Plant Physiol. (1985) 79, 672-6780032-0889/85/79/0672/07/$0 1.00/0

Presence of Heat Shock mRNAs in Field Grown Soybeans'Received for publication January 9, 1985 and in revised form August 2, 1985

JANICE A. KIMPEL*2 AND JOE L. KiEYDepartment ofBotany, University ofGeorgia, Athens, Georgia 30602

ABSTRACT

Our laboratory has extensively defined many parameters of the heatshock (HS) response in etiolated soybean (Glycine max [L.] Meff.)hypocotyls, including the identification of cDNA clones for mRNAsencoding several low molecular weight HS proteins. We have now inves-tigated the response of mature plants to a HS in a growth chamber andto high temperature stress under field conditions. Soybean plants showinduction ofHS mRNAs when the temperature of the chamber is rapidlyshifted from 28°C to 45°C. This temperature of induction is significantlyhigher than the optimal induction temperature for etiolated hypocotyls,probably reflecting the ability of mature plants to lower their leaftemperatures below the ambient air temperature through transpirationalcooling. Samples of soybean leaves were taken from an irrigated and anonirrigated field during a 24-h period when midday temperaturesreached 40°C. Several HS mRNAs were present in samples from bothfields, although the levels of these mRNAs were much higher in nonir-rigated leaves. This differential response ofHS mRNA steady state levelswas not a response to water stress, since water-stressed plants at 28°Cdid not induce HS mRNAS. Rather, these quantitative differences areprobably due to differences in actual leaf temperatures between irrigatedand nonirrigated leaves. The presence of these HS mRNAS in field-grown plants suggests that HS proteins are produced as part of thenormal plant response to high temperature.

In the last several years, this laboratory has been studying theheat shock response in etiolated soybean seedlings (8-13). Thisresponse is characterized by the induction ofa new set ofmRNAsand proteins after an abrupt increase in temperature of 8 to10°C. The proteins produced are a complex set of high mol wtand low mol wt polypeptides, referred to as HS3 proteins. Theseproteins and their mRNAs accumulate to significant levels dur-ing a 2-h HS, and this relative abundance of mRNAs has aidedin the isolation of cDNA clones for several low mol wt HSproteins (17). These clones have been used to define severalkinetic parameters of the accumulation of these heat shockmRNAs during different HS regimes (8-10).Of course, plants do not experience such abrupt temperature

shifts under field conditions. Instead, they experience daily,gradual fluctuations in temperature that are often greater than10°C. It is important, in determining the function(s) of HSproteins, to know whether this HS response can also occur in

'This work was supported by a research contract from AgrigeneticsResearch Corporation and Department of Energy DE AS09-80ER1078.

2Current address: Department of Botany and Plant Pathology, OregonState University, Corvallis, OR 97331.

3Abbreviations: HS, heat shock; DEP, diethyl pyrocarbonate; MOPS,3-(N-Morpholino) propanesulphonic acid: SSC, standard saline citrate;SSU, small subunit.

mature plants in the field. To begin to address this issue, threequestions have been posed: (a) In etiolated seedlings, does theinduction ofHS proteins and HS mRNAs require an abrupt shiftor can a gradual increase in temperature also induce them? (b)Do mature plants also exhibit a HS response; i.e. are HS mRNAsinduced during a heat shock treatment in a controlled environ-ment? (c) In the field, do plants experiencing heat stress alsoinduce the HS response?

In this report, evidence is presented that HS proteins and HSmRNAs do indeed accumulate in response to a gradual increasein temperature. Furthermore, mature soybean plants in the fieldor in a growth chamber produce these proteins and mRNAsduring periods of high temperatures.

MATERIALS AND METHODS

Plant Material. Etiolated seedlings were obtained by germi-nating soybean seeds (Glycine max [L.] Merr. cv Wayne) in rollsof moist Chem-pak at 28°C in a dark growth chamber. Two-d-old seedlings were used for both in vivo protein labeling studiesand RNA extractions. Cotyledons were removed from seedlings,and 10-g seedlings in 250-ml flasks were incubated in 40 mlincubation buffer (1% sucrose, 1 mM K-phosphate [pH 6.0], 50,gg/ml chloroamphenicol) in shaking water baths equilibrated atthe desired temperature.To obtain mature green plants, soybean seeds (cv Wayne) were

germinated in soil flats and maintained in a greenhouse for 3weeks. Plants were then moved to a well-lighted growth chamberon a 14-h photoperiod (24°C day/16°C night) for 1 week priorto the beginning ofan experiment. Plants were watered daily andgiven a dilute Hoagland nutrient solution weekly. Plants werethoroughly watered immediately prior to a HS treatment, exceptduring water-stress experiments. Plants were water-stressed onan empirically determined watering schedule that caused novisible wilting of the plants but resulted in significant growthreduction. Well-watered flats (25 x 50 cm) were watered with 1L of water daily. The water-stressed flats were watered with 0.25L daily, and this was supplemented with an additional 0.25 Levery 3rd d for 2 weeks. This mimicked the situation observedin field grown plants; nonirrigated soybeans were much smallerthan irrigated soybeans, although no wilting of these plants wasobserved during the sampling period.

In vivo Labeling of Etiolated Seedlings. Five seedlings (withcotyledons removed) were incubated under the indicated tem-perature regimes. After 15 min at the desired temperature forlabeling, 200 ,Ci of L-[3,4,5-3H(N)]leucine (140 Ci/mmol, NewEngland Nuclear) were added to the flask, and seedlings wereincubated for an additional 45 min. Protein extraction and one-dimensional SDS gel electrophoresis were carried out as describedpreviously (12).

Field Sampling. Irrigated and nonirrigated fields of soybean(cv Tracy) were sampled at the indicated times as follows. Leaveswere collected from several different plants in each field andimmediately frozen in liquid N2 at the collection site. Plantmaterial was returned to the laboratory on dry ice and stored at

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HEAT SHOCK mRNAs IN SOYBEANS

-70°C until use. The irrigated field was not actually irrigated onthe days that sampling was done, so there was no free-standingwater on the plants or the plots during the sampling period.RNA Extraction. For etiolated seedlings, poly(A)RNA was

extracted as described before (17). For green leaf tissue, thefollowing modifications were made: 15 g of leaf tissue washomogenized for each sample. After the fourthphenol:chloroform extraction, nucleic acids were precipitated inethanol. The pellet was then resuspended in 8.0 ml 10 mm Tris-HCI (pH 7.5), 1 mm EDTA (TE) and 0.5% sarkosyl, and theRNA was precipitated with 2 M LiCl. The RNA was resuspendedin TE and 0.1% SDS and precipitated in ethanol. Isolation ofpoly(A)RNA then proceeded as described for etiolated tissue(17).

Northern and Dot Blot Analyses. For Northern analysis,poly(A)RNA was electrophoresed in formaldehyde-containing2% agarose gels as described in Maniatis et al. (14). After electro-phoresis, RNA was blotted onto nitrocellulose as described byThomas (18). For dot blot analyses, 2-fold serial dilutions ofpoly(A)RNA were prepared in a small volume of sterile, DEP-treated water. Samples were denatured in two volumes of 50%(v/v) formamide, 6% (v/v) formaldehyde, 20 mM MOPS (pH7.0). Samples were then diluted with nine volumes of dilutionbuffer (11 x SSC, 6% [v/v] formaldehyde, 20 mM MOPS [pH

kD

92-

68-

12-_a go

7.0]) and applied to nitrocellulose previously equilibrated in 10x SSC. After applying all the samples, the nitrocellulose wasdried briefly and then baked for 2 h in an 80°C vacuum oven.

Probes for these Northern and dot blots were prepared byrandom primer labeling of cDNA inserts (5) or nick-translationof whole plasmids containing the cDNA inserts (15) using [a-32P]dATP (>3000 Ci/mmol, NEN). All blots were prehybridizedfor 4 h in hybridization buffer (50% formamide, 5 x SSC, 50mm phosphate [pH 7.0], 5X Denhardt's solution, 100 Ag/mlsalmon sperm DNA, 100 ,g/ml yeast RNA, 0.1% SDS). 32p_labeled probe was added to fresh hybridization buffer, and theblots were hybridized with probe for 24 h at 42°C.

Probes. The probes used in this study are cDNAs to HSmRNAs that were isolated as described by Schoffi and Key (17).pCE53 and pCE75, which hybridize to two families of mRNAsthat code for 15 to 18 kD proteins (7), were isolated by Dr. EwaCzarnecka and pFS2033, which hybridizes to a family ofmRNAscoding for 21 to 24 kD proteins, was isolated by Dr. Fritz Schoffi(17). The probe for the small subunit of soybean ribulose bisPcarboxylase, pSRS0.8, was obtained from Dr. Rich Meagher,Department of Genetics, University of Georgia, Athens, GA30602 (3).

RESULTSResponse of Etiolated Seedlings to a Stepwise Increase in

Temperature. Etiolated soybean seedlings were incubated in

______m hsps

1

std 28 31 34 37 40 42.5 45 47.5

*C

9 10

FIG. 1. In vivo labeling of soybean seedlings during a stepwise increase in temperature. Soybean hypocotyls were incubated for 1 h at 28C.Subsamples of tissue were extracted for protein, while the remaining tissue was incubated for another hour at 31 'C. Similarly, tissue was sampled atthe end of each hour as the temperature was increased stepwise to 34, 37, 40, 42.5, 45, then 47.5°C. [3H]leucine was added to each subsample duringthe last hour of incubation (see details in "Materials and Methods"). Each lane represents the spectrum of proteins that became labeled at theindicated temperature of incubation, following the stepwise increases to that temperature. Equal volumes of each extract were electrophoresed inSDS-12.5% polyacrylamide gels. The lanes labeled 28 to 42.5°C contained 30-35,000 cpm, the 45°C lane contained 20,000 cpm, and the 47.5°Cland contained 6,500 cpm. Lane 9, proteins labeled during the 8th h of incubation of 28°C, indicating that an 8-h incubation alone does not causechanges in protein synthesis; lane 10, representative labeling of proteins from hypocotyls heat-shocked for 2 h at 40°C; std, mol wt standards, withvalues indicated on left. The HS proteins (hsps) are identified by arrows on the right.

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Plant Physiol. Vol. 79, 1985

_m.A*

FIG. 2. Northern analysis of HS mRNAs duringa stepwise increase in temperature. Seedlings wereincubated under the temperature regime describedin the legend to Figure 1. At the end of each incre-mental temperature step (indicated at the bottom ofeach lane), poly(A)RNA was extracted, and 1 tgwas electrophoresed and analyzed by Northern blot-ting. For comparison, 0.5 ug poly(A)RNA fromseedlings heat-shocked for 2 h at 40C was electro-phoresed on the right (hs). The 32P-labeled cDNAprobe used for each blot is shown on the left. p53Hybridizes to a family of mRNAs that code for 15to 18 kD HS proteins; p75 hybridizes to anotherfamily of mRNAs coding for 15 to 18 kD HSproteins; and p2033 hybridizes to a family ofmRNAs coding for 21 to 24 kD HS proteins.

p75 -

T' 28 31 34 37 40 42.5 45 47.5 hs

A1i

shaking water baths for a series of step-increases in temperature(2.5 or 3°C/h). To examine changes in the pattern of newlysynthesized proteins, 200 ,Ci [3H]leucine was added to the flask15 min after the flasks had been moved to their final incubationtemperature (to allow time for the seedlings to equilibrate to thenew temperature). After 45 min ofincubation, the seedlings werecollected, and protein was extracted by homogenizing seedlingsin a Tris-HCl (pH 8.8) buffer containing 2% SDS, 2% mercap-toethanol, and I mM phenylmethylsulfonyl fluoride. The ho-mogenate was centrifuged for 30 min at 12, 1OOg and filteredthrough a layer of Miracloth. Figure 1 is a one-dimensionalelectropherogram in which equal volumes of tissue extract havebeen loaded into each lane. HS proteins are strongly inducedduring a stepwise increase in temperature, and the temperatureat which HS protein synthesis is first detectable is very similar tothe temperature at which a HS first induces these proteins (10).The minimum final temperature at which the response is firstfully expressed (40C) is also quite similar to the HS temperaturethat induces the maximal response (40°C for 2 h, see lane 10). Ifequal levels of radioactivity are loaded in each lane (data notshown; also see Altschuler and Mascarenhas [J]), the presence ofthe HS proteins at temperatures above 37°C is also quite prom-inent.During HS, a new set of highly abundant mRNAs are synthe-

sized that code for the low mol wt HS proteins. Using cDNA

clones of these mRNAs, it has been shown previously that thesemessages are not detectable in control tissue and that theyaccumulate to high levels during a 2-h HS at40C (17). Followingthe same regime of stepwise increases in temperature,poly(A)RNA was isolated from the treated tissues and monitoredusing Northern analysis (Fig. 2). The steady state level of HSmRNAs increases in parallel with the appearance of the HSproteins. The overall levels ofmRNAs induced by this treatmentare comparable to the level induced by a 2-h HS at 40C.HS Response of Soybean Plants in the Growth Chamber. One-

month-old soybean plants were maintained on a 14-h photope-riod (27°C/16°C day/night) in growth chambers. Plants werethen moved to a lighted chamber equilibrated at the desired HStemperature for 2 h to determine whether HS mRNAs are alsoinduced in the leaves of mature soybean plants. As shown inFigure 3, the temperature of maximal accumulation of HSmRNAs is higher than that found for etiolated seedlings. TheHS mRNAs are barely detectable at 40C (data not shown),accumulate to high levels at 45°C and, for some sets of HSmRNAs (i.e. those that hybridized to p53 and p75), are alsopresent at high levels at 50°C. These plants were well-wateredimmediately prior to the HS treatment, and no wilting wasobserved during the experiment.

Presence ofHSmRNAs in Field-Grown Soybean Plants duringa Period of High Temperatures. Due to the lack of adequate

p53 .*

p2033

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HEAT SHOCK mRNAs IN SOYBEANS

p53

p2033

p75

w-

i. I

28 45 50 IC

FIG. 3. Northern analysis ofHS mRNAs from mature soybeans heat-shocked in a growth chamber. Well-watered, mature soybean plants wereheat-shocked for 2 h at the indicated temperature in a lighted growthchamber. Poly(A)RNA was extracted, and 1 ug was electrophoresed andanalyzed by Northern blotting.

numbers of growth chambers, it was not possible to performexperiments involving a stepwise increase in temperature onmature plants. The data presented above, however, stronglysuggested that plants in the field might, synthesize these HSmRNAs and proteins during periods at high temperatures. InAugust 1983, Georgia had several days when midafternoontemperatures reached 38°C (100F) or above. Adjacent irrigatedand nonirrigated fields were sampled on the third consecutiveday of high temperature weather, and it had not rained for atleast 4 d prior to sampling. Leaf samples from several plantswere collected from each of the two fields at four times through-out a 24-h period. The air temperatures at the top of the canopywere recorded as follows: August 22, 10 AM, 320C; August 22, 2PM, 400C; August 22, 5 PM, 39 C; and August 23, 7 AM, 22C.Poly(A)RNA was extracted from harvested leaves and analyzedby Northern blots. The blots were hybridized with the cDNAprobes indicated in Figure 4. These three cDNAs, representativeof the majority of the low mol wt HS proteins, hybridized topoly(A)RNA from leaves of both the irrigated and the nonirri-gated fields, but clearly, the level of induction of these mRNAswas much higher for the nonirrigated leaves. These mRNAs wereidentical in size to the HS mRNAs induced by a 40'C treatmentofetiolated seedlings (shown on the left-hand portion ofthe blot).As a percentage of the poly(A)RNA, these HS mRNAs do not

appear to be as abundant in green leaf tissue as they are inetiolated hypocotyls.These differences in HS mRNA abundance in the field were

quantified using dot blot hybridization (Fig. 5). Over the 24-hperiod sampled, the accumulation of HS mRNAs was transient.The highest steady state levels for all of the HS mRNAs wereobtained by 2 PM. By 5 PM, the HS mRNA levels had declined,and, by 7 AM the next morning, the level of these mRNAs wasquite low. This overnight decline in HS mRNA levels parallelsresults obtained with etiolated seedlings. When heat-shockedseedlings are returned to 28C, HS mRNAs decay rapidly, witha half-life of about 2 h (9). Another poly(A)RNA, the SSUmRNA of ribulose- 1,5-bisP carboxylase (RuBPCase), was quan.-tified as an example of the response of a non-HS mRNA underthese field conditions (Fig. 5). In general, the level ofSSU mRNAin nonirrigated soybeans was much lower than the level inirrigated soybeans. The level increased during the evening hours,but it did not approach the level found in irrigated plants. Thedramatic decline in SSU mRNA in nonirrigated plants is prob-ably a response to both heat and water stress. However, thisresponse of SSU mRNA is probably not representative of allnon-HS mRNAs, since neither the total RNA (0.34-0.45 mg/gfresh weight) nor the poly(A)RNA (3.3-4.8 gg/g fresh weight)content ofthese leaves varied substantially between irrigated andnonirrigated plants.

Interaction of Heat and Water Stress in Plants Grown in aGrowth Chamber. One-month-old soybean plants in soil flatswere grown for 2 weeks under different watering regimes todetermine whether water stress alone could induce the transcrip-tion ofthe HS genes. Stressed plants, maintained under a limitedwatering regime at 28C, did not produce detectable levels ofHSmRNAs (Fig. 6). However, when these plants were incubated at40°C for 2 h, HS mRNAs were produced. At 40C, the amountof HS mRNAs accumulated was much higher in stressed plantsthan in well-watered plants. The steady state level was alsoinfluenced by the temperature of the heat treatment. For well-watered plants, the steady state level ofp53 and p75 HS mRNAswas highest at 50C. At this temperature, these plants showed novisible wilting, and they continued to grow normally when theywere returned to 28C. Water-stressed plants showed maximalaccumulation of HS mRNAs at 45C, and the absolute levelsobtained by these plants exceeded those obtained by well-wateredplants. While the stressed plants still accumulated detectableamounts of HS mRNAs at 50°C (data not shown), these plantssuffered severe wilting and did not recover when returned to28°C.

DISCUSSIONThe HS response has been studied in many organisms, and in

all cases, a new set of proteins is induced by an abrupt rise in thegrowing temperature of 8 to 10°C (for review, see Schlesinger etal. [16]). Few studies have addressed the issue of whether thisinduction ofHS proteins occurs in response to the magnitude ofthe shift in temperature, or whether it also occurs during gradualincreases in temperature (1-3). This is a particularly importantquestion for plants, which are obligately and daily exposed tofluctuations in temperatures that can be as much as 20 to 25Con sunny days. If HS proteins do have a function in protectingthe plant from heat injury, then one would expect to find themin tissues whenever the temperature exceeds normal, nonstress-ing values.

In etiolated seedlings, synthesis ofHS proteins was quite strongwhen, instead of a heat-shock, the temperature was increasedgradually by 2 to 3°C/h. This pattern of HS protein synthesiswas paralleled by the induction of HS mRNAs at temperaturesabove 34C. In a strictly analogous manner, mature plants, whichexhibited a typical HS response in the growth chamber, also

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KIMPEL AND KEY

irrigated

p53 -

non - irrigated

M

to it>p2033

p75

__

FIG. 4. Northern analysis ofHS mRNAs in field-grown soybeans. Poly(A)RNA was extracted fromfield samples collected at 10 AM, 2 PM, and 5 PM onAugust 22 and at 7 AM on August 23 from adjacentirrigated and nonirrigated fields. One zgpoly(A)RNA was analyzed by Northern blotting.For comparison, 0.5 1sg poly(A)RNA from etiolatedseedlings heat-shocked for 2 h was electrophoresedon the right (hs).

S10 2 5am pm pm am

10 2 5 7am pm pm am

accumulated HS mRNAs during a gradual increase in tempera-ture in the field (<2°C/h). Preliminary protein analyses indicatethat the HS proteins were also synthesized and accumulated inthese leaves (M. A. Mansfield, unpublished observations). Invitro translations of these poly(A)RNAs gave one-dimensionalprofiles on SDS-PAGE demonstrating the presence of the lowmol wt and high mol wt HS proteins (data not shown).The initial induction and the steady state levels ofHS mRNAs

are tightly correlated with specific temperature values. In etio-lated seedlings, HS mRNAs are first induced whenever thetemperature reaches 35C, either by a gradual, stepwise increaseor by a direct heat shock. Results from experiments on matureplants suggest that the induction of HS mRNAs is actuallydetermined by the temperature of the plant tissue rather thanthe ambient air temperature. This is demonstrated by the differ-ential response of nonirrigated and irrigated plants. It has longbeen recognized that plants can, through evapotranspiration,maintain leaf temperatures significantly lower than ambient airtemperatures. For soybeans, Jung and Scott (6) have shown thatleaf temperatures in nonirrigated soybean fields during warmsummer months stay quite close to the ambient air temperature.In contrast, leaftemperatures of irrigated soybeans are well belowambient. They state that the maximum differences between leaftemperatures of irrigated and nonirrigated soybeans were ap-proximately "5.5°C, and were usually found at midday and

midafternoon late in the season." Thus, we conclude that thequantitative differences in the steady state levels of HS mRNAsmeasured in irrigated and nonirrigated leaves is a direct reflectionofthe actual leaf temperatures experienced by these plants in thefield.

It is possible to argue that these differences in HS mRNAsobserved in the field samples are a response to water stress ratherthan heat stress. Since it was not possible to separate these stressesin the field, a water-stressed condition was simulated in thegrowth chamber. Water-stressed plants maintained at 28°C didnot show any induction ofthe HS mRNAs, indicating that thesemRNAs are not induced by water stress alone. In parallel withthe results obtained for the field plants, water-stressed plantsheat-shocked in the growth chamber for 2 h at 40 or 45°Cproduced higher levels of HS mRNAs than well-watered plants,undoubtedly reflecting the higher leaf temperatures in water-stressed plants. However, water-stressed plants did not survive50°C in the chamber, while well-watered plants survived andshowed a strong induction ofHS mRNAs.When seedlings are shifted to temperatures above 40°C, there

is very little incorporation of labeled amino acids into protein,although the proteins that are synthesized are largely HS proteins(9). In contrast, a stepwiseincrease in temperature allows aminoacid incorporation into protein to continue at fairly normal levelsthrough 45°C. This is analogous to the work of Lin et al. (12)

hs

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HEAT SHOCK mRNAs IN SOYBEANS

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E

0

1.0

50s ssu

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30k

20

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lOam 2pm 5pm 7am

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FIG. 5. Dot blot analyses of mRNAs from field-grown soybeans.Serial dilutions of poly(A)RNA (280-17.5 ng) were dot-blotted ontonitrocellulose. After hybridization to the indicated 32P-labeled probe (53,2033 or SSU, the small subunit of ribulose bisP carboxylase), each of thedots on the nitrocellulose filters were cut out and cpm hybridized weredetermined by liquid scintillation counting. The values obtained for eachsample were analyzed statistically to give a value for the slope of the bestfit line (cpm hybridized versus ng RNA; r > 0.99). These slope valueswere then plotted against the time of sampling, giving a graphic view ofthe changes in steady state levels of these mRNAs during the samplingperiod.

which demonstrated that substantial amino acid incorporationinto protein was observed at 45 and 47.50C when seedlings weregiven a prior HS at 40TC. Altschuler and Mascarenhas (1) havealso found that seedlings can be protected from death at poten-tially lethal temperatures by a prior mild HS or a gradual increasein temperature. Thus, there is a strong correlation between theproduction of HS proteins at nonlethal temperatures and theability of seedlings to maintain HS protein synthesis at otherwiselethal temperatures.A working hypothesis is that these two observations are

causally related; that is, the synthesis and accumulation of HSproteins at temperatures up to 40'C (in the case of etiolatedseedlings) allow the plant to maintain nearly normal metabolismat supraoptimal temperatures. Of course, such a hypothesis re-quires that these proteins are relatively stable; other work in thislaboratory has shown that, for etiolated seedlings, at least 80%of the HS proteins made during a 2-h HS are still present after a

IL

p53

p2033

p75

28 28S 40 45 45S 50

FIG. 6. Effect of water stress on HS mRNA levels in a growth cham-ber. Plants were water-stressed using an empirically determined wateringregime for a period of 2 weeks. At the end of this time, the growth of thestressed plants was significantly reduced, although these plants werenever allowed to wilt. Lane headings: 28, plants maintained in the growthchamber at 28C; 28S, water-stressed plants at 28C; 40, well-wateredplants heat shocked for 2 h at 40C; 45, well-watered plants heat shockedfor 2 h at 40C; 45, well-watered plants heat shocked for 2 h at 45°C; 50,well-watered plants heat shocked for 2 h at 50'C. Poly(A)RNA wasextracted from leaves at the end of each treatment, and I Ag waselectrophoresed and blotted for Northern analysis.

subsequent 21-h incubation at 28°C (8).To extrapolate this hypothesis to the mature plants in the field,

data are needed on the level of HS proteins present in theseleaves during high temperature stress. Currently, monoclonalantibodies are being prepared to the low mol wt HS proteins sothat such information can be obtained. If HS proteins have arole in protecting these plants in the field, one must explain thedecline in message level at 5 PM when air temperatures are stilloptimal for the HS response. In the case of the nonirrigatedplants, this decline in mRNA levels may reflect limitationsimposed on the entire transcriptional machinery by the waterstress itself, which would be maximal at mid to late afternoon.However, induction of HS mRNAs is also transient in theirrigated plants. It is possible that the decline in mRNA levelsreflects regulation of protein synthesis by the HS proteins them-selves. That is, as HS proteins accumulate to levels sufficient tofulfill their function, they also turn off their own gene transcrip-tion. Such a self-regulating role has been demonstrated for the70 kD HS protein of Drosophila (4). If this is the case for higher

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678 KIMPEL AND KEY

plants, it might seem surprising that, on the third consecutiveday of high heat stress, HS mRNAs are still induced, which thenimplies that sufficient levels of HS proteins are not alreadypresent. There is currently no data on the stability ofHS proteinsin mature, green tissue. It is possible that HS proteins are turnedover more rapidly in these plants, thereby limiting the deleteriouseffects that these proteins might have on normal gene transcrip-tion and message translation. The availability of antibodies toHS proteins will make it possible to begin to answer theseimportant questions on the regulation of the HS response.The response of plants in the field to several, simultaneously

imposed, environmental stresses must be complex. The induc-tion of HS mRNAs and HS proteins is not the only response.The decline in the steady state level of SSU mRNA indicatesthat other responses are occurring, but it is not yet clear to whichenvironmental trigger(s) the SSU mRNA is responding. In thecase ofthe HS mRNAs, the results presented here clearly indicatethat these mRNAs are produced in soybeans in response to tissuetemperature, and the absolute level of the response in matureplants is determined by the ability of leaves to regulate their owntemperature relative to ambient air temperature. The observa-tions that new gene transcription (HS mRNAs) and new proteinsynthesis (HS proteins) occur under stressful conditions arguestrongly for a protective role of the HS proteins in plants. Otherongoing work in this laboratory, including gene sequence analysisand production ofmonoclonal antibodies to HS proteins, shouldaid in elucidating the function of these proteins in the plant.

Acknowledgments-We thank Dr. D. A. Ashley, Department of Agronomy, forpermission to sample his soybean fields, and Dr. Glenn Galau, Department ofBotany, for the dot blot procedure. We are especially grateful to Dr. ElizabethVierling for her stimulating discussions and critical review of the manuscript.

LITERATURE CITED

1. ALTSCHULER M, JP MASCARENHAS 1982 Heat shock proteins and effects ofheat shock on plants. Plant Mol Biol 1: 103-115

2. BASZCZYNSKI CL, DB WALDEN, BG ATKINSON 1983 Temperature shifts withinthe normal growing range of maize lead to novel or enhanced synthesis ofheat shock proteins and their mRNAs. American Society for Cell. Biol.

Plant Physiol. Vol. 79, 1985

Meeting. J Cell Biol 97: Abstr 579, 153a3. BERRY-LoWE SL, TD MCKNIGHT, DM SHAH, RB MEAGHER 1982 The nucleo-

tide sequence expression and evolution of one member of the multigenefamily encoding the small subunit of ribulose-1,5-bisphosphate carboxylasein soybean. J Mol Appl Genet 1: 483-498

4. DiDoMENico BJ, GE BUGAISKY, S. LINDQuIsT 1982 The heat shock responseis self-regulated at both the transcriptional and post-transcriptional levels.Cell 31: 593-603

5. FEINBERG AP, B VOGELSTEIN 1983 A technique for labelling DNA restrictionendonuclease fragments to high specific activity. Anal Biochem 132: 6-13

6. JUNG PK, HD Scorn 1980 Leaf water potential, stomatal resistance, andtemperature relations in field-grown soybeans. Agron J 72: 986-990

7. KEY JL, WB GURLEY, RT NAGAO, E CZARNECKA, MA MANSFIELD 1985Multigene families of soybean heat shock proteins. In NATO AdvancedStudies Institute, 'Molecular Form and Function of the Plant Genome,"Renesse, The Netherlands, Plenum Press, New York. In press

8. KEY JL, JA KIMPEL, E VIERLING, CY LIN, RT NAGAO, E CZARNECKA, FSCHOFFL 1985 Physiological and molecular analyses of the heat shockresponse in plants. In BG Atkinson, DB Walden, eds, Changes in EukaryoticGene Expression in Response to Environmental Stress. Academic Press,New York, pp 327-348

9. KEy JL, CY LIN, E CEGLARZ, F SCHOFFL 1982 The heat shock response inplants: physiological considerations. In MJ Schlesinger, M Ashburner, ATissieres, eds, Heat Shock from Bacteria to Man. Cold Spring HarborLaboratory, Cold Spring Harbor, New York, pp 329-336

10. KEY JL, CY LIN, E CEGLARZ, F SCHOFFL 1983 The heat shock response insoybean seedlings. In L Dure, ed, NATO Advanced Studies Workshop onGenome Organization and Expression in Plants. Plenum Press, New York,pp 25-36

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