Roger J. Romani’s research while affiliated with University of California, Davis and other places

Bartlett' pears ( Pyrus communis L.) at two physiological stages, climacteric minimum or approaching the climacteric peak as achieved via storage for 2 or 8 weeks in air at 0C, respectively, were either ripened at 20C in air immediately or after exposure to 0.25% 02 for 4 days at 20C. Fruit stored for 2 weeks had relatively stable phosphofruc- tokinase (PFK), pyrophosphate : fru-6-P phosphotransferase (PFP), and pyruvate kinase (PK) activities but decreasing succinate dehydrogenase (SDH) activities during ripening in air. Similar fruit treated with 0.25% O 2 had slightly increased PFK, PFP, and SDH activities and decreased PK activity. Fruit stored for 8 weeks exhibited higher levels of PFK and PFP activity upon transfer to 20C, in accordance with their more advanced physiological state. In general, the enzymic changes in these fruit upon exposure to 0.25% O 2 and subsequent ripening in air were similar to those observed in the less-mature counterparts, most notable being an increase in mitochondrial SDH. Exposure of suspension-cultured pear fruit cells to hypoxia resulted in an accentuated rise in phosphoenolpyruvate carboxykinase activity and a dramatic rise in SDH activity upon transfer to air. Taken in concert, the enzymic analysis supports the hypothesis that the rise in succinate levels observed in hypoxic fruit tissues is the result of a partial reductive tricarboxylic acid cycle. Cytochrome oxidase activity did not change during hypoxia whereas soluble peroxidase decreased somewhat, perhaps a reflection of their Michaelis constants for O2.

To assess whether demethylation might play a role in the regulation of genes involved in ripening we investigated the changes in methylation levels of two ripening specific genes. DNA was extracted from tomato fruit (Lycopersicon esculentum, var. Castelemart) at progressive stages of ripening and digested with the isoschizomer pair HpaII/MspI. Southern blot analyses revealed a decrease in methylation of sequences containing the genes encoding polygalacturonase and cellulase coincident with the onset of ripening. No differences in methylation levels were observed when LHA1, a constitutively expressed gene not likely subject to changes in methylation, was used as a probe. It would appear that demethylation of specific genes may be involved in the epigenetic control of fruit ripening.

Bartlett' pears (Pyrus communis L.) that had been stored for either 2 or 8 weeks in air at 0C were placed under an atmosphere of 0.25% 0, (balance N 2 ) at 20C for 4 days then returned to air. Control pears were kept in air at 20C. Suspension-cultured `Passe Crassane' pear fruit cells in aging medium were treated similarly. During exposure of the fruit to 0.25% O 2 , loss of greenness and ethylene production were inhibited and CO 2 production substantially decreased. Pears that had been stored for 2 weeks at 0C ripened normally, while those that had been stored for 8 weeks at 0C failed to recover normal ethylene and CO 2 production upon transfer to air after a 4-day exposure to 0.25% O 2 at 20C. Most of the latter fruit were injured as indicated by skin browning. Acetaldehyde and ethanol content increased considerably with ripening of control fruit. Although 0.25% O 2 -treated fruit developed yet higher acetaldehyde and ethanol contents during treatment, the concentrations returned to or below normal during subsequent exposure to air. Pears exposed to 0.25% 0, had increased pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) activities that remained high after ripening in air for 6 days. Three ADH isozymes were discernible in the 0.25% O 2 -treated pears, whereas only one, ADHZ, was found in control fruit. These observations imply that preclimacteric pears are both less stressed during hypoxia and have greater potential for posthypoxia repair than pears of a more advanced physiological age. Increased posthypoxia respiratory and enzymatic activity and the elaboration of new ADH isoenzymes appear to be part of the repair response. Suspension-cultured pear fruit cells responded to the atmospheric changes very much like the S-week stored fruit and likely is a good model system to further study the effects of hypoxia on pear metabolism.

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.

The response of pear fruits and suspension-cultured pear fruit cells to 0% or 0.25% O 2 is being examined to evaluate the feasibility of using such atmospheres for postharvest insect control. These treatments inhibited ethylene production, had no effect on acetaldehyde content, and increased ethanol production in pears kept at 20C for 10 days. The blossom end area of pear fruits was more prone to anaerobiosis, as indicated by increased alcohol dehydrogenase activity and ethanol content. Pear fruit plugs showed increased respiration and ethylene production rates when skin was present compared to plugs without skin. Methods for measuring activity of alcohol dehydrogenase, pyruvate decarboxylase, and pyruvate kinase have been modified and optimized and will be used to determine changes in pear fruit tissue during low O 2 treatment and subsequent recovery in air.

Accurate and speedy measurement of cell growth and assessment of growth-related bioprocess kinetics are essential to the efficient and rational development of plant cell bioprocess engineering. Accurate measurement of cell growth in a plant cell bioreactor system has been a serious problem, in large part, due to the morphology and variation of plant cell lines, such as embryo cells, hairy root cells, and aggregated cells. In our study, several methods that could be used for evaluation of plant cell growth kinetics were compared using the same cell line (Pyrus communis L) medium. Based on our studies, we have concluded that the conductivity method of measuring growth kinetics of plant cell lines was the method of choice, especially for the purpose of bioprocess engineering application of plant cell cultures.

Suspension-cultured `Passe Crassane' pear fruit (Pyrus communis L.) cells in aging media were ventilated with air or air + 20% CO 2 for 4 days at 26C. Cells exposed to elevated CO 2 exhibited reduced respiration (0 2 consumption). Ethylene production of both air and CO 2 -treated cells also declined to barely discernible levels by day 3. Fructose 6-phosphate (F6P) accumulated, while levels of fructose l,6-bisphosphate (F1, 6-P 2 ), and activities of ATP and PPi phosphofructokinases (PFK and PFP) declined in response to elevated CO 2 . These results indicate an 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. The responses to elevated CO 2 levels of the cell suspension system and intact pear fruit ventilated with air + 10% CO 2 are compared, revealing a close similarity.

Ethylene production by apple discs is effectively inhibited by salicylic acid. Inhibition is pH dependent, being greatest from pH 3.5–4.5 and minimal at pH 6.5 and above. With 100 μM salicylic acid maximal inhibition, approximately 90%, is achieved in 3 h with an apparent Ki of 40 μM. At somewhat higher concentrations salicylic acid also inhibits the conversion of 1-aminocyclo-propane-1-carboxylic acid to ethylene by pear discs and mung bean hypocotyls. Salicylic acid interferes with action of the putative ethylene-forming enzyme and in this respect is somewhat more effective than cobalt ion. The inhibitory effects of salicylic acid and cobalt ion are not additive. Implications for the limits and locus of salicylic acid inhibition are discussed.

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.

The extinction point (EP), defined as the lowest O 2 concentration at which alcohol production ceases, was a useful concept in early interpretations of the Pasteur effect in fruit. However, ethanol is now known to be a normal constituent of many fruits under aerobic conditions. Therefore, we propose an alternative concept, the anaerobic compensation point (ACP), defined as the O 2 concentration at which CO 2 evolution is minimum. After 2 to 4 hr under various O 2 concentrations, the ACPs of mature-green pears ( Pyrus communis L.) and pear cell cultures are 1.6% to 1.7% and 1.1% to 1.3% O 2 , respectively. The ACP shifts to lower O 2 concentrations upon extended exposure of the cells to low-O 2 atmospheres and to higher O 2 concentrations as pear fruit mature physiologically or as the diffusion coefficient of cell suspensions is decreased. With O 2 diffusion coefficients of 4.4, 3.7, and 2.5 × 10 ⁻⁶ cm ² ·s ⁻¹ , the ACP effect is observed just below 1.3%, 3.0%, and 5.0% O 2 , respectively. Analogies between the responses of intact fruit and suspension-cultured cells to limiting O 2 are illustrated and use of the latter in assessing the response of pear cells to changing coefficients of diffusion is discussed.