Plant Physiol. (1978) 61, 806-811

Metabolic and Ultrastructural Changes in Winter Wheat duringIce Encasement Under Field Conditions'

Received for publication October 7, 1977 and in revised form January 17, 1978

M. KEITH POMEROY AND CHRISTOPHER J. ANDREWSResearch Branch, Agriculture Canada, Ottawa, Ontario KIA OC6 Canada

ABSTRACT

The effect of ice encasement on the physiological, metabol, andultrastnctural properties of winter wheat (Tritclm sestdvM L.) grownunder fiel conditios was examned by a lly encasing winter wheatin ice during early winter. Cold hadiess and survival of ice-encasedseedlngs declied less rapily In Kharkov, a cold-hardy cultivar than inFredrick, a less hardy cultivar. Ethanol did not accumulate in non-icedseedlngs, but increased rapidly upon applcatio of an ice sheet. Lacticacid accumulated in both cultivars during late autumn, prior to ice encase-ment, and elevated levels of lactic acid were maintained throughout thewinter In seedlngs from both iced and on-iced plots. Te rate of 0sconsumption of shoot tissue of seedlngs from non-iced plots remainedrelatively constant throughout the winter, but declined rapidy in seedlingsfrom ice encased plots. Major ultrastructural chages did not occur inshoot apex ceUs of non-iced winter wheat gs duri cold hardeningunder field conditions. However, the imposition of an ice cover In earlyJanuary resulted in a proiferation of the e l reticulm membranesystem of the cels, frequently resulting In the formation of concentricwhoris of membranes, often enclosing cytoplasmic organeles. Electron-dense areas within the cytoplasm which appeared to be associated with theexpanded endoplasmic reticulum were also frequently observed.

The cause ofoverwintering damage to winter cereals and foragecrops under field conditions is not completely understood. Ingeographical areas such as the Western Prairies where wintertemperatures are frequently extremely low and snow cover is oftensparse, soil temperatures are undoubtedly low enough to kill manycereal cultivars. In other areas with heavier snow cover, unseasonalthawing of snow followed by refreezing of fully hydrated plantsmay cause severe damage (16). In more northerly areas, thecontinuous insulating effect of snow usually prevents soil temper-ature from dropping much below the freezing point, and yet severewinterkill is frequently encountered. A number of studies (4, 6,24) have attributed damage under the latter conditions to theformation of ice sheets over the plants as a result of winter thawsor rain. Other investigations (3, 9, 21) have established the dele-terious physical effects of ice encasement on plants under labora-tory conditions. However, the specific cellular alterations associ-ated with injury under these conditions are not fully understood,although several studies (9, 22) have demonstrated the accumu-lation of CO2 in plants during ice encasement. Laboratory inves-tigations have shown that ethanol accumulates in winter cerealseedlings during encasement in ice (2, 5). Mitochondrial respira-tion declines slowly during icing, but a relatively high rate ofrespiration is still observed even after 50% of the seedlings havebeen killed (5).

'Contribution No. 998 of Chemistry and Biology Research Institute,Agriculture Canada.

Electron microscopic studies (20) have shown that the declinein viability of winter wheat seedlings encased in ice at -1 C isaccompanied by characteristic ultrastructural changes. ER in-creases markedly, often resulting in the formation of an elaborateseries of parallel membranes, either dispersed randomly through-out the cytoplasm or in the form of concentric whorls. However,the structural integrity of many cellular organelles is largelyunaffected even by prolonged ice encasement which results indeath of all plants. In contrast, exposure of wheat seedlings tonear lethal, subfreezing air temperatures results in severe ultra-structural disorganization of all cellular organelles.The present study was undertaken to examine metabolic and

ultrastructural changes associated with ice encasement injury un-der field conditions.

MATERIALS AND METHODS

Seed of winter wheat (Triticum aestivum L. cv. Kharkov andFredrick) was sown in plots on September 23, 1975, and September15, 1976 in a light sandy soil on the Central Experimental Farm,Ottawa, Ontario, Canada. A control plot was unaltered while soilwas banked up around plots for flooding and ice encasementtreatments. In all plots, thermocouples were inserted to 1-cmdepth. Two treatment plots were saturated with water using alawn sprinkler on November 23 and 29, 1976. On January 14,1976 and January 4, 1977, snow was removed from the plots forice encasement and water was added using a lawn sprinkler untila solid sheet ofice covered the entire plot. Snow was then shoveledback on to the plots to protect the plants from extremely lowtemperature. The second plot saturated with water in November,1976 was not encased in ice in January, 1977.

Plants were removed periodically (6) from the plots throughoutautumn and winter on days when the temperature was not colderthan -5 C. The seedlings in frozen blocks of soil were transferredto a cold room at 4 C, and after thawing overnight, survival andcold hardiness were determined (6). The thawed seedlings werewashed free of soil, placed in plastic bags and sealed, and put intoa freezer at 0 C. The temperature was reduced I degree C per hrand seedlings were removed at various temperatures and allowedto thaw overnight at 2 C. Seedlings were then transplanted toVermiculite at 20 C day (35,000 lux)/15 C night for 2 weeks andLD5o values were interpolated from survival and regrowth results.Plant and soil moisture were determined immediately by removingsamples from frozen blocks, and drying seedlings and soil samplesto constant weight at 80 C.

Respiratory activity of shoot tissue from untreated and ice-encased plants was determined on the basal 1-cm of seedlingshoots. The I-cm portions were cut into 2-mm segments, weighed,infiltrated with reaction media (19) under reduced pressure for 5min, and 02 consumption determined on 0. I-g lots using a Clark02 electrode (19). Ethanol content of seedlings removed directlyfrom frozen blocks was determined by the enzymic procedure ofLundquist (14), as previously described (2).

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ICE ENCASEMENT OF OVERWINTERING WHEAT

The accumulation of lactate in seedlings was determined by theenzymic procedure of Hohorst, using lactic dehydrogenase andNAD. Groups of five crowns, each with shoots and roots trimmedto 5 to 7 cm, were ground in a chilled mortar with 5.1% perchloricacid to a final volume of 6 ml. This was centrifuged at 5,000g for10 min, and l-ml aliquots of the supernatant were neutralized,sealed, and stored in the freezer up to I week for analysis (10). Acontrol, including all components of the reaction mixture exceptthe enzyme lactic dehydrogenase, was assayed with each sample.

For electron microscopy, shoot apices were dissected directlyinto fresh fixatives, either immediately after sampling or as soonas the samples had thawed. Fixation was carried out at 4 C eitherin aqueous 2% KMnO4 for 4 hr, or in 4% glutaraldehyde in 0.05M phosphate buffer (pH 7.4) for 4 hr followed by postfixation for16 hr in 2% osmic acid in the same buffer. The fixed material waswashed, dehydrated in acetone, embedded in Epon (13), sectioned,stained with uranyl acetate and lead citrate, and examined in aPhilips 300 electron microscope.

RESULTS

The results obtained in the first year (1975-1976) of this studyare presented in Figure 1 and Table I. Survival and cold hardiness(as measured by LD50 temperature) of both cultivars decreasedslightly during late winter in the non-iced plot, whereas markeddecreases in these parameters were observed in seedlings from theiced plots. By late February, survival of ice-encased Kharkov wasreduced to 60%o and hardiness had declined from -22 C to -12.6C, while survival of the less cold-hardy Fredrick was reduced to20o and hardiness had decreased from -16 C to -7.1 C. Thelevel of ethanol in seedling tissues increased substantially duringencasement in ice, while shoot respiration decreased in ice-encasedseedlings. These preliminary findings suggested that majorchanges occur in the physiology and biochemistry ofwinter wheatseedlings during encasement in ice under field conditions. It wasdecided, therefore, to conduct a second and more comprehensiveexamination of this phenomenon during the winter of 1976 to1977.

Soil moisture content declined steadily throughout the wintermonths of 1976 to 1977 in the untreated plot, from a high of about30Yo in November to a low of 13% in mid-February (Table II). It

-20

Lu

90-i

-16~

-12

-8

-4

0

NOV. DEC. JAN. FEB. MARCH APRIL

1975 1976FIG. 1. Change in cold hardiness (LD50, C) of Kharkov and Fredrick

winter wheat under natural field conditions and after artificial ice encase-ment during 1975 to 1976.

Table I. Survival, LD50 temperature, ethanol accumulation, and oxygenconsumption of non-iced and ice-encased winter wheat during 1975-76.

The ice encasement plot was shovelled free of snow, iced, and recoveredwith snow on January 14, 1976. Each value represents an average of at least3 determinations.

Sampling LD50 Ethanol OxygenDate Cultivar Survival Temperature Content Consumption

% C mg/g smoles/min.fresh wt mg dry wt

Jan 13/76 Kharkov (N)a 100 -22.0 0.17 1.6Fredrick (N) 100 -16.0 0.34 1.7

Feb 24/76 Kharkov (N) 100 -18.5 -b 2.1(I) 60 -12.6 1.17 1.4

Fredrick (N) 90 -12.5 -b 2.5(I) 20 - 7.1 1.01 1.6

a N denotes non-iced; I denotes iced. b Data not obtained.

then increased in March with initiation of the spring thaw andmelting of snow. In the ice encasement plot, which was flooded inNovember and iced in January, soil moisture remained relativelyconstant at about 35% throughout the winter (Table II). The largedifference in soil moisture between the treated and untreated plotswas probably due to the unusual absence of a significant amountof rain or thawing during this particular winter.The per cent plant moisture of both cultivars decreased abruptly

during autumn as a result of nearly a doubling of the dry matterof the seedlings (Table II). These reduced levels of moisture weremaintained throughout the winter, and did not decrease furtherwith decreasing soil moisture in the untreated plot. In the icedplot where soil moisture was greater, seedling moisture also wasconsistently slightly higher than in the control plot.

Overwintering injury ofwinter wheats was unusually low duringthe winter of 1976 to 1977. In the non-iced plot (Table II), and ina plot flooded in November but not iced in January (data notshown), greater than 90%o survival of both Kharkov and Fredrickwas observed. The application of an ice sheet, however, inducedsevere injury to plants of both cultivars. Injury occurred morerapidly in the less hardy Fredrick than in Kharkov, but by theend of March, survival of both cultivars was reduced to less than10%1o. Cold hardiness of winter wheats also was reduced markedlyby exposure to ice encasement treatment (Fig. 2). The hardinessof Fredrick in the plots flooded in November declined significantlyeven prior to application of the ice sheet, whereas the hardiness ofKharkov was not reduced significantly by this treatment. Coldhardiness decreased rapidly after icing and by mid-winter forFredrick and late winter for Kharkov, hardiness of survivingseedlings of both cultivars was reduced practically to zero. Thehardiness of seedlings in the control plot increased rapidly duringautumn, was maintained at a high level throughout early winter,and then declined during late winter and early spring (Fig. 2).

Ethanol did not accumulate in seedlings of either Kharkov orFredrick in the non-iced treatments during autumn and winter of1976 to 1977 (Table II). However, ethanol content of seedlingsincreased rapidly upon application of an ice sheet, and attained amaximum level in mid-February. It then decreased during March,with initiation of the spring thaw. Lactic acid also accumulated inoverwintering wheat seedlings, but considerable variation in itslevels was observed throughout the winter (Table III). Duringearly autumn, lactic acid was not detected in the seedlings of eithercultivar, but an appreciable increase in concentration was observedeven before the plots were flooded in November. This elevatedlevel was maintained throughout January with slightly highervalues in the iced than non-iced plots, but by mid-February, lacticacid had decreased in all treatments except ice-encased Fredrickwhich was severely damaged at this sampling time. Throughoutthe remainder of the winter and early spring, the level of lacticacid in all treatments remained lower than the maximum attainedwinter values.The effects of overwintering on the rate of 02 consumption of

\ ~~~~~~~NaturalKharkov \ Iced

Fredrick Natural0/~~~~~~~'Qced

Ice \\EIce d \\

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POMEROY AND ANDREWS

Table II. Effect of ice encasement on soil moisture and on moisture content and survival of winter wheat during 1976-77.

The ice encasement plot was saturated with water on November 29, 1976 and iced on January 5. 1977. Each valuerepresents an average ± the standard error of at least 3 determinations.

Sampling Date

Parameter Cultivar Oct 5/76 Nov 16/76 Jan 5/77 Jan 24/77 Feb 14/77 Mar 8/77 Mar 30/77

Soil Moisture (%)Untreated - 28.5 + 1.8 29.9 ± 2.3 20.3 ± 2.9 19.2 ± 2.4 13.0 ± 1.7 22.8 ± 0.4 cFlooded/Iced - - 36.8 ± 1.1 35.4 ± 0.9 35.6 ± 3.5 36.9 ± 0.8 33.9 ± 1.4 -

Plant Moisture Kharkov (N)a 87.4 + 0.5 78.9 ± 0.6 75.3 ± 0.3 77.4 ± 0.1 77.6 ± 0.2 80.2 ± 0.4 84.2 ± 0.2(% fresh wt) (I) - - 77.7 ± 0,3b 80.6 ± 0.3 82.9 ± 0.1 81.2 ± 0.5 85.3 ± 0.4

Fredrick (N) 89.7 + 0.1 82.1 ± 1.0 76.0 ± 0.6 80.0 ± 0.5 80.9 ± 0.2 81.9 ± 0.4 85.5 ± 0.2(I) - - 80.7 ± 0.3b 83.9 ± 0 85.9 ± 0 85.3 ± 0.4 87.2 ± 0.5

Survival Kharkov (N) 100 100 100 100 97.5 ± 1.2 98.3 ± 1.6 93.3 ± 3.3(W) (I) - - loob 97.5 ± 2.4 70.0 ± 4.0 76.0 ± 11.7 8.5 ± 3.1

Fredrick tN) 100 100 100 100 92.5 ± 2.6 87.5 ± 6.2 91.6 ± 8.3(I) - - loob 70.0 ± 12.5 2.5 ± 2.4 0 2.5 ± 1.9

a N denotes non-iced; I denotes iced. bThese values are for seedlings obtained prior to icing of the plot saturatedwith water on November 29, 1976. c flooded.

Table III. Overwintering changes in respiration and the levels of ethanol and lactic acid in non-iced and ice-encased winter wheat during 1976-77.

The ice encasement plot was saturated with water on November 29, 1976 and iced on January 5, 1977. Each valuerepresents an average ± the standard error of at least 3 determinations.

Sampling Date

Parameter Cultivar Oct 5/76 Nov 16/76 Jan 5/77 Jan 24/77 Feb 14/77 Mar 8/77 Mar 30/77

Ethanol Kharkov (N)a 0

(mg/g fresh wt) (I

Fredrick N O

Lactic Acid Kharkov (N) 0

(mg/g fresh wt) (I)Fredrick (N) 0

(I) -

0.05 ± 0.01 0 0 0.01 ± 0.01 0.02 ± 0 0.16 ± 0.08- ob 0.73 ± 0.22 4.58 ± 0.26 1.14 ± 0.31 0.11 ± 0.05

0 0.04 ±0 0 0 0.02 ± 0.01 0- 0.13 ± 0,04b 1.08 ± 0 2.67 ± 0.16 1.21 ± 0.11 0.07 ± 0

1.48 ± 0.44 1.92 ± 0.28 1.36 ± 0.24 0.40 ± 0.20 0.41 ± 0.11 0.79 ± 0.36- 2.08 ± 0.16b 2.20 ± 0.12 0.48 ± 0.12 1.16 ± 0.40 0.53 ± 0.13

1.64 ± 0.28 1.52 ± 0.16 1.72 ± 0.12 0.28 ± 0.18 0.38 ± 0.06 0.52 ± 0.25- 1.64 ± 028b 2.04 ± 0.20 3.16 ± 0.40 0.32 ± 0.10 0.19 ± 0.03

Oxygen Kharkov (N) 30.1 ± 2.0 34.1 ± 0.7 33.1 ± 0.5 31.1 ± 0.09 28.4 ± 1.2 33.7 ± 1.2 35.0 ± 0.2Consumption (I) - - 36.7 ± lOb 26.5 ± 0.8 8.3 ± 0.9 17.9 ± 1.0 26.9 ± 0.4(smoles/min. Fredrick (N) 29.3 ± 1.4 32.7 ± 1.0 31.1 ± 1.0 32.9 ± 1.2 30.7 ± 0.4 32.9 ± 0.7 28.1 ± 0.5mg fresh wt) (I) - 27.7 ± 0.4b 18.5 ± 0.6 7.3 ± 0.6 15.3 ± 0.3 16.8 ± 0.9

a N denotes non-iced; I denotes iced. b These values are forwith water on November 29, 1976.

-20 k

-16

-12

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Ice \Encased \

_

OCT. NOV. DEC. JAN. FEB. MARCH

1976 1977

FIG. 2. Change in cold hardiness (LDao, C) of Kharkov and Fredrickwinter wheat under natural field conditions and after artificial floodingand icc encasement during 1976 to 1977.

seedling shoot tissue from non-iced and ice-encased plots were

similar for Kharkov and Fredrick (Table III). 02 uptake inseedlings from the untreated plot remained relatively constantthroughout the sampling period. In contrast, 02 consumption

seedlings obtained prior to icing of a plot saturated

declined rapidly in seedlings encased in ice, and by mid-Februarywas reduced by approximately 75%. During March, 02 uptakeincreased in shoot tissue from ice encased seedlings until by theend of March it was approximately two-thirds that of non-icedcontrols. This marked increase in 02 consumption occurred despitethe fact that survival of ice-encased seedlings had been drasticallyreduced by this time.No major ultrastructural changes occurred in shoot apex cells

of non-iced winter wheat seedlings during cold hardening underfield conditions. Throughout the sampling period, the cells werecharacterized by large centrally located nuclei, copious mitochon-dria ofvarious sizes and shapes, numerous plastids, and occasionallipid bodies and dictyosomes (Figs. 3-5). ER was relatively abun-dant and usually present in cisternal form. The ER membraneswere more clearly visualized after fixation in KMnO4 (Figs. 4-6)than when fixed in glutaraldehyde-osmic acid (Fig. 3).The imposition ofan artificial ice cover in early January resulted

in characteristic changes in cellular ultrastructure of meristematicapical cells of ice-encased seedlings. The most prominent struc-tural alteration was a proliferation of the ER membrane system,frequently resulting in the formation of concentric whorls ofmembranes, often enclosing cytoplasmic organelles (Fig. 6). Asecond structural change often observed was the appearance ofmembrane-bound electron-dense areas within the cytoplasmwhich appeared to be associated with the expanded ER (Fig. 7).The formation of these structures was not observed in seedlingsfrom the non-iced plot (Fig. 5). In March when the soil had begunto thaw, concentric whorls ofmembranes were no longer observed,and the quantity and size of electron-dense areas also were greatlyreduced (Fig. 8). No evidence of the severe ultrastructural changesrecently shown (20) to be associated with freezing injury in winterwheat was observed in the present investigation.

Flooded

I-'aI-

C

0-I

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ICE ENCASEMENT OF OVERWINTERING WHEAT

FIG. 3 and 4. General structural features of apical cells of cold-hardened Kharkov winter wheat obtained from untreated field plots on November16, 1976. Fig. 3: fixation in glutaraldehyde-osmic acid (x 7,000). Fig. 4: fixation in KMnO4 (X 9,900). (cw): cell wall; (d): dictyosome; (er): endoplasmicreticulum; (1): lipid body; (m): mitochondrion; (n): nucleus; (p): plastid; (v): vacuole.FIG. 5, 6, and 7. Comparison of ultrastructural features of apical cells of Kharkov seedlings obtained on February 15, 1977, from untreated fieldplots (Fig. 5, x 8,000) and from plots flooded on November 29, 1976, and encased in ice on January 5, 1977 (Figs. 6, x 9,900, and 7, x 8,000). Fixationin KMnO4. (cw): cell wall; (ds): dense structure; (er): endoplasmic reticulum; (1): lipid body; (p): plastid; (ve): vesicles.FIG. 8. Ultrastructure of apical cells of Kharkov seedlings obtained from an ice-encased field plot on March 8, 1977. Fixation in KMnO4 (x 9,900).(cw): cell wall; (er): endoplasmic reticulum; (m): mitochondrion; (n): nucleus; (p): plastid.

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810 POMEROY AND ANDREWS Plant Physiol. Vol. 61, 1978

DISCUSSION

The winter of 1976 to 1977 was unusual in the study area inthat the normally expected winter thaw or rain did not occur,leading to nearly 100% survival in the control plots, and in otherplantings of cold-adapted wheat cultivars. In contrast, survival inadjacent plots which were artificially iced in January was reducedto less than 10o. The observations are generally in accord withearlier findings (4), but in the present study, the excellent negativecorrelation observed between survival and duration of ice encase-ment was undoubtedly due to a more effective icing treatmentthan was obtained in the earlier experiments.The level of soil moisture in the experimental plots did not

appear to have a significant effect on survival of winter wheatduring 1976 to 1977. Although soil moisture decreased markedlyin the untreated plot and remained high throughout the winter inthe flooded plots, survival in both the control and flooded plotswas greater than 90% for both Kharkov and Fredrick. In contrast,survival in the iced plot was reduced to very low levels. Theseobservations suggest that overwintering injury under ice and snowcover may not be related directly to soil moisture level, but ratherto biochemical changes associated with exposure of the plants topartial or total anaerobiosis.The accumulation of products of anaerobic respiration during

exposure of plants to ice encasement or flooding has been postu-lated to account for the observed injury of plants subjected tothese stresses. Encasement of winter cereal seedlings in ice undercontrolled environment conditions results in a rapid accumulationof ethanol (2), CO2 (21, 22), and lactate (unpublished data) withinthe tissues. Ethanol and lactate also accumulate during exposureof several species of plants to flooding (7, 23), a condition whichnecessarily precludes ice encasement. In the present investigation,ethanol content of the tissues increased only in seedlings whichwere subjected to anoxia by encasement in ice and its leveldecreased abruptly, concomitant with initiation of the spring thawwhen the plants were no longer completely enclosed in ice. Incontrast, lactate levels increased markedly in seedlings from theuntreated plot by mid-November, prior to exposure to conditionswhich would induce high levels of anaerobic respiration. Theenvironmental stress inducing the increased lactate levels is notknown but probably involves low temperature and elevated soilmoisture levels, normally occurring during late fall. However, theaccumulated lactate of early winter is not toxic to the plants, butthe high level of lactate in ice-encased Fredrick in late winter isassociated with almost total kill of these plants. Ethanol, whichaccumulates only in response to ice encasement, is present atconsistently high levels throughout the ice encasement period andmay be associated with the eventual death of plants of bothcultivars. Other studies on several plant systems have also shownthat lactate is formed more rapidly than ethanol in response toanaerobiosis (8, 12).The absence of significant change in the rate of 02 consumption

of seedling shoot tissue from the untreated plot throughout thesampling period is consistent with previously reported (5) resultsfrom controlled environment experiments. In those studies, nosignificant difference in the rate of respiration was observedamong four cereal cultivars of contrasting cold hardiness grownunder hardening and nonhardening conditions. The quantity ofmitochondrial protein was similar in seedlings grown at 2 and 24C, indicating that the quantity of mitochondria in the cells is notaltered during cold acclimation. These observations suggest thatrespiratory capacity of mitochondria remains constant in field-grown seedlings throughout autumn and winter.The decline in aerobic respiration of shoot tissue from ice-

encased seedlings is in accord with that observed in isolatedmitochondria from winter wheat encased in ice under controlledenvironment conditions (5). In contrast with the results obtainedwith mitochondria where 02 uptake continued to decline with

increasing injury due to ice encasement, shoot respiration ap-peared to increase markedly during late winter while field survivalcontinued to decrease. This apparent anomaly is not fully under-stood, but may be interpreted in one of two ways. Ice encasementinduced increase in 02 consumption by nonrespiratory processesin dead and injured cells may be stimulated upon thawing of theplants in spring. Alternatively, the observed increase in O2 uptakecould be due to an increase in the rate of normal respiratoryprocesses of cells not killed by the icing treatment, since in thisstudy no attempt was made to determine the extent of cellularinjury associated with death of the plants.The ultrastructural changes associated with ice encasement in

the field are similar to those observed under controlled environ-ment conditions (20). Proliferation of ER and the formation ofparallel arrays and concentric whorls of ER membranes occurredto a greater extent under controlled environment conditions, pos-sibly due to more complete ice encasement than can be obtainedunder field conditions. The dense regions associated with theconcentric whorls were more prominent in the field-collectedmaterial than in the controlled environment material, but therelationship of these structures to the membranes is uncertain.Other studies on many species of plants have shown that theformation of parallel arrays and concentric whorls of ER can beinduced by many types of stress, including mechanical effects,reduced 02 tension, CO2 atmosphere (25), dehydration (15), an-aerobiosis (1), chilling (17), and inhibition of respiration (18).Kimball and Salisbury (11) also observed the proliferation ofRER during exposure of three grass species to low temperature.This effect was not observed in the present study or in a previousinvestigation (20) on winter wheat seedlings grown at 2 C undercontrolled conditions. The precise function of the proliferated ERis not known, although it has been suggested that it may be anexpression of the adaptative mechanism protecting the cellsagainst anoxia (18), or involved in repair processes within stress-damaged cells (1).The results of this study have clearly shown that prolonged ice

encasement under field conditions is highly lethal to winter wheat.It was also demonstrated that ice encasement induces majorchanges in cellular metabolism and ultrastructure, including in-hibition of aerobic respiration, accumulation of ethanol, andproliferation of ER. The agent or combination of agents respon-sible for overwintering injury to winter cereals under ice sheetswas not identified, although the greatly elevated levels of ethanolsuggest that it may be involved in the processes leading to iceencasement injury.

Acknowkdgment-The authors wish to acknowledge the exoclent technical assistance of K. P.Stanley and P. C. Bonn.

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