Freezing of Barley Studied by Infrared · Freezing of barley (Hordeum vulgare), Hordeum murinum, and Holcus lanatus was studied using infrared video thermography. In the field, ice

Freezing of Barley Studied by InfraredVideo Thermography1

Roger S. Pearce* and Michael P. Fuller

Department of Biological and Nutritional Sciences, The University of Newcastle upon Tyne, Newcastle uponTyne NE1 7RU, United Kingdom (R.S.P.); and Department of Agriculture and Food Studies, Seale-HayneFaculty, University of Plymouth, Newton Abbot, Devon TQ12 6NQ, United Kingdom (M.P.F.)

Freezing of barley (Hordeum vulgare), Hordeum murinum, and Holcus lanatus was studied using infrared video thermography.In the field, ice could enter H. lanatus leaves through hydathodes. In laboratory tests with barley, initially 0.4% of the leafwater froze, spreading in alternate strips of high and low freezing intensity longitudinally at 1 to 4 cm s21, andsimultaneously spreading laterally at 0.3 cm s21. Similar results were obtained in the field with H. lanatus. A distinct second,more intense, freezing event spread slowly from the margins of the leaves toward the midrib. Organs of uprooted barleytested in the laboratory froze in this order: nucleated leaf, roots, older leaves, younger leaves, and secondary tillers. Whenice spread from one leaf to the rest of the plant the crown delayed spread to the roots and other leaves. There was a longerdelay above than below 22°C, helping to protect the crown from freezing during mild frosts. Initial spread of freezing wasnot damaging. However, the initial spread is a prerequisite for the second freezing event, which can cause damage. The routeof the initial spread of ice may be extracellular, drawing water from more gel-like parts of the cell wall.

The survival and performance of temperate wildand crop species depends on their ability to toleratesome degree of freezing. Understanding freezing tol-erance creates means to improve crop performanceand helps explain the distribution of wild species.Current strategies for achieving this understandingfocus on molecular analysis of cold acclimation(Pearce, 1999; Thomashow, 1999). However, it isequally important to understand the freezing processitself. This can identify similarities or differences be-tween species in how they are affected by freezing,and is essential for eventually differentiating aspectsof freezing that the plant does and does not control.

During freezing, substantial amounts of ice accu-mulate between the cells, growing at the expense ofwater drawn from inside the cells and thus dehydrat-ing them (Pearce, 1988; Pearce and Ashworth, 1992).The cells are killed when their dehydration toleranceis exceeded. It is clear that this will only happen to acell or tissue if freezing reaches that part of the plant.Understanding the spread of freezing within theplant is one factor helping to explain susceptibility ofdifferent plant parts to freezing damage and mayindicate ways of controlling or avoiding damage.

Different plant organs or tissues may freeze at dif-ferent temperatures. This is particularly well knownin woody plants and is explained by different icenucleation events in different parts of the plant andby barriers to spread of ice (Burke et al., 1976; Ash-worth, 1996; Wisniewski et al., 1997; Wisniewski and

Fuller, 1999). It is argued that ice forms in xylemvessels and then spreads to other parts of the plantthrough the vessels (Sakai and Larcher, 1987). Theidea of rapid spread through vessels is a reasonablesuggestion. There is direct evidence for ice propaga-tion through xylem tissue (Kitaura, 1967). Althoughthe rate of spread of freezing through stems can befast, of the order of 1 to 2 cm s21 (Single and Mar-cellos, 1981; Sakai and Larcher, 1987), direct evidencefor nucleation in vessels and spread through vesselsis lacking.

In cereals, stem nodes and the base of the rootspose a barrier to spread of ice, possibly related to xylemstructure (Single and Marcellos, 1981; Zamecnık et al.,1994). Proteins and polysaccharides with antifreezeproperties are present in cereal leaves and the crown,respectively (Olien and Smith, 1981; Griffith and Anti-kainen, 1996). On the other hand, a comprehensiveaccount of spread of freezing in cereals is lacking, yet isessential for understanding the relative roles of anyspecific features identified. Our purpose was to useinfrared video thermography (IRVT) to obtain a morecomprehensive account of spread of freezing in cere-als and to seek new and informative details.

When supercooled water freezes it causes a releaseof heat (an exotherm). Freezing is detected by theconsequent rise in temperature. Freezing after evenslight supercooling is detectable. The usual methodto study freezing has been by attaching thermocou-ples to parts of a plant and recording the time andtemperature at which exotherms occur. Though thishas provided important information (Burke et al.,1976; Sakai and Larcher, 1987), the approach haslimitations: it cannot identify the site of nucleation offreezing, is not ideal for identifying the pathway of

1 This work was supported by the Biotechnology and BiologicalScience Research Council (grant no. 321/JEI09421).

* Corresponding author; e-mail [email protected]; fax44–191–222–8684.

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spread of ice, and gives only partial information onrates of spread. In addition, thermocouples them-selves may induce freezing (Fuller and Wisniewski,1999).

IRVT has recently been used to study freezing(Wisniewski et al., 1997). Data obtained using IRVTcomprises video images in which false color indicatesthe temperature. IRVT provides detailed real-timeimages of the surface temperature of plant organsand allows the site of nucleation and initial spread offreezing to be observed. So far it has mainly beenapplied to tender species such as bean, tomato, andpotato, and to woody species such as rhododendronand fruit crops (Wisniewski et al., 1997; Carter et al.,1999; Fuller and Wisniewski, 1999; Wisniewski andFuller, 1999; Workmaster et al., 1999). These studieshave shown that freezing may occur first on the leafsurface and that ice enters leaves through stomata,but also that in some woody species freezing mayfirst occur inside the plant (Wisniewski et al., 1997;Wisniewski and Fuller, 1999). They have also verifiedthe importance of internal barriers to the spread offreezing (Carter et al., 1999; Workmaster et al., 1999).

IRVT studies have not included hardy herbaceousplants, nor any grasses or cereals, and have beenentirely based on laboratory studies. In a radiationfrost moisture condenses on the plant and freezes,and this may nucleate freezing in the plant. This isdifficult to achieve in the laboratory, and thereforenucleation is ensured by artificial methods. For thisreason it is important to include field experiments tohelp verify the ideas about plant freezing developedon the basis of laboratory experiments.

Thus we used IRVT to characterize freezing inwhole plants of barley (Hordeum vulgare) and in bar-ley and grass leaves (Hordeum murinum and Holcuslanatus), mostly in the laboratory, but also in nature,including determining rates of spread, spatial distri-bution, directions of spread, and sequences of organfreezing, and we drew conclusions about the possibleroutes of spread and role of barriers to spread.

RESULTS

Shoots of cereal plants usually require moisture ontheir surface to freeze in laboratory tests at temper-atures similar to those at which they freeze in thefield. To verify this in the present experiments eightwell-watered plants in pots, but having dry shootsurfaces, were cooled to a shoot temperature of24.0°C. No parts of the plants froze (data notshown). In addition, when ice or single droplets ofwater or of a suspension of ice-nucleation active(INA) bacteria were placed on leaves, freezing wasinitiated at the point of application (below), and notat any other point. Hence for shoot freezing to bestudied in the laboratory, water, ice, or a suspensionof INA bacteria had to be brought into contact withthe shoot surface.

Interpretation of the Images

A freezing event is exothermic and therefore itsinitiation can be detected as a rise in temperature.When the freezing event has finished, no more heat isreleased and the temperature falls. In each experi-ment the temperature range covered by the false-color scale (e.g. Fig. 1) was reset as cooling pro-gressed, to keep the temperature of unfrozen parts ofthe plant at the bottom-end of the temperature range,shown by cold-pink or blue. Thus freezing would bedetected by warming, giving a blue (if the specimenwas initially cold-pink) or white color. With furtherwarming the color would change again, to green,yellow, or red. Black indicates a temperature below,and pale pink indicates a temperature above, the setrange. It was important to identify the termination ofthe freezing event, when all the ice that could form atthat time in the experiment had formed, and whenthe specimen would recool. This was seen as achange in color toward the cold-end (blue or cold-pink) of the temperature scale.

More than one warming and recooling could occursequentially at one location. This would indicate thatonly part of the water that could freeze at the locationhad frozen during the first warming and recoolingevent, with more water freezing later.

Freezing of Whole Plants in the Laboratory

The effect on the spread of ice of simultaneousexposure of all parts of the plant to the same temper-ature was tested. To do this, young barley plantswere placed on cardboard or polystyrene and ice wasplaced in contact with the youngest fully expandedleaf of each plant, and then freezing was followedduring cooling.

Table I shows the time interval between ice reach-ing the region where the sheath base joins the stem atthe base of the plant, and the first occurrence offreezing in other organs. It therefore indicates thetime to spread across the stem at the base of thecrown and reach each of the organs. These time in-tervals differed significantly between organs, vary-ing from seconds to reach the roots, to minutes orabout 1 h to reach the youngest leaves and secondarytillers. The time-interval data for the acclimated (n 55) and nonacclimated plants (n 5 4) tested was notsignificantly different (P . 0.05; details not shown)and the data were therefore pooled in Table I. Theorder of time interval for spread to these organs was:roots , leaf older than the nucleated leaf , leavesyounger than the nucleated leaves and secondarytillers.

The time intervals varied between plants and amore precise account of the order in which freezingoccurred was obtained by documenting it separatelyfor each plant. Figure 1 is typical of the initiation andspread of freezing in the nine plants used in Table I:The leaf in contact with ice froze first, the ice spread

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rapidly and nucleated the roots, ice then spread tothe next older leaf and subsequently to the youngerleaves, and finally, it spread to secondary tiller. Thesame order occurred in the acclimated and nonaccli-mated plants tested (P . 0.05 in all comparisons;details not shown). In two of the plants used in TableI there were two leaves younger than the nucleatedleaf. Further tests were made of two more young barleyplants and of two reproductive wheat plants each withtwo leaves younger than the one nucleated. In all sixspecimens the leaf next youngest after the nucleatedleaf froze before the most recently emerged leaf.

Figure 1 also shows typical freezing of the leaves intwo stages. The first freezing event involved rapidspread of an area of slight warming. This recooledand subsequently, a second freezing event occurredthat was more prolonged than the first freezing eventand involved a larger rise in temperature. Thus, inFigure 1, leaf 3 showed the first freezing event (arrowin Fig. 1b), recooled (Fig. 1, c–e), and then a secondfreezing event occurred (Fig. 1, f–h). The same se-quence occurred in the other leaves, older andyounger than the first leaf to freeze (Fig. 1), and allleaves of the nine plants analyzed in Tables I throughIII, acclimated and nonacclimated, froze like this.Thus the same pattern of freezing occurred in eachleaf whether it was the first leaf to freeze, initiated byexternal ice or froze later, nucleated by ice spreadingwithin the plant.

Table I also shows that spread of ice across the stemat the base of the crown was much slower, between21.5°C and 22.0°C, than below 22.9°C. Temperaturealso affected the time between the first and secondfreezing event in the leaves: at and below 22.2°C itwas a few minutes, but at and above 21.9°C theaverage was 48 min (Table II).

The spread of the ice front in leaves was very rapidwithin each leaf: 1 to 3 cm s21 in leaf blades and leafsheaths of the main and secondary tillers (Table III).This did not differ significantly between field-grown(acclimated) and greenhouse-grown (nonacclimated)plants. However, spread was about twice as fast inyounger leaves than in older leaves. Again, spreadwas slower at 21.5°C and 22.0°C than at and below22.5°C, though the difference was smaller than theeffect of temperature on spread between organs.

Visible damage symptoms were assessed 7 d aftertesting. The lowest temperature that the plants inTables I through III were exposed to was 27.0°C. Thenonacclimated plants showed damage symptoms,whereas the acclimated plants did not.

Plants in pots or dug from the field and used withan intact root ball were also tested. In these labora-tory tests the soil was expected to cool more slowlythan the leaves and it was expected that the base ofthe crown embedded in the soil surface would there-fore also cool more slowly than the leaves. Therefore,it was predicted that ice nucleated in one leaf in eachplant would not initially spread to other leaves orother tillers. The first test was with four greenhouse-grown (nonacclimated) plants in which one drop of asuspension of INA bacteria was placed on one leaf ofeach plant. Figure 2A was typical; as expected, freez-ing in the nucleated leaf did not immediately spreadto other parts of the plants.

The plant surface in many climates acquires con-densed moisture early during a freezing night. Tosimulate this, three greenhouse-grown (nonaccli-mated) and three field-grown (acclimated) plantswere sprayed with deionized water, to run-off. Inthese tests the soil surface remained at or above21.0°C and did not freeze during the course of the

Table I. Time between the freezing front reaching the bottom of the sheath of the first leaf to freeze,and reaching other organs

Plants of barley cv Gleam were grown in either a greenhouse (nonacclimated: four plants) or in thefield (acclimated: five plants) and were analyzed in February 1999. Freezing was initiated by placing apiece of ice in contact with the youngest fully expanded leaf. Nine plants analyzed from three separatecooling runs, which each included acclimated and nonacclimated plants.

Organ to Which the FreezingFront Spread

Temperature of the ShootBasea When the Freezing

Front Reached ItTime 6 SE n

°C s

Roots 21.5 to 22.0 28.9 6 5.4 5Roots 22.9 to 23.0 2.61 6 0.57 4Next older leaf 21.5 to 21.9 125 6 48b 4Next older leaf 22.9 to 23.0 2.34, 6.32 2Next younger leaf 21.5 to 22.0 3.50 3 103 5

60.69 3 103

Next younger leaf 22.9 to 23.5 0.32 3 103 660.30 3 103

Secondary tiller 21.5 to 23.0 3.70 3 103 561.12 3 103

a The shoot base comprised the stem and the basal 1 mm of surrounding leaf sheaths. b Datarange from 38 to 268 s.

Freezing of Barley

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experiment. Below a leaf and droplet temperature of23°C, the droplets of water on the shoots froze spo-radically, some before the leaves on which they werefroze and some after. Most leaves froze during thecourse of the experiment. Figure 2B shows typicalresults. The leaves indicated froze at different times,and each froze by an initial spread of ice from at ornear the tip.

In these experiments using potted plants or plantsdug from the field and tested with root ball intact,freezing of a total of seven greenhouse-grown (non-acclimated) and 10 field-grown (acclimated) plantswas examined. Damage to the leaves was visuallyassessed 7 d after freezing. The lowest temperature ofthe leaves was 22.5°C to 25.0°C in different tests. Inno tests did leaves of the acclimated plants show

Figure 1. Sequential freezing of parts of a barleyplant during cooling in a controlled environ-ment chamber showing spread of ice throughthe plant, including the first (red arrows) andsecond freezing events in a number of leaves(leaves 2–5) and freezing of roots (black,double-headed arrow), a leaf sheath (s), andsecondary tiller (t). IRVT images are shown intime order from before the plant began to freeze(a) to when extensive freezing had occurred (j).k is a visible light image taken when freezingwas complete. Leaves are numbered from thesecond to the fifth leaf to emerge (the first leafwas removed before the test). The plant wasremoved from soil and placed flat on a corksurface and exposed to subzero cooling. Thestars in a and k mark two pins holding down astrip of plastic that gently held leaf 3 in contactwith ice (indicated by a single-headed blackarrow) to initiate freezing. Black bar (in a) 5 1cm. The color scale table (bottom right) indi-cates the temperature scale for each figure. Leaf3 froze first and showed two freezing events.The warming of leaf 3 in b (red arrow) comparedwith in a indicates freezing. The leaf then cooled(c–e). It warmed again (f), indicating a secondfreezing event, and reached a higher tempera-ture than in the first freezing event (compare gand h with b), and then cooled again (i). Rootsfroze second (red arrow in c). Leaf 2 froze next(red arrow indicates warming of the leaf in d)and also showed two freezing events: it cooled(e) and then warmed again (starting in f andcontinuing in g and h), finally cooling again. Thesheath of leaf 2 (s) also froze (second freezingevent shown in h–j). Leaf 4 froze next, icespreading up the leaf (red arrow in f; the secondfreeze is shown in g–h and cooling in i) and thelast leaf to freeze was leaf 5 (red arrow in g,cooled in h, second freezing event shown in i).The secondary tiller froze (red arrow in i; thesecond freezing event is shown in j).

Pearce and Fuller



damage symptoms. In nonacclimated plants no dam-age was seen when the lowest temperature reachedwas 22.5°C, whereas, in shoots that cooled to24.5°C, the leaves that froze showed damage symp-toms and those that did not freeze showed nosymptoms.

Freezing of Excised Leaf Blades in the Laboratory

When leaf blades were cut from the plants andimmediately cooled there was again a very rapidspread through the leaf of an area of slight warmingfollowed by recooling, and then a second and largerrise in temperature, which persisted for longer (Fig.2C). Whereas the first freezing event spread downthe leaf, the second freezing event did not start at onepoint and spread from there. Instead it started simul-taneously in many parts of the leaf.

Leaves from nonacclimated plants (grown in a20°C/15°C environment) were tested during twosuccessive freezing tests in which they were cooled to27.0°C. Because they were not acclimated, the leaveswere killed by the first exposure to 27.0°C (con-firmed by visible symptoms). During the first testthey froze typically, with an initial, very rapid spreadof an exotherm that quickly disappeared, followedshortly by a second, more intense exotherm (Fig. 2C).When thawed they had a water-soaked appearance.The leaves were stored for 3 h in a dark, humidenvironment before retesting. They remained water-soaked, indicating that the previous freeze killed theleaves and redistributed water, filling the extracellu-lar spaces. During the second test, freezing occurreddifferently from the first test. The first freezing eventwas much more intense (the leaf warmed much moreand for much longer) than in the preceding freezingtest, and freezing spread much more slowly downthe leaf (4.13 6 0.42 cm s21 in the first freezing testand 0.40 6 0.08 cm s21 in the second; n 5 6). Inaddition, although in the first test the second freezingevent occurred 11.5 6 0.8 s (n 5 6) after the firstfreezing event, in the second test there was only asingle extended freezing event (Fig. 2D).

Digital analysis was used to obtain more precisetemperature data than could be obtained directlyfrom the color video images. This confirmed that thefirst freezing event involved a much smaller rise intemperature and the rise lasted for a much shorter

Table III. Rate of longitudinal spread of the first freezing event in leaves of whole barley cv Gleamplants

Leaves and secondary tillers were analyzed from nine plants from three separate cooling runs.Experimental details were the same as in Table I. The data for leaf blades was analyzed by ANOVA totest for effects of temperature, growth environment, and the position of the leaf on the plant. ANOVAindicated significant differences (P , 0.01) between factors, and means were compared using confi-dence intervals.

Organs and Treatments Rate 6 SE95% Confidence

Intervaln

cm s21

Leaf bladesAll samples 2.36 6 0.22 30Effect of nucleation temperature

21.5°C to 22.0°C 1.64 0.78 922.5°C to 24.0°C 2.67 0.52 21

Effect of environmentGreenhouse (nonacclimated) 2.16 0.74 11Field (acclimated) 2.47 0.61 19

Effect of leaf position relative to nucleated leafNucleated leaf 1.90 0.43 10Next older leaf 1.57 1.21 6Younger leaf 3.02 0.69 14

Sheatha

All samples 1.2 6 0.42 6Tillersb

All samples 2.89 6 0.88 4a The warming due to the spread of ice within the leaf sheath was only clearly detectable for some

sheaths. b Only some plants had sufficiently large secondary tillers for accurate measurement.

Table II. Time in each leaf of whole barley cv Gleam plants be-tween the start of the first and second freezing events

Experimental details were the same as in Table I. Leaves wereanalyzed from 15 plants from three separate cooling runs, includingfour nonacclimated plants and 11 acclimated plants.

Temperature of the LeafWhen Freezing Began

Time 6 SE n

°C min

21.4 to 21.9 47.8 6 11.1 822.2 to 23.5 2.74 6 0.35 25

Freezing of Barley



Figure 2. Freezing of barley plants in pots and of excised leaves of barley during cooling in a controlled environmentchamber. IRVT images in each of B through G are shown in time order. In A and C through G, freezing was initiated byplacing a droplet of a suspension of INA bacteria on or touching the leaf surface. In B the plants were sprayed with watertill run-off. Black bars 5 1 cm. The color scale table (bottom right) indicates the temperature scale for each figure. A, Freezingof a greenhouse-grown (nonacclimated) plant of barley cv Gleam nucleated by a droplet of INA bacteria (black arrow)

(Legend continues on facing page.)

Pearce and Fuller



time than in the second freezing event. Less than0.37% 6 0.12% of leaf water froze during the initialspread of freezing (first freezing event, Fig. 3A). Thedigital analysis showed a clear separation in timebetween the first occurrence of freezing at differentplaces in the leaf, and this was observed during thefirst spread of ice in the first and second freezing tests(Fig. 3B). However, in the first test the second freez-ing event was clearly shown as occurring at the sametime in different parts of the leaf (Fig. 3B).

Details of freezing were studied by close-up exam-ination of leaf pieces of only a few centimeters length(Fig. 2E). These confirmed the rapid initial spread ofice and the higher more prolonged second freezingevent. In addition, they showed (n 5 10) that thewarming of the specimen during the initial freezingevent was not evenly distributed across the leaf sur-face. Instead, more warming occurred in narrowstrips down the leaf (equal numbers of leaves nucle-ated on the upper or lower surface with a drop ofINA bacterial solution gave the same appearance).This indicated some lateral localization of the initialfreezing event. The leaf pieces also showed that thesecond freezing event began at the edge of the leafand progressed toward the mid-rib (Fig. 2E, g–j).

In these experiments with short pieces of leaf,freezing was initiated by placing a drop of a suspen-sion of INA bacteria in the center of the leaf surfaceor touching one edge. This nucleated freezingthroughout the leaf, and not just along the centralregion or edge with which the drop was in contact.This made it possible to measure the rate of the initialspread of ice laterally across the leaf, as well aslongitudinally. The lateral spread was an order ofmagnitude slower than the longitudinal spread (Ta-ble IV).

Short pieces of leaf were also used to examinespread of ice in partially water-soaked samples. Par-tial water soaking was obtained by placing pottedplants of H. murinum in a growth chamber at 22°Cwith no lights and 100% relative humidity for 24 hbefore excising leaves and exposing them to subzerocooling. This resulted in one of two patterns of freez-ing. In five out of eight leaves tested, the initialspread of ice occurred in the usual way, by a rapidspread of ice, especially in strips. However, this wasimmediately followed by localized, more intense, butshort-lasting (1 to several s) freezing events (Fig. 2F),which continued to occur in new locations until theleaf had finished freezing. The time gap between the

Figure 2. (Legend continued from facing page.)placed on the surface of a leaf blade. In the image this leaf is warmer than the other leaves. It had already begun to freeze,whereas the other leaves (which remained cooler) had not. Note that freezing had progressed into the sheath (red arrow) ofthe freezing leaf, but not to the rest of the plant. At this stage in the experiment the soil surface (s) was 21.00°C (in the potshowing at the base of plant), whereas the unfrozen leaves were 23.0°C. Freezing did not spread to the rest of the plantduring the subsequent 75 min of observation, by when the soil surface had cooled to 22.6°C. B, Freezing of a field-grown(acclimated) plant of barley cv Gleam sprayed with water. The images show three leaves from one tiller. (The semicircle oflight pink at the bottom left of the image is due to the soil [s] in the plant pot.) In a, several leaves were freezing, indicatedby their warm color, including freezing of surface droplets (arrow heads). Later, these leaves had completed their freezing(b), but had not nucleated freezing of the youngest leaf (center of each figure). In this youngest leaf a droplet at its tip frozefirst (red arrow in b) and this initiated freezing in the whole leaf (warming shown in c). C and D, Successive exposure tofreezing of whole leaf blades excised from plants of barley cv Igri grown in a 20°C/15°C (nonacclimating) environment. InC the leaves were cooled to 27.0°C. They were then thawed and kept for 3 h in a dark humid environment, during whichthey remained water-soaked. In D they were then exposed again to subzero cooling. In C there were two freezing events.Freezing began from the droplet of INA bacteria (black arrow in a) and was first indicated by a localized warming (changein color from pink/blue to blue then to blue/white), which spread down the blade (red arrows in b and c). The area ofwarming spread to the base of the blade (d) and then cooled (e), indicating the end of the first freezing event. Shortly after,the second freezing event began throughout the blade (warming indicated by blue/white area in f, red arrowheads). The leafprogressively warmed further (white to yellow in g–i), indicating continuation of freezing, and then recooled (j and k),indicating completion of the second freezing event. In D there was one freezing event. The spread of warming was indicatedby the spread of the area of yellow and red down the leaf (b–h), beginning from the droplet of INA bacteria (black arrowin a). The warming of the leaf was greater than in the first freezing event in C, as indicated by the yellow and red false color.E, Typical details of a time-sequence of freezing in part of a leaf blade of barley cv Gleam. Freezing was initiated by a dropof a suspension of INA bacteria placed on the middle of the lower surface of the leaf, outside the upper edge of the field ofview of the IRVT images (m is a visible-light photograph of the leaf piece showing the droplet: black arrow). a shows the leafpiece just before freezing. Freezing first appeared as a wedge of warmer tissue (blue) in the upper part of the image (b). Thisspread down the leaf (c and d) and cooled (becoming pink in f). The second freezing event was indicated by progressivewarming in g through k (white to green to yellow) followed by recooling (to pink/blue in l). Note that the warming thatinitially spread down the leaf was most intense in narrow strips (red arrows in c and d). The second freezing event beganat the edge of the leaf (red arrowheads in g) and spread inwards from there (h–j). F and G, Details of freezing of excisedpieces of partially water-soaked leaf blades from plants of H. murinum (induced by 24 h in a 22°C, dark, humidenvironment). In F (typical of five out of eight of the leaves tested) initial warming spread in strips down the leaf (red arrowsin b and c). Immediately after, localized freezing events occurred (red arrowheads in d) and rapidly recooled. This wasfollowed by a period in which many localized freezing events, which lasted for varied times, occurred throughout the leaf(warmer patches in e). In G (typical of three out of eight of the leaves tested) there was initial spread of substantial warmingdown the middle of the leaf (b–e), and again, this was followed by many localized freezing events throughout the leaf (e).

Freezing of Barley



initial spread of ice and the first localized higher-intensity freeze was 1.96 6 0.82 s (mean 6 se; n 5 5)and the two shortest intervals were 0.40 and 0.56 s. In

three out of eight leaves tested the initial spread ofice was slow and localized to the mid-rib regionof the blade, and the warming in this initial spread of

Figure 3. Rise in temperature with time at one (A) or two (B) sites on leaf blades of barley cv Igri during typical freezing.Temperature was averaged at one (A) or two (B) lines crossing the image of the leaf, and averaged background temperaturewas subtracted. In B the two lines were spaced 4 cm apart, 6 and 10 cm, respectively, from the droplet of INA bacteria thatnucleated freezing in the leaf. A, First freezing event compared with the second freezing event. B, Initial stages in freezingat the two positions in the leaf. Results are shown for the same leaf during exposure to freezing for the first time (first freezingtest) or after cooling to 27.0°C and thawing, during exposure for the second time (second freezing test). In both tests icespread down the leaf from the site of nucleation. Thus, the position on the leaf that the ice reached first warmed first, andwarming occurred at the second site shortly after. Note that there was a shorter time gap between the start of freezing at thetwo sites in the first test (start of warming indicated by the single-headed bold arrows) than between the start of freezing atthe two sites in the second test (start of warming indicated by stars). This indicates the different rates of spread of ice in thetwo tests, 2.0 and 0.4 cm s21, respectively. Note also that in the first test the start of the second freezing event (indicated bythe double-headed bold arrow) began at the same time at both sites, and the subsequent curves of warming at the two sitesremained closely similar. On the other hand, in the second test the curves of warming at the two sites remained separate.

Pearce and Fuller



ice was much higher than usual. However, this wasagain immediately followed by localized spread lat-erally of the area of warming accompanied by, as inthe first type, localized high-intensity short-lastingfreezing (Fig. 2G).

Freezing of Lawn Grass Leaves in Situ

To verify the relevance of parts of the laboratoryobservations to Poaceae in the field, freezing ofleaves of H. lanatus in a lawn was studied during amild winter frost. Two leaves froze at 21.5°C andtwo at 22.5°C. All leaves showed an initial rapidspread of ice, seen as rapid spread of slight warminglasting for a short time (Fig. 4; Table V). At the highertemperature a second freezing event did not occurwithin the period of observation, whereas at thelower temperature a second freezing event occurredless than 1 min after the initial spread of ice. Theinitial spread of ice in two leaves was basipetal. Inone of these, droplets of water on the leaf surfacefroze sequentially, starting near the base of the leafand freezing droplets further up the leaf. When freez-ing of these surface droplets reached the tip, it nu-cleated freezing within the leaf. In the other leaf thetip was nucleated, causing spread of ice through theleaf, when ice spread on an adjacent frozen surfacetouching a droplet at the leaf tip (Fig. 4, A and C). Inthe other two leaves the initial spread of ice wasacropetal. In one leaf, when the ice spreading inter-nally reached the tip of the leaf, it froze a guttateddrop located there (Fig. 4B). In the three leaves wherefreezing of the droplet at the tip occurred, no differ-ence in time could be detected between the momentwhen the guttated droplet froze and the momentwhen freezing spread from or to the tip, indicating adirect connection between water freezing in the leafand the guttated drops.

DISCUSSION

IRVT showed freezing throughout the barley plant,allowing a comprehensive account of the spread ofice to be obtained: (a) The first and second freezingevents in leaves are distinct. The first involves a rapidlongitudinal spread of freezing, mainly in narrowstrips, involving only a small fraction of leaf water. Incontrast, the second freezing event spreads slowlyand locally, from the margin of the leaf toward themid-rib, and occurs simultaneously throughoutthe length of the leaf. It involves freezing of most ofthe leaf’s water, causing the intracellular dehydrationthat stresses the plant. (b) Above 22°C the initialspread of freezing through the crown is delayedmuch more than at lower temperatures. This sets animportant limitation on the ability of freezing initi-ated in one organ to spread further. Consistent withthis, our results also indicate that nucleation of freez-ing occurs independently in different leaves. Thusany function the barriers have in adaptation to freez-ing could be in protecting the crown rather thanpreventing spread to other leaves. (c) Without theinitial spread of ice from a point of nucleation, theplant will not experience freezing stress. Thus anystrategy to modify freezing behavior in plants mustaddress the route by which freezing spreads. Thewater that freezes during the initial spread of ice isprobably drawn from cell walls. If so, it is only afraction of the total water present in cell walls, andmay be drawn only from the most gel-like parts. (d)The species used acclimated only moderately in theenvironments used, so the results need not be typicalof all cereals or the most strongly acclimatory envi-ronments. Nevertheless, our experiments showed nodifferences in freezing behavior between the cold-acclimated and nonacclimated plants.

Natural Freezing

Freezing in the leaves during a natural frost wassimilar to that in leaves tested in the laboratory.There was an initial freezing event that comprisedrapid spread of ice, but freezing of only a fraction ofthe leaf water. When the temperature fell further,there was a second freezing event during whichmuch more water froze. The rate of spread of ice inthe first freezing event was also similar to in thelaboratory. Thus the more detailed analysis in thelaboratory tests was likely to be indicative of behav-ior and mechanisms in leaves in the field.

Laboratory experiments have shown that ice mayenter leaves through stomata (Wisniewski and Fuller,1999). However, at night, stomata will be closed. Inour field experiments done at night, the hydathodesat the tip of the leaves, through which water guttated,was an important channel for ice to spread into or outof the plant (Fig. 4). Laboratory tests indicated thatguttated water itself does not have a high nucleationtemperature (not shown), and in the limited obser-

Table IV. Rate of longitudinal and lateral spread of first freezingevent in excised youngest fully expanded leaves from plants ofbarley cv Gleam (grown in the greenhouse, nonacclimated, in thefield, acclimateda) and analyzed in February 1999

Freezing was initiated by freezing of a droplet of a suspension ofINA bacteria placed near the tip of each leaf. First spread of iceoccurred when leaves were at 22.5°C to 24.0°C.

Leaf Part and Direction ofSpread of Ice

Rate 6 SE n

cm s21

Whole leavesLongitudinal spread 2.85 6 0.18 7

Four-cm lengths of leafLongitudinal spread 2.18 6 0.18 10Lateral spread 0.31 6 0.08 10

a There was no significant difference in the data for the acclimatedand nonacclimated plants.

Freezing of Barley



vations in the field it was frozen by contact with icenucleated from elsewhere. Nevertheless, in our fieldexperiments droplets that froze on the leaf surfacedid not nucleate freezing in the leaf except when thedroplet at the leaf tip was nucleated.

Intercellular spaces in hydathodes are connected tothe termination of vascular strands. However, thereis not a free path of water between the guttating dropand the lumen of the xylem elements since thestrands terminate in tracheids (Esau, 1965). It is gen-

Figure 4. Freezing of leaves of H. lanatus in situin a lawn during a mild natural frost. Images ineach of A through C are shown in time order. Noartificial aid to nucleation of freezing was ap-plied. Black bar 5 1 cm. The color scale (topmiddle) indicates the temperature for the fig-ures. A, Spread of freezing on the leaf surface inwhich droplets initiated freezing in adjacentdroplets. This progressed toward the tip andfroze a droplet there that then initiated the firstfreezing event in the leaf. Numerous water drop-lets had condensed on the leaf surface (someindicated by the red arrowheads in a). Dropletsfroze outside the field of view and nucleateddroplets toward the base of the leaf (indicated bya warmer area at the left in b). Nucleation ofsurface droplets progressed sporadically, mainlyacropetally (c). When a droplet at the tip wasnucleated (red arrow in d) slight warming spreadbasipetally through the leaf (spread of the green,warmed, area from the tip toward the base ine–g). Other surface droplets subsequently froze(warmer areas in h), but eventually further sur-face freezing events ceased (i). A second freez-ing event within the leaf was not seen during theperiod of observation. B, The first freezing eventspread up the leaf and nucleated a guttateddroplet at the leaf tip. Warming spread up theleaf starting from outside the field of view (slightwarming indicated by white/blue at the bottomof b). This spread to the tip where it nucleatedfreezing in a guttated droplet. The red arrow-head in a indicates the droplet before freezing;the red arrow in c indicates the droplet at themoment of freezing; the guttated droplet contin-ued to emit heat in d through f, but had cooledagain in g. The leaf cooled slightly (d) followingthe spread of freezing to the tip of the leaf. Thesecond freezing event, indicated by warming,began at several points in the leaf (white andwhite/blue patches in e). This then spread andincreased in warmth (green and white areas in f)and then cooled (g), completing the second freez-ing event. C, Contact between frozen droplets onthe background surface and a droplet at the leaftip initiated the first freezing event in the leaf. Theleaf tip was touching a nearby surface on whichmoisture had condensed and was freezing. Whenfreezing of this moisture spread to droplets thattouched the tip of the leaf it initiated freezing inthe leaf (red arrow in b) and warming spreadbasipetally through the leaf (white and green ar-eas in b–d) and then cooled (e).

Pearce and Fuller



erally thought that there would be a delay in icecrossing into tracheids or crossing the end-walls ofvessels, due to the presence of pit membranes (Singleand Marcellos, 1981; Zamecnık et al., 1994). However,we could not detect any delay, which may indicatethat the initial rapid spread of ice through the leafwas not through the xylem vessels or that pore size inthese pit membranes was not restrictive.

Rates of Spread of Freezing in Leaves

The rate of longitudinal spread of freezing inthe leaf blades was rapid, between 1 and 4 cm s21,depending on age and freezing temperature, with anoverall average of 2.6 cm s21. These are among thefastest rates of spread recorded in plants, thoughearlier estimates are close; up to 2 cm s21 in nonac-climated wheat (Single and Marcellos, 1981), 1 cm s21

in mulberry (Kitaura, 1967), and 0.4 cm s21 inbarley seminal internodes and bean internodes(Zamecnık et al., 1994; Wisniewski et al., 1997).

The fastest rates of spread of freezing in plants aresomewhat faster than the linear growth rate of ice inwater, which at atmospheric pressure and when su-percooled by about 1° K, is 0.5 cm s21 (Hobbs, 1974).To obtain a rate comparable with the fastest found inplants, above 2 cm s21, supercooling of about 5° K isneeded (Hobbs, 1974). Allowing for a depression offreezing point of about 1° K (Levitt, 1980), this isseveral degrees more supercooling than occurred inour experiments. Growth of ice from the vapor phaseis much slower than in water, of the order of 1024 cms21 (Hobbs, 1974). Thus plants show unusual freez-ing behavior compared with physical experiments,speeding the spread of ice.

Rapid dissipation of the heat released from thevery small amount of water frozen during the initialspread of ice could explain this. Dissipation of theheat of fusion is a major factor determining the rateof growth of ice (Hobbs, 1974). The amount of waterthat froze during the initial spread of ice accountedfor less than 0.4% of the total leaf water. Consistentwith this explanation, the warming at the spreadingfront was much higher and the associated rate ofspread was slower in the water-soaked leaves, inwhich more water froze initially.

The Route of the Initial Spread of Ice in Leaves

The IRVT method used does not resolve ice forma-tion in or around individual cells. However, it didshow differences in freezing between adjacent areaswithin leaves at distances of less than 1 mm. Inaddition, it provided circumstantial evidence relatingto the precise locations of ice.

The initial longitudinal spread of freezing in leavesoccurred mainly in strips that could have corre-sponded to main ribs, and if so, this spread couldhave been associated closely with the vascular bun-dles. However, this need not indicate freezing in thevessels themselves. In Solanum and Brassica, freezingin petioles first occurred in vascular tissue and adja-cent intercellular spaces (Hudson and Idle, 1962).Several of our observations indicate that the initialspread of freezing was not through the xylem ele-ments themselves. Freezing withdraws water fromleaf cells, forming ice in the intercellular spaces(Pearce, 1988; Pearce and Ashworth, 1992). If the leafis damaged, then on thawing, this water remains inthe intercellular spaces causing a water-soaked ap-pearance. However, water would have remained inthe xylem vessels. Thus, one would expect to see thesame rapid initial spread of freezing in ribs in non-damaged, non-water-soaked leaves and in damaged,fully water-soaked leaves. However, the patterns ofspread (Figs. 2, C and D and 3B) and initial rates ofspread, which differed by a factor of 10, were quitedifferent. This may indicate that the initial longitudi-nal spread of ice was in both cases extracellular.

In five of the partially water-soaked leaves therewas a typical first freezing event involving rapidspread of freezing in strips down the leaf (Fig. 2F).However, these then initiated localized short-livedfreezing events in between under 1 s and a few seconds,again suggesting initial spread was extracellular.

The rate of lateral spread in leaves was an order ofmagnitude less than the rate of longitudinal spread.Minor vein connections run laterally between thelongitudinal veins. In festucoid grasses they occurwith a frequency connecting each pair of longitudinalvascular bundles of about 1 per mm (illustration inPate and Gunning, 1969) or more (illustration inEsau, 1965). The lateral veins do not form a linearpath across the leaf, instead, any ice spreading later-

Table V. Rate of longitudinal spread of first freezing event in fully expanded leaves of H. lanatustested in situ during a natural frost

No artificial aids to nucleate freezing were used.

Temperature of the Leaf WhenFreezing Began

Rate 6 SE Time Gap to Second Freezing Event n

°C cm s21

21.5 (n 5 2); 22.5 (n 5 2) 2.51 6 0.66 Second event not seen during 3 h in thetwo leaves which first froze at 21.5°C

4

22 and 38 s in the two leaves which firstfroze at 22.5°C

Freezing of Barley



ally through xylem vessels would have to take atortuous route, which might then partially explainthe slower rate of spread laterally than longitudi-nally. However, it is equally plausible that spreadoccurred outside the vascular bundles since paths oftravel at right angles to each other might lead todifferent rates of travel due the different structure ofthe leaf in the lateral and longitudinal directions.

The proportion of the leaf water that froze duringthe initial spread of ice was under 0.4%. Assumingthe initial spread of ice was extracellular, then thereare two sources of water for this initial freeze: thevapor phase in the gas spaces and the cell walls. Theformer would contribute a negligible amount: a fallin temperature from 0°C to 25°C would contributethrough condensation about 4 3 1025% of the leaveswater content (based on a change in water content ofthe vapor phase of 62.5 3 1026 mol L21 between 0°Cand 25°C). The proportion of the leaf water volumeaccounted for by water in the cell walls is about 6%(assuming the cell wall accounts for about 10% of theleaf fresh weight and that it has a water content of60%–70%). Thus much less than the full amount ofwater in the cell walls froze during this initial spreadof ice. The small pore sizes in plant cell walls, aver-aging 4 nm (Preston, 1974), would reduce the averagefreezing point of water in the wall to between 215°Cand 225°C (Ashworth and Ables, 1984). The con-verse will follow, that the vapor pressure of ice out-side the cell walls will not be low enough at temper-atures above these to draw water out from suchpores. On the other hand, water might be drawnfrom more gel-like parts of cell walls such as themiddle lamella or from water-rich wall layers foundin some fibers. If so, the distribution of these sourcesof wall water at the tissue and organ level couldinfluence the route of the initial spread of ice.

The Second Freezing Event

In leaves of freeze-stressed temperate cereals ice isextracellular (Pearce, 1988; Pearce and Ashworth,1992). The second freezing event accounts for most ofthe freezing in the leaf and therefore must compriseformation of this extracellular ice.

The ice formed during the first freezing event prob-ably initiates the second freezing event. In other re-spects, the two freezing events are separable. Whenthe first freezing event began above 22°C the occur-rence of the second freezing event could be delayed(using a slow cooling rate) for a considerable time.However, if the first freezing event occurred below22°C, the second freezing event followed within afew minutes or seconds. This indicates that the twofreezing events involved separate fractions of plantwater with different freezing points, one above 22°Cand one just below. This is consistent with the firstfreezing event being apoplasmic, whereas the secondinvolved drawing water from the symplast.

Other details confirm the distinctness of the twofreezing events. The first freezing event spread lon-gitudinally through the leaf and thus reached differ-ent parts of the leaf at different times, but the secondfreezing event began simultaneously at differentpoints along the length of the leaf (Figs. 2C and 3B).Also, the second freezing event began first at theedge and spread toward the center of the leaf fromthere, whereas the first freezing event involved lon-gitudinal spread across the leaf’s width (Fig. 2E).

Spread of Freezing through the Plant

In festucoid grasses the largest vascular bundlesoriginating in a leaf have major connections in thestem with the vascular bundles of the leaves insertedtwo nodes above and below, and are connected onlyindirectly via these main connections and via thenodal plexus with the leaves inserted one node toeither side (Hitch and Sharman, 1971). When thejoined vasculatures reach the base of the crown theyconnect with the root vasculature via the complexnetwork of the vascular transition zone. Thus if thespread of freezing were confined to the xylem ves-sels, at least parts of the rest of the shoot shouldfreeze before any of the roots, and leaves two-aboveand two-below the nucleated leaf should freeze be-fore the leaves adjacent to it. However, when thefreezing front reached the crown it always first initi-ated freezing in the roots, and the next leaf above thenucleated one froze before the one next above that.

Embolisms may form in vessels as a result of freez-ing, though the vessels can refill when thawed (Can-ny, 1997). The vascular system of angiosperms issegmented; vessels are terminated by tracheids atnodes, and this prevents the spread of embolismsbetween organs (Aloni, 1987). These termini are morefrequent in the vascular transition zone between theroot and shoot in hardy than in tender cereals (Aloniand Griffith, 1991), and spread of ice is delayed inthis zone (Zamecnık et al., 1995). The node is also abarrier to the spread of freezing in the reproductivestem of wheat. Single and Marcellos (1981) suggestedthat the barrier is the tracheal termini in the node,and that passage of ice across the node is in oraround living cells. The same pattern of spread oc-curs in the first barley leaf to freeze as in the barleyleaf that freezing spreads to last. This would be un-likely if spread were first in the vessels and thenoutside them.

Our results show that below 22.0°C the delay tospread of ice caused by these barriers is only a fewseconds, whereas above 22.0°C the delay may beprolonged or indefinite. Thus, in young barley plantsthe barriers are unlikely to be effective in reducingspread of ice throughout the plant except in themildest frosts. In many frost situations the base of theplant will tend to be warmer than the exposed partsof the leaves. This difference in temperature could, invegetative plants, also be an effective barrier delay-

Pearce and Fuller



ing spread from one leaf to the rest of the plant (Fig.2A). However, in the laboratory and in nature, indi-vidual leaves of barley are capable of freezing sepa-rately (Fig. 2B). Fuller and Wisniewski (1998) madesimilar observations in potato. Ice did not travel fromone shoot growing up from the tuber to another, be-cause of, it was suggested, insulation by the soil. Shootmorphology is quite different in potato and barley.Nevertheless, as in barley, leaves of potato sprayedwith water froze independently, thus structural orbiochemical barriers were important in both species.

Thus the role of these barriers in cereals, if theyhave one, would be to control freezing of the crown,rather than to protect leaves from freezing. This isinteresting because the crown is often as frost-sensitive or more sensitive than other parts of cerealand grass shoots (Tanino and McKersie, 1985; Shibataand Shimada, 1986) and the survival of the crown isessential for plant survival (Olien, 1967).

MATERIALS AND METHODS

Plant Material and Growth Environments

Barley (Hordeum vulgare cv Gleam) was grown in nonac-climating and cold-acclimating environments. The nonaccli-mating environment comprised a greenhouse at the Seale-Hayne campus of the University of Plymouth (Devon, UK)maintained with a minimum temperature of 10°C and sup-plementary lighting during an 8-h day. The acclimatingenvironment comprised a field site also at the Seale Haynecampus of the University of Plymouth (50° 329 N, 3° 349 W;January mean temperature of 5.7°C). At this site the dailyminimum and maximum air temperatures in the 7 d pre-ceding use of the plants in February 1999 varied from21.5°C to 28.8°C and from 6.4°C to 10.6°C, respectively.Barely cv Igri was grown in a nonacclimating environmentof 20°C/15°C (day/night temperature, 10-h photoperiod,250 mmol m22 s21 photon flux density). Hordeum murinumwas grown in a similar controlled environment using acold-acclimating temperature of 6°C/2°C. Holcus lanatuswas growing wild in a lawn in Newcastle upon Tyne, UK(55° 09 N, 1° 409 W; January mean temperature 3.7°C) andwas tested in February 2000. In the 7 d immediately preced-ing the test the daily grass minimum temperature variedbetween 21.2°C and 3.1°C and daily grass maximum tem-perature varied between 6.4°C and 12.0°C, as recorded at thenearby University of Newcastle Close House Field Station.

Freezing-Test Environments and IRVT

Freezing of barley cv Gleam was tested at Seale Hayne ina controlled environment radiation freezing chamber giv-ing a steady cooling profile and using a cooling rate ofbetween 1°C h21 and 3°C h21 (Fuller and LeGrice, 1998).Freezing of H. lanatus was observed in situ during a mildnatural frost in February 2000. Freezing of barley cv Igriand of H. murinum was done at Newcastle University in acontrolled environment chamber giving a steady coolingrate of 2°C h21 and a minimum temperature that could be

maintained for prolonged periods with less than 0.5°Cvariation.

An infrared imaging camera using a HgCdTe long-wavedetector (model 760, Inframetrics, North Billerica, MA) wasused to monitor exothermic events in the freezing plants(Wisniewski et al., 1997). Frame averaging of 16 frames s21

was used to smooth the image. The results were recordedon video tape as color and black-and-white images usingtwo recorders. Thermocouples placed in the field of view incontact with the background, but not touching the plantwere used as an independent check of the temperaturesrecorded. The temperature recorded by the camera wasalso standardized against an ice-water mixture at 0°C.

The image delivered by the camera used false color toshow the temperature. The temperature scale used covereda 2°C range from “cold” colors (pink and blue) to “warm”colors (yellow and red). The plant material was placedagainst a background chosen to be of a different tempera-ture to the plant, warmer or cooler (depending on thebackground material). This provided sufficient contrast forthe specimen to be visualized by the camera before it froze,and thus to position the plant in the camera’s field of viewand to focus the image. It was important to avoid con-founding warming of the plant due to any warming in theenvironment with warming due to heat released duringfreezing. Therefore we discounted any warming of theplant that was accompanied by warming in the back-ground. The color image facilitated visual detection ofexotherms with a temperature resolution of between 0.1°Cand 0.2°C.

The black-and-white recording was analyzed using theprogram Thermagram Plot (Inframetrics). This allowedquantification of freezing. Warming (in Kelvin) due tofreezing was detected by subtracting the average temper-ature of pixels in a 0.7-cm line in the background from theaverage temperature of pixels in a 0.7-cm line in the imageof the leaf. Freezing occurred during a period of slowcooling and raised the temperature of the sample by lessthan 1°K, and thus gave only small changes in the drivingforce (gradient of temperature between leaf and the con-denser of the cooling unit) for removal of heat throughoutthe test period. Thus the amount of water freezing wasproportional to the area under the graph of temperatureagainst time during freezing. The proportion of waterfreezing in the first freezing event was calculated as thearea under the graph of temperature against time duringthe first freezing event relative to the area under the wholegraph of first and second freezing event, from initiation offreezing to completion. A remaining small amount of un-frozen water would freeze subsequently as temperature fellfurther, and therefore this calculation gave a maximumestimate of the proportion of leaf water frozen in the firstfreezing event.

The video images were studied frame-by-frame to obtainthe time and location of start and finish of freezing events.The dimensions of excised leaves and of the organs ofplants used in the experiments was measured. From thisdata the rate of spread within organs and the time forspread between organs was calculated.

Freezing of Barley



Statistically significant effects were detected by t tests orin Table III, by ANOVA and estimates of 95% confidenceintervals.

After plants had been used in freezing tests they wereplaced in the greenhouse environment described aboveand assessed after 1 week for visual symptoms of damage:loss of turgor and dryness indicated killed regions andpremature yellowing of leaves indicated a sublethal effect.

INA Bacteria

The ice-nucleating bacterium, Pseudomonas syringaestrain Cit7 (Lindow, 1985), was grown for 72 h on plates ofKing’s medium B (King et al., 1954) at 20°C. The plates werethen stored at 4°C to induce expression of the ice-nucleationprotein. Cells were harvested with a loop and suspended insterile deionized water and stored on ice before use.

Received May 22, 2000; modified July 10, 2000; acceptedAugust 23, 2000.

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Documents

Freezing of Barley Studied by Infrared · Freezing of barley (Hordeum vulgare), Hordeum murinum, and Holcus lanatus was studied using infrared video thermography. In the field, ice