PHYSIOLOGIA PLANTARUM 82: S29--536, Copenhagen 1991

Prior temperature exposure affects subsequent chilling sensitivity

Mikal E. Saltveit, Jr.

Saitveit, M, E,, Jr, 1991, Prior temperature exposure affects subsequent chillingsensitivity, - Physiol, Plant, 82: 529-536,

The chilling sensitivity of small discs or segments of tissue excised from chilling-sensitive species was significantly altered by prior temperature exposure subsequentto holding the tissue at chilling temperatures as measured by a number of physiolog-ical processes sensitive to chilling. This temperature conditioning was reversible by anadditional temperature exposure before chilling, and mature-green and red-ripetomato tissue exhibit similar chilling sensitivities.Exposing pericarp discs excised from tomato fruit (Lycopersicon escuientum Mill, cv,Castelmart), a chilling-sensitive species, to temperatures from 0 to 37°C for 6 h beforechilhng the discs at 2,5°C for 4 days significantly altered the rate of ion leakage fromthe discs, but had no effect on the rate of ion leakage before chiUing and only aminimal effect on discs held at a non-chilling temperature of 12°C, Exposing chilling-sensitive tissue to temperatures below that required to induce heat-shock proteins butabove 20°C significantly increased chilling sensitivity as compared to tissue exposedto temperatures between 10 and 20°C. Rates of ion leakage after 4 days of chilling at2,5°C were higher from fruit and vegetative tissue of chilling-sensitive species {Cucu-mis sativus L, cv, Poinsett 76, and Cucurbita pepo L, cv. Young Beauty) that werepreviously exposed for 6 h to 32°C than from similar tissue exposed to 12°C, Exposureto 32 and 12°C had no effect on the rate of ion leakage from fruit tissue of chillingtolerant species {Malus domestica Borkh, cv. Golden Delicious, Pyrus communis L,cv, Bartlett), Ethylene and CO2 production were higher and iycopene synthesis waslower in chilled tomato pericarp discs that were previously exposed for 6 h to 32''Cthan the values from tissue exposed to 12°C for 6 h before chilling. Increased chillingsensitivity induced by a 6 h exposure to 32''C could be reversed by subsequentexposure to 12°C for 6 h.

Key words — Apple, carbon dioxide, cucumber, Cucumis sativus, Cucurbita pepo,ethylene, ion leakage, Iycopene, Lycopersicon escuientum, Malus domestica, pear,Pyrus communis, squash, tomato,

Mikal E, Saltveit, Jr,, Mann Laboratory, Dept of Vegetable Crops, Univ, of Cali-fornia, Davis, CA 95616, USA,

Introduction

Many plants of tropical and sub-tropical origin, andsome tissues from plants of temperate origin are injuredby expostire to non-freezing temperattires below 12°C(Bramlage 1982, Couey 1982, Lyons 1973, Saltveit andMorris 1990), Chilling-sensitive piants include avoca-dos, bananas, cucumbers, peppers and totnatoes, whilechilling-resistant plants include apples and pears. To-mato fhiit are particularly stisceptible to chilling injuryat the mattire-green stage when they are noenally har-vested and shipped. The response of tomato fruit to

Received 19 November, 1990; revised 30 March, 1991

37 Fhysiol, Plant. 82, t99t

chilling inury is similar to that of most chilling-sensitiveplants and is characterized by increased susceptibility toAlternaria rot, slow and aboornnal ripening, increasedrates of CO2 and C2H4 production and increased rates ofion leakage (Autio and Bramlage 1986, King and Lud-ford 1983, Morris 1982), Increased rates of solute andelectrolyte leakage occur in a varity of chilled tissue(Creencia and Bramlage 1971, Mtirata and Tatstimi1979), and have been used as measures of the increasedpermeability of the piasmalemma following chilling(Guinn 1971, Wright and Simon 1973),

Symptoms of chilling, injury that are induced by expo-

529

sure to chilling temperatures can be reduced in somechilling-sensitive tissues by modifying the pattem oftemperature esposuie, e,g, periodically warming thechilled tissue above the chilling temperature (Anderson1982, Cabrera and Saltveit 1990, Davis and Hofman1973), or by holding the tissue at temperatures near thechilling temperattire or above 35''C before chilling (Gra-ham and Patterson 1982, Lafuente et al, 1991), Forexample, expostire to cool, non-chilling temperaturesbefore chilling reduces chilling injtiry in fruit of bellpepper (McColloch and Worthington 1952) and grape-fruit (Chalutz et al, 1985, Hatton and Cubbedge 1983),tomato seedlings (King et al, 1982, Wheaton and Morris1967) and ornamental plants (Smith and McWiUiams1979), We recently showed that exposing excised 1-cmdiscs of cucumber cotyledons to 37°C for 6 h inducedheat-shock proteins and reduced chilling sensitivity asmeasured by reduced ion leakage (Lafuente et al,1991),

Changes in the chilling sensitivity of tomato fruit oc-cur naturally during diurnal fluctuations in temper-atures in the field (Saltveit and Cabrera 1987), Mattire-green totnato fruit harvested at sunrise (i,e, 0630 h),with a pulp temperature of 19'°C, were more resistant tochilling at 7°C for 7 days than were fmit harvested at1300 h or at sunset (i,e, 2030 h) with pulp temperattiresof 32 or 29°C, respectively (Saltveit and Cabrera 1987),Fruit harvested on cool, cloudy days during which thefruit were roughly the same temperature throughout theentire day, did not show significant differences in chill-ing sensitivity with time of harvest. Exposure to airtemperatures of 12,5 or 37°C (producing flesh temper-atures of 12 or 32°C, respectively) in the laboratory for 7h induced differences in sensitivity to 4 days of chillitigat 2,5°C that were similar to differences in fruit har-vested from the field at different temperatures- Shortterm exposures to 12 or 32°C in the laboratory had nosignificant effeet on the ripening of fruit exposed tonon-chilling temperatures.

The physiological responses of chilling-sensitive andresistant tissue to prior temperature exposures that alterchilling sensitivity have not been thoroughly studied.The object of this study was to characterize the effectsof prioi temperature exposure on the subsequent ex-pression of chilling injury symptoms in a number ofchilling-sensitive and resistant species, to examine thereversibility of this conditioning treatment and to deter-mine if changes in chilling sensitivity occur as tomatofruit tissue progresses from the mature green to thered-ripe stage of ripeness.

Materials and methods

Tissue preparation

Tomato frtiit {Lycopersicon escuientum Mill, cv. Castle-mart) were hand-harvested at the malure-green or red-ripe stage frona plants grown at the Vegetable Crops

field station in Davis, CA, under standard cultural prac-tices. Only uniform, healthy fruit with no external de-fects were used, Frtiit were washed in dilute comtnercialbleach (1:20 dilution of 5% sodium hypochlorite)., airdried under a transfer hood and 1-cm diam, pericarpdiscs were excised with a sterile stainless steel corkborer. Adhering locular material was trimmed away toproduce 3-mm-thick discs. Discs were washed 3 times inautoclaved deionized water, blotted dry and from 4 to 8discs were piaced epidermis down in 15 x 100-mmdiam, plastic Petri dishes. Replicate discs were from onefruit and were blocked for intensity of green coloracross treatments, since light-colored discs produce lessIycopene than dark green discs (Saltveit 1989b), Carewas also exercised to make sure that the discs remainedoriented with their epidermis surface down, since Iyco-pene synthesis was affected by the orientation of thediscs. Depending on the size ofthe experiment (i.e, thenumber of treatments and replicates), from 1 to 4 uni-form furit were used. An 80-nim diatn, disc of moistfilter paper was stuck to the top lid of the Petri dish toraise the humidity. Dishes were kept overnight at 20°Cin a flow of humidified, C^Hj-free air to allow dis-sipation of wound responses (MencareOi and Saltveit1988),

Pericarp discs were used because they offer manyadvantages over slices and whole tomato fruit. One-cmdiam, epidertnal pericarp discs are stnall and easily han-dled, yet they exhibit all the physiological changesfound in ripening whole fruit: a C2H4 and respiratoryclimacteric, chlorophyll degradation, Iycopene synthe-sis, development of characteristic aroma and softening(Edwards et al, 1983, Gross and Saltveit 1982), From 30to 60 discs of nearly identicai tisstie cati be preparedfrom a medium to large fruit. Solutions are easily ap-plied and absorbed through the cut surfaces, Asepticallyprepared discs ripen normally without significant in-vasion by pathogens.

Fruit of apple {Malus domestica Borkh, cv. GoldenDelicious), pear {Pyrus communis L, cv, Bartlett), cu-cumber {Cucumis sativus L, cv, Poinsett 76) and squash{Cucurbita pepo L, cv. Young Beauty) were obtainedfrotn local markets. Representative fruit from each lotwere held at 20°C and experimental results from that lotof fruit were discarded if any of the representative fruitdeveloped chilling, injury symptoms. One-cm diametercylinders of tissue were excised with a sterile stainlesssteel cork borer from the cortex of apple and pear fruit,and from the mesocarp of cucumber and squash fruit.The cylinders were cut into 3-mm thick discs which werewashed 3 titnes in sterile deionized water, blotted dry,and piaced in 15 x lOO-mm diam, plastic Petri dishes.One-cm apical root segments were exeised from 5-day-old squash (Cucurbita pepo L. cv. Young Beauty) seed-lings grown in vermictilite at 25°C under 18 h cool-whitefluorescent lights giving a photon fluence rate of 70limol m"'' s"' at plant level. The root segments wereplaced on moist filter paper in 15 x 100 plastic Petri

530 PhysioL Ptanti 82,1991

dishes. Dishes were kept overnight at 20°C in a flow ofhumidified, QH^-free air to allow dissipation of woundresponses.

Temperature treatmentsPericarp discs of mature-green tomato fmit were ex-posed to temperatures of 0, 5, 10, 15, 20, 25, 30, 32, 34and 37°C for 6 h followed by 4 days of exposure at either12 or 2,5''C, Temperature treatments were applied incontrolled temperature rooms under dim light by put-ting the Petri dishes between 30 x 30-cm plastic bagscontaining 1-1 of water that had equilibrated over 24 h tothe temperature of the room. The water bags helped tostabilize the exposure temperature and rapidly establishthe desired tissue temperattire. Preliminary experi-ments with a YSI model 47 Scanning Tele-Thermometerequipped with a small #421 temperature probe (YellowSprings Instrument Co,, Yellow Springs, OH, USA)showed that evaporative cooling from the moist filterpaper and tissue reduced tissue temperatures below theair temperature. Exposure temperatures were thereforeraised an appropriate amount to compensate for thisreduction and to prodtice the desired tissue temper-atures that were confirmed by periodic measurements.Weight loss by the tissue was less than 1,5% and notsignificantly different among any of the temperaturetreatments. Results from these preliminary experimentsindicated that optimal difference in chilling sensitivitywas produced by exposing tissue to 12 and 32°C, Con-trol discs were held at 12°C, the lowest non-chillingtemperature, to minimize physiologica! changes causedby ripening during the experiments.

Tissue was subjected to the following four temper-ature treatments: 1) exposed to 12°C for 6 h and held at12°C for 4 days, 2) exposed to 32°C for 6 h and held at12°C for 4 days, 3) exposed to 12°C for 6 h and held at2 ,5^ for 4 days, and 4) exposed to 32°C for 6 h and heldat 2,5°C for 4 days. Later experiments contained thefollowing two additional temperature treatments: 5) ex-posed to 32°C for 6 h and then to 12°C for an additional6 h, and held at 2,5°C for 4 days, and 6) exposed to WCfor 6 h and then to 32''C for an additional 6 h, and 2,5°Cfor 4 days.

Measuring chiUing injury

Chilling injttry was measured as increased ion leakagefor all tissues, and also as increased C2H4 and CO2production and decreased lyeopene synthesis for tomatotissue. Ion leakage was measured as increased electricalconductivity in a 0,3 M mannitol solution that was usedinstead of water to reduce osmotic stress on the tissue(Autio and Bramlage 1986, Saltveit 1989a, Vickery andBruinsma 1973), Tissite was removed from the holdingtemperature and warmed to 2O'"C for 0,5 h before beingplaced into a tared 100-tnl beaker. Beakers with tissuewere weighed, 30 ml of 0,3 M mannitol were added, andthe beakers were weighed. The beakers were shaken at2 cycles s ' between conductivity readings with an Ex-Tech Model 480 digital conductivity meter (Extech Inc,,Waltham, MA USA), The beakers were then heated toboiling for 5 min, cooled overnight at 0°C while beingshaken, wartned to room temperature and made to theirinitial weight with deionized water. After the beakerswere shaken for 1 h at room temperattire, the total

Fig, 1, Effects oftemperature and duration ofexposure on ion leakagefrom pericarp discs excisedfi'om mature-green tomatofruit. Discs were exposed totemperatures from 0 to 15°Cfor 4 days (large graph) andto 2,5°C for 0 to 8 days(insert). Data representtriplicate sampling. Thevertical error bars representthe SE of the mean.

8 -• 4 - '

•>

uT3

OU

6 -

r 4 -0)

u

2 -

L 1

K 1\ 1

I I 1 1 1 il 1 1

12

S

4

-

•jt,—

f0

•

2

t 1

4Days at

1 1

1 4 1 1

1 i t l|:

- -

6 82,5°C

I I 1 1

6 8 10Temperature

12 14

37' Physiot. Plant, 82, 199t 531

condtictivity was measured. Rates of ion leakage areexpressed as changes in the percent of the total conduc-tivity h^',

Prodtiction of COj and QH4 was measured by enclos-ing tisstie in glass jars at 20°C for an appropriate lengthof time for C2H4 and CO2 to accumulate to measurablelevels (abotit 2 h), One-ml gas samples of the headspacewere taken periodically and analyzed for C^H, by flameionization gas chromatography and for CO2 by infraredanalysis (Saltveit 1982),

Tomato discs were ripened at 20°C for 5 days in 20-1glass jars that were flushed with humidified air contain-ing 10 ul I"' C2H4, Dishes were stored at -20°C until thediscs were assayed for lycopene. Two discs from eachdish were weighed, homogenized with 10 ml of acetonefor 10 s in a Sorvall Omnimixer (Ivan Sorvall Inc,Newtown, CT, USA) at maximum speed, centrifuged atmaximum speed in an International clinical Model CL(International Equipment Co,, Needbam, MA, USA)table-top centrifuge and the absorbance of the clearsupernatant was measured at 503 nm, Lycopene con-centration is expressed as absorbance per gram freshweight.

Each experiment had at least 3 replicates per treat-ment and was repeated twice. Data within each experi-ment were subjected to an analyses of variance with 5%LSD values calculated to separate significantly differenttreatment means.

Results

Holding discs of tomato pericarp tissue at temperaturesfrom 0 to 12°C for 4 days resulted in a progressiveincrease in ion leakage with decreasing temperature

(Fig, 1), Tbe response declined linearly from 0 to 5°Cwitb a significant reduction in the slope occurring after7,5°C, The transition between chilling and non-chillingtemperatures occurs between 7,5 and 12°C for mature-green tomato pericarp discs, A temperature of 12°C waschosen for the non-chilling exposure and holding tem-perature because it was the lowest consistently non-chilling temperature. The cbilling temperature of 2,5°Ccaused a steady increase in tbe rate of ion leakage withtime after a delay of 2 days (insert in Fig, 1), A 4-dayexposure to 2,5°C was cbosen as the chilling treatmentbecause it was the shortest period tbat consistentlycaused significant increases in ion leakage, and a shortholding period would reduce ripening differences be-tween the two holding temperatures.

Exposing tomato discs to temperatures from 0 to 37''Cfor 6 h had no significant effect on subsequent ionleakage immediately after exposure (Fig, 2), Leakagefrom discs held at 12°C for 4,5 days after the 6 h expo-sure was roughly constant across exposure temperaturesfrom 15 to 37°C, and increased slightly from those discsexposed to 0 to 10°C for 6 h. In contrast, there weredramatic differences in tbe rates of ion leakage fromdiscs held at 2.5°C for 4,5 days after conditioning. Leak-age was higher from tissue previously exposed to thechilling temperatures of 0 and 5°C for 6 h than fromtisstie exposed to 10 and 15°C, The higher rate of leak-age may have been the result of the additional 6 h ofchilling at 0 or 5°C, It is difficult to imagine, however,how an additional 0,25 days of chilling could have such apronounced effect over that of 4,5 days. The highestrate of leakage was from tissue previously exposed to25, 30 and 32°C, while the lowest rate of leakage wasfrom tissue exposed to 34 and 37°C. We have previously

0•oou"(5o

u

10 20Temperature

30 40

Fig, 2, Effects of priortemperature exposure for 6h of mature-green tomatopericarp discs on theirsubsequent rates of ionleakage immediately aftereonditioning ( # # . ) andafter holding for 4,5 days at12°C (-A-A-) or at 2,5°C( - • - • - ) , Data representtriplicate sampling. Thevertical error bars representthe SE of the mean.

532 Physiol, Plant, 82, 1991

reported that the higher temperatures induce heat-shock proteins (Lafuenta et al, 1991), To accentuatedifferences, exposure temperatures of 12 atid 32°C wereselected for subsequent experiments.

Exposing mature-green epidermal tomato pericarpdiscs to 2,5°C for 4 days significantly increased the ratesof ion leakage and C2H4 and CO2 production during the2-h period immediately after they were warmed to 20°C,and decreased the lycopene concentration in discs heldfor an additional 4 days at 20°C in an atmosphere con-taining 10 ul r ' C2H4 (Fig, 3), Chilling caused thesechanges in tissue previously exposed to both 12 and32°C for 6 h, but the changes were significantly greaterfor discs exposed to 32 than to 12°C, In contrast, expos-ing discs to 12 or 32°C for 6 h had no significant effect ontheir response when they were held at the non-chillingtemperature of 12°C for 4 days.

While there were significant differences in all 4 meas-urements of chilling injury between discs exposed to 12and 32°C, the most pronounced effect of conditioning at12°C was on the rate of ion leakage (Fig, 3), Ion leakagefrom chilled discs conditioned at 32°C was almost 2-foldhigher than from discs conditioned at 12°C, Productionof C2H4 and CO, was increased by about 1,3- and 1,2-fold, respectively, while lycopene concentration was re-duced by around 20% by the 32°C conditioning treat-ment in comparison to the 12°C conditioning treatment.

The rates of ion leakage remained significantly higherfrom discs exposed to 32°C than from discs exposed to12''C prior to chilling for the four 1,5 h sampling periodstbat extended for 6 h from immediately after removal ofthe discs from the chilling temperature. Although therewas a decline in the rates of leakage from discs in alltreatments over this 6 h period, the statistically signif-

icant differences among the temperature conditioningand holding treatments remained the same.

Increases in conductivity during the 0,0 to 0,5 h pe-riod immediately following warming to 20°C exhibitedgreater variability within and among experiments thandid measurements made during other sampling periods.Because of the greater response of ion leakage than ofC2H4 and CO, production or lycopene concentratioti totemperattire conditioning (Fig, 3), ehanges in electricalconductivity during a 0,5 to 2,5 h period immediatelyafter warming to 20°C were ttsed as a measure of theextent of chilling injtiry in ali subsequent experiments.

The effect of temperature conditioning on chillingsensitivity was not confined to tomato pericarp tissue,but was also observed in tissue excised from other chill-ing-sensitive species (Tab, 1), In each of tbe chilling-sensitive tissues (i,e, cucumber, sqttash and tomato),exposure to 32°C for 6 h sigtiificantly increased the rateof ion leakage after 4 days of chilling at 2,5°C over thatof tisstie exposed to 12°C, Neither cotiditioning temper-ature had a significant effect on electrolyte leakage fromtissue held at the non-chilling temperature of 12''C, Incontrast to the pronounced effect of temperature condi-tioning on ion leakage from chilling-sensitive tissue,chilling-resistant tissue (i,e, apple and pear) showed nosignificant changes in ion leakage as the result of eitherthe exposure or holditig temperature. Within the exper-imental conditions, temperature conditioning uniquelyaffect the rate of ion leakage from chilling-sensitivetissue, and not from chilling-resistant tissue.

Surprisingly, pericarp discs from both mature-greenand red-ripe tomato fruit showed similar significant in-creases in the rate of ion leakage upon exposure to 32°Cover that at 12°C (Tab, 1), Conditioning at 32°C resultedin a 23% increase in ion leakage from chilled mature-

ICoRductivjty Ethylene Carbon dioxide Lycopene

Hg, 3, Effects of holding mature-green tomato epidertnal per-icarp discs at 12 or 2,5°C for 4 days after conditioning at 12 or32°C for 6 h on the rates of ion leakage and the production ofC2H4 and CO2 during tbe 2 h period immediately after warmingto 20°C, and on iycopene concentration after ripening for 4days at 2(fC in an atmosphere containing 10 ul 1 C2H4, Datarepresent triplicate sampling from two experiments. Barswithin each type of measurement that are associated with tbesame letter are not statistically different at the 5 % level.

Tab, 1, Effects of holding various tissues at 12 or 2,5°C for 4days after conditioning at 12 or 32°C for 6' h on the percent ioBleakage during a 0,5 to 2,5 h period immediately after warmingto 20°C, Data represent triplicate sampling from two experi-ments. Means for each tissue were separated by LSD at the 5 %level, and means followed by the same letter are not statisti-cally different at the 5 % level.

Conditioning temperatures

12°C

Holdingtemperatures

32°C

Holdingtemperatures

Tissue

Apple cortex discsPear cortex discsSquash root segmentsSquash mesocarp discsCucumber mesocarp discsTomato pericarp discs

Mature-greenRed-ripe

12°C

5,7a5,8a2,0c1,7c3,5c

3,9c1,8c

2,5°C

5,8a5,6a3,2b3,1b4,4b

4,3b2,0b

12°C

5,8a5,9a1,9c1,6c3,6c

3,7c1,7c

2,5°C

6,0a6,0a4,9a5,6.a4,9a.

5,3a2,7a

»ysioL Pianl, 82, t99I 533

green discs over that of discs conditioned at 12°C, whilea 35% increase was noted for discs from red-ripe fruit.

All 4 measurements of chilling injury were signif-icantly reduced when the mature-green tomato pericarpdiscs that had been exposed to 32''C for 6 h were sub-sequently exposed to 12°C for 6 h before chiDixig, Ionleakage was reduced to that of chilled discs conditionedonly at 12°C, For Q H j and COj production the reduc-tion was even greater, i,e, to the level of the non-chilleddiscs, Lycopene concentration was significantly in-creased to an amount halfway between the chilled andnon-chilled treatments. The increase in chilling sensitiv-ity brought on by exposure to 32°C for 6 h was appar-ently reversed by a 6 h period at 12°C, In contrast,additional exposure to 32°C for 6 h of discs first condi-tioned at 12°C for 6 b increased all 4 measuretnents ofchilling to that of discs initially exposed to 32°C,

The results of additional conditioning on ion leakagefrom mature-green and red-ripe pericarp discs areshown in Fig, 4, Mature-green and red-ripe tomatopericarp discs exhibited the previously described alter-ations in the rate of ion leakage when exposed to 32 or12°C before chilling at 2,5°C for 4 days. Holding discsthat had been exposed to 12 or 32°C for an additional 6h at 32 or 12°C, respectively, resulted In rates of ionleakage comparable to that from discs originally ex-posed to 32 or 12°C, respectively. The initial rate ofelectrolyte leakage during 0,0 to 0,5 h from all treat-ments was around 2-fold greater from red-ripe discsthan from mature-green discs. In contrast, leakage fromred-ripe discs during the normal 0,5 to 2,5 h samplingperiod was about 50% of that from matere-green discs.

•= 3

Mature-green Red-ripe

12

12

12

2.5

32

J2

32

2.5.

32

12

ZB

12 132 J

Comjit^»9ing |Tenqieratiires. [

HoWng 1TMrmoraturas *

" 12 T2

1 12 2.6

32 32

12 2.5

32

12

Z 5

12

32

2,5

Fig, 4, Effects of holding mature-green and red-ripe tomatoepidermal pericarp discs at 12 or 2,5°C for 4 days following 1 ofthe 6 temperature oonditioning treatments: 1) 12/12 = condi-tioned at 12°C for 6 h and held at 12°C for 4 days, 2) 12/2,5 =conditioned at 12°C for 6 h and held at 2,5°C for 4 days, 3)32/12 = conditioned at 32°C for 6 h and held at 12°C for 4 days,4) 32/2.5 = conditioned at 32°C for 6 h and then at 12°C for anadditional 6 h and held at 2,5°C for 4 days, and 6) 12/32/2,5 =conditioned at 12°C for 6 h and then at 32°C for 6 h and held at2,5°C for 4 days. Data represent triplicate sampling from twoexperiments. Bars within each maturity class that are associ-ated with the same letter are not statistically different at the 5%. level.

Discussion

Both field and laboratory data have shown that thechilling sensitivity of tomato fruit tissue is significantlyaffected by the temperature of the tissue prior to chill-ing (Saltveit and Cabrera 1987), In addition, the presentstudy has shown that exposure of several chilling-sensi-tive plant tissues to 32°C for 6 h increases their chillingsensitivity over tissue exposed to 12°C for 6 h as mea-sured by all 4 quantitative indicators of chilling injury.Chilling-resistant species were unaffected by any of thetemperature exposures or chilling treatments. Increasedrates of ion leakage were shown to be a sensitive mea-sure of the changes in chilling sensitivity induced byprior temperature exposure,

Tiie lower rates of ion leakage from discs of red-ripefruit than from discs of mature-green fruit were un-expected, since electrolyte leakage has been reported tobe around twice as great from red-ripe as from mature-green tomato tissue (King and Ludford 1983), This in-consistency perhaps resulted from my measuring ionleakage after a total of 5 days from excision (i,e. an 18 hincubation after excision, a 6-h conditioning treatmentand a 4-day holding period), rather than measuringleakage from freshly excised tissue as was done in theother study, I also observed that the rate of ion leakagefrom discs held at 12 or 20°C declined as the length oftime after excision increased.

The 32°C conditioning treatment by itself did notcause stress to the tissue sufficient to affect the rate ofripening or any of the measurements used to quantifychilling injury. While prolonged exposure to temper-atures around 32°C is koown to inhibit lycopene syn-thesis during tomato fruit ripening, our previous results(Saitveit and Cabrera 1987) agreed with those of Hall(1964) that short periods of exposure to high temper-atures do not significantly affect ripening or color devel-opment. Hall (1964) reported that holding whole to-mato fruit for 6 h at 32 to 43°C did not significantlyaffect either softening or color development during 7days of ripening at 20°C, It has been reported recentlythat mature-green tomato fruit held for 1 day at 40°Cproduced a normal respiratory and C2H4 climacteric andripened tiormally during subsequent storage at 25''C(Inaba and Chachin 1988), In addition, I have shownthat exposure to 32°C did not result in significantlyincreased COj or C2H4 production, increased ion leak-age or reduced iycopene content from treated tissue incomparison to tissue exposed to 12°C (Tab, 1 and Fig,3), Furthermore, the effect of the 32°C conditioningtreatment was not permanent, A 6-h exposure to 12°Cafter the 32°C exposure was sufficient to completelyreverse the 32°C effect (Fig, 4), The reversibility of the32°C conditioning treatment in chilled chilling-sensitivetissue, the failure of the 32°C conditioning treatment toaifect any non-chiiled tissue, and the normal ripetiing oftissue exposed, to higher temperatures for longer peri-ods of time all indicate that, by itself, the 6 h exposure

534 Pkysiot; Plant. 82, lWt

to 32°C did not cause a signficaet or persistent stress tothe tissues.

Since many symptoms of chilling injury involve aiter-ations of normal ripening, it is commonly thought thatripe fruit are more resistant to chilling injury than un-ripe fruit (Bramlage 1982, Couey 1982, King and Lud-ford 1983, Morris 1982), The fact is that ripe fruit mayappear more chilling-resistant simply because they cannot exhibit alterations in their already accomplishedprocesses of ripening. The ion leakage data presentedabove suggest that the physiological basis for chillinginjury in tomato fruit is equally present in both ripe andunripe pericarp tissue.

The chilling sensitivity of mature-green aod red-ripetomato pericarp discs was dependent on the last temper-ature to which they were exposed, A 6-h period oftemperature conditioning prior to 4 days of chillingresulted in a reversible change in a number of measure-ments of chilling injury in tissue from chilling-sensitivespecies. The reversibility of these temperature-inducedchanges in chilling sensitivity implies involvement of anactive physiological process rather than passive chatig-es.

One such passive change could be water loss. Waterloss is known to increase the content of sugar, prolineand ABA in many herbaceous plants, and concom-itantly increase chilling reistance (Lyons 1973, Wang1982), This possibility was eliminated by designing thetreatment containers to minimize water loss from thepericarp discs. In actuality, water loss did not differsignificantly among any of the temperature treatments.

Resistance of tomato plants to low temperature in-jury has been positively associated with the endogenouscarbohydrate content (King et al, 1988), The 32°C con-ditioning treatment possibly could have stimulated re-spiration or altered metabolism to deplete some impor-tant carbohydrate component to levels that increasedchilling sensitivity. However, the reversibility of theconditioning treatments rules out the possibility thatstimulated respiration was the primary cause. The possi-bility exists that a conversion of carbohydrates duringthe conditioning treatments (e,g, the inter-conversionof starch to sugars) was responsible for the changes inchilling sensitivity. For example, increasing the endoge-nous level of sugars by the application of 14 mAf sugarsolutions to excised tomato seedlings reduced subse-quent chilling injury (King et al, 1988), The protectiveeffect was most pronounced with sucrose, less with glu-cose and fructose and mannitol actually increased chill-ing injury, A positive relationship also exists betweenlevels of proline and chilling resistance, but it is notclear whether proline accutnulation was a result of chill-ing or a protective adaptation, These possibilities war-rant additional study.

Chilling resistance has also been related to increasedABA content (Markhart 1986), Increased ABA levelsinduced by a variety of stresses, including high temper-ature stress and exogenous application, have conferred

resistance to subsequent chilling treatments (Rikin et al,1976), ABA broadens the phase temperature range bydirectly affecting the fluid properties of the lipid biiayer(Markhart 1986), If the 32°C conditioning treatmentwas a sufficient temperature stress to induce synthesis ofABA, then the added ABA should have conferred pro-tection from chilling. The subsequent 6-h period of con-ditioning at 12°C that reversed the previous condition-ing effect could have allowed catabolism of ABA tonormal levels with increased chilling sensitivity. How-ever, the chilling responses of tissue conditioned at 32°Cand of tissue reconditioned at 12 after 32°C are in directopposition to those predicated on ABA changes. The32°C conditioned tissue which should have had the high-est ABA level and should, therefore, have had thehighest level of chilling resistance, was the most chilling-sensitive.

Many organisms, including bacteria, yeast, platjts andpoikilothermic animals maintain optimal membrane vis-cosity and function as the environmental temperaturevaries by altering the proportion of unsaturated andsaturated fatty acids in their phospholipids, a responsetermed "homeoviscous adaptation" (Sinensky 1974),For example, tomato fruit grown in cooler climates orduring cooler seasons are more resistant to chiUing in-jury than fruit grown under hotter conditions (Abdel-Maksoud et al, 1974), Such adaptation may be neces-sary to maintain the spatial orientation of membrane-bound enzymes and the permeability characteristics ofthe cellular membranes, since there is evidence thatchanges in the physical properties of cellular mem-branes at chilling tetnperatores can be a source of injur)'(Lyons et al, 1979),

Observed diurnal changes in chilling sensitivity mayresult from modification of the composition of plantmembranes to maintain a constant viscosity at differenttemperatures (Graham and Patterson 1982, Lyons et al,1979), One consequence of changes ie membrane com-position to maintain constant viscosity at different tem-peratures would be that phospholipids synthesized orincorporated into the membranes at higher temper-atures would exhibit progressively higher solid-to-liq-uid-crystalline phase transition temperatures (Chapmanet al, 1967), Therefore, membranes synthesized by cellsto produce the desired viscosity at 12°C would be muchless likely to undergo phase transition upon a 10°C dropto a chiUing temperature of 2,5°C than would mem-braties synthesized to produce a desired viscosity at32°C, These "high temperature" membranes wouldmore likely experience phase transition upon cooling30°C to a chilling temperature of 2.5°C, Additionalstudies on the physiological basis of temperature condi-tioning are in progress to differentiate among thesepossibilities.

Previous studies of the physiology and biochemistryof chilling injury have compared plants with differinglevels of chilling sensitivity. These types of comparisonstudies are hampered by the high degree of variability

Physiot, Ptant. 82, WM 535

among the cultivars or species being compared. Thissource of variability has been greatly reduced by in-ducing statistically significant differences in chilling sen-sitivity in excised tissue from the same fi'uit. Tissue,nearly identical except for a 6-h temperature exposurethat induced differences in chilling sensitivity, could becompared to detect differences too small to be observedin more heterogeneous tissue.

Acknowledgments - The author expresses his appreciation toMr Roberto M. Cabrera for assistance with preliminary experi-ments.

ReferencesAbdel-Maksoud, M, M., Aziz, A, B. A,, Adbel-Kader, A, S,

& Abdel-Samie, K. A, 1974. Influence of growing seasonand storage temperature on chilling injury of tomato fruit. -Egypt, J, Hortic. 1: 171-177.

Anderson, R, E, 1982, Long-term storage of peaches andnectarines intermittently warmed during controlled atmo-sphere storage, - J, Am, Soc, Hortic, Sci. 107: 214-216,

Autio, W. R, & Bramlage, W. ,1, 1986. Chilling sensit:ivity oftomato fruit in relation to ripening and senescence. - J,Am. Soc. Hortic, Sci. I l l : 201-204.

Bramlage, W. J. 1982, Chilling injury of crops of temperateorigin, - HortScience 17: 165-168,

Cabrera, R, M. & Saltveit, J., M, E, 1990, Physiological re-sponse to chilling temperatures of intermittently warmedcucumber fruit, - J, Am, Soc, Hortic. Sci, - 115: 256-261,

Chalutz, E,, Waks, J, & Schiffmann-Nadel, M, 1985. Reducingsusceptibility of grapefruit to chilling injury during coldtreatment. - HortSeience 20: 226-228.

Chapman, D., Williams, R, M, & Ladbrooke, B. D, 1967,Physical studies of phospholipids: VI, Thermotropic andlyotropic mesomorphism in some 1,2-diaeyl-phosphatidyl-cholines (lecithins). - Chem, Phys. Lipids 1: 445-475,

Couey, H. M, 1982, Chilling injury of crops of tropical andsub-tropical origin. - HortScience 17: 162-165,

Creencia, R, P, & Bramiage, W. J. 1971. Reversibility ofchilling injury to corn seedlings, - Plant Physiol. 47: 389-392.

Davis, P. L. & Hofman, R. C. 1973, Reduction of chillinginjury of citrus fruits in cold storage by intermittent warm-ing, - J, Food Sci. 38: 871-873.

Edwards, J. I,, Saltveit, Jr,, M, E. & Henderson, W. R. 1983.Inhibition of lycopene synthesis in tomato percarp tissue byinhibitors of ethylene biosynthesis and reversal with appliedethylene. - J, Am. Soc, Hortic. Sci. 108: 512-514,

Graham, D, & Patterson, B, D, 1982, Responses of plants tolow nonfreezing temperatures: Proteins, metabolism, andacclimation. - Annu, Rev.. Plant Physiol, 33: 347-372.

Gross, K, C, & Saltveit, Ir,, M, E, 1982. Galactose concentra-tion and metabolism in pericarp tissue from normal andnon-ripening tomato fruit, - J, Am, Soe. Hortic, Sci, 107:328-330,

Guinn, G, 1911, Chilling injury in cotton seedlings: ehanges inpermeability of cotyledons, - Crop Sci, 11: 101-102,

Hall, C, B, 1964, The effect of short periods of high temper-ature on the ripening of detached tomato fruits, - Proc,Am. Soc, Hortic, Sci, 84: 501-506,

Hatton, T, T, & Cubbedge, R, H. 1983, Preferred temperaturefor prestorage conditioning of 'Marsh' grapefruit to preventchilling injury a! low temperatures, - HortScienoe 18: 721-722,

Inaba, M, &Chac!iin,K, 1988, Influence of and recovery from

high-temperature stress on harvested mature green toma-toes. - HortScience 23: 190-192.

King, A, 1., Reid, M. S. & Patterson, B, D. 1982. Diurnalchanges in the chilling sensitivity of seedlings. - Plant Phy-siol. 70: 211-214.

- , Joyce, D. C, & Reid, M. S, 1988, Role of carbohydrates indiumal chilling sensitivity to tomato seedlings. - Plant Phy-siol. 86: 764-768.

King, M, M. & Ludford, P. M, 1983. Chilling injury andelctrolyte leakage in fruit of different tomato cultivars, - J,Am, Soc. Hortic. Sci, 108: 74-77.

Lafuente, M, T,, Belver, A,, Guye, M, G. SL Saltveit, Jr,, M.E. 1991, Effect of temperature conditioning on chillinginjury of cucumber cotyledons: Possible role of ABA andheat shock proteins, - Plant Physiol, 95: 443-449,

Lyons, J, M. 1973. Chilling injury in plants. — Annu, Rev,Plant Physiol, 24: 445-466,

- , Raison, J, K, & Steponkus, P. L, 1979. The plant mem-brane in response to low temperatures: An overview. - JnLow Temperature Stress in Crop Piants (J, M, Lyons, D.Graham and J, K, Raison, eds), pp. 1-24, Academic Press,New York, NY. ISBN 0-12-460560-5.

Markhart, A, H, 1986, Chilling injury: A review of possiblecauses, - HortScience 21: 1329-1333.

McColloch, L. P. & Worthington, J. T. 1952. Low temperatureas a factor in the susceptibility of mature-green tomatoes toAlternaria rot. - Phytopathology 42: 425^27,

Mencarelli, F. & Saltveit Jr,, M. E. 1988. Ripening of mature-green tomato fruit slices, - J. Am, Soc. Hortic. Sci. 113:742-745,

Morris, L, L, 1982, Chilling injury of horticultural crops: anoverview, - HortScience 17: 161-162,

Murata, T, & Tatsumi, Y. 1979, Ion leakage in chilled planttissues. — In Low Temperature Stress in Crop Plants (J. M.Lyons, D, Graham and J. K, Raison, eds), pp, 141-151,Academic Press, New York, NY. ISBN 0-12-460560-5,

Rikin, A,, Blumenfeld, A, & Richmond, E, 1976. Chillingresistance as affected by stressing environments and ABA.- Bot, Gaz. 137: 307-312,

Saltveit, Jr,, M. E, 1982, Procedures for extracting and analyz-ing internal gas samples from plant tissues by gas chroma-tography, - HortScience 17: 878-881.

- 1989a, A kinetic examination of ion leakage from chilledtomato pericarp disks, - Acta Hortic, 258: 617-622.

- 1989b, Effeet of alcohols and their interaction with ethyleneon the ripening of epidermal pericarp disks of tomato fruit,- Plant Physiol, 90: 167-174,

- & Cabrera, R, M, 1987, Tomato fruit temperature beforechilling influences ripening after chilling, - HortSdence 22:452-454.

- & Morris, L. L. 1990, Overview of chilling injury of horticul-tural crops. - In Chilling Injury of Horticultural Crops(Chien Yi Wang, ed,), pp, 3-15. CRC Press, Boca Raton,FL, ISBN 0-8493-5736-5.

Sinensky, M, 1974, HomeoviscoES adaptation - A homeostaticprocess that regulates the viscosity of membrane lipids inEscherichia coti. - Proc, Natl, Acad, Sd, USA 71: 522-525,

Smith, C. W, & McWilliams, E. L, 1979, Amelioration ofchilling injury in Maranta leucaneura and Scindaprus pictusby preconditioning, — HortScience 14: 439.

Vickery, R, S, & Bruinsma, J, 1973, Compartments and per-meability for potassium in developing fruits of tomato i^Ly-copersicon esculentum Mill,), - J, Exp. Bot, 24: 1261-1270,

Wang, C, Y, 1982, Physiological and biochemical responses ofplants to chilling, - HortScienee 17: 173-186,

Wheaton, T, A, & Morris, L, L, 1967, Modification of chillingsensitivity by temperature conditioning., - Proc, Am, Soc,Hortic, Sci, 91: 529-533,

Wright, M, & Simon, E, W, 1973, Chillinginjury in encumberleaves,-J, Exp, Bot, 24: 400-411,

Edited by T. C, Vogelmann

536 Physiot. Plant. 82, IWl