Protein Synthesis in Relation to Ripening of Pome Fruits ' Chaim Frenkel

Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. l"C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases durin,g the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment wit;h cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis. App)le, avocado, banana, za(l pear fruiits exh1ibit a marked increase in respliration rate, the clinmacteric. (luring ripening ( 16). It is (lurillg this perio(l of accelerated respiratory' activity that fruits softenl, lose chlorophyll. (levelop flavor and airomat. and iunider-go inuImierouis otlher phy'sical and chemical clhanIges associatedl x\ith ripening. One clhange that h1as attracted the attentioni of investigators is the observatioin by Hulmiie (12, 13) of a net increase in protein during the climiiacteric in alpples. This has generally been found for other climacteric fruits (17, 25), but not in all inxestigations ( 26). This coupled with numerous reports (6, 11, 15, 19. 22) of increased activity of several enzymes during ripening of climacteric fruits, has led to the speculation (9) that the increase in protein content may be the result of synthesis of specific enzymes involved in the ripening processes. Evidence of anmino acid incorporation into proteins of avocado at the earlv-climacteric stage, but sharply falling thereafter, led Richmond anid Biale (24) to propose the induction of enzymes wNhich catalyze the climiiacteric process and the final breakdown of the cell. This is an attractive hypothesis since the onset of the climacteric clearly delineates between cellular activities associated xvith growvth and maturation and those of ripening aind senescence in mannv fruits (1). Tihe objective of this investigation was to obtaini direct evidence of enhanced svx'thesis of specific enzy'mes during the respiratory climacteric of pomle fruits. Materials and Methods Physiologicallys miatture Bai-tlett l)ears (PYrtns comninunis, L. ) w\ere larv'ested and store(l n i airfor up to 2 imioniths prior to the ripening sttudies. Michigain Agricultural Exper-im-eiit Station Journa! .\rticlc No. 4322. Fruits emlployled later thlan 2 miionithls froml lharvest wvere maintained at 0) in a low O., atmosphere (ca. 2-3 %) achieved hv enclosing fruits in a sealed polyetllylene l)ag containinig hydrated limie. This wxxas necessary to inistlure normial rip)ening b)elavior following 3 or 4 mlonithis storage ( 18). Respfiratioi illMcasifrciciits. Oxygen ul)take aiid CO., production of intact fruit wvere measuredl at 2(° (lurinig the rip)ening stucdies wvith an automiatedl gas aaly'Sis systemii eillm)lo\ig aL Beckman paramiagnetic O., analyzer and an infra-red CO., anallyzer (8). R ip/clinl 1cl casic re cni ts. Parameters emlplove(l for ripening Nvere (legreening and softening. Chloroplyll content of the peel tissue was measured spectrophotometrically following acetone extraction. Softening of the cortical tissue was measured by a Magness-Tavlor fruit pressure tester. Ethvlenie Deteruiiinations. Ethylene Nvas deternined by gas chromatography employing a oneeightlh inclh X 6 foot colunln of Poropak Q and a flame ionization (letector. Ethvlene was collected from the airstream after passinig over the fruit durinig respiratory gas analysis or by extracting the internial atmosplhere. Proteitn Syntdi esis. To determline amino acid labeling of specific enzymes during the respiratory climacteric, procedures were employed to insure incorlporation of tracer aminio acid into sufficient fruit protein for subsequelnt purifi cati on and( analyses. To imieet this re(quirenment intact fruits xxere emlploved rcather th in tissue slices. Uniformly labeled 'C L-phenvIlalanine w,vas administered to intact fruit using a vacuuim infiltration technique. A hypodermic needle (containing a cleanouit Nxvire to prevent clogging) was inserted ilnto the central cavity region. The fruit wvith needle in place x<Vas aLttached to a syringe fitted into a rubber stopper and l)ositioned in a large moutlh glass clhamber. Solution xvaS introduced into the frulit throtuglh the sVi'imge as the clhamber xas e-acuated at ca. 1)8) lilm 1146 www.plantphysiol.org on December 30, 2017 Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved. FRENKEL ET AL.-PROTEIN SYNTHESIS AND FRUIT RIPENING Hg. This procedure provided for rather uniform distribution (as judged by dye distribution) of up to 10 ml of solution containing the amino acid throtughout the tissue of individual fruits within a 30 minute period. The infiltrating solution consisted of 0.35 M mannitol containing 0.4 ,c 14,C L-phenylalanine and 1 X 10-4 mM '2C L-phenylalanine per ml. 3H-uridine was incorporated in a similar manner to determine RNA synthesis. Various adjuncts were included as indicated in the text. Two replicates of 4 fruits each at various stages in the climacteric comprised a treatment series. At specified intervals following infiltration, longitudinal sections of each fruiit were removed and the cut surface of the remainder was overlayed with mvlar film. A composite 5 g sample of cortical tissue was obtained from the 4 fruits for determination of phenylalanine incorporation. The tissue was placed in 20 ml of 20 % TCA and heated to 1000 for 5 minutes, homogenized and separated by centrifugation into TCA-soluble and -insoluble fractions. The precipitate was washed twice with 5 % TCA containing 1 X 10-3 M plhenylalanine. The final precipitate was suspended in 20 ml of 1 N NaOH. Radioactivity of the trichloroacetic acid-soluble and -insoluble fractions was determined as described below. An acetone dried powder was made of the remainder of the 4 fruits utilizing the low temperature procedure of Clements (4) and was employed for polyacrylamide gel electrophoresis. Most of the experiments were repeated. Radioactivity Determinations. All radioactivitv measurements were made with a liquid scintillation spectrometer. The scintillation mixture consisted of: 4.5 g BBiOT [2,5-bis(2-(5-tert-butylbenzoxazolye) ) -theophene], 80 g naphthalene, 385 ml xvlene, 385 ml p-dioxane, 231 ml ethanol, and 37 g Cab-o-sil (thixotropic gel powder obtained from Cabot Corporation, Boston, Massachusetts). Counting efficiencies of 75 to 81 % were obtained with up to 1 ml of the variouis aqueous solutions or suspenisionis mixed with 15 nil of the scintillation solution. ;\Al counts were corrected for qutenclhing to obtain (lisintegrations per minutte (dpmn). Ele0rophoresis. Polyacrylaniiide gel was uised as the supporting media for disc gel or vertical slab electropboresis. Cul-rent was maintained at 8.8 nim per cm2 cross section for disc electrophoresis at 00 and at 12 volts per cm for vertical slab electrophoresis a-t 50 to 55°. Acrylamide running gels (6 and 7 %) and spacer gel for disc electrophoresis were prepared as described by Davis (5). Vertical slab gels were prepared by mixing the appropriate quantities of acrylamide, N,N-methylenebisacrylamide, tris, HCI, and water to prepare 106 ml of 7 or 6 % gel and adding 0.05 ml of N,N,N1,Nltetramethylethylenediamine, and 100 mg ammonium persulfate immediately before pouiring the solution into the gel chamber. Protein E.rt-action. The proteins in acetone dried powders (AP) of the fruit tissues were extracted by placing 500 mg of AP in the upper compartment of centrifugal filters (Gelman Instrument Company, Ann Arbor, Michigan) with 5 ml of a solution consisting of: 0.1 M K-phosphate buiffer, pIfr 9.0, 0.3 M mannitol, and 1 mm EDTA. After 1 hour of incubation at 00 with occasional stirring. the solution was separated from the residue by centrifugation at 2000 X g for 10 minutes. The residuie was re-extracted with 5 ml portions of the above soltution. Extracts obtained from singleor multiple extractions were employed for enzyme, electrophoresis, radioactivity or protein determinations. Exceptions are indicated in the text. Malic Enzymne Assay. Enzyme activity of various extracts was determined spectrophotometrically at 340 m.t (7). The standard assay consiste(d of: 0.1 M glycylglycine, 1.0 mm MnSO4, 0.16 mmi NADP, 3.0 mM L-nialate and up to 200 enzyme units at a pH 7.3 and 200. Localization of malic enzyme on acrylamide gels was acconiplished by incubating the gels immediately after electrophoresis in a solution of: 0.1 M glycylglycine, 0.05 M T,-malate, 1.0 mm MnSSO4 containing; 0.3 mg NADP, 0.8 mg m-nitroblue tetrazoliunm chloride (Nitro BT) and 0.14 mg phenazine methosulfate per ml at pH 7.3 and 200. The ratio of solution to gel volume was 1. Precipitation of the purple diformazan occurred on the gel at the site of malic enzvme within 2 hours.