Biochimica et Biophysica Acta, ! 121 (1992) i99-206 © 1992 Elsevier Science Publishers B.V. All rights r e . f r e d 0167-4838/92/$05.00

199

BBAPRO 34206

Plant thermal hysteresis proteins Maria E. Urrutia a.1, John G. Duman a and Charles A. Knight b Department of Biological Sciences. Unh'ersityof Notre Dame, Notre Dame, IN (USA) and h National Centerfor Atmospheric Research, Boulder. CO (USA) (Received 30 September 1991) (Revised manuscript received 23 December iqql )

Key words: Plant thermal hysteresis protein: Plant cold tolerance: Recrystallization inhibition; Thermal hysteresis protein

Proteins which produce a thermal hysteresis (i.e. lower the freezing point of water below the melting point) are common antifreezes in cold adapted poikilothermic animals, especially fishes from ice-laden seas and terrestrial arthropods. However, these proteins have not been previously identified in plants. 16 species of plants collected from northern Indiana in autumn and winter had low levels of thermal hysteresis activity, but activity was absent in summer. This suggests that thermal hysteresis proteins may be a fairly common winter adaptation in angiosperms. Winter stem fluid from the bittersweet nightshade, Solanum dulcamara L., also showed the recrystallization inhibition activity characteristic of the animal thermal hysteresis proteins (THPs), suggesting a possible function for the THPs in this freeze tolerant species. Other potential functions are discussed. Antibodies to an insect THP cross reacted on immunoelectroblots with proteins in S. dulcamara stem fluid, indicating common epitopes in the insect and plant THPs.

Introduction Thermal hysteresis producing antifreeze proteins lower the freezing point, but not the melting point, of water thus producing a difference between the freezing and melting points which is termed thermal hysteresis [1,2]. The unique, usually repeating, sequences of the antifreeze proteins with their preponderance of hydrophilic side chains (sugars in the glycoprotein antifreezes, hydrophilic amino acids in the others) apparently allow the proteins to hydrogen bond to the surface of a potential seed ice crystal, probably at step sites, and thereby inhibit crystal growth by forcing an increased radius of curvature of the advancing ice front. This effectively lowers the freezing point via the Kelvin effect [3,4]. Among a n ~ a l s , antifreeze proteins are most often found in freeze avoiding species where they function to lower the freezing point a n d / o r promote supercooling of body water. Antifreeze proteins

t Present address: Department of Basic Science, Univcrsidad National de Lujan. Argentina. Abbreviation: THP, thermal hysteresis protein. Correspondence: J. Duman. Department of Biological Sciences, University of Notre Dame. Notre Dame. IN 46556. USA

have been most thoroughly studied in cold water marine teleost (boney) fishes [1,2,5-8]. In addition, many terrestrial arthropods (including certain species of insects, spiders, centipedes and an Antarctic mite) also produce these proteins as a component of their suite of subzero temperature adaptations [9]. Levels of thermal hysteresis activity generally range from 1 to 1.5°C in fish and from 2 to 6°C in insects. In addition to depressing the freezing point without significantly lowering the vapor pressure, the antifreeze proteins can promote supercooling [10] and inhibit ice nucleators [9,11]. Another effect of the antifreeze proteins, which is a potential advantage to freeze tolerant organisms (those that can survive freezing of their extracellular water) is the recrystallization inhibition activity of both the fish [12,13] and insect [14] proteins, even at concentrations well below those required to detect thermal hysteresis activity. Recrystallization, the redistribution of ice crystal size (i.e. some crystals grow in size while others decrease) which commonly occurs during thawing and long term holding of aqueous solutions at high subzero temperatures, may cause tissue damage through mechanical disruption by growing crystals [ 15]. Because thermal hysteresis proteins (THPs) are so common in poikilothermic animals from cold regions, it seemed likely that these proteins may also have evolved in cold adapted plants. However, THPs have not previ-

20O ously been reported from plants. This study demonstrates the presence of THPs in several plant species. Materials and Methods

Plant material. Plants were collected from the field over the autumn and winter of '90-'91 in the vicinity of South Bend, Indiana (northern Indiana, southwestern Michigan) after the first killing frosts of the season, which occurred in mid-October, in the autumn collections emphasis was given to those plants, or portions thereof, which had apparently escaped freeze damage and had thus demonstrated some cold tolerance. Most early collections were made in old field habitats where the early frosts had their greatest impact. Most plants collected were herbaceous perennials. Fluid samples, ranging in volume from a few to several hundred microliters were collected from various organs. The stems, leaves of overwintering rosettes, roots, tubers, etc. as appropriate for the species were excised and the expressed fluid collected in a glass capillary tube. In some cases, especially with stems, the volume of the exudate expressed at the cut was increased by squeezing the stem. Note that it is impossible to know if the samples collected were primarily extracetlular or intracellular, apoplastic or symplastic, xylem, phloem or various combinations of these fluid compartments. Exudates were centrifuged at low speed to remove debris and the sample screened for the presence of thermal hysteresis activity. Thermal hysteresis acticity. The presence of thermal hysteresis (a difference between the freezing and melting points) activity in a sample was determined using the technique of DeVries [2]. A 3-6 /zl volume of sample was sealed in a I0 tzl glass capillary tube. A small seed crystal ( ~ 0.25 mm diameter)was formed in the sample by spraying with a spray-freeze (Cryokwik), and the sample placed in a refrigerated (alcohol) viewing chamber in which the temperature was finely controlled (+ 0.01°C). The seed crystal was viewed through a microscope. The temperature of the bath was raised 0.020C/5 min until the crystal disappeared, and this temperature was taken as the melting point of the sample. The bath temperature was then lowered a few hundredths of a degree below the melting point, another small crystal introduced in the sample, and the sample returned to the chamber. After equiIibration, the bath temperature was lowered slowly (0.05°C/2.5 rain) and the size of the seed crystal monitored. In samples without thermal hysteresis proteins, the crystal will immediately grow noticeably and thus the melting and freezing points are egsentially identical (within 0.02°C) as theory predicts. However, if thermal hysteresis proteins are present the crystal size remains stable as the temperature is initially lowered below the melting point. Eventually spear shaped crystals grow rapidly,

instead of the slow growth of hexagonal crystals common in the absence of antifreeze proteins. This temperature where crystal growth appears is taken as the hysteretic freezing point. Note that since a seed crystal is present this is not the supercooling point (i.e. nucleation temperature) of the sample. The difference between the melting and freezing points (thermal hysteresis gap) is dependent upon the specific activity and concentration of the antifreeze proteins. The hysteresis gap in the blood sera of winter fishes is usually ,--, 1.5°C, while in the hemolymph of certain overwintering insects it is usually 3-6°C. Values reported in this study are the averages of at least three measurements on a given sample. Recrystallization inhibition. Recrystallization inhibition activity, another characteristic of animal (both fish and insect) antifreeze proteins, was determined in fluid from the bittersweet nightshade, Solanum dulcamara L., using the 'splat cooling' technique of Knight et al. [13]. Samples (10 ~! volume) were frozen quickly at -80°C (dry ice temperature) and then annealed at - 8 ° C for varying periods. An absence of the usual increase in average crystal size after annealing at the higher temperature indicates recrystallization inhibition. Freeze tolerance. The ability of S. dulcamara to survive freezing of the body fluids (freeze tolerance) was determined in plants collected in mid-March. Supercooling points of cut tips ( ~ 7-8 cm lengths) were determined. The cut ends were treated with vaseline to prevent inoculative freezing, thermistors were attached to the stems, the stems placed in a refrigerated chamber and cooled at a rate of 0.25°C/min. The supercooling point temperature was identified by the release of the heat of fusion upon freezing. The stems were then held for 24 h at a temperature (-12°C) below the lowest supercooling point measured for any of the stems. The stems were removed from the freezing chamber and thawed at 4°C. The stems were then placed in rooting soil and placed in a growth chamber at 22°C and a 14L/10D photoperiod. The ability of the stem to root and to produce leaves within 3 weeks after freezing was taken as evidence of survivorship (i.e. freeze tolerance). Stems which had not been frozen were otherwi~ treated as described above and served as controls. hnmunoelectroblots. Polyclonal antibodies to purified insect (Tenebrio molitor) antifreeze proteins were used to probe immunoblots (Western blots) to determine whether these antibodies cross reacted with proteins in fluid collected from the bittersweet nightshade, Solanum dulcamara, thus providing evidence of whether the plant and insect thermal hysteresis proteins share common epitopes. Antiserum was raised in male New Zealand white rabbits [16,17]. Stem exudate from S. dulcamara was run on 7.5 and 10% SDS-PAGE gels

201 along with high- and low-molecular-weight markers (Bio-Rad), respectively. Portions of the gels were stained for protein with Coomassie blue [18]. Proteins were transferred from the remaining portion of the gel onto nitrocellulose paper (Bio-Rad, 0.45 pm) by standard eleetrophoretic blotting techniques [19], and the transblotted nitrocellulose paper was probed with the anti-Tenebrio antifreeze antiserum. Antigen-antibody complexes were visualized with an amplified (streptavidin-biotinylated) alkaline phosphatase conjugated goat anti-rabbit lgG detection system (Bio-Rad), run according to the suppliers instructions. Controls consisted of transblotted nitrocellulose paper treated with the second antibody, but not the primary anti-Tenebrio antifreeze antiserum. Pre-immune serum from the rabbit in which the anti-T¢;~ebrio antifieeze antibodies were raised was also used as a control. To further investigate the specificity of the cross reactivity of the anti-Tenebrio antiserum with S. dulcamara stem exudate, antiserum (10% v/v) was added to the exudate from winter collected stems and the effect of the antiserum on thermal hysteresis activity determined. Exudate to which Tris-NaCl buffer (25 mM Tris, 100 mM NaCI, pH 7.5; 10% v/v) was added sewed as a control. Proteinase and dithiothreitol treatments. To provide further evidence of the proteinaceous nature of the S. dulcamara thermal hysteresis factors, fluid collected from the stem of S. dulcamara in late November was treated with a proteinase or with dithiothrcitol (which breaks disulfide bridges), and the effects on thermal hysteresis activity checked. The proteinase was a nonspecific bacterial proteinase from Streptomyces griseus (Sigma, Type VI bacterial proteinase). Treatment with dithiothreitoI for reduction of disulfide bonds was conducted according to Konigsberg [20].

Results

Thermal hysteresis positice plants. The screening of plants from the vicinity of South Bend, Indiana which was conducted over the autumn and winter of '90-'91 identified sixteen thermal hysteresis positive plants. Table I shows the results of studies on one of these, the bittersweet nightshade, Solanurn dulcamara L. This plant develops as a small vine-like structure with woody stems. At the time of the first collection in mid-November, exudate taken from the primary growth stem of the previous summer had --- 0.45°C of thermal hysteresis. This level of hysteresis was maintained throughout the winter, decreased in the spring (3/7) and by late May the hysteresis gap had disappeared. Fluid from secondary browth stem consistently had less thermal hysteresis than did that from primary growth stem, however, because of the uncertain nature of the fluid compartment(s) contributing to the samples (See Materials and Methods.) these small differences in activity in different plant organs should not be emphasized. Fluid from berries, particularly from somewhat dehydrated berries, also had thermal hysteresis. S. dulcamara in the field lost their leaves by November, however, a plant which had been brought into the laboratory in mid-October and held at 3°C and a short photoperiod (10L/14D) for 3{I days retained its leaves. Table !I shows the thermal hysteresis activity in fluid from various organs of this plant. It is interesting that fluid from the leaves had thermal hysteresis activity since this suggests that the thermal hysteresis proteins function in the leaves in the early autumn prior to their dropping and/or the proteins are translocated from "the leaves to other regions ol the plant. Values for the secondary and primary growth stem were similar to those of field collected plants.

TABLE I

Comparisons of thermal hysteresis (melting point minus freezing point) of fluid from different regions of the bittersweet nightshade, Solanum dulcamara, collected from the field at t'arious times of the year from Not'ember. 1990 to May, 1991 Values are averages o f a m i n i m u m o f t h r e e m e a s u r e m e n t s o f a single sample. N u m b e r s in p a r e n t h e s e s indicate the n u m b e r of plants sampled on that collection date. W h e r e multiple s a m p l e s w e r e taken the values are m e a n s £ standard deviations. .

Date

Organ .

.

.

Freezing point

T h e r m a l hysteresis

(°C)

(°C)

(°C)

1"I/i 9 (2) "" 1 2 / 5 (2) 2/2.5 ( ! ) 3 / 7 (4) 5 / 2 4 (4) 1 2 / 5 (1)

-

-

11/19

- 1.12

0.45 __+0.02 0.40 ± 0.03 0.41 0.28 ± 0.05 0.02 +__0.00 0.21 0.16 0.37 4-__0.08

.

Stem (primary growth)

Stem (secondary growth) Berries (hydrated) Berries (dehydrated) ,,.

. . , .

Melting point

(1)

I 1/ 19 (3) , ,

,

1.87 + 0.{1I 1.29 ± 0.01 1.42 1.52 4- 0.06 0.75 + 0.04 1.06

- 2.26 ± 0.08 .

.

.

.

.

.

2.32 ± 0.04 1.69 ± 0.01 1.83 !.80 4- 0.05 0.77 ____0.05 !.27 - 1.28 - 3.03 ± 0.04

202 TABLE I!

Comparison of the thermal hystereds (melting point mintt.~freezing point) in fluid from carious organs of one indiridual Solanum duicamara which had been collected in mid-October 1990, brought into tit(' lahoratot3' and cold (3°Ct acclimated under a short photoperiod (IOL /14DI for I month Values are means + standard deviation of multiple samples (n = number in parentheses) taken from this one plant. Organ

Melting point (°C)

Freezing point (°C)

Thermal hysteresis (*C)

Stem ( p r i m a r y ) (3)

- 1.88±0.12 - ! .81 _+0.07

- 2.28±0.16

0.40±0.10

- 2.10 _+0.03 - i .3() ± 0.02 - 1.50 ± 0.08

0.29 ± 0.04 0.29 ± 0.02

Stem (secondary) (2) Leaf (4)

Petiole (2)

- ! .01 _+{).(}7 - 1.26 + 0.09

Thermal hysteresis proteins characteristically affect the shape of crystals which grow at the hysteretic freezing point [23]. Rather than the normal hexagonal crystals, growth in the presence of animal thermal t,~teresis proteins usually occurs as ~pear-like crystals, oi ~t low protein concentrations as bipyramidal crystals. As might be expected with the low thermal hysteresis activities present in S. dulcamara, and other plants to be discussed below, growth at the hysteretic freezing point often took the form of bipyramidal crystals. S. dulcamara stem fluid with thermal hysteresis activity of 0.330C was treated with a nonspecific bacterial proteinase or with dithiothreitol. Either treatment completely eliminated the thermal hysteresis activity, demonstrating that the thermal hysteresis factor(s) is proteinaceous and that it contains disulfide bonds which are required for activity. The hysteresis gap of -., 0.40°C in S. dulcamara primary growth stem was greater than that generally seen in other thermal hysteresis positive plants. However, as noted earlier these small differences should probably not be emphasized. Values for a single individual of the narrow leaved plantain, Plantago lanceoiata L., collected Oct. 31 are shown in Table I!I. This level of thermal hysteresis activity was maintained in subsequent collections made through colder weather in December (Table IV), but P. lanceolata collected in June had lost activity.

0.24 ± 0.01

The thermal hysteresis found in the wood aster, Aster cordifolius L., collected Oct. 31 is presented in Table V. The plants were still flowering at this time and had been only minimally damaged by the frosts. Table VI presents data which demonstrate thermal hysteresis activity in several additional plants. Levels of activity range from approx. 0.20 to 0.35°C. Since most of the plants listed are dicots, the two grasses Poa annua L. (speargrass) and P. pratensis L. (Kentucky blue grass) are perhaps noteworthy. However, not many monocots were tested during the screening process, and thus the lesser number of thermal hysteresis positive monocots identified may simply represent sampling bias. Several of the plants listed are common weeds, including a second plantain species (Piantago major L.), chickweed (Steilaria media L. Viii.) and dandelion (Taraxacum officinale Weber). Thermal hysteresis was also present in three crop plants known to have cold tolerance capabilities. Young cabbage and Brussels' sprout plants which were cold acclimated (4°C) under a short photoperiod in the laboratory had activity. Also the axillary buds ('eyes') of tubers of potato, Solanum tuberosum L. (var., Keenan), which were dug out of the ground in a field plot in midFebruary had activity. Unfortunately, fluid was not taken from the tubers themselves at the time of collection, however, fluid taken from the tubers after they had been held in the laboratory at 40C for ~ 2 months lacked activity. Also, newly emerged potato plants sampled in May did not have activity. Thus, it may be that

TABLE Iii

Compari.~ions of thermal hysteresis (melting point minus freezing point) in fluid from rarious orgattv of one mdit'idual narrow/era ed plantain, Plantago lanceolata, collected from the field on October 31, 1990

TABLE IV

Values are means zl: standard deviatkm of multiple samples ( n = number in parentheses) taken from this one plant.

Thermal hysteresis (melting point minus freezing point) in stems of the narrow lea~'edplantain, Plantago lanceolata, collected from the field at rarious times of the year from late October 1990 to June 1091

Organ

Values are means ± standard deviation. Numbers in parenthesis indicate the number of plants sampled on that date.

Melting point

Freezing point

(°c)

(*c)

Thermal

hysteresis

(°C) Root (3)

- 0.63 _+0.06

- 0.84 ± 0.07

Stem (2) Lateral Bud

- 0.69 + 0.01

-0.97±0.01

0.21 +0.01 0.28 ± 0.04

(3) Leaf (2)

-0.61 _+0.08

- 0.96 ± 0.05 - 0.91 _ 0.117

0.35 _+0.08 0.215= (I.02

- 0.70 +_0.05

Date

Melting point (°C)

Freezing point (°C)

Thermal hysteresis

10/31 (3) 11/26(7) 12/6(41 6 / 7 (3)

-0.62+0.03 -0.89+0.13 -0.96_+0.30 - 0.45 _+0.02

-0.93+0.06 -!.11+{).12 - !.22_+0.22 - 0.47 _-20.02

0.31 +0.05 0.22±0.06 0.26_+0.09 0.02 ± 0.00

203 TABLE V Thermal hysteresis (melting point minus freezing point) in carious organs of the w~m¢l aster, Aster cordifolius, collected from the field October 31, 1990 Values are means_slandard deviations. Numbers in parentheses indicate the number of plants sampled. Organ

Melting point (°C)

Freezing point (°C)

Thermal hysteresis (°C)

Slem (4)

- 0.75 + 0.03

Root (4)

-0.66±0.13

Leaf(4)

-0.404-0.01 - 1.08_+0.03

- 0.95 _+0.00 -0.84±0.18 -0.524-0.02 - !.394.0.01

0.20 + 0.03 0.184-0.05 0.I24.0.03 0.31 _+0.01

Flower (2)

only the axiilary b u d s on the o v e r w i n t e r i n g t u b e r s in the soil have activity. Several t h e r m a l hysteresis positive species (T. officihale, P. m a j o r a n d as m e n t i o n e d b e f o r e the nightshade, potato, a n d P. lanceolata) w e r e tested for activity in the s u m m e r . Activity was not f o u n d in any species t e s t e d d u r i n g the w a r m m o n t h s . Recrystal/ization inhibition. A c o m m o n feature o f a n i m a l t h e r m a l hysteresis p r o t e i n s is t h e i r ability to inhibit recrystallization. To d e t e r m i n e w h e t h e r a plant t h e r m a l hysteresis protein can do likewise, cxudates t a k e n from the stems o f both w i n t e r and s u m m e r bittersweet n i g h t s h a d e were c h e c k e d for recrystallization i n h i b i t i o n activity. Fig. 1 c o m p a r e s rccrystallization in d r o p l e t s o f w i n t e r a n d s u m m e r collected S. dulcamara a n d distilled water. S a m p l e s were frozen rapidly by i m p a c t onto a l u m i n u m at - 8 0 ° C a n d then a n n e a l e d for 6 h at - 8 ° C . Note that the grain size in the w a t e r a n d in the s u m m e r n i g h t s h a d e s a m p l e s is m u c h l a r g e r t h a n that of the w i n t e r n i g h t s h a d e ,

d e m o n s t r a t i n g recrystailization i n h i b i t i o n in the latter. T h e w i n t e r n i g h t s h a d e s a m p l e was d i l u t e d 1 / 1 0 a n d t h e r e f o r e the recrystallization i n h i b i t i o n activity o f the u n d i l u t e d s a m p l e would be even m o r e potent. T h e lack o f recrystallization inhibition in the s u m m e r sample coincides with the a b s e n c e of t h e r m a l hysteresis activity in the s u m m e r . Freeze tolerance. T h e ability o f S. dulcamara to survive freezing was assessed in m i d - M a r c h . T h e term i n i of p r i m a r y growth stems (7.5 cm length) were collected from p l a n t s in the field a n d t h e i r supercooling points d e t e r m i n e d . T h e m e a n s u p e r c o o l i n g point ( n = 12) was - 8.5 +_ 2.8 (range = - 4.0 to - 11.5°C). T h e stems were t h e n held for 24 h at - 1 2 ° C , a t e m p e r a t u r e below the lowest s u p e r c o o l i n g point, and h a n d l e d as d e s c r i b e d in M a t e r i a l s a n d Methods. H a l f o f the stems rooted a n d p r o d u c e d leaves, d e m o n s t r a t ing their t o l e r a n c e to freezing at - 12°C. W h i l e - 12°C is not an especially cold t e m p e r a t u r e note that this test was c o n d u c t e d in m i d - M a r c h , r a t h e r late in the winter for this area, a n d it is t h e r e f o r e likely that the plants w o u l d have t o l e r a t e d m o r e e x t r e m e t e m p e r a t u r e s in mid-winter. Immunoblots. Fig. 2 shows the results of imm u n o b l o t s ( W e s t e r n ) o f S. dulcamara s t e m exudate run on 10% S D S - P A G E , electroblotted to nitrocellulose p a p e r a n d p r o b e d with anti-Tenebrio antifreeze protein a n t i s e r u m . F o u r protein b a n d s were c o m m o n to the control ( p r o b e d only with the ~econd antibody, i.e. lacking the p r i m a r y anti-insect a n t i f r e e z e antis e r u m ) and the test blot and thus these r e p r e s e n t the presence of e n d o g e n o u s p h o s p h a t a s e a n d / o r nonspecific b i n d i n g o f the s e c o n d antibody. ( W e s t e r n blots p r o b e d with the p r e - i m m u n e rabbit s e r u m likewise showed only t h e s e s a m e four b a n d s . ) However, six

TABLE V! Thermal hysteresis (melting point minus freezing point) in fluid from caritnls plants Number in parentheses indicate the number of plants sampled. Values are means, if multiple plants were sampled, +standard deviation. Measurements on all individual samples were replicated a minimum of three times. Species

""

Date

"" Organ

Melting point (cC)

Freezing point (°C)

Thermal hysteresis , , ,

Poa annua'iii Poa pratensis (1) Taraxacum officinale (1) Hydroph~'llum :'irginianum ( 1) Stellaria media (7) Brassica oleracea cabbage (3) * B r u s s e l ' s s p r o u l (2) * V/o/a sp. (2) Euphorbia serpens (1) Alliara petiolata (1) Barbarea rulgaris (1) Plantago major (1) Solanum tuberoslon (3) • Cold acclimated (5°0 for 4 months.

1i / 8 12/4 12/3 I 1/7 I 1/8 10/31 12/3 I !/21 12/16 ! 1/21 2/15

blade blade leaf root leaf & stem leaf leaf leaf leaf & stem stem leaf stem Tuber stem bud

-

1.12 0.60 0.64 0.64 0.58 _+0.02 - 0.96 ± 0.05 - 1.134.0.03 -0.98 4.0.01 - 0.45 - I. 12 -0.65 - 1.16 - 0.22 _+0.02

-

1.35 0.93 0.87 0.87 0.79 _+0.04 - I. 174. 0.03 - 1.37 ± 0.04 - i.21 + 0.00 - 0.65 - !.42 - 1.00 - !.42 - 0.42 ± 0.02

0.Z~ 0.33 0.23 0.23 0.21 ± 0.05 0.21 + 0.08 0.24 _+0.01 0.23 _ 0.00 0.20 0.30 0.35 0.26 0.20 +_0.02

204

A

•

71:. ~:~

:

"~ ":" "'l*; ' ~gk :,-.... .~..'" ~ -~.

•

.)

~

~." e-..

al

-

41mill.

i ,r J l l l l D

....-. :._- 2

'."lllP

:. :' (,,.\

. . .

-

Fig. 2, Western blots from 10% SDS-PAGE gels of winter S. dulcamara stem fluid probed with anti-Tenebrio (insect) antifreeze protein a n t i , r u m (primary antibody) demonstrating cross reactivity of the antibodies with certain S. dutcamara proteins. Lane B shows the normal Western blot probed with the primary antibody followed by second antibody (amplified, streptavidin-biotinylated, alkaline phosphatase conjugated goat anti-rabbit lgG detection system). Lane A was treated only with the second antibody and thus serves as a control for endogenous phosphatase and/or nonspecific binding of the ~ c o n d antibody. Lines between the two lanes identify common bands in the two lant:s. Lines to the right of lane B identify protein bands unique to lane B (i.e. those with which the primary antibody has complexed). Origin is at the top.

rfp -..'2~'.~,~-~,~"-I-.-~

~:~,~,~..,~.

~:~.

.~.~,

~''7_ .

~

,

i~..

~,,

........

....

~:

,

.....

.:~

.

. . . . .

Fig. I. Demonstration of recr~stallization inhibition activity in stem fluid of winter S,lanum dulcamara. Droplets (top = distilled water; center --= fluid from stem of winter S. dulcamara: bottom = fluid from stem of summer S. duicamara) 'splat cooled' at -80°C and then annealed at - 8 ° C for 6 h. Note the smaller grain size in the winter ,*i. dulcamara fluid, indicating the presence of recrystallization inhibititm activity, and the absence of this activity in the summer sample and in the water. Diameter of splats is ~ ] cm.

other bands were unique to the test blot probed with the primary anti-insect antifreeze antiserum, indicating that these have epitopes in common with the insect THP. These have molecular masses of 140, 62, 47, 40, 34 and 31 kDa, as determined by ti,ese blots from 10% SDS-PAGE gels and also from 7.5% SDS-PAGE (data not shown) using low and high molecular weight markers, respectively. Both fish [8] and insects [9] typically produce multiple sizes of THPs with similar primary structures. Therefore the apparent presence of six THPs in S. duicamara is not unusual. However, the larger nightshade THPs are considerably bigger than the Iargest known animal THP which is ~ 34 kDa [8]. Addition of 10% (v/v) anti-Tenebrio antiserum to S. dulcamara stem exudate with thermal hysteresis activity of 0.29°C eliminated the activity. This result establishes the specificity of the antiserum for the S. dulcamara THPs. Discussion With the exception of water trapped in certain gels [21], thermal hysteresis activity is known to occur only

205 in the presence of the thermal hysteresis producing proteins which function as antifreezes in many poikilothermic animals. Therefore, even the comparatively low levels of thermal hysteresis reported in the plants studied in this survey are highly suggestive of the presence of thermal hysteresis proteins. Also, the typical bipyramidal shape of growing crystals in the plant fluids with low thermal hysteresis, along with the recrystallization inhibition activity of winter collected S. dulcarnara stem fluid, indicate the presence of thermal hysteresis proteins. Elimination of thermal hystere:;is activity in S. dulcamara stem fluid by treatment with a bacterial proteinase also demonstrates the proteinaceous nature of the plant thermal hysteresis factor, while the inactivation of the factor by treatment with dithiothreitol indicates the presence of disulfide bonds which are required for activity. THPs with disulfide bonds are known to occur both in fish [7,8] and in insects [9]. Evidence for the presence of similar epitopes in the S. dulcamara and an insect (Tenebrio molitor) THP is provided by Western blots (Fig. 2) which demonstrate proteins in S. dulcamara to which anti-insect antifreeze antibodies cross react and by the elimination of thermal hysteresis activity in stem exudate to which the anti-insect antifreeze antiserum was added. While thermal hysteresis proteins have not previously been reported in plants, the number and phylogenetic diversity of thermal hysteresis positive plants identified in this study suggest that these proteins may be fairly common in cold adapted angiosperms. Kurkela and Franck [22] recently described a gene in Arabidopsis thaliana L. which is induced by cold acclimation. The deduced 6.5 kDa polypeptide product of this gene was described as having sequence homologies to winter flounder (fish) antifreeze protein. At this time it is not known whether this Arabidopsis protein is a thermal hysteresis protein. So-called 'freezing inhibitors" were described in freeze tolerant plants by Oiien and collaborators [23,24]. These should not be confused with thermal hysteresis proteins. These 'freezing inhibitors' are polysaccharides (not proteins) which modify 'ice structure' after nucleation has occurred. They apparently do not inhibit the initiation of freezing. Some years ago Dr. Olien kindly provided one of the authors (J.G.D.) with a sample of 'freezing inhibitor'. The sample did not have thermal hysteresis activity. The function of the THPs in plants is not obvious. Most antifreeze protein producing animals are freeze avoiding and these proteins function to depress the freezing point a n d / o r promote supercooling. While the nightshade was the only plant in this study identified as being freeze tolerant, it is likely that all or most of the others are also freeze tolerant as this is the case with most overwintering cold tolerant plants from temperate regions [25]. This, along with the low thermal

hysteresis activity of the plants, indicates that the function(s) of the thermal hysteresis proteins in plants differs somewhat from that in most animals. An additional problem associated with assigning a function to the plant THPs is that we know little about their location within the plant. The fluid samples which we collected from various excised regions of plants can only be used to determine the presence or absence of thermal hysteresis activity in the plant. It may be that the Iow activities in our samples result from dilution during the collection procedure of a low volume high activity fluid compartment by a larger volume fluid compartment which lacks activity. In spite of these problems, the absence of both thermal hysteresis, and the more sensitive recrystallization inhibition activity, in the summer indicates that the THPs function as a low temperature adaptation. The recrystallization inhibition activity in stem fluid from freeze tolerant winter S. dulcamara suggests one possible function since recrystailization can cause freeze damage [15], although recrystallization damage has not yet been reported in plants. Other possible functions of the plant THPs are indicated by the studies of Cutter et aI. [26] which showed that introduction, by vacuum infiltration, of winter flounder antifreeze protein into the leaves of canola decreased both the amount of water frozen at any given temperature and the rate of ice crystal formation. Both of these effects might be advantageous in a freeze tolerant species. it was reported recently that glycoprotein antifreezes from Antarctk. fish protect pig oocytes from freeze damage [27]. Wi,le the mechanism of this cryoprotection is uncertain, the authors suggested that the glycoproteins interact with and protect the cell membrane. Interestingly, the glycoproteins also stabilize the pig oocyte membrane at low, but above freezing, temperatures [28]. Unpublished data (Urrutia, Van Lieshout and Duman) from immunofluorescence histological studies, using the primary antibodies to insect antifreeze proteins which cross react with the thermal hysteresis proteins of S. dulcamara (Fig. 2), have identified THPs on the plasma membranes of certain cell types in the stem of S. duicamara. Also, isolated S. dulcamara plasma membrane preparations demonstrate thermal hysteresis activity. THPs on cell membranes, or intracellular THPs, could protect the intracellular fluid from being seeded by extracellular ice, an important protection since intracellular freezing is usually lethal. Although the cell membrane is generally considered to provide a barrier to such seeding, this may not always be the case, particularly if membrane damage ensues as temperatures continue to decline after initial freezing. THPs on the membrane may inhibit seeding across the cell membrane. This protection provided by THPs would be in addition to other

206 cold acclimation-induced cell membrane changes [29,301. To summarize the above, admittedly somewhat speculative, discussion, possible functions of THPs in freeze tolerant plants are recrystallization inhibition, reduced rate of crystal growth, decreased percentage of water frozen at a particular temperature, protection of the cell membrane and prevention of intracellular ice formation. While this study does little to identify the function(s) and location of THPs in plants, the finding that these unique proteins are apparently common in cold adapted plants suggests that they constitute an important and previously unstudied component of the suite of low temperature adaptations of plants. Acknowledgements The National Center for Atmospheric Research is supported by the National Science Foundation. References ! DeVries, A.L, (1972) Science 172, 1152-1155. 2 DeVries, A.L. (1986) Methods Enzymol. 127, 293-303. 3 Raymond, J.A., Wilson, P.W. and DeVries, A.L, (1989) Proc. Natl, Acad. Sci. 86. 881-885. 4 Knight, C.A., Clleng, C.C. and DeVties, A.L. (1991) Biophys. J. 59, 409-418. 5 Feeney, R.E. and Burcham, T.S. (1986) Annu. Rev. Biophys. Chem. 15. 59-78. 6 Davies. P.L, Hew, C.L. and Fletcher, G.L. (1988) Can. J. Zool. 66, 261 i-2617. 7 Davies, P.L. and Hew, C.L. (1990) FASEB J. 4, 2460-2468. 8 Cheng, C.C. and DeVries, A.L. (1991) in Life Under Extreme Conditions (di Prisco, G , ed.), pp. 1-14. Springcr-Verlag, Berlin.

9 Duman, J.G., Xu, L., Neven, L.G.. Tursman, D, and Wu. D.W. 11991) in ln~cls at Low Temperature (Lee, R.E. and Denlinger, D.L,. eds.)o pp. 94-127. Chapman and Hall. I1| Zachariassen. K.E. and Husby, J.A, (1982) Nature 298, 865-867. 11 Parody-Morreale, A., Murphy, K.P., DiCerca E., Fall, R., DeVries, A.L. and Gill. SJ. (1988) Nature 333, 782-783. 12 Knight, C.A., DeVries. A.L. and Oolman, L.D. (1984) Nature 308, 295-296. 13 Knight, C.A., Hallet, J. and DeVries. A.L. 11988) Cryobiology 25, 55 -60. 14 Knight, C.A. and Duman, J.G. (1986) Cryobiology 23, 25b-262. 15 Mazur, P. (1984) Amer, J. Physiol. 247, C125-C!42. 16 Wu, D.W., Duman, J.G. and Xu, L. (1991), Biochim. Biophys, Acta 1076. 416-420. 17 Xu, L., Duman, J.G.° Goodman, W.G. and Wu, D.W. (1992) Comp. Biochem. Physiol. B, in press. 18 Faitbanks. G,. Stek. T.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2606-2617. 19 Towbin, H.T., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 20 Konigsberg, W.H. (1972) Methods En~mol. 25. 185-188. 21 Bloch, R., Waiters, D.H. and Kuhns, W. (1963) J. Gen, Physiol. 46, 605-615 22 Kurkela, S. and Franck, M. (1990) Plant Molecular Biology 15, 137-144. 23 Shearman, L.L., Olien, C.R., Marchetle, B.L. and Ever~n, E.H. (1973) Crop. Sci, 13, 514-518. 24 Olien, C.R. (1965) Cryobiology 2, 47-54. 25 Sakai, A. and l.,archer,W. (1987) 321 pp., Springer-Verlag. 26 Cutter, A.J., Saleem, M., Kendall, E., Gusta, L.V., Georges, F. and Fletcher, G.L. (1989)J. Plant Physiol. 135, 351-354. 27 Rubinsky, B., Afar, A. and DeVries, A.L. (1991) Cryo-Lett. 12, 93-106. 28 Rubinsky, B., Arav, A., Mattioli, M. and DeVries, A.L, (1990) Biochem. Biophys. Acta 173, 1369-1374. 29 Steponkus, P.L. and Lynch, D.V. (1989) J. Bioenerg. Biomembr. 21, 21-41. 30 Steponkus, P.L., Lynch, D.V. and Uemura, M. (199{))Phil.Trans. R. Soc. Lond. B 326, 571-583.