cellular senescence, radiation damage to and compensatory ... · climacteric pear fruit cells...

Plant Physiol. (1968) 43, 108Q-lNO

Cellular Senescence, Radiation Damage to Mitochondria, and theCompensatory Response in Ripening Pear Fruits'

Roger J. Romani, Ida K. Yu, Lily L. Ku, L. Karl Fisher, and Nancy DehganDepartment of Pomology, University of California, Davis, California 95616

Received February 5, 1968.

Abstract. A compensatory response, viz. in vivo recovery from radiation damage to mito-chondria, ocours in preclimacteric pear fruits (Pyrus communis L.) -treated with ionizingradiation. The compensatory response is absent or markedly impaired in senescent fruitsirradiated at or near the climacteric peak. 'Senescent cells failed to reoover from harmfulaffects of radiation on: 1) mitoohondrial yield, 2) in vivo incorporation of amino acids intomitochondrial protein, and 3) mitochondrial respiratory control and ADP/O. A diminishedresponse to "split-dose" irradiation and a delayed rate of recovery confirmed the degeneracyand loss of compensatory power with cell age.A loss of restorative activity, especially in mitochondria that supply the celil with essential

energy, may underlie the more obvious signs of cumulative stress that accompany cellularsenescence. Use of ionizing radiation as an investigative tool and the molecular implicationsof radiation damage, recovery, and cellular senescence are discussed.

It is generally accepted that the climacteric phaseof ripening fruits is well suited for the study ofcellular senescence (1). Since a surge in respira-tory activity is the dominant, readily measurable phe-nomenon, mitochondria are implicated as the sites ofcausal events. This view was embodied in the earlywork of Millerd et al. (19) with the resultingpostulate that uncoupling of oxidative phosphoryla-tion permitted an increased respiratory rate. Alongsimilar lines, Hulme (7) and Pearson and Robertson(21) attributed the rise in respiration to the avail-ability of phosphate acceptor (ADP) resulting fromincreased protein synthesis and accelerated demandsfor energy. However, with specific reference tomitochondrial protein, data on the yield of particulateprotein (27) and on the incorporation of amino acidsinto mitochondrial protein (28) indicate a declinein synthetic activities as fruit reach the climactericpeak.

Richmond and Biale (23), Sacher (37), andespecially Young and Biale (44) have questioned theprevalence of "uncoupling" or "ADP control" inripening fruit cells. Moreover, the work of Lanceet al. (10), Hulme et al. (8,9) and the experimentsreported below, reveal that mitochondria remainfunctionally sound and "cotipled" throughout theclimacteric phase.

1 This investigation was supported by the UnitedStates Atomic Energy Commission, Contract AT (11-1)-34, Project No. 112, Report No. UCD34P112-32.

2 "Preclimacteric" and "climacteric minimum" will beused interchangeably as will "climacteric" and "climactericpeak."

The persistence of active, phosphorylating mito-chondria implies their continued essentiality to thesenescent cells. More specifically, energy producingmitochondria may be vital to compensatory reactionsessential for the survival of aging cells (38, 43).In this context, a change in the ability of mitochon-dria to compensate for stress, temporal or otherforms, is a more critical age-dependent transitionthan changes in mitochondrial activity per se.

We have previously shown that massive, in vivoirradiation of preclimacteric pear2 fruits results in aloss and subsequent recovery of mitochondrial pro-tein and an actual enhancement of mitochondrialactivity (32). Similarly irradiated fruit cells alsoexhibit a suppression and recovery in the capacity toincorporate amino acids into mitochondrial protein(29). UTtilizing respiratory control (RiC) as anindex to mitochondrial function, it was furtherdemonstrated that RC is lost and then restored, invivo, following massive irradiation of preclimactericpear fruit (34).

Evidence has therefore been provided that pre-climacteric pear fruit cells retain a capacity to repairradiation damage to mitochondria. At the sametime, radiation stress also provided a tool with whichto assess the level of compensatory activity as afunction of cell age. Accordingly, with each radio-biological study experiments were also carried oututilizing pears at or near their climacteric peak. Wepresent these age-comparative aspects in this paperand purport to demonstrate a marked decline incompensatory power with progressive senescence andthe approach of the climacteric peak.

Preliminary reports of this work have appearedelsewhere (30,35).

1089 www.plantphysiol.orgon September 21, 2020 - Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

PLANT PHYSIOLOGY

Materials and MethodsTreatmenit of Fruiit. 'Bartlett' and 'Bosc' pears

(Pvruts cornmutnis L.) were obtained from localorchards and stored for several days at 0 to 20 beforeuse. \Vith the exception of "split-dose" experimentsfor which fruits were taken directlv from coldstorage, all samples were first placed in respirationjars at 20{ and CO, production measured (3). Ateither their climacteric minimum or near their cli-macteric peak, pears were taken from the jars andirradiated with cobalt-60 gamma rays. A uniformityof absorbed dose within ±10 % was assured byprior ferrous sulfate dosimetry (33). Dose ratesover the period of these experiments ranged from0.28 to 0.35 'Mrad/hr. Temperature during irradia-tion ranged from 0° to 50. After exposure the fruitwere either kept chilled (00-50) for the isolation ofmitochondria or returned to respiration jars at 200.

Split-Dose Irradiation. A normal irradiationtreatment was given except that only a portion of thetotal dose was administered at 1 time. Between doseincreimieints the fruits were returned to respirationjars at 200.

Isoiation of Ilitochondria. For determinationsof mitochondrial vield, peeled, grated, and homog-enized pear tissue was first lyophilized, and thenreconstituted prior to the isolation of mitochondria.Details of the isolation procedure have been givenelsewhere (27). "Yield" was defined as the amountof proteini in the once-washed mitochondrial pellet,exxpressed as a percent of the protein, b)othnmito-chonidrial anid soluble, remaining in suspeInsioni afterthe first low speed centrifugation. Hence, anomalousresults from differential cell-breakage were largelyavoided.

Freslh tissue and added precautions were neces-sarv to obtain coupled mitochondria from pears, orapples ('34). Use of polyvinylpyrrolidone (PVP),as described by Hulme et al. (8) and carefully con-trolled pH during gentle maceration were primeessentials. \Viskich (42) also reported similar re-quirements for apple mitochondria. In brief, ap-proximately 60 g of peeled, cold, pear tissue weregently cut and macerated into 180 ml of isolationmediunm consisting of 0.5 M sucrose, 0.05 M tris(pH 6.8), 0.006 M EDTA, cysteine (2 ,moles/gmtissue), 0.5 % PVP (40,00 MW), and 1 mg/mlbovine serum albumin. The pH was monitored con-tinnally durinig maceration and kept between 6.8 and.0 with dropwise additions of KOH.

Centrifugations, first for 10 minutes at 1200 X gad theln 15 minutes at 18,000 X g, were used torenm'ove debris and to sedinment the mitochondria.The centrifugations were repeated to obtain once-washed and once-clarified mitochondria.

Incorporation of Amino Acids. Procedures havebeen reported in detail elsewhere (29). Tissueslices were first incubated for 4 hours in a buffersolution containing 14C-leucine (0.03 ,,c per ml).Mitochondria were then isolated from the slices and

the mitochondrial protein precipitaited with trichloro-acetic acid and resolubilized in base. After a secondcycle of precipitation and solubilization in NH41OH,the final protein solution was place(d on lens paperand dried in preparationi for liquid scintillationcounting.

Assays. UtilizatioIn of 02 by the mitochondriawas measure(l with a polarographic oxygen electrode(34). Respiratory control (RC) was determinedas prescribed by Chance and Williams (2). Tonormalize the slight increase in RC knowln to occurwitlh each successive addition of ADP (34) State 3and State 4 rates were always calculated at the third(penultimate) addition of ADP. The final additionof ADP resulted in a renewed State 3, assuring a

sufficiency of O., in the reaction mixture.Proteins were assayed with a modification of the

Lowry method (18).

ResultsRespiration of Whole Fruit. \With most fruits

and vegetables irradiation causes a burst of respira-tory activitx (15, 16). Pertinent to this study isour earlier observation that the magnitude of therespiratory rise is markedly reduced when fruit isirradiated at or near the climacteric peak (25, 31).Typical post-irradiation respiratory rates as affectedby dose [0.25 avid 1 millioin rad (Mrad) and cli-macteric state at time of irradiation, are shown infigure 1. The respiratory rates of pears irradiatedat the climacteric minimuimii undergo increases of300 to 400 %. In conitrast, siiilar doses applied ator near the clinmacteric peak elicit onlv a 20 to 40 %increase. The respiratory rise caused by 1.0 Mraldlis transient, leading rapidly into a suppressed activity.This transition from sustained to declining post-irradiation respiratory rates generally occurs inbetween 0.4 and 0.6 1\rad (25) and cotuld be con-

s 40H

U-

(9x 30

-J2 20

10

_ rMINIMUM, r..- RISE - , PEAK-_-rPOST-

\ V 0

2~ ~ ~ ~~~~~04_ _

0 0~ ~2 3 4 5 6 7

DAYS

8 9 10 11 12

FIG. 1. Respiratory activity of Bartlett pears irradi-ated at either the climacteric minimum or near the cli-macteric peak as indicated by the swiggle arrows. Un-irradiated controls (O-- -0), 0.25 Mrad dose (*-*),1.0 Mrad dose ( *-----). Approximate stages of theclimacteric are indicated at the top.

1090

..o0

www.plantphysiol.orgon September 21, 2020 - Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved.


ROMANI ET AL.-SCENESCENCE, MITOCHONDRIA AND RADIATION RECOVERY

z

w

0

a.

8

r

0 7z

0

o 6

5

-J

5 4

I I !_

A B

0

0

-U-M

.."S

I ,I 11 I,.0 2 3 4 5 6 0 2 3 4 5 6

DAYS AFTER IRRADIATIONFIG. 2. Yield of mitochondrial protein from pear

fruit tissue that had received 0.3 (0 ) Mrad or0.6 (E---E) Mrad of ionizing radiation at the climactericminimum (A) or near the climacteric peak (B). Thevield of mitochondria from nonirradiated pears, (0---0).Yiel,d = particulate protein expressed as a percent oftotal protein remaining in t-he supernatant fraction afterthe first low-speed centrifugation. Each point in partA is the average of 6 separate isolations and in part Bthe average of 7 isolations. Methods for statisticalanalyses of the data were discussed elsewhere (32).

sidered as a first sign of excessive intra-cellulardamage.

Yield of Mlitochondria. A statistically significantdecrease and subsequent recovery of mitochondrialprotein occurred following irradiation of preclimac-teric pears with 0.25 or 0.3 Mrad (32). Higherdoses (0.6-1.0 Mrad) caused an even greater lossof mitochondrial protein with no recovery (32).The impact of increased senescence on the mito-chondrial yield is illustrated in figure 2. In contrastto preclimacteric pears (fig 2A), only minor changesin mitochondrial yield were noted following irradia-tion at or near the climacteric peak (fig 2B).Moreover, at no time following the irradiation ofclimacteric pears was there a sign of recovery in

mitochondrial protein. Indeed, statistical analyses ofthe yield data from climacteric fruit revealed that,with only 2 exceptions (0.3 Mrad, day 6; and 0.6Mrad, day 2), the differences in mitochondrial yieldas a result of radiation treatment were nonsignificant.

The unresponsiveness of older fruit cells in termsof mitochondrial yield correlates with the suppressedrespiratory response (fig 1). For the climactericfruit used in the yield determinations, the respiratoryresponses to 0.3 and 0.6 Mrad were not only minorbut also indistinguishable.

Incorporation of Amino Acids. The influencesof irradiation and cell age on mitochondrial yields,noted above, are substantiated by data on the rate ofamino acid incorporation into mitochondrial protein.We have previously shown that there is 1) a markeddecline in rate of incorporation as fruit reach theirclimacteric peak (28); 2) a suppression and subse-quent recovery in incorporation rate following a0.25 Mrad radiation dose to preclimacteric pears(29); and 3) a more drastic suppression with norecovery following a 1.0 Mrad dose to similar pre-climacteric fruit (29). The question remained asto the effects of fruit age at time of irradiation.Data from a typical experiment examining the age-effect are shown in table 1.

An approximately 50 % inhibition caused by 0.25Mrad was reversed within 24 hours in preclimactericcells, there was no such reversal in pear cells neartheir climacteric peak. A 1.0 Mrad dose resulted ina greater and irreparable loss of activity regardlessof the climacteric state.

Recovery of Mitochondrial Functions. The per-turbations implied in the preceding experimentscalled for an assessment of their effects on mito-chondrial activity. For this purpose RC, A;DP/O,and Qo, (protein) were used as indices. A con-sistent pattern of suppression and recovery of theseftunctional properties was noted following the irradia-tion of preclimacteric pears (34). However, asshown in figure 3, the performance of mitochondriafrom pears irradiated at the climacteric peak wasquite different.

Each oxygen electrode trace (fig 3) was obtainedwith mitochondria from fruit given an identical,1.0 Mrad radiation dose. Representative fruit sam-ples were macerated and mitochondria isolated eitherimmediately after exposure or after 24 hours at 200.Oxidative activity was suppressed and RC completelveliminated by the 1.0 Mrad dose regardless of climac-teric stage. However, after 24 hour mitochondria

Table I. Incorpor(ation of 14C-Leucine into Mitochondrial Protein as a Function of Radiation Dose, Stage of CellSenescence, and Time Interval Between Radiation Exposure and Introduction of Labeled Amino Acids

Radio-specific activityRadiation Preclimacteric Near climacteric peak2

dose 1 hrl 24 hr 1 hr 24 hrMrad cPm/mg protein X 10-20 55.7(-+±1.4) 63.4(+±0.5) 40.3 (±4.6) 36.6(+±0.4)0.25 24.8(+±0.4) 60.7( ±0.7) 14.2 (+4.7) 10.4(4±0.7)1.0 6.0(±+1.8) 3.4(±+0.5) 2.7( ±0.5) 2.6(±+0.9)Approximate time interval between irradiation of the pear fruit and preparation of tissue slices. Values are theaverage ('cpm/mg protein) in 2 separate incubations of pear tissue slices.

2 Pears were irradiated approximately 24 hours before they would 'have reached their climacteric peak,

1091

a



-1'1LANTr 1P YSIOI.O(X'

FIG. 3. Effects of a 24 hour delay in the isolationof mitochondria from pears irradiated with a 1.0 Mraddose at the climacteric peak (upper 2 traces) or at theclimacteric minimum (lower 2 traces). Repeated addi-tions of 0.3 ,umole ADP indicated by the slant lines.Numbers along trace refer to rate of 0.2 uptake in ,ul0,/hr. Reaction mixtuire: 1.5 mmole sucrose, 60 ,umolesP (pH 6.8), 30 ,amoles a-ketoglutarate, 0.03 p.nole DPN,0.1 pimole thiamiiine p)yrophosphate, 3 /Amoles MgCl,, 0.013,Lmole CoA, 3 mg bovine serum albumin. Final vollme-3 ml + 0.03 ml with each addition of ADP. Temlera-ture 250 The RC of unirradiated controls run at thesame times (O and 24 hr) were: climacteric mim. 2.3 +0.3 aind climacteric peak 2.7 ± 0.4.

in preclimacteric fruit had recovered RC and oxida-tive activity to approximately control levels (legend,fig 3). There was no sign of recovery bv mito-choildria in cliniacteric fruit.

Data from a more comprehensive experiment aregiven in taable II. In preclimacteric pears radiationdaillage was not only countered lut over-comnl)ensate(l,re.sultinig in anl RC overshoot, e.g. RC values of 7.5and 10 collparecl to a maxinmumll of 3.9 for the non-irradiated controls. The radiobiological implicationsof the overshloot and repair kinetics are discussedelsewhere ('36). For the purpose of assessing theeffects of seinescence it is significant that there was110 overslhoot in RC by mitochondria in climactericpeak fruit. Even after 0.25 Mrad RC was neverrestored to control levels.

Table III contains the ADP/O and Qo2 (protein)values obtained concomitantly with the RC data.With only 1 exception, the ADP/O values following0.25 Mrad indicate a reasonably efficient conservationlof energy from the oxidation of a-ketoglutarate.despite radiation injury or climacteric state. A1.O-Mrad dose, however, results in a marked loweringof the ADP/0, and a subseqtuent recovery only inpreclimacteric cells.

Qo., (protein) valtues establish the general levelof metabolic activity. However, co-precipitation ofvariable amounts of inactive protein during cen-

trifugation of the mitochondria render these valuesunreliable as indicators of mitochondrial condition.This is reflected by the variability in Qo2 values(table III). Nonetheless, it is significant that some

oxidative activity persists after 1.0 Mrad, with a

sign of considerable post-irradiation recovery bymitochlondria in preclimacteric fruit.

Responise to Split-Doses. Classical "split dose"techniques have been used to demonstrate the pres-

ence of mitochondrial repair, or de novo synthesis.in irradiated frtuit cells (36). The basic assumptionin "split-dose" experiments is that repair, or re-

constitution, must have occurred in the intervalbetween dose increments when the overall radiationtolerance is increased (4). Data from a set ofexperiments comparing the response to split-dosesbv mitochondria in preclimacteric and climactericpears are summn<arized in figure 4. A l.5 MArac (losewas require(d to completely obliterate RC (Qo..state 3 : Qo., state 4 = 1) in niitochondria from thislot of preclimacteric pears (fig 4, upper graph).However, wheni administered in 3 closes of 0n5 Mradleach, witlh 24 hours interveniing l)etween incremlients,the samiie total, 1.5-Mrad, dose resulted ill onllv a

)50 % decrease in RC. These results are in markedcontrast witb the perfornmanice of mitochondria insimilar pears irradiated at their climacteric peak(fig 4, lower graph ). Not only was tile radiationtolerance lowered and RC completely lost after only0.75 Mrad, but partial repair could be demonstratedolnly if the second dose was limited to 0.25 Mracl.

Table II. In vivo ReCc7.er! of Respiratory Control by Pear Fruit Mitochondria as Affected by the Climacteric Stageat Time of Irradiation

Respiratory control2Time after Climacteric minimum Climacteric peakirradiation' 0 0.25 1.0 Mrad 0 0.25 1.0 Mrad

Days Ratio RatioJ2.4 7.5 1 3.1 2.2 1

l 4.1 10 3.9 3.2 12 3.6 4.1 2.8 2.8 20(4 3.9 ( )3

5-6 3.7 (...) 4.9 2.7

Fruit were held at 200 for the indicated period of time, Thilled for approximately 2 hours, and the mitochondriaisolated.

2 Conditions of assay for resl)irators control given in legend of figuire 3.Inistances where frtuit were discarded because of excessive radiation damage and/or decay.

109)'2

', ..C'E-ICC .,

y---

--1-

BARTLETT PEARSA., '-.CCS BE- ll. [q' 01'. . --

9



ROMANI ET AL,.-S(CENl,ES'ICENCE, MITOCHIONDRIA AND RADIATION RECOVERY

Table III. ADP/O and Qo2 (Protein) Values Obtained During the Oxidation of a-Ketoglutarate by MitochondriaIsolated at Different Time Intervals After Irradiation of Whole Pear Fruit

irradiation Climacteric minimum Climacteric peakTime after' 0 0.25 1.0 Mrad 0 0.25 1.0 Mrad

Days ADP/020 2.7 2.8 <13 3.0 2.8 <.131 3.1 4.2 3.0 2.8 <12 2.7 2.8 2.2 2.7 2.3 4

4 2.5 (.)5-5 2.7 (...) 3.3 2.5

Qo2 (Protein) 20 24 37 4 57 39 41 50 30 85 19 202 52 29 36 28 29 4

4 43 (.)5-6 97 (...) 23 40

Fruit were held at 200 for the indicated neriod of time. c,hilled for arroximatelv 2 hours. and the mitochondria

34

isolated.Conditions of assay for ADP/O and Qo2 (protein) given in legend of figure 3.Low ADP/O values are not discernible in the absence of RC.Instances where fruit were discarded because of excessive radiation damage and/or decay.

0

I-z0

2

a 0 24 24b 0 24 48

FIG. 4. Comparing the response to "split doses" bymitochondria in preclimacteric pear cell,s (upper graplh)and in climacteric pear cells (lower graph). Respiratorycontrol (RC) valeus are for mitochondria isolated from:1) nonirradiated pears, 2) pears irradiated in single ex-

posures, or 3) pears irradiated in Mrad increments as

indicated at the top of each ibar. On the abscissa, line a

= time between doses, line b = cumulative time fromfirst exposure.

Discussion

Use of Ionizing Radiation. Since ionizing radia-tion was utilized as an investigative tool, it is per-haps well to consider certain characteristics ofradiation damage before discussing the experimentalresults. Setlow and Pollard (39) have definedimportant biophysical aspects of radiation damageand Romani (26) has reviewed these aspects inrelation to the high dose irradiation of plants andplant parts. The following points are especiallyrelevant to the present experiments. 1) Damagefrom ionizing radiation is all pervasive and uniformlydistributed, e.g. assuming a mitochondrial volume of4 u3, at an absorbed dose of 0.1 Mrad each organelleis the site of approximately 7 X 105 ionizing events.2) Damage is at the molecular level and, if notexcessive, amenable to biochemical (molecular) re-pair mechanisms. 3) The amount of cellular damage(radiation dose) can be readily quantitated to in-cipient, reparable, or irreparable levels.

Thus, as opposed to age-stress, ionizing radiationhas defined temporal and spatial parameters. More-over, it can be administered in fixed increments.

Of added importance to this study is the effect ofradiation in ameliorating the influences of ripeningchanges on homogenization procedures and the isola-tion of organelles. As discussed by Hanson (6)and by Romani et al. (27), analyses of temporal,quantitative, or functional changes in mitochondriaare subject to the vagaries of isolation techniques.The problem is rendered even more difficult by thephysical and chemical changes that occur in ripeningfruits. Radiation is effective in 2 ways. First,obvious physical and textural alterations in the tis-sues result from the doses used. This imposedtextural change will have its own effect on macera-tion procedures, diluting and somewhat normalizingthe impact of ripening changes. Second, and more

0

Peok - C/lmacteric

Q5

Q5QL25

I 0.75

1093



PLANT PHT YSIOLOGY

imnportant, radiation damage elicits a cellular response(mitochondrial repair or de novo synthesis) thattakes place in vivo preceding and thus largelyobviating isolation artifacts.

Stress anid th/e Co(openisatory Reactions of Senes-cent Cells. As postulated by Oldfield (20) everystress affecting the cell induces repair processeswlhichl tend to restore the normal state. Samis (38)outlines the role of compensatory repair processes incountering age-stress. Data demonstrating increasednucleic acid and protein metabolism in the liver ofaging rats are presented by \Wulff et al. (43) as

siggns of accelerated synthetic activity compensatingfor age-stress. However, in the sanme paper, it isacknowledged that evidence also exists in supportof a diametrically opposed relationship between agingan(l synthetic reactions. As noted in the introductionto this paper, a sinlilar divergency exists regardingthe (jIuestion of protein synthesis in seniescent fruiitcells.

Ouir experiments emphasize the loss in capacityof senescent pear cells to repair, or in some wavconpl)ensate for radiation damage to mitochondria.This phenomenon w\as evidenced by several criteriaincltluding 1) the overall respiratory response of thecells, 2) qjuantitative changes in mitochondria, 3)rate of amiiino acid incorporation into mitochondrialprotein, 4) time course of the mitochonidrial recoveryof RC, and 5) recovery following a split radiationdose. In all cases the climacteric respiratory patternserved in the selection of the 2 "age-states," climac-teric minimum anid climacteric peak. Clearly, atsomiie point during this time-span pear fruit cells loosetheir capacity to compensate for radiation damageto mitoclhondria.

The existence of a fundamental tranlsition inmloribulnd cells from adequate to inade(quate compen-

satory meclhanisnms could explain some of the con-

flicting reports cited by WVulff ('43) and in theintrodcuctioni to this paper. As a case in point, the(lecrease in mitoclhondrial vield as pear fruit reachthe climacteric peak was, as reported (27), preceded

a sliglht increase in yield during the first portionof the climlacteric rise. Similarly, Riclhmond andMiale (23) reported a rise in protein synthesis duringthe early phases of the climacteric prior to a declinein sy ntlhesis as fruit reached the climacteric peak.

Cellular Senescence anid the Respiratory Cli7nac-teric. The age-depen(lenit transitions noted abovehave beein substantiated in repeated experiments with

pear fruit obtained from different regions, and inthe caise of mitoclhondrial functions, with 2 pear

varieties, Bartlett and Bosc. While it is acceptedthat the climacteric respiratory pattern is coincidentwith a senescent phase, it is questionable that therespiratory pattern is an absolute guide to physio-logical age. For instance, the production of volatilesdtiring the respiratory climacteric in peaches ismarkedlys altered bv the stage of maturity at harvest('14). Cultural conditions during growth and thepost-harvest treatment of fruit may well affect the

subsequent cellular response to age (ripening), orother forms of stress. It should not be surprising,therefore, if age-related cellular functions reflectsome variability vis a vis the climacteric. \Ve havenoted a variability in the radiation tolerance of mito-chondria in pears that were, presumably, at similarpreclimnacteric stages; for instance, a loss of RC at1.0 'Mrad in one lot of fruit (table II) and (at 1.5Mtrad (fig 4) in another. This observation does notdetract from the age-related loss of compensatoryactivity which is normalized within a given experi-mlent and sample of fruit. It does suggest, however,that radiation stress maiy prove useful in unmiaskingtransitions in other cellular processes that prece(leand perhaps control the onset of the climacteric phase.

Molecutlar A4spects. In another study attentionwas called to the overshoot in compensatory activitvresulting in a 2 to 3 fold higher RC for the "re-covered" mitochond(Iria as op)posed to organelles fromiiunirradiated tissues (36). The occurrence of thisovershoot, in concert with increased cellular respira-tory activitV, suggests tllat recovery of RC is medi-ated by energy requiring processes leading to therepair of damaged sites or de novo synthesis ofmitoclhondria to replace the injured organelles. VainSteveenick and Jackman ('41 ) also observed a lossand subsequent reconstitutioni of mitochondria fol-lowing slicing of storage tissues, whlile Lee andChasson (13) noted an increase in mitochondrialmaterial with "aging" of potato discs. WVith regar(lto both studies it is important to distinguish betweenlocalized mechanical injury as occurs in the prepara-tion of tissue slices and all-pervasive, moleculardlamage from radiation. A distinction should alsobe made between "aging" of tissue slices in vitroas opposed to nornmal physiological senescence and(leatlh.

It has been proposed that cellular senescence isthe final phase of genetic expression (5, 17, 40) andhence, subject to the control mechanisms implicit incurreint theories of "molecular biology." To theextent that compensation of radiation damage in-volves protein synthesis it too is subject to the sameor similar control mechanisnms. The interrelation-ship betwveen protein synthesis and radiation repairis reinforced by the observatioin that almost completeinactivation of 70 anid 80S ribosomes ( 12, 22) occursin the same (lose raange (0.6-1.2 Mrad) that resuiltsin irreparable injury to pear nlitoclhondria. Pearribosonmes are 80S (11), and moreover, a rapid(lecliine in their synthlesis (Ku and Romani, unpub-lished observation) has been foundl coincident withthe final portion of the climacteric rise. Richmondand Biale (24) have also reported a marked declinein RNA synthesis as avocados reach the climactericpeak.

Concluding Remarks. Our findings indicate thata critical intracellular transition occurs during thelatter portion of the climacteric rise in senescentfruit cells leading to a degeneracy in compensatorypower. Because of the apparent involvement of pro-

10(94



ROMANI ET AL.-SCENESCENCE, MITOCHONDRIA AND RADIATION RECOVERY

tein synthesis, the transition may stem from a de-generacy in RNA synthesis, as suggested by Rich-mond and Biale (24), or from a programmed changeat any one of the many control points manifest incurrent molecular theory of protein and RNA syn-thesis.

As it applies to mitochondria, a degeneracy incompensatory activity may be especially critical ifthe organelles are to supplv the senescent cell withenergy to counter age-stress.

Literature Cited

1. BIALE, J. B. 1964. Growth, maturation, and sene-scence in fruits. Science 146: 880-88.

2. CHANCE, B. AND G. R. WILLIAMS. 1955. Respira-tory enzymes in oxidative phosphorylation. I. Ki-netics of O., utilization. J. Biol. Chem. 217: 383-93.

3. CLAYPOOL, L. L. AND R. M. KEEFER. 1942. Acolorimetric method for CO., determination in res-piration studies. Proc. Am. Soc. Hort. Sci. 40:177-86.

4. ELKIND, M. M. AND H. SUTTON. 1960. Radiationresponse of mammalian cells grown in culture. I.Repair of X-ray damage in surviving Chinesehamster cells. Radiation Res. 13: 556-93.

5. HAHN, H. P. VON. 1966. A im-odel of "regulatory"aging of the cell at the gene level. J. Gerontol.21: 291-94.

6. HANSON, J. B. 1963. The yield of mitochondrialmaterial from maize roots. Physiol. Plantarum16: 814-21.

7. HULME, A. C 1954. Studies on the nitrogen me-tabolism of apple fruits. The climacteric rise inrespiration in relation to changes in equilibriumbetween protein synthesis and breakdown. J. Exptl.Botany 5: 159-72.

8. HULME, A. C., J. D. JONES, AND S. C. WOOLTORTON.1964. Mitochondrial preparations from the fruitof the apple. I. Phytochemistry 3: 173-88.

9. HULME, A. C., M. J. C. RHODES, AND L. S. C.WOOLTORTON. 1967. The respiration climactericin apple fruits: some possible regulatory mechan-isms. Phytochemistry 6: 1343-51.

10. LANCE, C., G. E. HoBsON, R. E. YOUNG, AND J. B.BIALE. 1966. Metabolic processes in cytoplas-micparticles of the avocado fruit. VII. Oxidativeand phosphorylative activities throughout the cli-macteric cycle. Plant Physiol. 40: 1116-23.

11. KIT, L. (LiM) AND R. J. ROMANI. 1966. Ribo-somes from pear fruit. Science 154: 408-10.

12. KUCAN, Z. 1966. Inactivation of isolated E.schcri-chia coli ribosomes by ga-mma radiation. RadiationRes. 27: 229-36.

13. LEE, S. G. AND R. M. CHASSON. 1966. Aging andthe development of enhanced respiration in potatotuber tissue. Physiol. Plantarum 19: 194-98.

14. List, L. AND R. J. ROMANI. 1964. Volatiles anidthe harvest maturity of peaches and nectarines. J.Food Sci. 29: 246-53.

15. MASSEY, L. J., JR., D. F. TALLMAN, AND Z. I. KEI-TESZ. 1961. Effects of ionizing radiations on

plant tissues. V. J. Food Sci. 26: 389-96.

16. MAXIE, E. C. AND K. E. NELSON. 1959. Physio-logical effects of ionizing radiation on some de-ciduous fruits. Final Rept. Contract 115, Quarter-master Food Container Inst. (Chicago)

17. MEDVEDEV, ZH. A. 1964. The nucleic acids in de-velopment and aging. In: Advances in Geronto-logical Research. B. L. Strehler, ed. AcademicPress, Niew York, Vol. 1. p 181-202.

18. MILLER, G. L. 1959. Protein determinations forlarge numbers of samples. Anal. Clhem. 31: 964.

19. MILLERD, A., J. BONNER, AND J. B. BIALE. 1953.The climacteric rise in fruit respiration as coIn-trolled by phosphorylative coupling. Plant Phy-siol. 28: 521-31.

20. OLDFIELD, D. G. 1967. Cvtokinetics. Bull. Math.Biophys. 29: 597-603.

21. PEARSON, J. A. AND R. H. ROBERTSON. 1954. Thephysiology of growth in apple fruits. VI. Thecontrol of respiration rate and synthesis. Aus-tralian J. Biol. Sci. 7: 1-17.

22. POLLARD, E. 1961. The action of ionlizinlg radiatioInon the celluilar synthesis of protein. In: The InitialEffects of Ionizing Radiations on Cells. R. J. C.Harris, ed. Academic Press, New York. p 75-90.

23. RICHMOND, A. AND J. B. BIALE. 1966. Protein andnucleic acid metabolism in fruits. I. Studies ofamino acid incorporation during the climacteric risein respiration of the avoca(lo. Planit Physiol. 41:1247-53.

24. RICHMOND, A. AND J. B. BIALE. 1967. Proteinand nucleic acid metabolism in fruits. II. RNAs3 nthesis during the respiratory rise in the avo-cado. Bioohim. Biophys. Acta 138: 625-27.

25. ROMANI, R. J. 1964. Radiation physiology offruit-respiration during anid immediately followingkilorad doses of ionizing radiation. RadiationBotany 4: 299-307.

26. ROMANI, R. J. 1966. Bioclhemical responses ofplant systems to large doses of ionizing radiation.Radiation Botany 6: 87-104.

27. ROMANI, R. J., R. W. BREIDENBACH, AND J. G.VAN KooY. 1965. Isolation, yield and fatty acidcomposition of intracellular particles from ripeningfruits. Plant Physiol. 40: 561-66.

28. ROMANI, R. J. AND L. K. FISHER. 1966. De-creased synthesis of mitochondrial protein in thesenescent cells of pear fruit. Life Sciences 5:1187-90.

29. ROMANI, R. J. AND L. K. FISHER. 1966. Mito-chondrial resistani-ce to massive irradiation in vivo.IV. Suppression and recovery in rate of aminoacid incorporation into mitochondrial protein. Arch.Biochem. Biophys. 117: 645-49.

30. ROMANI, R. J., L. K. FISHER, AND I. K. YU.1966. Recovery from intracellular radiation dam-age as a function of cell age. Gerontologist 6: 42.

31. ROMANI, R. J., J. VAN KooY AND B. J. ROBINSON.1961. Gamma irradiation of fruit-preliminaryphysiological studies. Food Irradiation 2: All-A13.

32. ROMANI, R. J., L. L. Ku, L. K. FISHER, ANDJ. G. VAN Kooy. 1966. Mitochondria: Increase inoxidative capacity and quantitative recovery aftermassive doses of ionizing radiation. RadiationRes. 28: 257-65.

33. RoMANI, R. J.. B. J. ROBINSON, H. L. RAE, E C.MAXIE, AND N. F. SOMMER. 1963. Radiation of

1095



PLANT PHYSIOLOGY

fruit-physical methlods. RadiatioIn Botany 3: 345-50.

34. RONIANI, R. J. AND I. K. Yu. 1966. Mitochoni-drial resistanice to massive irradiation in vivo. II1.Suppression and recovery of respiratory control.Arch. Biochem. Biophys. 117: 638-44.

35. ROMANI, R. J. AND I. K. Yu. 1967. Mitochon-drial respiratory control, radiation injury, and theclimacteric in fruit. Plant Physiol. 42: S-14.

36. ROMANI, R. J. AND I. K. Yu. Mitochondrialresistanc-e to massive irradiation in vivo. V. Repairand the repair overshoot. Arch. Biochem. Biophys.In press.

37. SACHER, J. A. 1966. Permeability characteristicsand amino acid incorporation during senescen-ce(,ripening) of banana tissue. Plant Physiol. 41:701-08.

38. SAMIS, I. V., JR. 1966. A concept of biologicalaging: The role of compensatory processes. J.Theoret. Biol. 13: 236-80.

39. SETLOW, R. B. AND E. C. POLLARD. 1962. Molecu-lar Biophvsics. Addison-Wesley, Reading, Massa-clhusetts.

40. SINEX, F. M. 1966. Biochemistry of Aging. Per-spectives Biol. Med. IX: 208-24.

41. VAN STEVENINCK, R. F. M. AND M. E. JACKMNAN.1967. Respiratory activity anid morphologx ofmitochondria isolated from whole and sliced stor-age tissues. Australian J. Biol. Sci. 20: 749-60.

42. WISKICH, J. T. 1966. Respiratory control by iso-lated apple mitochonidria. Nature 212: 641-42.

43. WULFF, 0. J., H. V. SAMIS, JR., AND J. A. FAL-ZONE, JR. 1967. Metabolism of rihonluicleic acidin young and old rodents. In: Gerontological Re-search. B. L. Strelhler, ed. Acadenmic Press, NewYork. p) 37-96.

44. YOUNG, R. E. AND J. B. BIALE. 1967. Phosphory-lation in avocado fruit slices in relation to therespiratory climacteric. Plant Phvsiol. 42: 1357-62.

1096



cellular senescence, radiation damage to and compensatory ... · climacteric pear fruit cells...

Documents