ABSTRACISalicylic acid inhibited ethylene formation from ACC in self-buffered (pH 3.8) pear (Pyrus communis) cell suspension cultures with a K1'PP 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.Applications of SA2 and ASA to plants have been shown to influence a wide variety of biological processes including flower stimulation (12), vegetative bud formation (4), adventitious root initiation (13), disease resistance (25), stomate function (15), and heat production (24). Recently Leslie and Romani (16) further demonstrated that these compounds strongly reduce the conversion of ACC to ethylene in pear cell suspension cultures, suggesting they inhibit EFE, the putative terminal enzyme in ethylene biosynthesis. Rapid inhibition, proportional to the concentration of SA or ASA in the medium, maximized within 2 h and was followed by a slower reversal requiring a period of hours to days. This paper further characterizes this inhibition and compares SA activity to that of several previously demonstrated EFE inhibitors. under continuous light at room temperature. Experimental additions or manipulations were made following a minimum 1-h equilibration period. ACC was routinely added to increase ethylene production and amplify the effects of SA or other inhibitors. It has been shown, however, that SA inhibits both endogenous and ACC-stimulated ethylene production (16). MATERIALS AND METHODSIn experiments calling for pH adjustment all culture aliquots were supplemented with 40 mm phosphate buffer and pH was adjusted with HCI or KOH as needed. At this level the P04 did not itselfaffect ethylene production. For SA removal, the cultures were centrifuged (International model HN, swinging bucket rotor) at 1000g for 5 min, the supernatant discarded, and the cells gently resuspended in medium of appropriate pH without SA.Ethylene measurements were a modification of the procedure used by Puschman and Romani (21). The 125 mL culture flasks were flushed with a vigorous air flow and capped for 30 min with rubber septa. Six mL head space samples were collected by syringe and ethylene concentrations measured by flame ionization gas chromatography using a Carle model 211 analytical gas chromatograph fitted with an alumina column held at 80°C and employing N2 as a carrier gas.Respiration readings employed an IR CO2 analyzer (H...
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.
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.
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.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. The major advantages of using conductometry as the biosensing technique for measurement of plant cell growth kinetics are:(1) The method is very economical and efficient and gives accurate, reliable, and reproducible measurements, while amenable to continuous on-line monitoring and process control. (2) It is a noninvasive method which does not adversely affect the plant cells or the bioreactor operation. (3) The kind of plant cell lines or their morphology do not affect the method itself.For the development of plant cell culture technology, accurate and rapid measurement of cell growth and assessment of growth-related bioprocess kinetics are essential to the rational development of plant cell bioprocess engineering. Although the plant cell culture system appears to be similar to a microbial cell culture system, there are important differences between the two, and the best method of assessing the plant cell growth kinetics should be carefully examined and evaluated. The major differences include cell size, aggregation of plant cells, change in plant cell physiology for its primary and secondary metabolisms, rheological properties of the medium, and requirement of plant cells for complex nutrients.Measurement of cell growth in plant cell cultivation devices presents a serious problem in some cases. There are various plant cell lines and cell morphology that con- tribute to this problem. There are embryo cells, hairy root cells, aggregated cells, and others that are employed in plant cell culture systems for varying purposes. In each case the determination of growth rate requires different methodology.There are several methods of evaluating growth kinetics of plant cells. Selected examples include fresh cell weight, settled volume, packed cell volume, cell optical d e n~i t y ,~-~ cell size and n~m b e r ,~ nitrogen protein c~n t e n t ,~,~~-~~ nucleic acid,11*'3-'6 organic matters," mitotic index, I4 electrical r e~p i r a t i o n ,~~"~~~ and pH measurement.2' In addition, concentrations of substrate and extracellular product have also been used for such a purpose of selecting the best method of studying growth kinetics, especially from the bioprocess engineering point of view.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 this study, the method tha...
,Most studies concerning the effects of the gas composition of the atmosphere surrounding fruit have been directed toward the response of the combined changes of oxygen (3) is suggested.For the avocado, a fruit which exhibits a climacteric pattern of respiration, it was shown by Biale (2) that low oxygen reduced the respiratory activity during the preclimacteric period andl the (luration of the preclimacteric period was prolonged. Within the range of 2.5 to 21 % oxygen the time required to reach the climacteric peak was extended in proportion to the decrease in oxygen tension and the intensity of respiration at the peak was reduced. No significant stimulation of respiration wvas observed by concentrations of oxygen above 35 %. However, the cumulative carbon dioxide production from the time of picking until the climacteric peak was not changed by the treatment at any level of oxygen. In the case of the banana, another fruit with a climacteric pattern, it was found by Kidd and West ( 11 ) that storage in 2.5 and 5.0 % oxygen did not materially decrease the rate of ripening. On the other hand, Leonard (13) observed a reduction in CO, liberation by fruit stored in oxygen concentrations lower than air and no effect in concentrations higher than air.Lemons did not exhibit the climacteric pattern of respiration after picking when stored in air or at oxygen tensions below that of air, as shown in the sttudies of Biale and Young (5). The respiratory 1 Received revised manuscript Dec. 20, 1961. activity under air decreased slightly during storage. Reduction of oxygen in the atmosphere surrounding the fuit reduced the rate of respiration in proportion to the oxygen concentration in the range of 21 to 5 % oxygen. Carbon dioxide evolution increased at oxygen levels below 5 %, indicating the similarity of the behavior of lemons with other fruits characterized by a critical oxygen concentration. The storage life was extended and the (lecomposition of chlorophyll in mature green lemons was delayed by lowered oxygen.The effect of added carbon dioxide on respiratorv activity of fruits at a particular oxygen concentration has been studied little, largely due to technical difficulties. Limited data are available on the banana. By the use of the katharometer method Gane (9) observed a suppression of the climacteric and reduction of respiratory activity in an atmosphere of 10 % carbon dioxide and 10 % oxygen. Wardlaw (20) subjected unripe bananas to clifferent combinations of oxygen and carbon dioxide. On the basis of gas analysis he concluded that there was a 50 X redluction in rate of respiration over a wide range of 02 and CO2 concentrations as compared with air. His (leterminations were limited to green fruit and iwere done in a closed system in which the gaseous composition could not be kept constant. The use of analytical methods described in the first paper (21) of this series has enabled us to study the effect of carbon dioxide at several oxygen levels on the respiratory activity and storage behavior. The responses to C...
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