitatum grown in liquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mM was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cycloheximide and actinomycin D. Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilized as a substrate since labeled ethylene was produced from [¹⁴C]methionine.     

Gravitropism in Higher Plant Shoots: I. A ROLE FOR ETHYLENE

It has long been known that applied ethylene can redirect the gravitropic response, but only occasionally has it been suggested that ethylene normally plays a role in gravitropism. Two inhibitors of ethylene synthesis [Co²⁺ and aminoethoxyvinylglycine (AVG)] and two inhibitors of ethylene action (Ag⁺ and CO₂) were shown to delay the gravitropic response of cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) stems. Gentle shaking on a mechanical shaker does not inhibit the gravitropic response, but vigorous hand shaking for 120 seconds delays the response somewhat. AVG and Ag⁺ further delay the response of mechanically stimulated plants. AVG delays the response of defoliated and of decapitated plants. Plants laid on their side and restricted so that they cannot bend upward store both bending energy and gravitropic stimulus; they bend immediately when released from restriction (stored energy) and continue to bend for some hours after (stored stimulus). AVG retards the storage of bending energy but not of stimulus. In gravitropism, graviperception may first stimulate ethylene evolution, which may then influence bending directly, or responses involving ethylene could be more indirect.     

Increased Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon Aeration: Involvement of an NADH Oxidase in Oxidative Stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H₂O₂ oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Carbohydrates Stimulate Ethylene Production in Tobacco Leaf Discs : II. Sites of Stimulation in the Ethylene Biosynthesis Pathway

Galactose, sucrose, and glucose (50 millimolar) applied to tobacco leaf discs (Nicotiana tabacum L. cv `Xanthi') during a prolonged incubation (5-6 d) markedly stimulated ethylene production which, in turn, could be inhibited by aminoethoxyvinylglycine (2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid) (AVG) or Co²⁺ ions. These three tested sugars also stimulated the conversion of l-[3,4-¹⁴C]methionine to [¹⁴C]1-amino-cyclopropane-1-carboxylic acid (ACC) and to [¹⁴C]ethylene, thus indicating that the carbohydrates-stimulated ethylene production proceeds from methionine via the ACC pathway. Sucrose concentrations above 25 mm considerably enhanced ACC-dependent ethylene production, and this enhancement was related to the increased respiratory carbon dioxide. However, sucrose by itself could directly promote the step of ACC conversion to ethylene, since low sucrose concentrations (1-25 mm) enhanced ACC-dependent ethylene production also in the presence of 15% CO₂.