During the Development of the Respiration Climacteric

It is shown that a sequential development of a serie-s of enzyme systems occurs in the peel of the apple as the respiration climacteric develops in the wvhole fruit. The sequence of development of these systems, i.e. acetate incorporation into lipid, production of ethylene, inoorporation of amino acid into protein and, finally, the decarboxylation of added malate (ma,late effect) is the same as that shown earlier for the short term (24 hr) aging of peel discs from pre-climacteric apples. As these systems appear in the initial discs from fruit passing through the climacteric they gradually cease to increase during the 24 hour aging period. Uptake studies show that none of the changes in these systems can be due solely to changes in the permeability of the tissue over the climacteric period. On the basis of these results it is tentatively suggested that the aging of discs from pre-climacteric tissue might provide a model system for a detailed study of the physiological and biochemical changes occurring during the climacteric of apple fruits. It is almost 40 years since Kidd and West attempted to explain the climacteric rise in respiration which precedes visible ripening in apples in terms of a change in "organization resistance" or permeability of the tissue (11). This hypothesis, based on studies of changes in membrane permeability, has been revived in recent years and become 1 of the 2 main theories advanced to explain the series of biochemical changes which are now known to be associated with the climacteric in apples as well as various other fruits. Bain and Mercer (1) found that changes in the inner structure of the chloroplasts had already occurred by the time climacteric rise had begun and as the rise progressed there was also a disorgan,ization of the ultrastrtcture of the protoplasts. Their resuilts were con.sistent witlh ain increase in the permeability of the cells especially in the early stages of the climacteric. Rho(les an-ld WVooltorton (19) slhowed that considerable disorganization of chloroplast lamellae accompaniedl the loss of chlorophyll in the peel of the fruit during the climacteric period. Sacher (25) from his work on the banana concluded that the initiation of permeability changes may be construed as a cause of the onset of the climacteric. At the climacteric peak hiis tissue was "essentially 100 % free space to mannitol, sucrose, fructose, and chloride". In general agreement with this hypothesis, Young and Biale (26) from studies of 32P-uptake by discs of the avocado pear, concluded that the respiratory climacteric is initiated by the failure of the cellular membranes to maintain their permeability characteristics. The other major theory concerning the climacteric originated in the discovery of a net increase in protein (as nitrogenotus material insoltuble in 80 % ethanol) in apples during the climacteric. the increase not taking place under conditions which suppress the development of the climacteric (7). A similar increase in protein was found in avocados and tomatoes by Rowan et al. ('24). 'More recentlv, Richmond and Biale (22) have shown that in avocados, while there appears to be a period of increased protein synthesis during the early stages of the climacteric, there is no direct relationship between protein synthesis and increased O.-uptake throughout the climacteric period. This was implicit for apples in the earlier work of -lutlme (7) in which .1n increase in the ratio of rate of respiratioin to protein conteint (the R/P ratio) xwas demonstrated and linkied wvith the stiggestioni that a change in the pattern of proteini to a more enizNimaticallv active tpl)e mliglht occuir (diring the climacteric rise, a sNntthesis of the 'enllzmes of ripening'. In(lee(l, (dring the p)ast few vears inicreases in thle activity of several imiitochioii(Irial (6. 12, 13) aind soluble enzymne systemls (2, 8, 9, 14, 19, 27) have been reported. These clhanges do not appear to be dule to the removal of enzvmic inhibitors and are not associated with nmarke(d changes in the levels of pyridine nucleoti(les in the tissue (20). Tn the early stages of the respiration climacteric in avocados, Richmond and Biale (23) showed that there was a stimulation in the rate of svnthesis of ribosonmal and messenger RNA. Over the climacteric period as a whole in apples, Looney and Patterson (15) found an increase in total RNA. Previous reports from this laboratory (4 5 21) have described various changes wlhiclh occuir when discs from pre-climacteric apples are aged by incubation in soluition for time periods up to 24 hoLurs. 1154 www.plantphysiol.org on September 22, 2017 Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved. If tl I'E ET AL. .X I''lABOLI,I(' C HANGE;IS IN API'P'LE. P'E1 115 In this earlier work clhanges in 4 enlzymiie systems, naiiiely increase(d incorporationi of '4C acetate into lipid, the development of the capacity of the discs to produce ethylene, the increased incorporation of labeled valine into protein and finally increased capacity of the discs to decarboxylate added malate [the 'malate effect' (3, 17)], were demonstrated. In the present paper we vill consider these same enzvme systems in relation to 2 further processes ic. A) changes occurring in the whole fruit during the climacteric rise in respiration as measured bv the activitv of discs of peel tissue taken from fruit at various stages of the climacteric and assayed immediately as 'initial' (liscs, and B) the further changes that occur wlheln such discs from fruit in the climclcteric plhase al-e subsequently aged for periods ulp to 24 hours. Materials and Methods The fruit ise(l was taken froml a group of 32 Cox's Orange Pippin apple trees growing on Mallinig TX rootstocks in an orchard at Burlingham Horticultural Station, Norfolk, England. Samples of 20 apples of uniform size were stored in glass vessels at 12° (see table I). A stream of CO.,-free air was passed through the vessels at a constant rate and facilities were incorporated into the apparatus so that measurements of CO, and ethylene production bv the fruit could be made at intervals (5, 9). During the period of storage of up to 35 days the respiration of the fruit was monitored daily and samples taken for analysis at suitable intervals as the respiration climacteric developed. Discs of peel were prepared from the apples after they had been surface sterilized by the methods previously described (21). Discs studied immediately after removal from the apple were called "initial discs" while "aged discs" were those incubated aerobically for periods up to 24 hour-s in 0.05 M KH.,PO at pH 4.5 containing 50 /Ag/ml of chloramphenicol (ternmed CAP/plhosphate medium). TIhe measurement of the basic re)spiration, the -malate (lecarboxylating capacity of the discs (manometrically anid radiochemically), the measuremenit of the tuptake and incorporation of 1-14C-acetate, U-'4,C-valine and uptake of 32P04 were as previously described (4, 21). In the incorporation of U_14Cvaline into protein we have shown that at least 90 % of the measured radioactivity was due to peptide-bonded radioactive valinie (21). In the experiment in wlhich the effect of cycloheximiiide on the uptalke and incorporation of valine into protein was situdied, discs from apples at the clinmatcteric peak were aged for 6 houirs in CAP/phosphate nmedium. Then batches of 20 discs were incubated for 1 hour in CAP/phosphate medium contain,ing 1 uc of IJ 1'C-valine (6.9 mc mmole-1) and various concentrationis of cycloheximide (as indicated in fig 5) at 25°. At the end of this period the uptake of valine and the incorporation into protein were (letermiiined as previously described (21). Chloramphenicol was used in the aging an(l incubation medium to minimize growth of microorganisms. We have already shown that it does not itself, at the concentrations used, inhibit the activity of the peel discs (21). Microbial contamination of the discs has been investigated, and the total contamination was found to be in the region of 104 organisms/g tissue; this did not increase significantly with aging.