Ethylene and Wound-Induced Gene Expression in the Preclimacteric Phase of Ripening Avocado Fruit and Mesocarp Discs.

Whereas intact postharvest avocado (Persea americana Mill.) fruit may take 1 or more weeks to ripen, ripening is hastened by pulsing fruit for 24 h with ethylene or propylene and is initiated promptly by cutting slices, or discs, of mesocarp tissue. Because the preclimacteric lag period constitutes the extended and variable component of the ripening syndrome, we postulated that selective gene expression during the lag period leads to the triggering of the climacteric. Accordingly, we sought to identify genes that are expressed gradually in the course of the lag period in intact fruit, are turned on sooner in response to a pulse, and are induced promptly in response to wounding (i.e. slicing). To this end, a mixed cDNA library was constructed from mRNA from untreated fruit, pulsed fruit, and aged slices, and the library was screened for genes induced by wounding or by pulsing and/or wounding. The time course of induction of genes encoding selected clones was established by probing northern blots of mRNA from tissues variously treated over a period of time. Four previously identified ripening-associated genes encoding cellulase, polygalacturonase (PG), cytochrome P-450 oxidase (P-450), and ethylene-forming enzyme (EFE, or 1-aminocyclopropane-1-carboxylic acid synthase), respectively, were studied in the same way. Whereas cellulase, PG, and EFE were ruled out as having a role in the initiation of the climacteric, the time course of P-450 induction, as well as the response of same to pulsing and wounding met the criteria[mdash]together with several clones from the mixed library[mdash]for a gene potentially involved in preclimacteric events leading to the onset of the climacteric. Further, it was established that the continuous presence of ethylene is required for persisting induction, and it is suggested that in selected cases wounding may exert a synergistic effect on ethylene action.

Ethylene-Mediated Posttranscriptional Regulation in Ripening Avocado (Persea americana) Mesocarp Discs.

Discs of avocado (Persea americana) fruit (15 x 3 mm thick) kept in a stream of moist air ripen within 72 h. Following cutting, a modest evolution of wound ethylene that dissipates in 24 h is followed by a burst of autocatalytic ethylene production associated with a respiratory climacteric, much as in the intact fruit. Aminoethoxyvinylglycine (AVG), an inhibitor of ethylene synthesis, and 2,5-norbornadiene (NBD) and Ag+, inhibitors of ethylene action, inhibit disc ripening, as does 2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic auxin. On the other hand, none of the foregoing agents except Ag+, at concentrations that delay or prevent ripening, suppress the induction of four ripening-related genes encoding cellulase, polygalacturonase (PG), cytochrome P-450 oxidase (P-450), and ethylene-forming enzyme (EFE, or 1-aminopropane-1-carboxylic acid oxidase), respectively. Whereas Ag+ fully inhibits the production of cellulase and PG mRNAs, it has little effect on the induction of EFE and P-450 mRNAs. Cellulase and PG enzyme activities are absent in extracts of discs treated with AVG, NBD, or 2,4-D, as are antigenically detectable cellulase and PG proteins. The strong appearance of ripening-related mRNAs in discs inhibited from softening by ethylene antagonists suggests posttranscriptional control by ethylene. Similarly, inhibition of ripening by 2,4-D without suppression of mRNA induction suggests translational control. Whether ethylene inhibits transcription or postttranscriptional events or both depends on its concentration.

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 Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit.

When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A(+)) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.

Site-specific binding of a nuclear factor to the carrot extensin gene is influenced by both ethylene and wounding.

Experiments conducted in vitro using the electrophoretic mobility shift assay have shown that a single region of the extensin gene of carrot (Daucus carota L.) interacts with a protein factor designated Extensin Gene Binding Factor-1 (EGBF-1) present in nuclear extracts obtained from carrot roots. This interaction is sequence-specific as judged by the failure of other plant gene sequences to compete with the extensin gene for EGBF-1 binding. The EGBF-1 activity is organspecific, not being expressed in nuclear extracts obtained from carrot leaves or stems. Both ethylene treatment and wounding of roots are shown to have a controlling influence on the expression of EGBF-1 activity in nuclear extracts. These results demonstrate that at least three distinct signals: ethylene treatment, wounding, and development, are important in determining the activity of EGBF-1 in nuclear extracts, and indicate a role for EGBF-1 in stress-related signal transduction and the regulation of extensin-gene expression.

Identification of a wound-induced inhibitor of a nuclear factor that binds the carrot extensin gene.

Following wounding of carrot (Daucus carota L.) roots, the activity of a nuclear factor (EGBF-1) that binds a 5'-region of the carrot extensin gene declines to undetectable levels within 48 h. Mixing of nuclear extracts from wounded roots with nuclear extracts from unwounded roots has demonstrated the existence of a wound-induced inhibitor of EGBF-1. Inhibition of EGBF-1 DNA-binding activity by nuclear extracts from wounded roots is shown to be specific for EGBF-1, and to be destroyed by heat treatment. In addition, inhibition is saturable and occurs rapidly. Active EGBF-1 can be reconstituted from its inhibited state by renaturation of proteins from mixed extracts following denaturation by boiling in sodium dodecyl sulfate and 2-mercaptoethanol, and electrophoretic separation, indicating that inhibition is dependent upon the reversible interaction of EGBF-1 with a titratable factor. However, EGBF-1 activity could not be detected in nuclear extracts from wounded roots following denaturation and electrophoretic separation. Inhibitory activity was not detectable in nuclear extracts from roots that had been trated with ethylene. The action of the inhibitor indicates one possible mechanism for the control of EGBF-1 activity in carrot roots following wounding.

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 O(2). 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 O(2) 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.

Comparative effects of ethylene and cyanide on respiration, polysome prevalence, and gene expression in carrot roots.

Treatment of carrot roots (Daucus carota L.) with 10 microliters per liter ethylene in O(2) evokes a three- to four-fold increase in polysome prevalence and associated poly(A)(+) RNA. The increase in polysome prevalence is attended by a similar change in CO(2) evolution. The increase in polysomal poly(A)(+) mRNA constitutes primarily a generic increase in constitutive mRNAs as assayed by in vitro translation. However, changes in the relative abundance of several in vitro translatable ethylene specific mRNAs do occur.Cyanide, at concentrations which inhibit cytochrome oxidase, initiates a respiratory rise very similar in kinetics and magnitude to that evoked by ethylene. Moreover, the combined treatment with cyanide and ethylene evokes a respiratory response resembling that caused by ethylene or cyanide alone. Nevertheless, cyanide, in the presence of ethylene, significantly inhibits the increase in polysome prevalence and new gene expression associated with ethylene treatment of carrot roots. Separation of in vitro translation products by one-dimensional and two-dimensional gel electrophoresis shows that several new in vitro translation products appear in cyanide-treated carrots different from those evoked by ethylene. Engagement of the less energy efficient alternative electron transport path by cyanide may be responsible for inhibition of the normal ethylene associated increase in polysome prevalence and new gene expression. The implications of these results on regulation of respiratory metabolism are discussed and compared with the results for similar experiments with avocado fruit (Tucker and Laties 1984 Plant Physiol 74: 307-315) in which cyanide does not inhibit an ethylene educed increase in polysome prevalence and change in gene expression.

Interrelationship of Gene Expression, Polysome Prevalence, and Respiration during Ripening of Ethylene and/or Cyanide-Treated Avocado Fruit.

Upon initiation of ripening in avocado fruit (Persea americana Mill. cv Hass) with 10 microliters/liter ethylene, polysome prevalence and associated poly(A)(+) mRNA increase approximately 3-fold early in the respiratory climacteric and drop off to preclimacteric levels at the peak of the respiratory climacteric. The increase in poly(A)(+) mRNA on polysomes early in the respiratory climacteric constitutes a generic increase in constitutive mRNAs. New gene expression associated with ripening is minimal but evident after 10 hours of ethylene treatment and continues to increase relative to constitutive gene expression throughout the climacteric. The respiratory climacteric can be temporally separated into two phases. The first phase is associated with a general increase in protein synthesis, whereas the second phase reflects new gene expression and accumulation of corresponding proteins which may be responsible for softening and other ripening characteristics. A major new message on polysomes that arises concomitantly with the respiratory climacteric codes for an in vitro translation product of 53 kilodaltons which is immunoprecipitated by antiserum against avocado fruit cellulase.Cyanide at 500 microliters/liter fails to affect the change in polysome prevalance or new gene expression associated with the ethylene-evoked climacteric in avocado fruit. Treatment of fruit with 500 microliters/liter cyanide alone initiates a respiratory increase within 4 hours, ethylene biosynthesis within 18 hours, and new gene expression akin to that educed by ethylene within 20 hours of exposure to cyanide.

Cellulase gene expression in ripening avocado fruit: The accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection.

A cDNA library was constructed from poly(A)(+)RNA of ripe avocado fruit. Colony hybridization identified a number of ripening specific clones of which one, pAV5, was shown to be specific for cellulase. Hybrid selection with pAV5 provided a message from ripe fruit that on in vitro translation yielded a polypeptide of 53kD, comigrating with purified avocado cellulase on SDS polyacrylamide gel electrophoresis. The translation product was selectively immunoprecipitated by antiserum to purified avocado cellulase. Immunoblotting of unripe and ripe avocado fruit extracts following SDS-PAGE showed a plentiful immunoreactive polypeptide in ripe fruit, and essentially none in unripe fruit. Hybridization of pAV5 to poly(A)(+)-RNA from unripe and ripe avocado fruit demonstrated that there is at least a 50-fold increase in the cellulase message concentration during ripening. Thus, the expression of cellulase enzyme activity during ripening is regulated by the appearance of mRNA coding for cellulase rather than by either translational or post-translational control mechanisms.

Ethylene-regulated gene transcription in carrot roots.

The plant hormone ethylene elicits many biochemical changes in target tissues. To investigate ethylene effects on expression of genetic information, cDNA clones corresponding to ethylene-induced carrot root mRNAs were constructed and isolated. RNA dot blot analysis showed that for the three clones studied peak cytosolic mRNA prevalence occurred at 21 h of treatment followed thereafter by rapid messenger decay. DNA filter excess hybridization to in vitro synthesized nuclear RNA showed that the ethylene-induced mRNA increase is engendered by transcription of previously quiescent genes. The kinetics and magnitude of changes in mRNA prevalence parallel changes in transcriptional activity; therefore the ethylene effect is primarily at the level of transcription. In vivo pulse labelling with [(35)S]-methionine showed that between 18 and 27 h of ethylene treatment a 2.5 fold increase in translational efficiency occurred for one message studied. The resulting protein is the predominant protein synthesized in carrots treated with ethylene for 27 h. Thus ethylene seemingly exerts multiple regulatory controls on the expression of genetic information.

Membrane-Associated NAD-Dependent Isocitrate Dehydrogenase in Potato Mitochondria.

The oxidation isotherms for citrate and isocitrate by potato (Solanum tuberosum var. Russet Burbank) mitochondria in the presence of NAD differ markedly. Citrate oxidation shows positively cooperative kinetics with a sigmoid isotherm, whereas isocitrate oxidation shows Michaelis-Menten kinetics at concentrations up to 3 millimolar, and cooperative kinetics thereafter up to 30 millimolar. In the absence of exogenous NAD, the isocitrate isotherm is sigmoid throughout. The dual isotherm for isocitrate oxidation in the presence of exogenous NAD reflects the operation of two forms of isocitrate dehydrogenase, one in the matrix and one associated with the inner mitochondrial membrane. Whereas in intact mitochondria the activity of the membrane-bound enzyme is insensitive to rotenone, and to butylmalonate, an inhibitor of organic acid transport, isocitrate oxidation by the soluble matrix enzyme is inhibited by both. The membrane-bound isocitrate dehydrogenase does not operate through the NADH dehydrogenase on the outer face of the inner mitochondrial membrane, and is thus considered to face inward. The regulatory potential of isocitrate dehydrogenase in potato mitochondria may be realized by the apportionment of the enzyme between its soluble and bound forms.

Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria.

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms.

Ethylene regulation of gene expression in carrots.

The respiration of carrot (Daucus carota L.) roots is stimulated by ethylene. Polyribosomes were shown to proliferate concomitantly with the increase in respiration, and the extent of polyribosome augmentation was closely correlated to the amount of respiratory stimulation. In addition to the increase in quantity, ethylene caused a 2-fold increase in the average polyribosome size, suggesting tighter packing of ribosomes on RNA. In vitro translation of carrot polyadenylylated RNA with the rabbit reticulocyte lysate system followed by electrophoresis of the resulting translation products showed that ethylene treatment results in the appearance of new mRNAs.

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 O(2) 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 O(2), with or without peeling. The effect of ethylene plus O(2) 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 O(2) selectively enhances the alternative path. It is proposed that the gross rise in respiration evoked by ethylene is implemented by a system with an O(2) requirement much higher than that of cytochrome oxidase, while the ethylene-induced development of the alternative path depends on a system of still higher O(2) requirement.

Potentiating effect of pure oxygen on the enhancement of respiration by ethylene in plant storage organs: a comparative study.

A number of fruits and bulky storage organs were studied with respect to the effect of pure O(2) on the extent and time-course of the respiratory rise induced by ethylene. In one group, of which potato (Solanum tuberosum var. Russet) and carrot (Daucus carota) are examples, the response to ethylene in O(2) is much greater than in air. In a second group, of which avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii Lambert var. Valery) are examples, air and O(2) are equally effective. When O(2)-responsive organs are peeled, air and O(2) synergize the ethylene response to the same extent in parsnip (Pastinaca sativa), whereas O(2) is more stimulatory than air in carrots. In the latter instance, carrot flesh is considered to contribute significantly to diffusion resistance. The release of CO(2), an ethylene antagonist, is recognized as another element in the response to peeling.The potentiating effect of O(2) is considered to be primarily on ethylene action in the development of the respiratory rise rather than on the respiration process per se. On the assumption that diffusion controls O(2) movement into bulky organs and the peel represents the major diffusion barrier, simple calculations indicate that the O(2) concentration in untreated organs in air readily sustains respiration. Furthermore, in ethylene-treated organs in pure O(2), the internal O(2) concentration is more than enough to maintain the high respiration rates. Skin conductivity to O(2) is the fundamental parameter differentiating O(2)-responsive from O(2)-nonresponsive fruits and bulky storage organs. The large preceding the earliest response to ethylene, as well as the magnitude of the ethylene-induced respiratory rise, is also controlled by permeability characteristics of the peel.

Gene expression during fruit ripening in avocado.

The poly(A) (+)RNA populations from avocado fruit (Persea americana Mill cv. Hass) at four stages of ripening were isolated by two cycles of oligo-dT-cellulose chromatography and examined by invitro translation, using the rabbit reticulocyte lysate system, followed by two-dimensional gel electrophoresis (isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of the resulting translation products. Three mRNAs increased dramatically with the climacteric rise in respiration and ethylene production. The molecular weights of the corresponding translation products from the ripening-related mRNAs are 80,000, 36,000, and 16,500. These results indicate that ripening may be linked to the expression of specific genes.

Disulfiram inhibition of the alternative respiratory pathway in plant mitochondria.

Disulfiram (tetraethylthiuram disulfide) was found to be a potent and selective inhibitor of the alternative respiratory path of plant mitochondria. The onset of inhibition by disulfiram takes several minutes and the inhibition is not readily reversed by washing, nor by metal ions. By contrast, thiols such as dithiothreitol not only reverse, but also prevent, disulfiram inhibition. Inhibition by disulfiram and by hydroxamic acids are not mutually exclusive. Structural analogs of disulfiram are far less potent inhibitors, with the exception of bisethyl xanthogen. Inhibition is due to disulfiram, per se, and not to its reduction product, diethyldithiocarbamate, a powerful chelator. Accordingly, the inhibitory effect of disulfiram is considered to involve the formation of mixed disulfides with one or more sulfhydryl groups in the alternative path. Disulfiram does not act as an electron sink diverting electron flow from oxygen.Disulfiram inhibition was observed only with isolated mitochondria or submitochondrial particles. In intact cells or tissues either a failure to absorb disulfiram, or its dissipation in the cytosol, precludes inhibition. In vitro, bovine serum albumin reduces disulfiram inhibition by complexing free inhibitor.The binding of (35)S-disulfiram by cyanide-resistant mitochondria displays the same kinetics as disulfiram inhibition. A comparison was made of (35)S-disulfiram binding by cyanide-sensitive and cyanide-resistant potato mitochondria. Cyanide-resistant mitochondria were obtained from ethylene-treated potato tubers. Incorporation of label proved essentially the same in both types of mitochondria, suggesting that the disulfiram-sensitive component of the alternative path is present in untreated potato tubers, and is not induced by ethylene.

Alternative path mediated chloride absorption in cyanide-resistant tissues.

The ability of two cyanide-resistant tissues-aged potato slices and fresh preclimacteric banana slices-to take up chloride in the presence of cyanide has been established. Extensive inhibition of chloride uptake by cyanide and chlorbenzhydroxamate together indicates that chloride absorption in the presence of cyanide is supported by respiration mediated by the alternative path. The partial inhibition of respiration-dependent chloride uptake by chlorbenzhydroxamate alone is independent of its effect on the alternative oxidase, and points to inhibition of the transport process per se.