Antarctic Plants List

Characterisation of anti-freeze activities in Antarctic plants1 | Journal of Experimental Botany

The Deschampsia antharctica and Collobanthus quenzensis are the only plants that have inhabited the Maritime Antarctic characterised by permanent low ambient air humidity and common frost..... In order to gain an understanding of how the plants can withstand year-round freeze conditions, the frost protection effect was investigated in Apoplast® extract from non-acclimatized and cool acclimatized Antarctic plants.....

Observation of the form of icecrystals growing in dilutions of the extract revealed that D. antharctica had anti-freeze action, but C. quitenensis did not..... D. Antarctic showed anti-freeze agent activities in non-acclimatized state and this activities rose after acclimatization..... Antarctic D. frost protection was unstable to protect against protein lysis and high temperatures, over a broad pH area and associated with more than 10 kilodia of molecule mass.....

The results show that D. antharctica generates anti-freeze protein that is excreted in the apoplasty..... In the SDS-PAGE study, the anthropoplastic extract from the D. antharctica 13 polypeptide..... The conclusion is drawn that D. antharctica accumulated AFSs as part of its freeze toleration mechanisms..... It is also the first system in which anti-freeze agent activities have been monitored to be constituent.....

The Antarctic hair grass (Deschampsia of the Poaceae ) and the Antarctic pearl word (Colobanthus quensis of the Caryophyllaceae) are the only two plants that have colonised the Antarctic marine landscape (Lewis Smith, 2003). As the Antarctic marine environments are constantly cool, these perennials must live at a constantly low temperatures (Day et al., 1999; Alberdi et al., 2002; Lewis Smith, 2003)......

Though D. antharctica and C. quenzensis are covered by powder for 6-7 month a year, they are often subjected to freeze-thaw cycling in the summers when the average daytime temperatures are 4 °C (Day et al., 1999; Lewis Smith, 2003)..... These two plants can be grown in summers because they keep about 30% of the optimum net synthesis at 0°C (Xiong et al., 1999).

Throughout the long summers, D. antharctica also collects the high amounts of saccharose and propylene required to maintain high resistance to stresses (Bravo et al., 2001). Astonishingly, D. antharctica and C. quinensis react very differently to icy temperature. Non-acclimatized D. antharctica has a LT50 of -12 C under controled circumstances, and plants acclimatized to cool survives -26 C (Bravo et al., 2001).

C. quensis, on the other hand, is not freeze-tolerant. When winters approach in moderate climates, perennials cause a number of changes in structure and biochemistry that increase their frost stability but also slow down their habit. Unlike plants from moderate areas, Antarctic plants are always in a hard state, as shown by the fact that in hot summers they have the same type of LT50 as plants acclimatized under lab condition (Bravo et al., 2001).

The results show that Antarctic plants need to thrive and multiply, while at the same time showing high levels of abstractionism. Therefore, it is of interest to study not only the mechanisms of freeze toleration but also the control of this phenomenon in Antarctic plants. Numerous wintering species, among them invertebrates, aquatic plants, fungus and bacterial, are accumulating anti-freeze protein (AFPs) that freeze to the faces of icecrystals and retard their proliferation (Duman and Olsen, 1993; Ewart et al., 1999; Griffith and Yaish, 2004).

It is not yet clear what the particular importance of ASPs is in plants that remain freeze-dried. D. antharctica as well as C. quenzensis should contain ion recrystallisation regulators in homogeneousates of aboveground tissue (Doucet et al., 2000). To determine whether amylopectin is present in Antarctic plants, it is important to investigate the antifreezing effect in such a way that it is clear whether the protein actually binds to the icy surfaces.

The aim of this research was therefore to investigate anti-freeze activities with the Antarctic icecrystal modifier which clearly differentiates whether the two Antarctic vessel plants are producing protein that binds to glaciers. and Colobanthus Quietensis (Kunth) Bartl. Non-acclimatized plants grew at 15 C at a photonic flow of 200. ?mol m-2 sec-1 photosynthesis supplied by a mix of cold whites fluorescents and incandescents for 21 hours a day.

The plants were acclimatized by placing them in a growing room at 4 C, 200 ?mol photon m-2 s-1 and a day length of 16 hours for 3 wards. Appoplastic protein was derived from foliage, as described by Hon et al (1994). Briefly, the sheets of D. antharctica were sliced into 1.5 cm length and C. quinensis sheets into 0.5-1.

Blot the leaves in a 20 ml cylinder which was placed in a 50 ml centrifugal vial and centrifugated at 4 ºC for 30 minutes at 9000 grams and 7000 grams at D. antharctica and D. quensis. The overall content of AP was determined in the extract using the Bradford technique (Bradford, 1976), which Bio-Rad Laboratories (Mississauga, Ontario, Canada) used as the default binder for buffering cow sarcoma.

All of the alquots of the APAPOPLASTIC EXTRACT of both types were ultrafiltrated with a Centricon YM10 with 10,000 molecules (Millipore Inc., Bedford, MA, USA). Store all Apoplastische Extkte at -20 C until analyzed. In order to test the thermal equilibrium, aliquoted Apoplasttic extract from D. antharctica plants acclimatized to low temperatures were placed in microjoint vials and placed in a tempered bain-marie at 20, 40, 60 or 100 °C for 30 minutes.

Every pipe was put on hold and the anti-freeze effect was examined immediately. Influence of pH on anti-freeze activities was analyzed by addition of 1 vol 4x concentrates of Apoplast extracts to 3 vol. solutions with 50 mM Tris-HCl or Tris-Base, according to pH.

Extract was kept for 10 minutes at 20 C at a certain pH value, then put on hold and the frost protection effect examined. The susceptibility to protease was measured by the addition of Proteinase K or Pronase E (Sigma Chemical Co., St. Louis, MO, USA) to Apoplasttic Extract in a total of 1 mg ml-1 protease solution.

Extract was prepared at 20 C and the anti-freeze effect was tested every 30 minutes until complete elimination. The anti-freeze activities were investigated in 10 nl specimens of Apoplastische extract by qualitative observation of the Morphologie of glacial crystal growing in solutions (DeVries, 1986; Hon et al., 1994).

During this test, round icecrystals cultivated in solutions show no frost protection effect, while hexagonal icecrystals indicate the existence of an inorganic blocker for the growing of ices. SDS-PAGE was used to denature and separate protein using Bio-Rad's Mini Protean II system according to the Laemmli methodology (1970).

Both Antarctic plants have very small foliage, making it hard to obtain high apoptotic extracty. It appears that the D-Antarctic has a powerful foliar texture, as it was possible to raise the radial centrifuge up to 9000 grams to gather the low-damaged Apoplasttic Liquid, as the clear, colorless extract obtained shows.

Sheets of D. antharctica gave about 25 ?l of aapoplastic liquid g-1 of virgin matter with a proteinaceous of 0. 15 ?g ?l-1 in unacclimatized and 0. 28 ?g ?l-1 proteins in child-acclimatized sheets. However, it was only possible to centrifugate C. quinensis at 7000 grams without damaging the leaf.

Only 10 ?l of the Apoplastyan liquid g-1 of C. quenensis sheets was obtained and the plasma content was similar in excerpts from non-acclimatized and child-acclimatized sheets (0. 082 and 0. 086 ?g proteins ?l-1, respectively). Therefore, the harvest of freshwater based APAPOPLASTIC proteins was 8 times higher than that of cold-acclimatized C. quensis.

To compare: Secale corereale can be centrifugated at only 800-2000 grams to obtain Apoplastische liquids, since higher centrifuge strengths result in verdant liquids, which indicate a symmetric contaminant (Hon et al., 1994; Yu et al., 2001). Hexagonal shape icecrystals, typical for the growth of glaciers in the midst of AFP ( "DeVries", 1986), produced by freeze coating of D. anarctica.

It is interesting to note that the aperoplastic liquid from sheets of non-acclimatised and cool acclimatised plants showed an antifreezing effect (Fig. 1). Concentration of D. antharctica excerpts by ultrasfiltration did not inhibit icing in the low molecule fractions of the D. antharctica low polymer extraction, which went through the 10 kilodia cutoff ultrasfiltration membranes (Fig. 2A).

The proportion of more than 10 kiloda in molar mass was however 4-fold concentrations and showed a higher frost protection effect, which was shown by more proliferation along the C-axis of the crystall (Fig. 2B). On the other hand, the aperoplastic extract from non-acclimatized and cool acclimatized quinensis sheets showed no retardation of icecrystal formation, but only round, shallow cristals (Fig. 1).

No frost protection activities of C. quensis were observed after ultra-filtration (Fig. 2C). The results show that anti-freeze activities in the D. antarctic were associated with a proportion of more than 10 kilodia1. Frost protection in Apoplastische excerpts of antarctic herbs. Microcrystals of individual icecrystals were investigated in 10 nl specimens of Apoplast extract from leafs of non-acclimatized and child-acclimatized plants of D. antharctica (D. a.) and C. quinensis (C.q.).

Hemming of glacial proliferation was seen in both non-acclimatized and child-acclimatized D. antharctica Apoplasttic extract, but no inhibiting effect was seen in C. quinensis extract. Frost protection in Apoplastische excerpts of antarctic herbs. Microcrystals of individual icecrystals were investigated in 10 nl specimens of Apoplast extract from leafs of non-acclimatized and child-acclimatized plants of D. antharctica (D. a.) and C. quinensis (C.q.).

Hemming of glacial proliferation was seen in both non-acclimatized and child-acclimatized D. antharctica Apoplasttic extract, but no inhibiting effect was seen in C. quinensis extract. Anti-freeze activities in the D. antarctic are associated with substances larger than 10dkDa. Ultrafiltrations were used to concentrate apolytic extract of the D. antharctica (D. a.) and C. quenensis (C. q.), which had been acclimatized at low temperatures, until the volumes were quadruple.

A) The throughflow of D. antharctica was lacking anti-freeze action, while B) the concentrate showed a higher anti-freeze action, suggesting that the action was associated with more than 10 kilodiaolumoles. No anti-freeze effect was found in 4-fold concentrations of quensis essences. Anti-freeze activities in the D. antarctic are associated with substances larger than 10dkDa.

Ultrafiltrations were used to concentrate apolytic excerpts of the D. antharctica (D. a.) and C. quensis (C. q.), which were acclimatized at low temperatures, until the volumes were quadruple. A) The throughflow of D. antharctica was lacking anti-freeze action, while B) the concentrate showed a higher anti-freeze action, suggesting that the action was associated with more than 10 kilodiaolumoles.

No anti-freeze effect was found in 4-fold concentrations of quensis essences. Degree of anti-freeze protection activities was measured between non-acclimatized and cool acclimatized D. Antarctic plant essences by recompartment. Non-acclimatized leafs lose their frost protection effect entirely at a 1:5 thinning, while the aperoplastic leafs extracted from chilled plants show a certain frost protection effect even at a 1:10 thinning ((Fig. 3).

Therefore, the antifreezing effect in Apoplasty extract of sheets acclimatized to low temperature was 2-5 time higher than in non acclimatized sheets. Cryogenic acclimatization enhances frost protection in the D. Antarctic. Appoplastic extract of non-acclimatized and cool acclimatized D. antharctica were thinned with 1, 5 or 10 vol. high-purity HPLC aqueous solution and subsequently tested for anti-freeze effect.

Kalt-acclimatized extract thinned 10 times still had very little acidity, while the acidity was reduced in non-aclimatized specimens thinned 5 times. Cryogenic acclimatization enhances frost protection in the D. Antarctic. Appoplastic extract of non-acclimatized and cool acclimatized D. antharctica were thinned with 1, 5 or 10 vol. high-purity HPLC aqueous solution and subsequently tested for anti-freeze effect.

Kalt-acclimatized extract thinned 10 times still had very little acidity, while the acidity was reduced in non-aclimatized specimens thinned 5 times. To show that anti-freeze activities are associated with APAPOPLASTIC PROTEINES, a range of tests were conducted to assess the susceptibility to proteases and the dependency of anti-freeze activities on temperatures and pH using anti-freeze activities with PAPOPLASTIC EXTRACT from D. ANNARCTICA, which has been acclimatized to CLL.

After incubating the excerpt with protease K for 6 hours or with pronase for 30 minutes, the anti-freeze function was fully removed (Fig. 4A). The anti-freeze action in the thermal trial was reduced at 40 C and fully reversed after 30 minutes of inoculation at 60 C (Fig. 4B).

SDS-PAGE shows that pronase eliminates all existing peptides for 30 minutes (Fig. 5B). In the Antarctic D., the decrease in anti-freeze effect due to protective measures and the denaturing of the skin with high temperatures indicated that the anti-freeze effect was associated with the protein present in the Apoplasttic Extractions.

Susceptibility of the anti-freeze to protectases, pH value and pH value. The susceptibility of APAPOPLASTIC proteins to protectedase activities was measured by the addition of 1 mg ml-1 K or pronase K to apoplast extract from D. ANARCTITA plants acclimatized to low temperatures and incubated at 20ºC for 6 hours and 30 minutes, respectively.

The BSA was investigated as a controlling profile lacking anti-freeze activities. Apoplasty using D. antharctica plants with low acclimatization was performed in a tempered bain-marie at 20, 40, 60 and 100 ºC for 30 minutes. The frost protection effect was not effective at above 40ºC. The effect of pH on anti-freeze activities was analyzed by the addition of 1 vol 4x concentrates of Apoplastin to 3 vol. buffed solution prepared with TRIS-HCl or TRIS-Base, according to pH.

Anti-freeze activities were present from pH 3. 0 to pH 10. 0 and were higher at alkali pH. Subsequent to each session, specimens were tested for anti-freeze activities. Susceptibility of the anti-freeze to protectases, pH value and pH value. The susceptibility of APAPOPLASTIC proteins to protectedase activities was measured by the addition of 1 mg ml-1 K or pronase K to apoplast iced D. ANARCTITA plant extract and incubation at 20ºC for 6 hours and 30 minutes, respectively.

The BSA was investigated as a controlling profile lacking anti-freeze activities. Apoplasty using D. antharctica plants with low acclimatization was performed in a tempered bain-marie at 20, 40, 60 and 100 ºC for 30 minutes. The frost protection effect was not effective at above 40ºC. The effect of pH on anti-freeze activities was analyzed by the addition of 1 vol 4x concentrates of Apoplastin to 3 vol. buffed solution prepared with TRIS-HCl or TRIS-Base, according to pH.

Anti-freeze activities were present from pH 3. 0 to pH 10. 0 and were higher at alkali pH. Subsequent to each session, specimens were tested for anti-freeze activities. The anti-freeze activities in the D. Antarctic are caused by the presence of Apoplastische Proteine. A. Protein from non-acclimatized (NA), non-acclimatized (CA), and 4x concentrate of CA D. antharctica (4×CA) were denatured and severed by SDS-PAGE.

A polypeptide in the D. antharctica Apoplasty extract was decomposed by pronase E (ProE) and was not affected by incubation at pH 10. 0 (S, supernatant level and pH, 15 000 grams of pellets, after centrifugation). The anti-freezing effect seen in APPLASTIC EXTRACT corresponding to the treatment described in (A) and (B).

The anti-freeze activities in the D. Antarctic are caused by the presence of Apoplastische Proteine. A. Protein from non-acclimatized (NA), non-acclimatized (CA), and 4x concentrate of CA D. antharctica (4×CA) extracts were deratured and SDS-PAGE was used to separate them. A polypeptide in the D. antharctica Apoplasty excerpts was decomposed by pronase E (ProE) and was not affected by incubation at pH 10. 0 (S, supernatant level and pH, 15 000 grams of pellets, after centrifugation).

The anti-freezing effect seen in APPLASTIC EXTRACT corresponding to the treatment described in (A) and (B). In order to test whether a chance anti-freeze effect could be demonstrated by a particular type of proteinaceous proteins, the form of icecrystals cultivated in a 1 mg ml-1 BSA negatively controlled 1 mg ml-1 BSA aqueous solutions was investigated. BSA showed round cristals (Fig. 4A) in all tests, which does not indicate blockage of icecrystal-growing.

The anti-freeze activities were constant over the entire pH area of 3 to 10 (Fig. 4C), although the Apoplastische liquid was visible cloudy at pH 9 and 10. Therefore, a specimen of the Apoplast iced at pH 10 was centrifugated and the anti-freeze action of the pellets and excess was tested with SDS-PAGE.

The pellets did not produce any proteinaceous material (path P, Fig. 5B), but the proteinaceous material was left in the overhang ( path S, Fig. 5B), which showed antifreezing effect (Fig. 5C). Appoplastic extract of non-acclimatized D. antharctica sheets included 0. 15 ?g albumin ?g-1, in comparison to 0. 28 ?g albumin ?g-1 in cool-acclimatized sheets.

At the same volume, the non-acclimatized plants' appoplastic excerpt showed only five readily identifiable 36, 32, 30, 22 and 10 kilodia of polypeptide in the same molar weight. A further eight polyypeptides with Apparent Muscle Weights in the range of 10 to 74 thousand DNA were identified in aperoplastic excerpts from D. antarctica sheets acclimatized to low temperatures (Fig. 5A).

The increase in ultra-filtration led to a higher anti-freeze effect, which is indicated by a larger C-axis increase in the icecrystal in 4x concentrations of apoplast icecrystal ((Fig. 5C). Astonishingly, some of the highly apparently bulk interactions in the concentrate extracts, in particular the 43 kiloda polyypeptide (cf. Lane's CA and 4 CA, Fig. 5A), declined.

Due to the restricted sampling scope, it was not possible at this point to determine single FAPs within the extract. In the present report, the peculiar type of vessel plants successfully colonised the Maritime Antarctic showed various freeze resistant mechanism. The anti-freeze effect was demonstrated by D. antharctica leaf extract, while C. quinensis leaf extract, even at 4-fold concentrations (Fig. 1), did not produce an albumin level equivalent to that of the D. anarctica.

The Antarctic hair grass frost protection effect was not exceptionally high. Thus, for example, aperoplastic extract from chilled acclimatized hibernated ray obtained by similar methods was watered 1:15 to 1:24 before loosing its anti-freeze effect (Hon et al., 1994; Stressmann et al., 2004), while chilled acclimatized D. antharctica extract looses its anti-freeze effect at a 1:10 thinning ( (Fig. 3), corresponding to about 0.03 mg of aperoplastic protein ml-1.

The results are in line with earlier reporting on frost protection activities in plants. Antifreezing effects are found in only about half of wintering plants in moderate areas (Duman and Olsen, 1993; Doucet et al., 2000), suggesting that there is more than one frozen glacial plant change path.

Many monocotyls, especially in the Poaceae genealogy, which contain grains such as barsley, grain, oats and oats (Antikainen and Griffith, 1997) and grass ( "Duman and Olsen, 1993), have been described as anti-freeze. However, many hibernating dicotyledons lacked antifreezing effect (Duman and Olsen, 1993; Antikainen and Griffith, 1997; Doucet et al., 2000), so that it was not so unexpected that there was no antifreezing effect in C. quenensis Apoplastische ext. (Figs. 1, 2).

Anti-freeze protein levels in D. antarctica's D. frost resistant plant are in line with earlier research, which showed that this type is very frost resistant: the temperature of the NT50 of non-acclimatized plants is around -12°C, while the temperature of plants acclimatized to low temperatures is -26°C. Conversely, quinensis does not accept the build-up of icing in its tissue and does not raise its value of approximately -5°C during exposure to temperature.

In Doucet et al (2000), totally dissolvable D. antharctica and C. quinensis extract, when mixed with 30% saccharose, 50 mM tris-HCl, pH 7.4, 20 mM Ascorbat and 10 mM EDTA, showed a loss of capacity to prevent the recrystallisation of egg when thinned to 0.05 and 0.1 mg ml-1 proteins.

Recrystallisation of D. antharctica remained at 95 C, but after a pronase breakdown the recrystallisation retardant had a decreased level of activitiy, while the C. quenensis retardant was resistant to both thermal and protective use. Since these properties differ from those of CFPs derived from D. antarctica's leafoplast, it is possible that the recrystallisation-inhibiting substances detected by Doucet and his team (2000) were non-specifically active nonspecific recrystallisation peptides (Knight et al., 1995) or heat-soluble protein and solute normally localised in the sulfate.

Alternatively, the molecule release from the symblast was able to stabilise the apoplastyic protein and avoid denaturing by overheating. APAPOPLASTIC EXTRACT apapoplastic extract from non-acclimatized plants of D. antharctica showed a significant ability to block the growing of icecrystals (Fig. 3). To the best of the authors' knowledge, this is the first article on the constituent anti-freeze effect in plants.

To be effective, all recent research has shown that anti-freeze agents are only effective when plants are accustomed to low temperature and shorter periods (Urrutia et al., 1992; Marentes et al., 1993; Duman and Olsen, 1993; Griffith and Yaish, 2004). An anti-freeze regulating effect is the production of ethene in reaction to coldness and dryness, which in turn triggers the enrichment of ARPs ('Yu et al., 2001).

Since D. antharctica developed on the Antarctic Isles under the selective pressures of low temperatures (Day et al., 1999; Alberdi et al., 2002; Lewis Smith, 2003), the type may have purchased regulative components for the constitutional expressing of AFPs. D. antharctica also shows an anti-freeze inductive action in reaction to low temperatures and short day lengths (16 hours d-1) (Fig. 1), suggesting that it still reacts to coldness even after generation of continual low temperatures growing.

Ambient is not the only selected power causing changes in the control of frost protection activities, as C. quietensis has developed a different approach to withstand frost and yet still remain alive under the same environment. Whilst the frost stability of Arctic species surviving in ice-rich sea water at -1.8°C is solely due to the AFP ( (Marshall et al., 2004), it does not adequately illustrate the ability of Antarctic plants to withstand much cooler tempera tures.

Instead, AMPs are probably only one part of the complexity of the freeze toleration mechanisms in Antarctic plants, which involves the enrichment of high levels of saccharose and non-structural carbohydrate and stress-related protein such as dehydrin during refrigeration acclimatization (Bravo et al., 2001; Zúñiga-Feest et al., 2003; Olave-Concha et al., 2004).

Furthermore, it has been shown that dehydrines can also have an anti-freeze effect (Wisniewski et al., 1999; Griffith and Yaish, 2004). Overall, these adjustments to the freeze can partly explains the viability of D. antharctica and why this particular type of grassland inhabits the Antarctic areas. Differences in frost protection activities between D. antharctica and C. quitenis may mirror different frost damage prevention policies that appear to be similarly effective for plants colonising marine areas in the Antarctic.

Additional research is needed to investigate how anti-freeze protection activities are sustained in the non-acclimatized D. antarctic. We would also like to monitor the anti-freeze activities of D. antharctica in the area where the population of D. antharctica is growing (Day et al., 1999; Lewis Smith, 1994; Gerighausen et al., 2003), as the Antarctic is experiencing tragic local global warming {Simpson, 2000; Karentz, 2003}.

They thank the Ministerio de Educación, Chile (MECESUP UCO 9906), who financed the LAB's trip to Canada, the Instituto Antártico Chileno (INACH) for their logistic assistance and permission to obtain plants from a special protection area on Robert Island, and the Natural Science and Engineering Research Council of Canada (MG).

Also we thank Lynn Hoyles and Nidhee Jadeja, University of Waterloo, for the cultivation of the plants and the performance of frost protection investigations. Eco-physiology of Antarctic plants. Enrichment of frost protection proteins in freeze-tolerant cereal. Refrigeration-caused, barley-purified dihydrin cycloprotective action. Resistant to low temperatures in Antarctic angio-perms. Propagation of Antarctic plants in reaction to heating and UV-B emission reductions in the fields.

Glycopeptide and peptide antifreeze: Interaction with ices and waters. Distributing and characterizing the recrystallisation inhibitors in plants and Lichens from Great Britain and the Antarctic Ocean. Thermic hybridization proteinaceous activities in bacterial, fungal and phylogenetic plants. Composition, functioning and development of anti-freeze protections. Enlargement of plants on an Antarctic island: a result of climatic changes?

Extra-cellular icing in freeze-tolerant plants. Frost protection in wintering plants: a history of two wintering plant species. Immunolocalisation of freeze toleration associated protein in the nucleoplasma and nucleoplasma of coronary tissue. Antarctic environment changes: ecologic effects and reactions. The recrystallisation of egg by means of insects' thermic protein hystersis: a possible cyoprotective part.

Non-equilibrium anti-freeze peptide and recrystallisation of iron. Splitting of structurally neutral protein during the formation of the bacteriophagus T4. in Antarctica. Plants as bio-indicators of Antarctic climate change. A Lin C, Thomashow MF genes encode a strong cycloprotective activeypeptide. Protein accumulates in the apoplastic of the ryegrass leaf during acclimatisation to the freezing temperatures.

Hypoactive anti-freeze protein in a seafood. Enhanced coloration of protein in acrylamide polygels with Coomassie Brilliant Blue G-250 and R-250 for clear backgrounds and nano image sensitivity included. Anti-freeze protein is excreted by frost protection cell cultures. It is an anti-freeze protein and chilinase from chilled rayrops.

Vegetable thermic hystereseproteins. Ethylene-induced frost protection in frost protection of frost protection in leaf.