Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 ± 2°C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (−)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.     

Ethylene as an endogenous inhibitor of root regeneration in tomato leaf discs cultured in vitro

We examined ethylene effects on root regeneration in tomato leaf discs cultured in vitro. Applied ethylene or Ethephon did not stimulate rooting in the leaf discs. In the presence of indoleacetic acid. 5 × 10^-6M, these substances significantly inhibited root formation. Ethylene production (nl C₂H₄· (24 h)^-1. flask^-1) was positively correlated with increased IAA concentrations at various times during the culture period and, as a consequence, with the rooting response after 168 h. However, separate testing of equimolar concentrations of seven different auxins and auxin-like compounds showed no positive correlation between the rate of ethylene production and subsequent rooting response. Aeration of gas-tight flasks containing leaf discs and absorption of ethylene evolved from the discs by mercuric perchlorate in gas-tight flasks or pre-treatment of leaf discs with AgNO₃ significantly enhanced IAA induced root regeneration. Thus, these studies indicate that ethylene is not a rooting hormone per se. Furthermore, ethylene (whether applied externally or synthesized by the tissue) does not appear to account for the ability of auxin to stimulate rooting.     

Effects of exogenous ethylene on ethylene production in citrus leaf tissue

Exogenous ethylene stimulated ethylene production in intact citrus (Citrus sinensis L. Osbeck cv. “Washington Navel”) leaves and leaf discs following a 24-hour exposure. Studies with leaf discs showed that ethylene production decreased when ethylene was removed by aeration. The extent of stimulation was dependent upon the concentration of exogenous ethylene (1-10 microliters per liter). Silver ion blocked the autocatalytic effect of ethylene at concentrations of 0.5 millimolar and lower, but increased ethylene production at higher concentrations. The stimulating effect of ethylene resulted from the enhancement of both 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. Whereas autocatalysis was evident following 24 hours incubation, autoinhibition of wound- and mannitol-induced ethylene production was observed during the first 24-hour incubation. Ethylene treatment during this period resulted in a marked decrease in ACC levels and ethylene production rates. Furthermore, in leaf discs treated for 24 hours with ethylene, ethylene production rates increased greatly during the first 2 hours after removal of exogenous ethylene by aeration. This increase was eliminated if the discs were transferred to propylene instead of air, indicating that the autocatalytic effect of ethylene is counteracted by its autoinhibitory effect. It is suggested that autocatalysis involves increased synthesis of ACC synthase and the enzyme responsible for the conversion of ACC to ethylene, whereas autoinhibition involves suppression of the activity of these two enzymes.     

Ethylene-induced Isocoumarin Formation in Carrot Root Tissue

The concentrations of 3-methyl-6-methoxy-8-hydroxy-3,4-dihydroisocoumarin (MMHD) formed in carrot roots inoculated with certain fungi or treated with indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, or 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were related to the amount of ethylene produced by the root tissue. Ethylene applied exogenously in concentrations above 0.3 ppm induced the formation of MMHD in carrot root discs. Continued production of MMHD required the continued presence of ethylene. The amounts of MMHD in the discs were reduced by CO₂, an inhibitor of ethylene action, and by reduction of the partial pressure of ethylene in fungus-inoculated or 2,4,5-T-treated carrot root discs. The results indicate that ethylene is required for the induction of MMHD formation by carrot root tissue.     

Endorhizal and Exorhizal Acetylene-reducing Activity in a Grass (Spartina alterniflora Loisel.)-Diazotroph Association

Earlier studies indicated that bacteria responsible for nitrogenase activity of some grasses are located inside the roots. Those studies were conducted with excised roots in which a long, unexplained “lag phase” occurred before initiation of nitrogenase activity. When hydroponically maintained Spartina alterniflora Loisel. was incubated in a two-compartment system with acetylene, ethylene was produced following, at most, a 2-hour lag in both the upper (shoot) and lower (roots + water) phases. Ethylene production in the upper phase not attributable to leaf-associated acetylene-reducing activity or to diffusion of ethylene from around the roots is considered to represent “endorhizal acetylene-reducing activity,” the internally produced ethylene diffusing into the upper phase via the lacunae. Ethylene produced in the lower phase is designated “exorhizal acetylene-reducing activity.” The endorhizal acetylene-reducing activity, in comparison to exorhizal activity, was relatively insensitive to additions of HgCl₂, NH₄Cl, or carbon sources to the lower phase. Post-lag acetylene-reducing activity of roots excised from plants growing in soil responded to additions in a manner similar to that of endorhizal acetylene-reducing activity, whereas post-lag acetylene-reducing activity of rhizosphere soil responded in a manner similar to that of exorhizal acetylene-reducing activity.     

Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for hormonal regulation of plant growth

Activity of arginine decarboxylase in etiolated pea seedlings appears 24 hours after seed imbibition, reaches its highest level on the 4th day, and levels off until the 7th day. This activity was found in the apical and subapical tissue of the roots and shoots where intensive DNA synthesis occurs. Exposure of the seedlings to ethylene greatly reduced the specific activity of this enzyme. The inhibition was observed within 30 min of the hormone application, and maximal effect—90% inhibition—after 18 hours. Ethylene at physiological concentrations affected the enzyme activity; 50% inhibitory rate was recorded at 0.12 microliters per liter ethylene and maximal response at 1.2 microliters per liter. Ethylene provoked a 5-fold increase in the K_m^app of arginine decarboxylase for its substrate and reduced the V_max^app by 10-fold. However, the enzyme recovered from the inhibition and regained control activity 7 hours after transferral of the seedlings to ethylene-free atmosphere. Reducing the endogenous level of ethylene in the tissue by hypobaric pressure, or by exposure to light, as well as interfering with ethylene action by treatment with silver thiosulfate or 2,5-norbornadiene, caused a gradual increase in the specific activity of arginine decarboxylase in the apical tissue of the etiolated seedlings. On the basis of these findings, the possible control of arginine decarboxylase activity by endogenous ethylene, and its implication for the hormone effect on plant growth, are discussed.     

Involvement of wound and climacteric ethylene in ripening avocado discs

Avocado (Persea americana Mill. cv Hass) discs (3 mm thick) ripened in approximately 72 hours when maintained in a flow of moist air and resembled ripe fruit in texture and taste. Ethylene evolution by discs of early and midseason fruit was characterized by two distinct components, viz. wound ethylene, peaking at approximately 18 hours, and climacteric ethylene, rising to a peak at approximately 72 hours. A commensurate respiratory stimulation accompanied each ethylene peak. Aminoethoxyvinyl glycine (AVG) given consecutively, at once and at 24 hours following disc preparation, prevented wound and climacteric respiration peaks, virtually all ethylene production, and ripening. When AVG was administered for the first 24 hours only, respiratory stimulation and softening (ripening) were retarded by at least a day. When AVG was added solely after the first 24 hours, ripening proceeded as in untreated discs, although climacteric ethylene and respiration were diminished. Propylene given together with AVG led to ripening under all circumstances. 2,5-Norbornadiene given continuously stimulated wound ethylene production, and it inhibited climacteric ethylene evolution, the augmentation of ethylene-forming enzyme activity normally associated with climacteric ethylene, and ripening. 2,5-Norbornadiene given at 24 hours fully inhibited ripening. When intact fruit were pulsed with ethylene for 24 hours before discs were prepared therefrom, the respiration rate, ethylene-forming enzyme activity buildup, and rate of ethylene production were all subsequently enhanced. The evidence suggests that ethylene is involved in all phases of disc ripening. In this view, wound ethylene in discs accelerates events that normally take place over an extended period throughout the lag phase in intact fruit, and climacteric ethylene serves the same ripening function in discs and intact fruit alike.     

The Influence of Auxin and Ethylene on Chromatin-directed Ribonucleic Acid Synthesis in Soybean Hypocotyl

Soybean seedlings treated with ethylene exhibited small increases in ribonucleic acid content in the elongating section of the hypocotyl. Chromatin isolated from the elongating section of ethylene-treated seedlings showed a 35 to 60% increase in the capacity for RNA synthesis. The ethylene-induced response was saturated at 1 microliter/liter of ethylene and was fully expressed after 3 hours. Auxin caused marked accumulation of RNA and DNA in the elongating and basal tissue of the hypocotyl. Chromatin isolated from these auxin-treated tissues showed an 8- to 10- fold increase in RNA synthetic capacity as measured in vitro. Ethylene added with auxin reduced the auxin enhancement of nucleic acid synthesis in the elongating and basal tissues. Both ethylene and auxin treatment of the seedlings inhibited nucleic acid accumulation and chromatin activity in the apical tissue. Ethylene did not appear to mediate the auxin effects on nucleic acid synthesis in soybean hypocotyl with the possible exception of inhibition in the apical tissue.     

Differential counteraction of ethylene effects by gibberellin a(3) and n(6)-benzyladenine in senescing citrus peel

Treatment of mature citrus fruit (Citrus sinensis) with ethylene induced rapid chlorophyll destruction, a rise in respiration, a release of free amino acids, an accumulation of reducing sugars, and an appearance of phenylalanine ammonia lyase activity. Gibberellin A₃ (GA₃) and N₆-benzyladenine (BA) opposed the effects of ethylene on chlorophyll, amino acids, and to a lesser extent, reducing sugar levels. The ethylene-induced respiratory rise was only slightly modified by GA₃ and BA. Phenylalanine ammonia lyase activity was not affected by GA₃.     

Selective enhancement of alternative path capacity in plant storage organs in response to ethylene plus oxygen: a comparative study

The respiration rise in bulky storage organs induced by ethylene plus pure O₂ is accompanied by an increase in the CN-resistant respiration, or alternative path. Whereas a lesser respiratory stimulation in response to ethylene is demonstrable in air and increased by peeling, ethylene-induced alternative path development depends on the synergistic effect of pure O₂, with or without peeling. The effect of ethylene plus O₂ is evident, whether untreated parent organs yield CN-sensitive or CN-resistant fresh slices. Alternative path capacity and maximal cytochrome oxidase-mediated electron transport have been separately estimated. Ethylene plus O₂ selectively enhances the alternative path. It is proposed that the gross rise in respiration evoked by ethylene is implemented by a system with an O₂ requirement much higher than that of cytochrome oxidase, while the ethylene-induced development of the alternative path depends on a system of still higher O₂ requirement.     

Light Stimulation of Ethylene Release from Leaves of Gomphrena globosa L

The effect of light and CO₂ on both the endogenous and 1-aminocyclopropane-1-carboxylic acid (ACC)-dependent ethylene evolution from metabolically active detached leaves and leaf discs of Gomphrena globosa L. is reported. Treatment with varying concentrations of ACC did not appear to inhibit photosynthesis, respiration, or stomatal behavior. In all treatments, more ethylene was released into a closed flask from ACC-treated tissue, but the pattern of ethylene release with respect to light/dark/CO₂ treatments was the same.

Leaf tissue in the light with a source of CO₂ sufficient to maintain photosynthesis always generates 3 to 4 times more ethylene than tissue in the dark. Conversely, the lowest rate of ethylene release occurs when leaf tissue is illuminated and photosynthetic activity depletes the CO₂ to the compensation point. Ethylene release in the dark is also stimulated by CO₂ either added to the flask as bicarbonate or generated by dark respiration. Ethylene release increases dramatically and in parallel with photosynthesis at increasing light intensities in this C₄ plant. Ethylene release appears dependent on CO₂ both in the light and in the dark. Therefore, it is suggested that the important factor regulating the evolution of ethylene gas from leaves of Gomphrena may be CO₂ metabolism rather than light per se.

     

Mediation of a plant response to malformin by ethylene

Malformin and ethylene stimulate abscission of the primary leaves of Phaseolus aureus Roxb. in the dark, and abscission stimulation by both compounds is inhibited by indeleacetic acid and CO₂. Ethylene production by malformin-treated buds is stimulated within 4 hours. and up to 8 days, after treatment. Malformin-induced growth disturbances in P. vulgaris L. and abscission in P. aureus are considered mediated by ethylene. Although root curvatures of Zea mays L. are induced by both malformin and ethylene, and malformin is inhibited by CO₂, ethylene production is not stimulated by malformin. A role of ethylene in root curvatures induced by malformin is neither proposed nor disproved.     

Autoinhibition of Ethylene Production in Citrus Peel Discs : SUPPRESSION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHESIS

Wound ethylene formation induced in flavede tissue of citrus fruit (Citrus paradisi MacFad. cv. Ruby Red) by slicing was almost completely inhibited by exogenous ethylene. The inhibition lasted for at least 6 hours after removal of exogenous ethylene and was then gradually relieved. The extent of inhibition was dependent upon the concentration of ethylene (1 to 10 microliters/liter) and the duration of treatment. The increase in wound ethylene production in control discs was paralleled by an increase in 1-aminocyclopropane-1-carboxylic acid (AAC) content, whereas in ethylene-treated discs there was little increase in ACC content. Application of ACC completely restored ethylene production in ethylene-pretreated discs, indicating that the conversion of ACC to ethylene is not impaired by the presence of ethylene. Thus, autoinhibition of ethylene synthesis was exerted by reducing the availability of ACC. Ethylene treatment resulted in a decrease in extractable ACC synthase activity, but this decrease was too small to account for the marked inhibition of ACC formation. The data indicate that autoinhibition of ethylene production in citrus flavede discs results from suppression of ACC formation through repression of the synthesis of ACC synthase and inhibition of its activity.     

Shoot Inversion Inhibition of Stem Elongation in Pharbitis nil: A Possible Role for Ethylene-Induced Glycoprotein and Lignin

Inversion of the upper shoot of Pharbitis nil results in the inhibition of elongation in the inverted stem. The objective of the present study was to determine how shoot inversion-induced gravity stress inhibited elongation and to elucidate the possible role of ethylene-induced glycoprotein and lignin in this process. Determinations of hydroxyproline, peroxidase, phenylalanine ammonia-lyase (PAL), phenol, and lignin content/activity were carried out by appropriate spectrophotometric methods. It was found that inversion and Ethrel treatments of upright shoots caused significant increases in hydroxyproline content, peroxidase, and PAL activity in 12 hours and in phenol and lignin contents in 24 hours. All of these increases except for that of cytoplasmic peroxidase activity were partially reversed by AgNO₃, the ethylene action inhibitor. It is concluded that possible cross-linking associated with the accumulation of ethylene-induced hydroxyproline-rich glycoprotein and lignin may be responsible for the later stages of cessation of elongation in the inverted Pharbitis shoot.     

Chitinase in bean leaves: induction by ethylene,purification, properties,and possible function

Ethylene induced an endochitinase in primary leaves of Phaseolus vulgaris L. The enzyme formed chitobiose and higher chitin oligosaccharides from insoluble, colloidal or regenerated chitin. Less than 5% of the total chitinolytic activity was detected in an exochitinase assay proposed by Abeles et al. (1970, Plant Physiol. 47, 129–134) for ethylene-induced chitinase. In ethylene-treated plants, chitinase activity started to increase after a lag of 6 h and was induced 30 fold within 24 h. Exogenously supplied ethylene at 1 nl ml^?1 was sufficient for half-maximal induction, and enhancement of the endogenous ethylene formation also enhanced chitinase activity. Cycloheximide prevented the induction. Among various hydrolases tested, only chitinase and, to a lesser extent, β-1,3-glucanase were induced by ethylene. Induction of chitinase by ethylene occurred in many different plant species. Ethylene-induced chitinase was purified by affinity chromatography on a column of regenerated chitin. Its apparent molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis was 30,000; the molecular weight determined from filtration through Sephadex G-75 was 22,000. The purified enzyme attacked chitin in isolated cell walls of Fusarium solani. It also acted as a lysozyme when incubated with Micrococcus lysodeikticus. It is concluded that ethylene-induced chitinase functions as a defense enzyme against fungal and bacterial invaders.     

Anomalous Temperature Dependence of Seedling Development in Some Soybean (Glycine max [L.] Merr.) Cultivars: Role of Ethylene

In Clark and Shelby soybean (Glycine max [L.] Merr.) seedlings, hypocotyl elongation was inhibited and hypocotyl swelling and root dry weight were increased by a temperature of 25 C. At 20 and 30 C, development was normal, as was development of Hawkeye and Mandarin soybean seedlings at all three temperatures. Dry matter distribution at 9 days indicates that inhibition of hypocotyl elongation is not due to a lack of translocation from cotyledons, but to a diversion of dry matter from hypocotyl to root. Ethylene evolution by Clark seedlings at 25 C exceeds that at 20 and 30 C. At all three temperatures, Mandarin seedlings' ethylene evolution is at the same low rates as those of Clark at 20 and 30 C. Clark's enhanced rate at 25 C precedes the deceleration of hypocotyl elongation occurring at 5 days. The abnormal effects of a temperature of 25 C on Clark seedlings' development is partially reversed by CO₂ and GA₃ and can be duplicated in Mandarin by applying ethylene. In Clark, effects of the temperature can be further accentuated by indoleacetic acid, which stimulates ethylene evolution, and by applying ethylene to the seedlings. It is concluded that the temperature-induced symptoms, similar in most respects to the well known “triple response” of legume seedlings, are caused by abnormally high levels of ethylene in tissues of the anomalous cultivars.     

Stimulation of ethylene production in citrus leaf discs by mannitol

Wound ethylene formation induced in leaf tissue of citrus (Citrus sinensis [L.] Osbeck cv. “Washington Navel”) by excision was significantly stimulated by mannitol after a lag period of about 6 hours. The extent of stimulation was dependent upon the concentration of mannitol (10 to 100 millimolar). This increased ethylene production was not simply due to osmotic effect or water stress as other osmoticums tested failed to exert such an effect. The stimulatory effect of mannitol resulted from both the enhancement of 1-aminocyclopropane-1-carboxylic acid (ACC) formation and the conversion of ACC to ethylene. The effect on the latter step was particularly pronounced in aged discs. The use of labeled mannitol showed that it was taken up by the leaf discs, utilized for respiration, and metabolized to sucrose, but no radioactivity was detected in the ethylene.     

Turnover of 1-aminocyclopropane-1-carboxylic Acid synthase protein in wounded tomato fruit tissue

Ethylene production in plant tissues declines rapidly following induction, and this decline is due to a rapid decrease in the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, a key enzyme in ethylene biosynthesis. To study the nature of the rapid turnover of ACC synthase in vivo, proteins in wounded ripening tomato (Lycopersicon esculentum) fruit discs were radiolabeled with [³⁵S]methionine, followed by a chase with nonradioactive methionine. Periodically, the radioactive ACC synthase was isolated with an immunoaffinity gel and analyzed. ACC synthase protein decayed rapidly in vivo with an apparent half-life of about 58 min. This value for protein turnover in vivo is similar to that previously reported for activity half-life in vivo and substrate-dependent enzyme inactivation in vitro. Carbonylcyanide-m-chlorophenylhydrazone and 2,4-dinitrophenol, potent uncouplers of oxidative phosphorylation, strongly inhibited the rapid decay of ACC synthase protein in the tissue. Degradation of this enzyme protein was moderately inhibited by the administration of aminooxyacetic acid, a competitive inhibitor of ACC synthase with respect to its substrate S-adenosyl-l-methionine, α,α′-dipyridyl, and phenylmethanesulfonyl fluoride or leupeptin, serine protease inhibitors. These results support the notion that the substrate S-adenosyl-l-methionine participates in the rapid inactivation of the enzyme in vivo and suggest that some ATP-dependent processes, such as the ubiquitin-requiring pathway, are involved in the degradation of ACC synthase proteins.     

Headspace ethylene accumulation on Stizolobium hassjoo hairy root culture producing L-3,4-dihydroxyphenylalanine

The addition of 1-aminocyclopropane-1-carboxylic acid (ethylene precursor), or 2-chloroethylphosphonic acid (ethephon, an ethylene-releasing compound) decreased root dry weight and l-DOPA (l-3,4-dihydroxyphenylalanine) accumulation in hairy root cultures of Stizolobium hassjoo. The inhibition caused by ethephon-mediated ethylene release was alleviated by 0.5 mg CoCl₂ l^–1 as an inhibitor of ethylene biosynthesis. The action of ethylene was inhibited by 1.5 mg AgNO₃ l^–1. Ethylene thus lowers hairy root formation and l-DOPA production; CoCl₂ decreases ethylene formation leading to a considerably improved root dry weight and l-DOPA production.     

Differential control of ethylene-induced gene expression and respiration in carrot roots

Ethylene treatment of carrot roots elicits a developmental program encompassing an increase in respiration rate and changes in gene expression. Both phenomena are potentiated when ethylene is administered in O₂. Our previous studies showed that both respiration and a number of ethylene specific mRNAs increase together in response to ethylene through some 21 hours, whereas thereafter respiration continues to rise, while the level of induced mRNAs drops. Herein we ask whether an experimentally effected drop in the respiration rate within the first 21 hours caused by the withdrawal of ethylene, or substitution of air for O₂ in the continued presence of ethylene, is linked to a drop in the level of ethylene-induced mRNA. Quantitative estimation of two ethylene evoked mRNAs by dot blot hybridization with appropriate cDNA clones has shown that under the specified treatment the induced mRNA levels remain constant while the respiration rate drops, suggesting that gene expression, as reflected in induced mRNA levels, and respiration rate are separately regulated facets of the ethylene response.