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Immunological Assays of Freezing Tolerance in Barley using Antifreeze Proteins Antisera

Cheol Ho Hwang1, Dae In Kim1 and Jeong Gon Kim2

1School of Bioresources Science, Dankook University, Cheonan, 330-714, Korea ,
151, Seodun-dong Gwonseon-gu Suwon Gyeonggi-do, Korea, Email


In order to measure an antifreezing tolerance, accumulation of antifreeze proteins upon cold acclimation in apoplast was analyzed. As Dongbori1ho was cold-acclimated for 3 to 74 days there was an abrupt increase in apoplastic proteins up to 30 days and then a gradual decrease. Among the known antifreeze proteins, CLP produced in E. coli. and TLP purified from apoplast were used to generate antisera that allow to measure and to localize the proteins in leaves of barley. The CLP of 27.7 kDa and TLPs of 6, 26, 27, 30, and 31 kDa were increased in their amounts in apoplast as cold treatment being longer. There was a correlation among the amounts of those proteins accumulated in apoplast and freezing tolerance as shown in field and ion leakage tests for five cultivars. The deposit of CLP was localized in the marginal area and the area adjacent to leaf vascular bundle in an increasing manner according to duration of cold acclimation but no variation was observed in terms of it's distribution. Based on the close correlation between levels of antifreeze proteins and degrees of freezing tolerance, the immunological methods were used to develop estimate a freezing tolerance in barley.

Media summary

Immunological assays of freezing tolerance in barley.

Key Words

Antifreeze protein, Apoplast, CLP(chitinase like protein), TLP(thaumatin like protein), CA(cold-acclimation), GLP


A freezing injury in plant results from an ice nucleation and it's growth within apoplast. The resulting ice of bigger size breaks the adjacent cells in irreversible manner so that the cells in plant tissue become injured permanently (Griffith et al. 1992). In order for plant to protect against freezing stress, antifreezing proteins are accumulated in apoplast during cold acclimation (Pearce and Ashworth 1992; Hwang 1995). The formation of oligomeric complexes with CLP, TLP, and GLP was known to bind to surface of ice and to inhibit further addition of water molecules to the growing ice so that the amounts of the antifreeze proteins determine the degrees of tolerance against freezing (Chun and Griffith 1998). With aims on development of an assay for freezing tolerance in plants, antisera against the antifreeze proteins were produced and used for quantitative estimation of the proteins in five cultivars of barley with different degrees of tolerance.


Plant Materials and Cold Acclimation

Five cultivars of barley, Dongbori1ho(D), Olbori(O), Saechalssalbori(S), Albori(A), and Doowonchapssal-bori(Do), ranged from high to low in freezing tolerance, were provided by National Institute of Crop Science, Suwon (Table 1). Once reaching at 1.8 ~ 2 leaves stage, the plants were subjected to 6/4℃ (day/night for 8/16 hours) for cold acclimation of 0, 3, 10, 20, or 40 days.

Ion Leakage Test

Ion leakage test of the leaf from the same sets of plant were frozen at -2, -6, -10, -14℃ and the leakage from damaged cells was measured using spectrometer at 265nm (Sulc and Kenneth 1991). Based on the total amounts of ions released from the tissues broken by autoclaving, the relative levels of freezing-induced leakage per each tissue were estimated.

Extraction of Apoplast Proteins

The sliced leaf tissues were vacuum-infiltrated with apoplast solution(20 mM ascorbic acid, 20 mM CaCl2) at room temperature for 30 minutes. And the apoplast proteins were extracted by centrifugation at 760g for 30 minutes. And an acetone precipitation of the proteins was performed to concentrate and the concentration was adjusted by dissolving the pellet in 10 ㎕ per 1 g of d fresh tissue and stored at -20℃ before used (Hon et al. 1994).

Preparation of Polyclonal Antiserum Against CLP and TLP

The cDNA for CLP was isolated from cold-acclimated Dongbori1ho and constructed in pET32 vector (Novagen) and introduced to BL21(DE3). A treatment of IPTG induces a synthesis of the protein in the cells and total cell proteins were extracted and separated in 15% SDS-PAGE and purified by gel-elution. The TLP of 16 kDa was purified from total apoplastic proteins from the cold acclimated Dongbori1ho using a 15% SDS-PAGE and gel-elution methods. 100㎍ of the proteins was mixed with a 500㎕ of Freund's adjuvant (Sigma) before injecting into a rabbit for four times at a weeks' interval.

Western Analysis and In situ Immunolocalization

An equal volume of apoplastic proteins extracted from the equal fresh weight of leaves was separated in 15% SDS-PAGE and probed with CLP, TLP- antiserum and normalized by apoplastic proteins from Saechalssalbori(S), Doowonchapssalbori(Do) as an internal standard. The primary antiserum of either TLP or CLP at a dilution of 1:10,000 and the secondary anti-rabbit IgG conjugated with AP (Promega) at a dilution of 1:7,500 were used. For immunolocalization, cold acclimated leaf tissues were fixed and embedded in paraffin before being sectioned in 10㎛. Each section was then treated with the pre-immune serum as a negative control and with Anti-CLP (antifreeze protein) serum.


In an attempt to quantify a freezing tolerance, the levels of ion leaked from leaves upon freezing were measured in five cultivars of barley (Sulc and Kenneth 1991). As the longer cold acclimation is treated, the less amounts of ion leakage were observed. It indicated that a cold pre-treatment increased the freezing tolerance in all of the five cultivars tested (Figure 1). However there were variations in degree of the tolerance among cultivars and the differences were shown to be correlated with what the field test had shown in terms of survival under freezing conditions (Table 1).

Table 1. Degree of freezing tolerance measured from field test among different cultivars of barley (NCES, 1999~2000).

Degree of Freezing Tolerance

cultivars of barley



Dongbori1ho (D)



Olbori (O)


Saechalsalbiri (S)



Albori (A)



Doowonchapsalbori (DO)


*: data is not available but expected as 100% based on Cho et al, (1979).

Figure 1. Ion leakage test of barley under non acclimation(A), 3 days(B), 40 days(C) after cold acclimation.

Even among non-acclimated samples, some variations in freezing tolerance were also observed but only Albori showed a significant difference in freezing tolerance (Figure 1). The CLP of 42.5 kDa was synthesized in E. coli at maximum rate in 3 hours after an ITPG treatment (Figure 2 A). A primary antiserum produced against the CLP was confirmed to be specific by western analysis of apoplastic proteins from Dongbori1ho in which a CLP of 27.7 kDa was increased as cold acclimation progressed (Figure 3 A).

Figure 2. A, Expression of CLP fused with thioredoxin protein using a pET-32 expression system in E. coli. In 4 hours after an IPTG treatment, a maximal expression of CLP proteins is shown to be achieved. B, Concentration of extracellular proteins accumulated in apoplast of Dongbori1ho leaves as the time increased for cold acclimation.

Figure 3. A, Western analysis of the apoplastic proteins with CLP antiserum from leaves of Dongbiri1ho as the time for cold acclimation increased. B, Western analysis of apoplastic proteins accumulated during 40 days' cold acclimation. Using CLP antibody among 5 cultivars of barley; D(Dongbori1ho), O(Olbori), S(Saechalsalbori), A(Albori), DO(Doowonchapsalbori), Sc(non-acclimated of Saechalsalbori as an internal control to compare between each of membranes tested).

A close correlation observed between amounts of the CLP and the duration of cold acclimation (Figure 2B; Figure 3A; Hwang et al. 2000). The amounts of CLP accumulated among five different cultivars of barley of different degrees of the tolerance were measured in attempt to see a correlation. The total proteins, in parallel to the CLP of 27.7 kDa, were found to be increased in apoplast as being cold-acclimated longer. On day 40 after cold acclimation, five cultivars showed the increasing levels of CLP in apoplast in the same order of Dongbori1ho, Olbori, Saechalsalbori, Albori and Doowonchapsalbori as having reported from field test of freezing tolerance (Figure 3B and Table 1).

The time course analysis of western hybridization with TLP antiserum showed a gradual increase of apoplast proteins but there were various induction patterns of individual TLP of different sizes though they commonly reacted with the TLP antiserum. With an emphasis on a TLP of 16 and 30 kDa that was used as antigen, their accumulation reached to a maximum level at day 10 (Figure 4A).

Figure 4. A, Western analysis using TLP antibody of the apoplastic proteins from leaves of Dongbori1ho cold acclimated in different time period. B, Western analysis of apoplastic proteins accumulated during 40 days' cold acclimation using CLP antibody among 5 cultivars of barley; D(Dongbori1ho), O(Olbori), S(Saechalsalbori), A(Albori), DO(Doowonchapsalbori), DOc(non-acclimated of Doowonchapsalbori as an internal control to compare between each of membranes tested).

Five cultivars of barley showed the amount of total TLPs accumulated in apoplast in an order of Dongbori1ho, Olbori, Saechalsalbori, Albori and Doowonchapsalbori as reported from a field test of freezing tolerance (Figure 4B). This may suggest a possibility of CLP and TLP as a molecular marker for freezing tolerance as long as plant undergoes cold acclimation for 40 days. There were many clues showing that a CLP is involved in both cold acclimation and freezing tolerance. An in situ immunolocalization was performed to see any qualitative difference such as distribution of CLP in leaf tissue.

Figure 5. In situ immunohistochmical localization of CLP antiserum and preimmuno serum in transverse sections of leaf margin and vein at 0 day and 40 days after cold acclimation. (A)~(E), CA 0 day with CLP antiserum; (F)~(J), CA 40 days with pre-immunoserum; (K)~(O), CA 40 days with CLP antiserum. (A), (F), (K), Dongbori1ho; (B), (G), (L), Olbori; (C), (H), (M), Saechalsalbori; (D), (I), (N), Albori; (E), (J), (O): Doowonchapsalbori

As shown in Figure 5, there were much more signals for CLP found to be accumulated in margin of the bottom side of leaf acclimated for 40 days comparing to the non-acclimated. In the light of the fact that the empty space in bottom side make it more vulnerable to freezing than the upper side piled with palisade layer, the presence of highly concentrated CLP in the lower side of leaf may provide protection from freezing. In facts, the similar observation was reported also in rye by Mervi et al (1996). When comparing both the amount and distribution of CLP, there was no clear difference observed in distribution of CLP among five cultivars of barley acclimated for 40 days.


In order to measure a freezing tolerance in barley, western analysis and in situ immunological methods had been applied to see both qualitative and quantitative differences of antifreeze proteins, CLP and TLP. The results showed that the levels of the proteins accumulated in leaf apoplast were correlated with those of antifreezing tolerance as observed with field and ion leakage tests. The CLP was found to increase in areas of margin and around vein as the plants were cold-acclimated but no difference was observed among cultivars by in situ localization (Figure 5).Also based on both western analysis and Sandwich ELISA (data not shown), we were able to confirm that the amount of the protein induced by a cold acclimation could be a quantitative indicator for freeze tolerance in plants.


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