PHYSIOLOGIA PLANTARUM 89: 117-124. 1993Printed in Denmark - all rights reserved

Heat shock increases chilling tolerance of mung bean hypocotyltissue

Graham G. Collins, XunLi Nie and Mikal E. Saltveit, Jr

Collins, G.G., Nie, X. and Saltveit, M E . Jr. 1993. Heat shoek increases chillingtolerance of mung bean hypocotyl tissue. - Physiol. Plant. 89: 117-124.

The effects of heat shock on the chilling tolerance of mung bean [Vigna radiaia (L.)Wilczek] seedling tissue were studied by using two measurements of chilling injury:increased l-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and soluteleakage. ACC oxidase activity (measured as ACC-induced ethylene production) offreshly exeised mung bean hypocotyl segments was highly dependent on the temper-ature at which the seedlings were grown. However, this highly temperature-depend-ent level of ACC oxidase activity was probably a wound response since it was almostentirely eliminated by incubating the excised segments at 2()°C for 3 h. In contrast,heating of excised segments to 40°C for up to 4 h resulted in a time-dependentincrease in ACC oxidase activity which was sensitive to cycloheximide, indicatingrapid protein synthesis during the heat treatment. ACC oxidase activity tell sharplyduring subsequent chilling at 2.5°C. After 3 days of chilling, all treated segments,regardless of their initial ACC oxidase activity, showed a decline to the same iowactivity level and ACC oxidase activity continued to fall slowly for up to 9 days at2.5°C. Hypocotyl segments excised from seedlings held at 15°C showed no change insolute leakage, but leakage increased rapidly when seedlings were either chilled at2.5°C or heated to 32°C (just below the heat shock temperature). Chill-inducedleakage from non-heat-shocked segments increased steadily with chilling durationand was unaffected by cycloheximide concentration up to day 6. Within the elevatedrate of leakage on day 9, however, leakage was lower from segments exposed to 10and 50 \iM cycloheximide. Solute leakage was markedly reduced for up to 9 dayswhen segments were heat shocked at 40°C for 3 or 4 h with or without 10 ^Mcycloheximide, but the presence of 50 [xM cycloheximide caused an initial doubling ofsolute leakage and a 3-fold increase after 3 days of chilling. Cycloheximide preventedformation of heat shock protection against chilling from the start at 50 ^iM and after 9days at 10 \iM. These results indicate that the protection afforded by heat shockagainst chilling damage is quantitative and probably involves protein synthesis.

Key words- ACC oxidase activity, cycloheximide, mung bean, O|,i. solute leakage.Vigna radiata.

G.G. Collins, Dept of Horticulture, Viticulture and Ocnology, Waite Campus, Univ.of Adelaide, Glen Osmond, South Australia, 5064, Australia: X. Nie and M. E.Saltveit, Jr (corresponding author), Mann Laboratory, Dept of Vegetable Crops,Univ. of California, Davis, CA 95616, USA.

ethylene production, with the greatest amounts.of ethy-Introduction I^P^ being produced by the most chilling-sensitive spe-Many plants which have evolved in tropical and semi- cies (Chen and Patterson 1985, Guye et al. 1987). Thetropical areas show tissue injury after a period of expo- tissues of chilling-sensitive plants which have been ex-sure to nonfreezing temperatures below 12°C (Wang posed to chilling temperatures may respond by either an1982, Saltveit and Morris 1990). The imposition of low immediate increase in their production of ethylene, astemperature stress on plants which are chilling-sensitive in the primary leaves of bean (Wright 1974), or by ausually results in an increase and subsequent decrease in decrease, followed by a rapid increase when they are

Received 15 December, 1992; revised 17 May, 1993

Physiol. Plant. 89, 1993

transferred to warmer temperatures, as in cucumberepidermal tissue (Wang and Adams 1980). In the lattercase, ethylene production during the chilling period wasvery low.

The final steps in ethylene biosynthesis are the con-version of S-adenosylmethionine to 1-aminocyclopro-pane-1-carboxylic acid (ACC) by ACC synthase and theconversion of ACC to ethylene by ACC oxidase(Adams and Yang 1977). Wang and Adams (1980)found that cycloheximide inhibited the stimulation ofethylene production which followed the rewarming ofchilled tissues and that the activities of both ACC syn-thase and ACC oxidase were reduced by cycloheximide.Further work by Wang and Adams (1982) showed thatinhibitors of RNA synthesis had no effect on the activityof ACC synthase or the level of ACC. These resultswere interpreted as indicating that transcription pro-ceeds during the chilling period, but that translation isnot able to proceed until the tissue is warmed. Thecapacity of chilled tissue to show accelerated ethyleneproduction during subsequent warming decreases as thetime the tissues are held at the chilling temperatureincreases (Wang and Adams 1982). This feature sug-gests that the activity of ACC oxidase, a membrane-bound enzyme, has been affected. Thus, the use ofACC oxidase activity as an indicator of chilling damagemay be limited to the first few days of chilling.

The increase in solute leakage, which usually accom-panies chilling, is interpreted as resulting from chilling-induced membrane damage (McCullough and Simon1973, Murata 1990). In some plants, the amount ofsolute leakage can be significantly decreased by priorexposure of the tissue to high temperatures before chill-ing. Saltveit (1991) measured the rate of ion leakagefrom tomato pericarp disks which were conditioned atvarious temperatures and then subjected to incubationat 2.5°C for 4 days. Ion leakage was markedly reducedin the tissues which were conditioned at 32°C or greater.Lafuente et al. (1991) exposed cucumber cotyledondisks to either 12.5 or 37°C for 6 h followed by 4 days at2.5°C. The tissue conditioned at the higher temperaturewas found to be more resistant to chilling injury, asindicated by reduced chilling-induced ion leakage.

Since both ACC oxidase activity (Etani and Yoshida1987, Field and Barrowclough 1989) and solute leakage(Autio and Bramlage 1986, McCollum and McDonald1991. Saltveit 1991) are commonly used as indicators ofchilling injury, the present paper reports on their useful-ness as measures of chilling injury in mung bean hypo-cotyls. Cycloheximide has been reported to decreasethe effect of heat shock at 4()°C on mung bean seedlingsby decreasing their thermotolerance to 45°C (Chen etal. 1986). Accordingly, we report on the amelioration ofchilling injury by heat shock treatments and the effect ofcycloheximide on decreasing this induced protection tochilling.

Abbreviation - ACC, 1-aminocyclopropane-l-carboxylic acid.

Materials and methods

Germination of mung beans

Seeds of mung bean [Vigna radiata (L.) Wilczek] weresterilized in 0.26% sodium hypochlorite (1:20 dilutionof Bleach, All Pure Chemical Company, Tracy, CA,USA) for 10 min. The seeds were rinsed four times indistilled water, allowed to imbibe in aerated 1 mMCaSO4 overnight, and germinated in vermiculite for 4days in the dark at 25°C. When excised hypocotyl seg-ments were used, two 1-cm hypocotyl sections were cutfrom each etiolated seedling 4 days after germinationand incubated under the conditions described.

Determining conditions for measuring soiute leakage

Seedlings were placed in light-tight metal boxes ingrowth rooms at various temperatures. An air flow of ca100 ml min ' was passed continuously through eachbox. After the appropriate incubation period, two 1-cmhypocotyl sections were cut from each seedling starting1 cm below the plumular hook (Kevers et al. 1989).

About 2 g of hypocotyl segments were harvested andweighed and placed in 10 ml of 0.3 M mannitol (Saltveit1991) in a 2()-ml glass container. The container wascovered with aluminum foil and incubated at room tem-perature (ca 22°C) with constant shaking at 60 cyclesmin '. The conductivity of the solution was read at30-min intervals up to 180 min. Total conductivity of thetissue was determined either by freezing the tissue andsolution at -20°C overnight and thawing it. or by auto-claving it for 20 min and allowing the containers to cool(McCollum and McDonald 1991). Deionized water wasadded to the autoclaved containers to bring them backto their initial weights.

Determining conditions for measuring ACC oxidase activity

About 1 g of hypocotyl segments was harvested andweighed and placed in a 10-ml plastic syringe cappedwith a rubber serum stopper. The syringe contained 2ml of 5 mM 2-[N-morpholino]ethanesulfonic acid(MES)/KOH buffer, pH 6.8, 1% glucose and ± 5 mMACC (Sigma, A-3903, Lot 79F4053) (Etani and Yoshida1987). Syringes ± tissue and ± ACC were included ascontrols. The syringes were adjusted to 10 ml, coveredwith aluminum foil and incubated horizontally at roomtemperature with constant shaking at 60 cycles min '.At 1, 2 and 3 h, 1-ml gas samples were removed as thevolume of the syringe was reduced by an equivalentvolume. The amount of ethylene was estimated by gaschromatography (Saltveit 1982) and adjusted for valuesfrom the controls and for syringe volume and tissueweight.

ACC oxidase activity (measured as ACC-inducedproduction of ethylene) of mung bean hypocotyl seg-ments was measured at intervals over a period of 3 hand was found to be relatively constant (data not

118 Physiol. Plant. X9. 1993

shown). In all experiments, ACC-induced ethylene pro-duction was measured after the first 2 h of incubation inthe reaction mixture.

Temperature effects on intact seedlings

Seedlings were incubated in the dark at hypocotyl tem-peratures of 5, 15, 24 and 32°C and then either main-tained at these temperatures or transferred to 2.5°C.Hypocotyl temperature was measured with a small tem-perature probe. After the appropriate duration, hypo-cotyl segments were excised for the immediate determi-nation of solute leakage and ACC oxidase activity.

Temperature effects on isolated hypocotyl segments

About 4 g of 1-cm hypocotyl segments were harvested,weighed and placed in 15 x 100 mm diameter Petridishes containing a 9-cm diameter disc of Whatman No.1 filter paper and 2 ml of 5 mM MES/KOH buffer, pH6.8, and 0, 10 or 50 tM cycloheximide. The Petri disheswere sealed with Parafilm and immersed in a water bathat 40°C for up to 4 h. After the treatment, the segmentswere rinsed several times in deionized water, drained ontissue paper and sealed in fresh Petri dishes containing adisk of filter paper and 2 ml of 5 mM MES/KOH buffer,pH 6.8. The tissue was then either assayed immediatelyfor ACC oxidase activity and solute leakage or stored insealed Petri dishes at 2.5°C and assayed at intervals upto 9 days. An additional treatment consisted of tissuewhich was unheated and stored at 15°C.

10 20 30Incubation time (h)

40 50

Eig. 1. ACC oxidase activity of hypocotyl segments assayedimmediately after excision from 4-day-old etiolated mung beanseedlings incubated at either 2.5. 15 or 32°C for up to 48 h. Thevertical line indicates the 5% LSD value of 16 nl g ' h '.

ethylene g ' h ' for segments excised from seedlings attime zero to 80 nl g~' h ' for segments from seedlingsincubated at 32°C for 6 h and decreased to 52 and 34 forseedlings incubated at 15 and 2.5°C, respectively, for 6h. At 6 h, the enzyme activity had a Qm of approxi-mately 1 and an activation energy for the enzyme of3 700 J mol ' as calculated from the Arrhenius plot(Fig. 2).

ACC oxidase activity decreased about 65% for allincubation temperatures over the period from 6 to 48 h;reductions were from 80 to 27 nl ethylene g ' h ' at32°C, from 52 to 19 nl ethylene g ' h ' at 15°C and from34 to 12 nl ethylene g ' h ' at 2.5°C (Fig. 1). The declineat 32°C was linear from 6 to 48 h, while the declines at15 and 2.5°C had reached their final rates by 24 h. Theproduction of ethylene in the absence of ACC wasalways less than 5 nl g ' h ' and was not affected bytemperature (data not shown).

ResultsSolute leakage by mung bean hypocotyls

The rate of solute leakage from excised hypocotyl seg-ments was higher during the first 30 min of incubationthan during the following 150 min. The earlier, morerapid rate was assumed to represent leakage primarilyfrom the apoplast, whereas the subsequent slower ratewas leakage from the symplast (Saltveit 1989). The rateof leakage from the symplast was calculated as the dif-ference in conductivity between 30 and 120 min and isexpressed as a percentage of the total conductivity.

The total solute concentration of the hypocotyl seg-ments was estimated after the tissue was either frozen/thawed or autoclaved. The conductivity of the solutionbathing autoclaved tissue was 40% greater than that forfrozen/thawed tissue, and the coefficient of determina-tion between the two methods was 0.96.

Seedling temperature and ACC oxidase activity

There was a marked effect of temperature on ACCoxidase activity of hypocotyl segments assayed imme-diately after excision from 4-day-old seedlings incu- .gg^e^j, ^^^^^^^ immediately after excision from 4-day-oldbated at 2.5, 15 and 32°C for vanous periods ot time etiolated mung bean seedlings incubated at either 2.5. 15 or(Fig. 1). ACC oxidase activity increased from 60 nl 32°C for 6 h.

Incubation temperature [1/K (x 10 ) ]

Physiol. Plant. 89. t993 119

0 486 24Incubation time (h)

Eig. 3. Solute leakage by hypocotyl segments assayed imme-diately after excision from 4-day-old etiolated mung bean seed-lings incubated at either 2.5, 15 or 32°C for up to 48 h. Thevertical line indicates the 5% LSD value of 1.3% of total con-ductivity.

Seedling temperature and solute leakage

The amount of solute leakage by hypocotyl segmentswas sensitive to the temperature at which the seedlingswere incubated. Leakage remained relatively constantat 4% of total conductivity (as determined by freezingand thawing the excised hypocotyl segments) from seed-lings incubated at 15°C for up to 48 h (Fig. 3). Incontrast, leakage from seedlings incubated at 32°Cshowed a 2-lold increase from 4.6% of total conduc-tivity at an incubation time of 6 h to 9.1% of totalconductivity at 24 h. A slight increase to 9.6% occurredby 48 h. Leakage from seedlings incubated at 2.5°Cremained about 5% during the first 24 h and then in-creased by around 50% to 7.4% of total conductivity by48 h.

In contrast to the immediate effect of various in-cubation temperatures, solute leakage from hypocotylsegments excised from seedlings incubated at either 15,24 or 32°C for 6 h and then chilled at 2.5°C for 3 daysshowed a uniformly high amount of solute leakage ofabout 8% (data not shown).

Heat shock and ACC oxidase activity in hypocotyl segments

Whereas hypocotyl segments excised from whole mungbean seedlings incubated at various temperatures for 6 hshowed high levels of ACC oxidase activity (Fig. 1),excised segments sealed into Petri dishes and incubatedfor 3 h at 20°C produced little or no ACC-inducedethylene (Fig. 4; 0 days, 3 h, 20°C, no CHX). However,ACC oxidase activity was greatly stimulated by incu-bating hypocotyl segments at 40°C for 3 or 4 h. Therates assayed in hypocotyls immediately after excisionincreased from 1.3 to 56.5 to 110 nl ethylene g ' h ' asexposure to 4()°C increased from 0 to 3 to 4 h, respec-tively. Subsequent exposure of excised hypocotyl seg-

120

100

80

60

20

0 -

No CHX lOfjMCHX

Days at 2.5°C

Eig. 4. ACC oxidase activity of hypocotyl segments excisedfrom 4-day-old etiolated seedlings and incubated in sealedPetri dishes at 2t) or 40°C for 3 or 4 h in 1.5 ml of 5 mMMES/KOH buffer, pH 6.8, ± 10 [iM cycloheximide (CHX).Measurements were made after various durations of chilling at2.5°C. The vertical line indicates the 5% LSD value of 14 nl g 'h ' .

ments from all treatments to chilling at 2.5°C resulted ina rapid decrease in ACC oxidase activity to a commonrate of 12 nl ethylene g ' h ' (range 7 to 18) for alltreatments on day 3. Activity continued to decline in alltissues until it approached zero after 9 days of chilling(Fig. 4).

Hypocotyl segments incubated for 3 days at 15°Cwere light brown at the cut edges and exhibited 8 timesthe activity of ACC oxidase compared to the tissueincubated for 3 days at 2.5°C (data not shown). By 9days, the segments at 15°C were brown throughout, hadan unpleasant smell and had an extremely high soluteleakage of 29% of the total solutes.

20"C 40"C

[2 ] No CHX

10/jMCHX

^ 50f/MCHX

3 6 9Days at 2.5°C

Fig. 5. Solute leakage by hypocotyl segments exeised from4-day-old etiolated seedlings and incubated in sealed Petridishes at 20 or 40°C for 3 or 4 h in 1.5 ml of 5 mM MES/KOHbuffer, pH 6.8, ± 10 or 50 (xM cycloheximide (CHX). Measure-ments were made after various durations of chilling at 2.5°C.The vertical line indicates the 5% LSD value of 4.5% of totalconductivity.

120 Physiol. Plant. 89, 1993

Heat shock and solute leakage by hypocotyl segments

The amount of solute leakage from excised hypocotylsegments which received no heat treatment increased10-fold from 1.6 to 16.1% of total conductivity over aperiod of 9 days at 2.5°C (Fig. 5). The increase wasuniform for the first 6 days over the 0, 10 and 50 iMtreatments. By day 9, however, the rate of increasediverged with the 0 [iM treatment being greater and the50 \.iM being less than the continuing rate of increaseshown by the 10 tM treatment. In contrast, solute leak-age by hypocotyl segments which were heated to 40°Cfor 3 h before they were chilled at 2.5°C showed only a2-fold increase from 3.0 to 6.0% of total conductivityduring the 9 days. A heat shock of 4 h at 40°C produceda similar result (data not shown).

After 9 days at 2.5°C, the non-heat-shocked segmentswere light brown at the cut ends, whereas the segmentsheat shocked for 3 or 4 h were still white throughout.

Cycloheximide and ACC oxidase activity in hypocotylsegments

The stimulation of ACC oxidase activity which occurredduring incubation of hypocotyls at 40°C was greatlyinhibited by 10 piM cycloheximide (Fig. 4). In the pres-ence of 10 \iM cycloheximide, ACC-induced ethyleneproduction decreased by 40% from 57 to 34 nl g ' h^'and by 76% from 110 to 27 nl g^' h ' during a 3- and 4-hheat shock at 40°C, respectively. Cycloheximide wasintroduced 1 h prior to, and during, the heat treatment.Control tissue was treated the same way as the tissueincubated for 3 h, while tissue incubated for 4 h at 40°Cwas in contact with cycloheximide for an additionalhour.

Cycloheximide and solute leakage by hypocotyl segments

Solute leakage from non heat-shocked segmentssteadily increased over 6 days of chilling from around2.3 to 8.7% of total conductivity (Fig. 5). The steadyrise continued to 11.0% of total conductivity on day 9for segments treated with 50 \iM cycloheximide, butshowed a more rapid increase to 13.7 and 16.3% of totalconductivity for segments treated with 10 and 0 \iMcycloheximide, respectively. The control segments de-veloped a marked browning at the cut edges whichextended back for several millimeters. This response,presumably indicating polyphenoloxidase activity, wasless evident at a concentration of 10 jxM cycloheximideand absent at 50 [LM.

A concentration of 10 jiM cycloheximide had no ef-fect on the rate of solute leakage by heat-shocked hypo-cotyl segments during the first 6 days of chilling at 2.5°C(Fig. 5). After 9 days, the segments which had received3 h of heat shock in the presence of 10 \iM cyclohexi-mide had about double the rate of solute leakage com-pared either to the rate on day 6 (10.9 vs 5.2% of total

conductivity), or to segments exposed to the same treat-ment without cycloheximide on day 9 (10.9 vs 5.9% oftotal conductivity).

Heat shocking segments in the presence of 0 or 10 [iMcycloheximide increased leakage around 35% from 2.2to 3.0% of total conductivity, while heat shocking in thepresence of 50 \iM cycloheximide increased leakageabout 150% to 5.6% of total conductivity. The rate ofleakage increased another 80% for segments heatshocked in the presence of 50 [iM cycloheximide from5.6 to 10.1% of total conductivity after 3 days of chillingand then increased by 13% to 11.4% of total conduc-tivity by day 9. This effect was significantly greater thanfor segments heat shocked in the presence of 0 or 10 |j.Mcycloheximide. A similar effect occurred after 6 days ofchilling, but after 9 days, solute leakage was the samefor both concentrations of cycloheximide.

DiscussionSeedling growth temperature effects ACC oxidase activity andsolute leakage

The activity of ACC oxidase assayed immediately afterexcision of mung bean hypocotyl segments from seed-lings which had been transferred from 25°C to varioustemperatures for 6 h was greatly influenced by tempera-ture (Fig. 1). The Q,,, for the reaction was approxi-mately 1, suggesting that the increase in ethylene pro-duction was due to de novo synthesis rather than toincreased activity of a pre-existing enzyme. Field andBarrowclough (1989) found that ethylene production bybe'an leaf tissue was affected by prior temperature expo-sure. They report that tissue previously held at 2.5°Cproduced virtually no ethylene. The maximum rate ofproduction was from leaf tissue previously held at 35°C,while production declined to zero for tissue previouslyat 45°C.

An Arrhenius plot of the rate of ACC-induced ethy-lene production showed an activation energy of 3 700 Jmol ' (Fig. 2). In another experiment (data not shown),seedlings were incubated at 15. 24 and 32°C for 6 h priorto chilling at 2.5°C for a further 42 h. The activationenergy for ACC oxidase at the end of the chilling periodwas 3 900 J mol '. The similarity of the two values foractivation energy before and after chilling suggests thatthe same pathway for ethylene production operates atboth ambient and chilling temperatures for mung beantissue, as has been reported for cucumber (Wang andAdams 1982). Field (1985) reported that the activationenergy for ethylene production by chilling-sensitiveplants was approximately doubled at temperatures be-low their transition point.

The initial rise in ACC oxidase activity in hypocotylsof mung bean seedlings from 0 to 6 h was followed by ageneral decline in activity with time of incubation of theseedlings for up to 48 h (Fig. 1). This decline in activitywas presumably due to a decrease in the ability of the

Physiol. Plant. 89, 1993 121

tissue to synthesize ACC oxidase as it increased in age,as has been reported for lupins (Prez-Gilabert et al.1991).

When intact seedlings were incubated for up to 48 hat 15°C there was no change in the amount of soluteleakage from the excised hypocotyl segments, indicatingthat this temperature was not damaging to the tissue.However, solute leakage from the segments increasedafter the seedlings had been incubated at 32°C for 24 hor at 2.5°C for 48 h (Fig. 3). This suggests that whilemembrane permeability is sensitive to chilling tempera-tures, it is even more sensitive to temperatures justbelow those which induce heat shock proteins.

Saltveit (1991) conditioned pericarp disks from ma-ture-green tomatoes at temperatures from 0 to 37°C for6 h. Chilling-induced solute leakage was found to begreatest for the disks conditioned at 0 to 10°C and at 25to 32°C before they were chilled at 2.5°C for 4 days.Although measurements of ion leakage immediately af-ter the 6-h conditioning treatment failed to detect anysignificant differences among the treatments, it is pos-sible that the disks heated at 25 to 32°C had alreadyincurred some membrane damage that was exacerbatedby subsequent chilling and caused the increase in ionleakage.

Hypocotyl temperature and ACC oxidase activity and soluteleakage

Isolated hypocotyl segments were used to furtherexamine the temperature responses of ACC oxidaseactivity and solute leakage because evaporation fromseedlings prevented them from consistently reaching40°C in the growth chamber and because of growth andaging of the treated hypocotyl region during subsequentincubation of the seedlings for up to 9 days.

In contrast to the high level of ACC oxidase activityfound in hypocotyl segments excised from seedlings thathad been incubated for 6 h at various temperatures(Fig. 1), the activity of ACC oxidase in hypocotyl seg-ments incubated at 20°C for 3 h after excision wasalmost zero (Fig. 4). This suggests that much of theACC oxidase activity shown in Fig. 1 was produced denovo as a result of wounding during excision of thehypocotyl segments from the seedlings and that thiswound-induced ACC oxidase activity is temperaturesensitive. Incubation of isolated hypocotyl segments for3 h at 2()°C before they were assayed for ACC oxidaseactivity probably allowed the tissue to recover from thewound response, and the ACC-induced ethylene pro-duction then dropped to the low level. This implies thatboth the induction and the destruction of ACC oxidaseactivity are extremely rapid processes.

The synthesis of ACC from S-adenosylmethionine,the rate-limiting step in the production of ethylene(Boiler et al. 1979), was shown by Wang and Adams(1982) to be inhibited at low temperatures. However, inthe experiments reported here, ACC was supplied and

therefore the inhibitory effect of chilling that we mea-sured is on the activity of ACC oxidase.

Guye et al. (1987) found that the ACC oxidase ac-tivity in Phaseolus species was reduced when the tissuewas chilled, and the effect was found to be more pro-nounced for those species which were considered to bethe most chilling sensitive.

The conversion of ACC to ethylene is reported torequire oxygen and to be promoted by carbon dioxide(Field 1990). Because the hypocotyl segments weresealed in Petri dishes it could be argued that the tissueswere oxygen starved and that this contributed to thereduction in ACC oxidase activity. This was shown notto be the case when hypocotyl segments which werestored in sealed Petri dishes at 15°C for 3 days de-veloped ACC oxidase activity more than 8-fold higherthan comparable segments stored at 2.5°C. However,promotion of the conversion of ACC to ethylene bycarbon dioxide may have occurred.

Heat shocking excised hypocotyl segments at 40°Cbefore chilling them at 2.5°C markedly inhibited thechilling-induced increase in solute leakage compared tothat of unheated segments. Reduced chilling sensitivitycould be detected as early as 3 days after the commence-ment of chilling, and after 9 days, solute leakage fromunheated segments was more than 1.5-fold greater thanthat from heat-shocked segments (Fig. 4). A heat shockeffect of this duration has not been reported previouslyin isolated plant tissue.

Cycloheximide and ACC oxidase activity and solute leakage

Both ACC accumulation and ethylene production areinhibited by a concentration of 50 [iM cycloheximide incucumber epidermal tissue (Wang 1989), indicating thatcontinuous protein synthesis is necessary for the pro-duction of ethylene. The results presented in the pre-sent paper indicate that ACC oxidase activity is in-ducible in mung bean hypocotyl tissue by wounding andby heat shock. The removal of hypocotyl segments fromintact seedlings produced a rapid temperature-depend-ent increase in ACC oxidase activity in the segmentswhich could be attributed to a wound response. Whenthe hypocotyl segments were allowed to recover over aperiod of 3 h, ACC oxidase activity fell to near zero, butthen increased to high levels when the tissue was in-cubated at 40°C. The presence of 10 \iM cycloheximidereduced the amount of ethylene produced during theheat shock by as much as 76% indicating the require-ment for protein synthesis (Fig. 4). There was littleeffect of cycloheximide on segments which were notheat shocked.

The rapid increase in the development of ACCoxidase activity during heat shock raises the question ofwhether the ethylene produced provides a trigger forthe production of heat shock proteins.

Solute leakage by unheated hypocotyl segments in-creased from about 2% of total solutes to about 16%

122 Physiol. Plant. 89. 1993

after 9 days of chilling (Fig. 5). Cycloheximide had noeffect on this trend until sometime after the 6th day. Byday 9, the amount of solute leakage by the cyclohexi-mide-treated tissue had decreased compared to the con-trol, and the reduction was significant for 50 [iM cyclo-heximide. The control tissue showed marked browning,suggesting the activity of polyphenoloxidase, which wasgreatly reduced at 10 |xM cycloheximide and absent at50 \iM. The development of this effect 9 days after thehypocotyl segments had been exposed to cycloheximideand then extensively washed and incubated on freshmedia suggests that these procedures may have beeninsufficient to remove all of the cycloheximide taken upby the cells.

Compared to the effect of cycloheximide on ACCoxidase activity (Fig. 4), solute leakage was almost un-affected by 10 |xM cycloheximide during the first 6 daysof chilling (Fig. 5). However, after 9 days at 2.5°C, thehypocotyl segments which received a 3-h heat shock inthe presence of 10 (xM cycloheximide showed a changein membrane permeability (Fig. 5). This change inmembrane permeability was detected as a significantincrease in solute leakage more than 80% greater thanthat of the tissue which received a 3-h heat shock in theabsence of cycloheximide. In contrast, the hypocotylsegments which received a 4-h heat shock at 40°C con-tinued to show a low level of solute leakage both in thepresence and the absence of cycloheximide for up to 9days of chilling (results not shown).

Heat-shocked hypocotyl segments showed a remark-able resistance to chilling damage. If we assume thatpart or all of this induced chilling resistance is related tothe synthesis and accumulation of heat shock proteins,then a partial inhibition of protein synthesis by 10 \iMcycloheximide would account for the breakdown inchilling resistance between 6 and 9 days of chilling at2.5°C because of the reduced amount of heat shockprotein in the tissue. In contrast, additional heat shockprotein produced during exposure to 40°C for an addi-tional hour (a total of 4 h) could account for the addedprotection to over 9 days of chilling. This idea Is furthersupported by the response of hypocotyl segments heatshocked in the presence of 50 \iM cycloheximide (Fig.5). Treatment with this high level of cycloheximide dur-ing heat shock caused the induced chilling resistance tobreak down sometime during the first 3 days of chilling,presumably because of the reduced accumulation ofheat shock proteins.

These findings indicate that some minimal amount ofheat shock protein is necessary to initiate protectionagainst chilling damage and that the duration of theprotection depends upon the amount of protein pro-duced.

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Edited by T. C. Vogelmann

124 Physiol. Plant. 89. 1993