Metabolically driven self-restoration of energy-linked functions by avocado mitochondria: general characteristics and phosphorylative aspects.

To assess the restorative capacity of isolated avocado (Persea americana) fruit mitochondria, the organelles were first aged in the absence of an energy source at 25 degrees C for several hours until respiratory control and oxidative phosphorylation were greatly diminished or totally lost. Energy-linked functions were then gradually restored over a period of several hours after the addition of substrate. Restoration of respiratory control resulted from both an increase in state 3 and a decrease in state 4 respiratory rates. Either alpha-ketoglutarate or succinate served as restorants, each with distinctive rates of recovery in state 3 and state 4 respiration. ATP also served as a restorative agent but not as effectively as metabolizable substrate. ATP synthase activity was modulated by stress and restoration but neither the extent nor the rate of change was sufficient to constrain state 3 rates. Orthophosphate was released from the mitochondria during substrate-deprived stress. Restoration of phosphorylation preceded that of RC with phosphate uptake and phosphorylation being evident immediately upon the addition of substrate. During restoration [(32)P]orthophosphate was incorporated into several organic fractions: phospholipid, ATP, a trichloroacetic acid-precipitable mitochondrial fraction, and an organophosphate that accumulated in the medium in relatively large amounts. The organophosphate was tentatively identified as a hexosephosphate. Incorporation into ATP and the putative hexosephosphate continued unabated beyond the point of maximum restoration. Phosphate metabolism thus appears to be a necessary but not sufficient precondition for mitochondrial restoration and maintenance. Based on the recovery kinetics of the various phosphorylated components, the mitochondrial-bound fraction appears to be most directly linked with restoration. Results are discussed with reference to specific characteristics and components of self-restoration and to possible underlying mechanisms. We suggest that a degree of self-restoration is consistent with the quasi-autonomous nature of mitochondria and that this intrinsic capacity may be pivotal to the respiratory climacteric in senescent fruit cells and to cellular homeostasis in general.

Inhibition of ethylene biosynthesis by salicylic Acid.

Salicylic acid inhibited ethylene formation from ACC in self-buffered (pH 3.8) pear (Pyrus communis) cell suspension cultures with a K(1) (app) of about 10 micromolar after 1 to 3 hours incubation. Inhibition appeared noncompetitive. Among 22 related phenolic compounds tested, only acetylsalicylic acid showed similar levels of inhibition. Inhibition by salicylic acid was inversely dependent on the pH of the culture medium and did not require a continuous external supply of salicylate. When compared to known inhibitors of the ethylene forming enzyme, cobalt, n-propyl gallate, and dinitrophenol, inhibition by salicylic acid most closely resembled that by dinitrophenol but salicylic acid did not produce the same degree of respiratory stimulation. Results are discussed in terms of other known effects of salicylic acid on plants, pH-dependency, and the possible influence of salicylic acid on electron transport.

Respiration and protein synthesis in nongrowing cultured pear fruit cells in response to ethylene and modified atmospheres: a model system for fruits postharvest.

The respiration of pear fruit (Pyrus communis L. Passe Crassane) cells was monitored after subculture into an auxin-free, mannitol-enriched medium in which the cells remained viable but did not grow. Respiration rates were affected by the presence or absence of sucrose in the medium even though the cells retained reserves of sucrose and starch. Provided the medium contained respirable carbohydrate, exposure to ethylene (1-10 microliters per liter) increased the respiration rate with some acceleration of cell death. In the range from 10 to 2% oxygen by volume, the respiration rate of the cells decreased with oxygen concentration resulting in some prolongation of cell life. Thus, in their responses to ethylene and modified atmospheres, the cells reflected the behavior of harvested fruits. Having defined conditions under which respiration rate could be varied without apparent influence on the quiescent state of the cells, we sought a connection between maintenance respiration and protein turnover. Relative rates of protein synthesis were assessed by measuring ribosome distribution between monosomes and polysomes. In general, the higher the respiration rate the higher the proportion of polysomes supporting the thesis that protein turnover is a variable component of maintenance metabolism. Protein turnover in cells incubated in the presence or absence of sucrose was measured as retained alpha-amino-(3)H following a pulse of (3)H(2)O. Turnover was shown to be a quantitatively important component of the maintenance budget and to be more rapid in cells in media supplemented with sucrose through the chase period. The experiments illustrate that cultured cells may be used to explore aspects of the maintenance metabolism of resting or senescent cells that are not amenable to study in bulky fruit tissues.

Effects of Elevated CO(2) Concentrations on Glycolysis in Intact ;Bartlett' Pear Fruit.

Mature intact ;Bartlett' pear fruit (Pyrus communis L.) were stored under a continuous flow of air or air + 10% CO(2) for 4 days at 20 degrees C. Fruit kept under elevated CO(2) concentrations exhibited reduced respiration (O(2) consumption) and ethylene evolution rates, and remained firmer and greener than fruit stored in air. Protein content, fructose 1,6-bisphosphate levels, and ATP:phosphofructokinase and PPi:phosphofructokinase activities declined, while levels of fructose 6-phosphate and fructose 2,6-bisphosphate increased in fruit exposed to air + 10% CO(2). These results are discussed in light of a possible inhibitory effect of CO(2) at the site of action of both phosphofructokinases in the glycolytic pathway, which could account, at least in part, for the observed reduction in respiration.

Salicylic acid: A new inhibitor of ethylene biosynthesis.

Salicylic acid and acetylsalicylic acid at concentrations of 10(-6)M to 10(-4)M effectively inhibit ethylene production by pear cell suspension cultures. Results suggest these acids act by blocking the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene.

Effects of low temperature and respiratory inhibitors on calcium flux in plant mitochondria.

The effects of low temperature on uptake and release of (45)Ca(2+) were studied with sound, well-coupled mitochondria extracted at room temperature from avocado (Persea americana Mill, cv Fuerte) fruits. Low Ca(2+) concentrations (10 micromolar) were employed to simulate physiological conditions. At 25 degrees C, the rate of Ca(2+) uptake decreased with time, whereas at 5 degrees C the initial rate, though lower, remained linear. As a consequence total uptake at 5 degrees C was substantially greater than at 25 degrees C for periods greater than 5 min. Preincubation of mitochondria at 5 degrees C enhanced subsequent Ca(2+) uptake at 25 degrees C. Ca(2+) uptake was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and by ruthenium red, but neither KCN nor salicylhydroxamic acid separately or together had any major inhibitory effect. Preloaded mitochondria held for 60 min in a Ca-free medium lost little Ca(2+) at 25 degrees C and none at 5 degrees C, except in the presence of ruthenium red or CCCP.

Cycloheximide stimulation of cyanide-resistant respiration in suspension cultures of senescent pear fruit cells.

Pear fruit cells undergoing a period of senescence in auxin-deprived media develop a substantial cyanide resistant respiration in response to the addition of 0.7 to 3.5 micromolar cycloheximide. The inhibitor does not affect overall cellular repiratory activity and titrations with salicylhydroxamic acid reveal that only a minor portion, about 10%, of the alternate pathway is utilized by the cycloheximide-treated senescent cells. The alternate respiratory pathway appears to be of mitochondrial origin but is not induced by chloramphenicol.

Effects of aminoethoxyvinylglycine and countereffects of ethylene on ripening of bartlett pear fruits.

Pear fruits (Pyrus communis L. var. Bartlett) were treated with solutions containing aminoethoxyvinylglycine (AVG) using a modified vacuum infiltration method that introduced 4.3 milliliters solution per 100 grams tissue. At concentrations of 1 millimolar, AVG strongly inhibited ethylene production and delayed for 5 days the respiratory climacteric and accompanying ripening changes in skin color and flesh firmness. AVG was less effective in inhibiting the ripening of more mature fruits. Fruit infiltrated with 5 millimolar AVG had not begun to ripen 12 days after initiation of ripening in the controls. When treated with ethylene the inhibited fruit exhibited a climacteric rise in respiration, softened, and became yellow. Treatment of the AVG infiltrated fruits with ethyelne for 24 hours resulted in no recovery in endogenous ethylene production, but in a stimulation of protein synthesis measured as a 200% increase in leucine incorporation by excised tissue and a 74% increase in the percentage of ribosomes present as polysomes.

Senescence of Pear Fruit Cells Cultured in a Continuously Renewed, Auxin-deprived Medium.

Auxins and cytokinins support cell division in tissue and cell cultures. In cytokinin-independent pear (Pyrus communis) cells, omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from the medium for two successive transfers leads to rapid cell lysis, unless the osmolarity is raised to 0.4 molar with mannitol. Use of this system (nutrients plus mannitol minus 2,4-D) for the study of cell senescence was explored both in batch culture and in a system designed to permit medium renewal without withdrawal of live cells.In both systems, an initial period (1-6 days) of limited increase in cell number is characterized by a continuous decrease in the respiratory activity and in protein and RNA synthesis to very low basal rates. In batch culture, cell death occurs after 13 to 15 days with little or no change in metabolic activity, or in protein and RNA synthesis. With renewal of cell medium, death is slightly delayed and is preceded by a burst in RNA synthesis followed by a notable increase in protein synthesis. Cycloheximide inhibition of protein synthesis is transient and its effect on cell longevity variable. Nonetheless, in all instances cell death is preceded by a burst in protein synthesis. Actinomycin D (1.6 micromolar) did not significantly affect protein synthesis but delayed RNA synthesis and cell death. The possible roles of auxin, osmoticum, and macromolecular synthesis in cellular senescence and death are discussed.

The maintenance of respiratory control at 25 degrees C by mitochondria from various animal and plant sources.

1. Mitochondria from four different animal and five different plant sources exhibit a wide variation in their capacity to maintain respiratory control at 25 degrees C. 2. Beef heart mitochondria, and pear and avocado fruit mitochondria exhibit the longest retention of respiratory control extending to periods of approximately 80 and 40 hr, respectively.

Ripening and in vitro retention of respiratory control by avocado and pear mitochondria.

The retention of respiratory control ("survival") by mitochondria held at 25 C was studied in relation to the ripening of two varieties of avocado (Persea americana Mill. var. ;Fuerte' and ;Hass') and one variety of pear (Pyrus communis. L. var. ;Bartlett') fruit. The survival of avocado mitochondria increased from 8 to 10 hours when isolated from unripe, preclimacteric fruit, to 48 hours when isolated from fully ripe, postclimacteric fruits. Although rates of alpha-ketoglutarate oxidation, respiratory control, and ADP/O decreased somewhat in the postclimacteric phase, survival per se was not affected. Pear mitochondria survived for more than 30 hours regardless of the physiological age of the source.Exposure of postclimacteric avocado mitochondria to a preclimacteric supernatant fraction curtailed their survival. The harmful effect of some unknown substance(s) in the preclimacteric avocado supernatant fraction was confirmed by utilizing pear mitochondria as an independent test system.

"Survival" of mitochondria in vitro: physical and energy parameters.

Isolated mitochondria have been maintained active and coupled for 72 hours at 25 C. Survival (retention of respiratory control) is a function of incubation temperature and dependent upon aeration and substrate. ATP does not entirely substitute for substrate, indicating a need for products of active metabolism other than energy. An improvement in respiratory control is often observed during the first several hours of incubation. Sedimentation and resuspension at 24-hour intervals prolonged survival. As revealed by electron microscopy, mitochondria maintained their basic structure during a 72-hour period at 25 C.Survival is a dynamic, energy-requiring process and must be distinguished from so-called "aging" of organelles at ice temperatures. As a manifestation of partial autonomy, survival may prove useful in assessing aspects of mitochondrial function and the mitochondrial-cellular interrelationship.

The ribosomes of pear fruit. Their synthesis during the climacteric and the age-related compensatory response to ionizing radiation.

Maceration at liquid nitrogen temperatures, use of poly-vinylpyrrolidone, and careful pH control are essential to the isolation of ribosomes and polysomes from deciduous fruit tissue. Characteristics of the ribosomes and constituent RNA are described. The distribution of ribosomes among monomer and polymer forms remains relatively constant until fruit reach the climacteric peak, after which there is a notable decline in the polymeric forms. In contrast to the relative stability in size distribution there is a marked change in ribosomal turnover during the climacteric rise. A transitory increase in rate of ribosomal synthesis is followed by a rapid decline coincident with the final portion of the climacteric rise. No incorporation of radioactive base into ribosomes could be detected once fruit had reached the climacteric peak.Coincidence of radioactivity with the ribosomal RNA on methylated albumin-Kieselguhr chromatograms and complete inhibition of ribosomal RNA synthesis by actinomycin D confirm that radioactive nucleotide was incorporated into newly synthesized ribosomes. Data are presented to distinguish between a cellular response to injury, as may result from the preparation of tissue slices, and the effects of physiological age. Superimposed intracellular radiation injury stimulates the synthesis of new ribosomes and underscores a major transition in the dynamics of the ribosomal system coincident with the climacteric rise.