- Email: [email protected]
Accumulation and disappearance of dehydrins and sugars depending on freezing tolerance of winter wheat plants at different developmental phases
Journal of Thermal Biology 27 (2002) 55–60
Accumulation and disappearance of dehydrins and sugars depending on freezing tolerance of winter wheat plants at different developmental phases I.V. Stupnikova*, G.B. Borovskii, N.V. Dorofeev, A.A. Peshkova, V.K. Voinikov Siberian Institute of Plant Physiology and Biochemistry, Siberian Division of Russian Academy of Sciences, Irkutsk P.O.Box 1243, 664033, Irkutsk-33, Russia
Abstract Several heat-stable dehydrins with mol. wts of 209, 196, 66, 50 and 41 kD characteristic of hardened winter wheat (Triticum aestivum L.) plants were detected by PAGE and Western blotting. These proteins together with sugars were accumulated under field conditions during low temperature adaptation and disappeared during spring. Their content depended on developmental phase and was connected with plant winter tolerance. Seemingly, more tolerant winter wheat plants have more effective mechanism of additive action of these protecting compounds. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Triticum aestivum L.; Heat-stable plant proteins; Dehydrins; Hardening; Deacclimation
1. Introduction Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. It is provided by different biochemical mechanisms which are known to involve the induction of genes encoding stress proteins, increased levels of sugars, enhancement of antioxidative mechanisms, changes in lipid composition, etc. (Thomashow, 1999; Borovskii et al., 1999; Storz and Imlay, 1999). These processes were activated by low temperature signal alone and in most cases low temperature dehydration that are deleterious for plants. It appears that dehydration in turn induce expression of dehydration-inducible genes and production of plant hormone abscisic acid (ABA) which also triggers the transcription of ABA-inducible genes (Shinozaki and YamaguchiShinozaki, 1997). Among the COR-proteins encoded by the COR-genes dehydrins family presents a particular
*Corresponding author. Fax: +7-3925-510754. E-mail address: irina@sifibr.irk.ru (I.V. Stupnikova).
interest because of they cryoprotective property. Dehydrins (dhns) have unusually hydrophilic, heat stable nature. They have been hypothesized to function by stabilizing large-scale hydrophobic interactions such as membrane structures or hydrophobic patches of proteins, acting essentially as a surfactant (Close, 1997). A number of studies have established positive correlation between drought and cold stress tolerance and dehydrin accumulation in a different plant species (Labhilili et al., 1995; Close, 1996; Pelah et al., 1997). Also correlation was established between other cryoprotectants, for example water-soluble sugars, and frost tolerance in various plant species (Vagujfalvi et al., 1999). The carbohydrates as other cryoprotectants, such as amino acids, glycinebetaine, polyamines, are hypothesized to act synergistically to stabilize macromolecules, thereby stabilizing the protoplasm (Close, 1996). However, available information about accumulation and disappearance of the protecting compounds is presented widely but is dispersed. Particularly, its observation concern with experiments conducted under field conditions, where all complex of nature factors acts. In this connection, the aim of the present work was to find proteins with immunochemical affinity to dehydrins
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 5 - 8
56
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
among the heat-thermostable protein fraction of winter wheat crowns and to elucidate whether joint accumulation and disappearance of these proteins and sugars are related to winter tolerance of plants of winter wheat (T. aestivum L.) at different developmental phases under field conditions. It presents particularly interest because of climate of East Siberia is severe. Especially dangerous period is the spring when frequent returns of low freezing temperatures take place.
2. Materials and methods Crowns of winter wheat plants (Triticum aestivum L., var. albidum, cv. Irkutskaia ozimaia) were used in the study. This genotype is winter-resistant and highly productive under the severe climatic conditions of the East Siberia. All experiments were carried out in the field, and seeds were sown at different times: August 15, 25 and September 5. Crowns or etiolated underground stem segments (depending on the phase of plant development) were sampled in the field. The part of these was frozen in liquid nitrogen, and brought to the laboratory where they were processed for protein extraction. Other samples were dried and analysed on content of total water-soluble carbohydrates. All samples were collected every 15 days throughout the entire period of fall and spring growth (starting November) and also monthly in the winter. Plants sown in August had time to tiller before winter, whereas plant sown on September 5 developed only the third leaf. Plants sown on August 15 had three to five tillers and, on August 25 developed one or two tillers. Autumnal growth of plants sown at different times lasted for 68, 58, and 47 days, respectively. For the region of the studies the early frosts are characteristic. The coldest month is January, with an average daily temperature varying from 218C to 288C. In the year of our field experiments, stable snow cover was established on November 13, and its height was 19 cm. After snow thawing at the end of March/ beginning of April, the daily air temperature varied from 128C to 178C. During this period, the temperature of the soil surface could change during the day by 408C (from a night temperature of 138C to a day temperature of 278C). Plant winter resistance was determined as the number of plants surviving in spring. Three places for plant counting, with a total area of 1 m2, were marked in autumn on each experimental plot. Total water-soluble carbohydrates were determined according to Dische (1967). Water-soluble heat-stable and total proteins were extracted from crowns as previously described by Lin et al. (1990) with modification (Stupnikova et al., 1998). Total and heat-stable
proteins were separated electrophoretically. Protein concentrations of samples were determined according to Esen (1978). Proteins were subjected to polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) using a mini-Protean PAGE cell (Bio-Rad, USA) according to manufacturer’s instruction. On each lane, 15 mg of protein were loaded. Western blotting and immunodetection were carried out as described by Timmons and Dunbar (1990) using anti-dehydrin antibodies (1 : 1000 dilution), kindly provided by Close (Close et al., 1993). In order to examine the specificity of antibodies we use the antiserum pre-blocked with dehydrin consensus peptide (Werner-Fraczek and Close, 1998). Western blot images were analysed by Sigma Scan Pro Software (Sigma Chemicals, USA). Relative content of proteins were estimated in the conventional units. All the experiments were performed in three replicates; the figures illustrate representative electrophoregrams; the table represents the average numbers of surviving plants for six years (1990–96) and their standard errors.
3. Results There was a great difference on overwintering between the winter wheat plants of cv. Irkutskaia ozimaia that were sown at different times: August 15, 25 and September 5 (Table 1). When plants were sown later, they had time to produce only one or two tillers or they were at the phase of three leaves, and such plants survived much better than plants sown on August 15. Field cold acclimation of winter wheat plants resulted in the accumulation of several heat-stable proteins with mol. wt. of 209, 196, 66 and 50 kD (Fig. 1A). Westernblotting with antibodies to dhns was carried out on the SDS-extracted winter wheat crowns heat-stable proteins. This technique revealed that COR-polypeptides with mol. wt. of 209, 196, 66 and 50 kD were related to dhns (Fig. 1B). At the same time, there is some amount increase of another dhn with mol. wt. of 41 kD (Fig. 1B). It was not detected on electrophoregrams (Fig. 1A). It is probably due to its small content and also high sensitivity of immunoblotting.
Table 1 Plant overwintering as dependent on the date of sowing Date of sowing
Surviving plants, % (average values for six years)
August 15 August 25 September 5
35 1.6 55 2.5 51 1.7
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
57
Fig. 1. SDS-electrophoresis (A) and Western blotting with antibodies to dehydrins (B) of winter wheat crowns heat-stable proteins. Proteins were extracted from freshly sampled crowns frozen in liquid nitrogen. Electrophoresis was run on 10% SDS-PAGE. Proteins were stained with Coomassie R-250 (A). Proteins were transferred to membrane and probed using anti-dehydrin antibodies (B). Mol. wt. of proteins are indicated in the center.
All winter wheat plants of different ages accumulated the dhns during fall. The protein spectrum of these plants did not change throughout entire winter and spring until April (Fig. 2A and B). However, amount of dhns depended on developmental phase. Before winter, the younger plants (sown on September 5 and August 25) accumulated less heat-stable COR-proteins than plants sown on August 15. Thus, morphologically more advanced plants (sown on August 15) contained more dhns (Fig. 2A). In the April (after temperature increasing and snow thawing), quantitative changes in dhns occurred. By the middle of April (sampling on April 16, 1996), differences between plants sown at different times became more distinct. By this time, the pattern of dhns was opposite to that observed in autumn. Thus, plants sown earlier, which accumulated more these proteins before winter came, lost these proteins in spring more rapidly and returned to the unhardened state. In contrast, the content of heat-stable dhns in younger plants was significantly higher than in morphologically more advanced plants (Fig. 2B). In May, no qualitative or quantitative differences were observed between plants sown at different times, except for 50 kD protein; however, the differences in the amounts of this protein were also small (Fig. 2C). Cold hardening also resulted in significant changes in the carbohydrate content (Fig. 3A). The plants of different ages responded similarly to cold, with a continuous increase in sugar content during autumnal field adaptation. This process was accompanied by gradual decreasing of average daily temperature
(Fig. 3A). At the same time, the degree of carbohydrates accumulation distinguished between the plants sown at different times. The plants sown on August 15 accumulated less sugars, that ones sown on August 25 and September 5. At the end of winter and during the spring the content of the carbohydrates gradually decreased what was accompanied by slow uneven increasing of average daily temperatures and frequent returns of low freezing temperatures (Fig. 3B). Finally, at the end of spring the plants sown earlier, having low level of overwintering (Table 1), had significantly less sugars than plants sown later (August 25 and September 5).
4. Discussion In the East Siberia, among the other components of winter resistance the tolerance to freezing temperatures are major for agricultural plants. So biochemical changes during almost all period of fall, winter and spring growth of winter plants are directed to acquisition and conservation of low temperature persistence. Plants are known to have different tolerance depending on phase of their ontogeny (Ingram and Bartels, 1996). As follows from our results, younger plants sown on August 25 and September 5 survived much better than morphologically more advanced plants sown on August 15 (Table 1). In this connection, it was proposed that development and loss of plant cryotolerance under field conditions are related to the accumulation and
58
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
Fig. 2. Heat-stable dhns extracted from crowns of winter wheat plants of different ages. Plant were sown on August 15, and 25 and September 5 and sampled on October 30 (A), April 16 (B), and May 4 (C). Electrophoresis and Western blotting was carried out as described in the legend to Fig. 1. Mol. wt. of proteins are indicated above density tracings.
disappearance of cryoprotectors for example such as dhns and carbohydrates. Indeed our results showed that winter wheat plants accumulated dhns and sugars during autumn adaptation which according Guy (1990) is accompanied by increasing of freezing tolerance. However, dynamics of dhns and sugars accumulation during autumn were not similar (Figs. 2A and 3A). Before winter, the younger plants sown on August 25 and September 5 accumulated less heat-stable dhns than plants sown on August 15, but maximal amount of carbohydrates. It was depended on autumnal gradual decreasing temperatures. Therefore biosynthesis of dhns was suppressed, but rates of sugar accumulation were increased (Figs. 2A and 3A). It apparently point to particular importance of carbohydrates as cell osmoregulators, cryoprotectants and participating in vitrification of protoplasm during preparatory period to winter, known as hardening. Unlike autumnal pattern of dhns and sugars accumulation the dynamics of its disappearance in plants during
spring deacclimation were similar (Figs. 2B and 3B). Thus, more tolerant young winter wheat plants of cv. Irkutskaia ozimaia contained higher concentrations of these compounds (Figs. 2B, C and 3B). The available data show connection between plant winter resistance and content of dhns and sugars in crowns during the spring (Table 1, Figs. 2B and 3B). Since the winter wheat survival in East Siberia is determined by the plant tolerance in spring period the concentration of these protecting compounds in this time seemingly is vital. Their joint autumnal accumulation and especially simultaneous disappearance in the spring confirms the suggestion about additivity or synergism between dhns and sugars (Close, 1996). The mode of sinergistical action is not clear entirely. Several investigators proposed an interaction between sugars and dhns (Blackman et al., 1992). At the same time, most of workers adhere to the exclusion theory (Arakawa et al., 1993; Close, 1996). In any case, as our results indicate, the more tolerant young plants, on
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
59
Fig. 3. Accumulation (A) and disappearance (B) of total water-soluble sugars in crowns of winter wheat plants of different ages during autumnal, winter and spring development. 1}sowing on August 15, 2}sowing on August 25, 3}sowing on September 5, 4}air average daily temperature. Values are in milligrams water-soluble carbohydrates/milligrams dry weight.
the stage of third leaf or one – two tillers before winter, have most effective mechanism of additive action of dhns and sugars which protects against freezing injures.
5. Summary The results obtained showed that firstly, heat-stable COR-proteins with mol. wts of 209, 196, 66, 50 and 41 kD, are related to dehydrins. These COR-proteins together with sugars were accumulated during autumnal adaptation. They seemingly act sinergistically to protect and stabilize cell structures and protoplasm. Secondly, connection between plant overwintering and content of these protecting compounds during spring was found. It appears that mechanism of additive action of dhns and sugars is more effective in the more tolerant plants.
Acknowledgements This research was funded by a grant from the Russian Foundation of Basic Research (project 99-04-48121). We sincerely thank Dr. T. J. Close for his generous gift of the dehydrin antibodies.
References Arakawa, T., Prestrelski, S.J., Kenney, W.C., Carpenter, J.F., 1993. Factors affecting short-term and long-term stabilities of proteins. Adv. Drug Delivery Rev. 10, 1–28.
Blackman, S.A., Obendorf, R.L., Leopold, A.C., 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol. 100, 225–230. Borovskii, G.B., Stupnikova, I.V., Peshkova, A.A., Dorofeev, N.V., Voinikov, V.K., 1999. Heat-stable proteins of winter wheat seedlings and crowns. Russ. J. Plant Physiol. 46, 678–683. Close, T.J., Fenton, R.D., Moonan, F., 1993. A view of plant dehydrins using antibodies specific to the carboxy terminal peptide. Plant Mol. Biol. 23, 279–286. Close, T.J., 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol. Plant. 97, 795–803. Close, T.J., 1997. Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol. Plant. 100, 291–296. Dische, Z., 1967. Total colour reactions. In: Kochetkov, N.K. (Ed.), Methods in carbohydrate chemistry. Mir, Moscow, pp. 21–22. Esen, A.A., 1978. A simple method for quantitative, semiquantitative and qualitative assay of protein. Anal. Biochem. 89, 264–273. Guy, C.L., 1990. Cold acclimation and freezing tolerance: role of protein metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41, 187–223. Ingram, J., Bartels, D., 1996. The molecular basis of dehydration tolerance in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 47, 377–403. Labhilili, M., Joudrier, P., Gautier, M.-F., 1995. Characterization of cDNAs encoding Triticum durum dehydrins and their expression patterns in cultivars that differ in drought tolerance. Plant Sci. 112, 219–230. Lin, C., Guo, W.W., Everson, E., Thomashow, M.R., 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol. 94, 1078–1083.
60
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
Pelah, D., Wang, W., Altman, A., Shoseyov, O., Bartels, D., 1997. Differential accumulation of water stress-related proteins, sucrose synthase, and soluble sugars in Populus species that differ in their water stress response. Physiol. Plant. 99, 153–159. Shinozaki, K., Yamaguchi-Shinozaki, K., 1997. Gene expression and signal transduction in water-stress response. Plant Physiol. 115, 327–334. Storz, G., Imlay, J.A., 1999. Oxidative stress. Curr. Opinion in Microbiol. 2, 188–194. Stupnikova, I.V., Borovskii, G.B., Voinikov, V.K., 1998. Accumulation of heat-stable proteins in winter wheat seedlings during hypothermia. Russ. J. Plant Physiol. 45, 744–748.
Thomashow, M.F., 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 571–599. Timmons, T.M., Dunbar, B.S., 1990. Protein blotting and immunodetection. Methods Enzymol. 182, 679–688. Vagujfalvi, A., Kerepesi, I., Galiba, G., Tischner, T., Sutka, J., 1999. Frost hardiness depending on carbohydrate changes during cold acclimation in wheat. Plant Sci. 144, 85–92. Werner-Fraczek, J.E., Close, T.J., 1998. Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map positions. Theor. Appl. Genet. 97, 220–226.
Accumulation and disappearance of dehydrins and sugars depending on freezing tolerance of winter wheat plants at different developmental phases I.V. Stupnikova*, G.B. Borovskii, N.V. Dorofeev, A.A. Peshkova, V.K. Voinikov Siberian Institute of Plant Physiology and Biochemistry, Siberian Division of Russian Academy of Sciences, Irkutsk P.O.Box 1243, 664033, Irkutsk-33, Russia
Abstract Several heat-stable dehydrins with mol. wts of 209, 196, 66, 50 and 41 kD characteristic of hardened winter wheat (Triticum aestivum L.) plants were detected by PAGE and Western blotting. These proteins together with sugars were accumulated under field conditions during low temperature adaptation and disappeared during spring. Their content depended on developmental phase and was connected with plant winter tolerance. Seemingly, more tolerant winter wheat plants have more effective mechanism of additive action of these protecting compounds. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Triticum aestivum L.; Heat-stable plant proteins; Dehydrins; Hardening; Deacclimation
1. Introduction Many plants increase in freezing tolerance upon exposure to low nonfreezing temperatures, a phenomenon known as cold acclimation. It is provided by different biochemical mechanisms which are known to involve the induction of genes encoding stress proteins, increased levels of sugars, enhancement of antioxidative mechanisms, changes in lipid composition, etc. (Thomashow, 1999; Borovskii et al., 1999; Storz and Imlay, 1999). These processes were activated by low temperature signal alone and in most cases low temperature dehydration that are deleterious for plants. It appears that dehydration in turn induce expression of dehydration-inducible genes and production of plant hormone abscisic acid (ABA) which also triggers the transcription of ABA-inducible genes (Shinozaki and YamaguchiShinozaki, 1997). Among the COR-proteins encoded by the COR-genes dehydrins family presents a particular
*Corresponding author. Fax: +7-3925-510754. E-mail address: irina@sifibr.irk.ru (I.V. Stupnikova).
interest because of they cryoprotective property. Dehydrins (dhns) have unusually hydrophilic, heat stable nature. They have been hypothesized to function by stabilizing large-scale hydrophobic interactions such as membrane structures or hydrophobic patches of proteins, acting essentially as a surfactant (Close, 1997). A number of studies have established positive correlation between drought and cold stress tolerance and dehydrin accumulation in a different plant species (Labhilili et al., 1995; Close, 1996; Pelah et al., 1997). Also correlation was established between other cryoprotectants, for example water-soluble sugars, and frost tolerance in various plant species (Vagujfalvi et al., 1999). The carbohydrates as other cryoprotectants, such as amino acids, glycinebetaine, polyamines, are hypothesized to act synergistically to stabilize macromolecules, thereby stabilizing the protoplasm (Close, 1996). However, available information about accumulation and disappearance of the protecting compounds is presented widely but is dispersed. Particularly, its observation concern with experiments conducted under field conditions, where all complex of nature factors acts. In this connection, the aim of the present work was to find proteins with immunochemical affinity to dehydrins
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 5 - 8
56
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
among the heat-thermostable protein fraction of winter wheat crowns and to elucidate whether joint accumulation and disappearance of these proteins and sugars are related to winter tolerance of plants of winter wheat (T. aestivum L.) at different developmental phases under field conditions. It presents particularly interest because of climate of East Siberia is severe. Especially dangerous period is the spring when frequent returns of low freezing temperatures take place.
2. Materials and methods Crowns of winter wheat plants (Triticum aestivum L., var. albidum, cv. Irkutskaia ozimaia) were used in the study. This genotype is winter-resistant and highly productive under the severe climatic conditions of the East Siberia. All experiments were carried out in the field, and seeds were sown at different times: August 15, 25 and September 5. Crowns or etiolated underground stem segments (depending on the phase of plant development) were sampled in the field. The part of these was frozen in liquid nitrogen, and brought to the laboratory where they were processed for protein extraction. Other samples were dried and analysed on content of total water-soluble carbohydrates. All samples were collected every 15 days throughout the entire period of fall and spring growth (starting November) and also monthly in the winter. Plants sown in August had time to tiller before winter, whereas plant sown on September 5 developed only the third leaf. Plants sown on August 15 had three to five tillers and, on August 25 developed one or two tillers. Autumnal growth of plants sown at different times lasted for 68, 58, and 47 days, respectively. For the region of the studies the early frosts are characteristic. The coldest month is January, with an average daily temperature varying from 218C to 288C. In the year of our field experiments, stable snow cover was established on November 13, and its height was 19 cm. After snow thawing at the end of March/ beginning of April, the daily air temperature varied from 128C to 178C. During this period, the temperature of the soil surface could change during the day by 408C (from a night temperature of 138C to a day temperature of 278C). Plant winter resistance was determined as the number of plants surviving in spring. Three places for plant counting, with a total area of 1 m2, were marked in autumn on each experimental plot. Total water-soluble carbohydrates were determined according to Dische (1967). Water-soluble heat-stable and total proteins were extracted from crowns as previously described by Lin et al. (1990) with modification (Stupnikova et al., 1998). Total and heat-stable
proteins were separated electrophoretically. Protein concentrations of samples were determined according to Esen (1978). Proteins were subjected to polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) using a mini-Protean PAGE cell (Bio-Rad, USA) according to manufacturer’s instruction. On each lane, 15 mg of protein were loaded. Western blotting and immunodetection were carried out as described by Timmons and Dunbar (1990) using anti-dehydrin antibodies (1 : 1000 dilution), kindly provided by Close (Close et al., 1993). In order to examine the specificity of antibodies we use the antiserum pre-blocked with dehydrin consensus peptide (Werner-Fraczek and Close, 1998). Western blot images were analysed by Sigma Scan Pro Software (Sigma Chemicals, USA). Relative content of proteins were estimated in the conventional units. All the experiments were performed in three replicates; the figures illustrate representative electrophoregrams; the table represents the average numbers of surviving plants for six years (1990–96) and their standard errors.
3. Results There was a great difference on overwintering between the winter wheat plants of cv. Irkutskaia ozimaia that were sown at different times: August 15, 25 and September 5 (Table 1). When plants were sown later, they had time to produce only one or two tillers or they were at the phase of three leaves, and such plants survived much better than plants sown on August 15. Field cold acclimation of winter wheat plants resulted in the accumulation of several heat-stable proteins with mol. wt. of 209, 196, 66 and 50 kD (Fig. 1A). Westernblotting with antibodies to dhns was carried out on the SDS-extracted winter wheat crowns heat-stable proteins. This technique revealed that COR-polypeptides with mol. wt. of 209, 196, 66 and 50 kD were related to dhns (Fig. 1B). At the same time, there is some amount increase of another dhn with mol. wt. of 41 kD (Fig. 1B). It was not detected on electrophoregrams (Fig. 1A). It is probably due to its small content and also high sensitivity of immunoblotting.
Table 1 Plant overwintering as dependent on the date of sowing Date of sowing
Surviving plants, % (average values for six years)
August 15 August 25 September 5
35 1.6 55 2.5 51 1.7
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
57
Fig. 1. SDS-electrophoresis (A) and Western blotting with antibodies to dehydrins (B) of winter wheat crowns heat-stable proteins. Proteins were extracted from freshly sampled crowns frozen in liquid nitrogen. Electrophoresis was run on 10% SDS-PAGE. Proteins were stained with Coomassie R-250 (A). Proteins were transferred to membrane and probed using anti-dehydrin antibodies (B). Mol. wt. of proteins are indicated in the center.
All winter wheat plants of different ages accumulated the dhns during fall. The protein spectrum of these plants did not change throughout entire winter and spring until April (Fig. 2A and B). However, amount of dhns depended on developmental phase. Before winter, the younger plants (sown on September 5 and August 25) accumulated less heat-stable COR-proteins than plants sown on August 15. Thus, morphologically more advanced plants (sown on August 15) contained more dhns (Fig. 2A). In the April (after temperature increasing and snow thawing), quantitative changes in dhns occurred. By the middle of April (sampling on April 16, 1996), differences between plants sown at different times became more distinct. By this time, the pattern of dhns was opposite to that observed in autumn. Thus, plants sown earlier, which accumulated more these proteins before winter came, lost these proteins in spring more rapidly and returned to the unhardened state. In contrast, the content of heat-stable dhns in younger plants was significantly higher than in morphologically more advanced plants (Fig. 2B). In May, no qualitative or quantitative differences were observed between plants sown at different times, except for 50 kD protein; however, the differences in the amounts of this protein were also small (Fig. 2C). Cold hardening also resulted in significant changes in the carbohydrate content (Fig. 3A). The plants of different ages responded similarly to cold, with a continuous increase in sugar content during autumnal field adaptation. This process was accompanied by gradual decreasing of average daily temperature
(Fig. 3A). At the same time, the degree of carbohydrates accumulation distinguished between the plants sown at different times. The plants sown on August 15 accumulated less sugars, that ones sown on August 25 and September 5. At the end of winter and during the spring the content of the carbohydrates gradually decreased what was accompanied by slow uneven increasing of average daily temperatures and frequent returns of low freezing temperatures (Fig. 3B). Finally, at the end of spring the plants sown earlier, having low level of overwintering (Table 1), had significantly less sugars than plants sown later (August 25 and September 5).
4. Discussion In the East Siberia, among the other components of winter resistance the tolerance to freezing temperatures are major for agricultural plants. So biochemical changes during almost all period of fall, winter and spring growth of winter plants are directed to acquisition and conservation of low temperature persistence. Plants are known to have different tolerance depending on phase of their ontogeny (Ingram and Bartels, 1996). As follows from our results, younger plants sown on August 25 and September 5 survived much better than morphologically more advanced plants sown on August 15 (Table 1). In this connection, it was proposed that development and loss of plant cryotolerance under field conditions are related to the accumulation and
58
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
Fig. 2. Heat-stable dhns extracted from crowns of winter wheat plants of different ages. Plant were sown on August 15, and 25 and September 5 and sampled on October 30 (A), April 16 (B), and May 4 (C). Electrophoresis and Western blotting was carried out as described in the legend to Fig. 1. Mol. wt. of proteins are indicated above density tracings.
disappearance of cryoprotectors for example such as dhns and carbohydrates. Indeed our results showed that winter wheat plants accumulated dhns and sugars during autumn adaptation which according Guy (1990) is accompanied by increasing of freezing tolerance. However, dynamics of dhns and sugars accumulation during autumn were not similar (Figs. 2A and 3A). Before winter, the younger plants sown on August 25 and September 5 accumulated less heat-stable dhns than plants sown on August 15, but maximal amount of carbohydrates. It was depended on autumnal gradual decreasing temperatures. Therefore biosynthesis of dhns was suppressed, but rates of sugar accumulation were increased (Figs. 2A and 3A). It apparently point to particular importance of carbohydrates as cell osmoregulators, cryoprotectants and participating in vitrification of protoplasm during preparatory period to winter, known as hardening. Unlike autumnal pattern of dhns and sugars accumulation the dynamics of its disappearance in plants during
spring deacclimation were similar (Figs. 2B and 3B). Thus, more tolerant young winter wheat plants of cv. Irkutskaia ozimaia contained higher concentrations of these compounds (Figs. 2B, C and 3B). The available data show connection between plant winter resistance and content of dhns and sugars in crowns during the spring (Table 1, Figs. 2B and 3B). Since the winter wheat survival in East Siberia is determined by the plant tolerance in spring period the concentration of these protecting compounds in this time seemingly is vital. Their joint autumnal accumulation and especially simultaneous disappearance in the spring confirms the suggestion about additivity or synergism between dhns and sugars (Close, 1996). The mode of sinergistical action is not clear entirely. Several investigators proposed an interaction between sugars and dhns (Blackman et al., 1992). At the same time, most of workers adhere to the exclusion theory (Arakawa et al., 1993; Close, 1996). In any case, as our results indicate, the more tolerant young plants, on
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
59
Fig. 3. Accumulation (A) and disappearance (B) of total water-soluble sugars in crowns of winter wheat plants of different ages during autumnal, winter and spring development. 1}sowing on August 15, 2}sowing on August 25, 3}sowing on September 5, 4}air average daily temperature. Values are in milligrams water-soluble carbohydrates/milligrams dry weight.
the stage of third leaf or one – two tillers before winter, have most effective mechanism of additive action of dhns and sugars which protects against freezing injures.
5. Summary The results obtained showed that firstly, heat-stable COR-proteins with mol. wts of 209, 196, 66, 50 and 41 kD, are related to dehydrins. These COR-proteins together with sugars were accumulated during autumnal adaptation. They seemingly act sinergistically to protect and stabilize cell structures and protoplasm. Secondly, connection between plant overwintering and content of these protecting compounds during spring was found. It appears that mechanism of additive action of dhns and sugars is more effective in the more tolerant plants.
Acknowledgements This research was funded by a grant from the Russian Foundation of Basic Research (project 99-04-48121). We sincerely thank Dr. T. J. Close for his generous gift of the dehydrin antibodies.
References Arakawa, T., Prestrelski, S.J., Kenney, W.C., Carpenter, J.F., 1993. Factors affecting short-term and long-term stabilities of proteins. Adv. Drug Delivery Rev. 10, 1–28.
Blackman, S.A., Obendorf, R.L., Leopold, A.C., 1992. Maturation proteins and sugars in desiccation tolerance of developing soybean seeds. Plant Physiol. 100, 225–230. Borovskii, G.B., Stupnikova, I.V., Peshkova, A.A., Dorofeev, N.V., Voinikov, V.K., 1999. Heat-stable proteins of winter wheat seedlings and crowns. Russ. J. Plant Physiol. 46, 678–683. Close, T.J., Fenton, R.D., Moonan, F., 1993. A view of plant dehydrins using antibodies specific to the carboxy terminal peptide. Plant Mol. Biol. 23, 279–286. Close, T.J., 1996. Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol. Plant. 97, 795–803. Close, T.J., 1997. Dehydrins: a commonalty in the response of plants to dehydration and low temperature. Physiol. Plant. 100, 291–296. Dische, Z., 1967. Total colour reactions. In: Kochetkov, N.K. (Ed.), Methods in carbohydrate chemistry. Mir, Moscow, pp. 21–22. Esen, A.A., 1978. A simple method for quantitative, semiquantitative and qualitative assay of protein. Anal. Biochem. 89, 264–273. Guy, C.L., 1990. Cold acclimation and freezing tolerance: role of protein metabolism. Ann. Rev. Plant Physiol. Plant Mol. Biol. 41, 187–223. Ingram, J., Bartels, D., 1996. The molecular basis of dehydration tolerance in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. 47, 377–403. Labhilili, M., Joudrier, P., Gautier, M.-F., 1995. Characterization of cDNAs encoding Triticum durum dehydrins and their expression patterns in cultivars that differ in drought tolerance. Plant Sci. 112, 219–230. Lin, C., Guo, W.W., Everson, E., Thomashow, M.R., 1990. Cold acclimation in Arabidopsis and wheat. Plant Physiol. 94, 1078–1083.
60
I.V. Stupnikova et al. / Journal of Thermal Biology 27 (2002) 55–60
Pelah, D., Wang, W., Altman, A., Shoseyov, O., Bartels, D., 1997. Differential accumulation of water stress-related proteins, sucrose synthase, and soluble sugars in Populus species that differ in their water stress response. Physiol. Plant. 99, 153–159. Shinozaki, K., Yamaguchi-Shinozaki, K., 1997. Gene expression and signal transduction in water-stress response. Plant Physiol. 115, 327–334. Storz, G., Imlay, J.A., 1999. Oxidative stress. Curr. Opinion in Microbiol. 2, 188–194. Stupnikova, I.V., Borovskii, G.B., Voinikov, V.K., 1998. Accumulation of heat-stable proteins in winter wheat seedlings during hypothermia. Russ. J. Plant Physiol. 45, 744–748.
Thomashow, M.F., 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 571–599. Timmons, T.M., Dunbar, B.S., 1990. Protein blotting and immunodetection. Methods Enzymol. 182, 679–688. Vagujfalvi, A., Kerepesi, I., Galiba, G., Tischner, T., Sutka, J., 1999. Frost hardiness depending on carbohydrate changes during cold acclimation in wheat. Plant Sci. 144, 85–92. Werner-Fraczek, J.E., Close, T.J., 1998. Genetic studies of Triticeae dehydrins: assignment of seed proteins and a regulatory factor to map positions. Theor. Appl. Genet. 97, 220–226.