ISSN 10214437, Russian Journal of Plant Physiology, 2010, Vol. 57, No. 6, pp. 859–864. © Pleiades Publishing, Ltd., 2010. Original Russian Text © N.A. Gumilevskaya, M.I. Azarkovich, 2010, published in Fiziologiya Rastenii, 2010, Vol. 57, No.6, pp. 918–924.
RESEARCH PAPERS
Identification and Characterization of Dehydrins in Horse Chestnut Recalcitrant Seeds N. A. Gumilevskayaa and M. I. Azarkovichb a
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 9778018; email: m
[email protected]
b
Received January 27, 2010
Abstract—The fraction of heatstable dehydrins cytosolic proteins from mature recalcitrant seeds of horse chestnut (Aesculus hippocastanum L.) was studied in the period of their dormancy and germination in order to identify and characterize stressinduced dehydrinlike polypeptides. In our experiments, in tissues of dor mant seeds, dehydrin was identifies by immunoblotting as a single bright band with a mol wt of about 50 kD. Lowmolecularweight heatstable proteins with mol wts of 25 kD and below 16 kD, which were abundant in this fraction, did not crossreact with the antibody. Dehydrin was detected in all parts of the embryo: in the cells of axial organs, cotyledon storage parenchyma, and petioles of cotyledonary leaves. This indicates the absence of tissuespecificity in distribution of these proteins in the horse chestnut seeds. Dehydrins were detected among heatstable proteins during the entire period of stratification and also radicle emersion. Dur ing radicle emergence, not only the fraction of heatstable proteins was reduced but also the proportion of dehydrins in it decreased. In vitro germination of axes excised at different terms of stratification also resulted in dehydrin disappearance. When growth of excised axes was retarded by treatments with ABA, cyclohexim ide, or αamanitin, dehydrins did not disappeared from the fraction of heatstable proteins. When excised axes were germinated in vitro in the presence of compounds, which did not affect their growth or stimulated it (dehydrozeatin, glucose), this resulted in dehydrin disappearance. This means that dehydrin metabolism is closely related to the process of germination. Dehydrin in the horse chestnut seeds could crossreact with the antibody against ubiquitin, which can indicate the involvement of ubiquitination in the process of dehydrin degradation during germination via the proteasome system. The analysis of total proteins of the homogenate from horse chestnut seeds revealed, along with a 50kD heatstable dehydrin, one more component with a mol wt of 80 kD, which was located in the fraction of heatsensitive proteins and was named as a dehydrin like protein. It was demonstrated that dehydrins in horse chestnut seeds represented only a very small fraction of heatstable cytosolic proteins. The role and function of major heatstable proteins in horse chestnut seeds are yet to be studied. Keywords: Aesculus hippocastanum, recalcitrant seeds, dehydrins, seed dormancy, germination. DOI: 10.1134/S1021443710060154
INTRODUCTION Dehydrins are stressinduced proteins belonging to the complex and poorly studied D11 family of hydro philic heatstable proteins from the group of late embryogenesis abundant (LEA) proteins. According to modern notion, they are synthesized in response to cell dehydration occurring at drought, salt stress, cold acclimation, treatments with some phytohormones (ABA, for example), and during seed maturation. Dehydrins were detected in tissues of gymnosperms and angiosperms, in trees and grasses, in vegetative organs and seeds. In numerous studies, by immunolo calization dehydrins were found in different cell parts: nucleus, cytoplasm, cytoskeleton, and mitochondria. It was shown that dehydrins are absent from immature embryos and young seedlings. In recent years, dehy drins are studied very intensively, which resulted in the
appearance of numerous reviews and research papers [1–6]. It is believed that, in orthodox seeds, dehydrins favor the development of tolerance to osmotic stress at seed dehydration during their maturation. It turned out that recalcitrant seeds also could synthesize dehy drins; however, they remain sensitive to water loss. In this connection, the investigation of dehydrin func tions, properties, and distribution in recalcitrant seeds becomes actual. The data available so far indicate that dehydrins are present in some but not all recalcitrant seed species. They appear in response to lowtemper ature stress, an increase in the ABA content, and nat ural or artificial limited dehydratation. In most cases, dehydrins are represented by a group of lowmolecular heatstable polypeptides. In the horse chestnut seeds,
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several dehydrins with mol wts of 12, 14, 18, 30, and 55 kD were detected [7]. There was suggested that in recalcitrant seeds, dehydrins could provide for the protection of cell structures against damaging action of low temperature and dehydrating factor. Earlier, we have studied and described some spe cific physiological and biochemical features of horse chestnut seeds; we have characterized the proteome of axial organs and cotyledons, demonstrated the main tenance of translational capacity in the cells of excised axes and cotyledons in dormant seeds during stratifica tion; we also established that excised axes have no own dormancy and are capable to grow in vitro [8–12]. With a knowledge of the protein composition in axial organs and cotyledons, we tried to elucidate which polypeptides are dehydrins, how they are dis tributed in embryo tissues, and how keeping moist seeds under low abovezero temperature (stratifica tion) and germination affect dehydrin content. This was the objective of this work. To this end, SDSPAGE was used for protein separation and immunoblotting for dehydrin identification. MATERIALS AND METHODS Plant material. Fallen seeds of horse chestnut (Aes culus hippocastanum L.) were collected in the dendrar ium of the Main Botanical Garten of Russian Acad emy Science and in Moscow parks. Both freshly col lected seeds and those subjected to stratification in moist sand at 5°С in darkness were used. Before exper iment, seeds were sterilized with calcium hypochlorite and washed in running water. Axes, cotyledon pieces, and petioles were excised by hand using a scalpel, fro zen or used immediately for analysis. Subcellular fractionation of the homogenates of axes, cotyledons, and their petioles was performed by differen tial centrifugation, as was described earlier [8]. Frozen axes (5 axes, the total average weight is 250–300 mg) were homogenized in 10 ml of 0.25 M sucrose dissolved in 0.05 M Tris–HCl (pH 7.2) containing 0.01 M Mg ace tate, 0.025 M KCl, 7 μg/ml of pepstatin, 5 μg/ml of leupeptine, and 1 mM PMSF (phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 1000 g for 10 min. Aliquots of the supernatant were used as a source of total protein; the rest extract was centrifuged at 20000 g for 20 min for obtaining subcellular struc ture fractions. Proteins of the postmitochondrial extract (cytosol) were separated into heatstable and heatsensitive fractions in dependence of their behav ior at heating at 75°С for 10 min. Coagulated heat sensitive proteins were removed by centrifugation, whereas proteins remained in the solution were pre cipitated with TCA. All protein samples for electro phoresis were prepared from TCAinsoluble material. Protein content in samples was determined as described in [13]. Samples were put on filter paper Watmann 3MM, fixed in 10% TCA, stained with
Amido Black, washed from excess dye; dye bound with protein was eluted from paper and its absorption was measured at 620 nm. Protein content was determined using the calibrating curve built with BSA as a stan dard. SDSPAGE was performed under reducing dissoci ating conditions on gel plates (0.2 × 12 × 12 cm) with the 10–20% PAAG gradient and also on 12.5% PAAG. Electrophoresis was run for 16 h at 10 mA and room tem perature or for 5 h at 30 mA in the refrigerator. Prestain protein markers (Fermentas) were used to monitor protein migration during electrophoresis. After electrophoresis termination, the apparatus was dismantled, concentrating gel and separating gel below the front line, marked by bromphenol blue band, were removed. Immunoblotting. Moist or submerged protein transfer to membrane was performed as described in [14–16]. Transfer occurred for 16–18 h in the refrigerator at 30–40 V and 20mA. After transfer, gel was stained with Coomassie blue R250 to evaluate the completeness of transfer from the gel. Proteins on membrane were stained with Ponceau S for 5–10 min at room temper ature (staining is reversible). Thereafter membrane was rinsed with distilled wated until background destaining and marked positions of transferred pro teins and markers. 3% dry defatted milk (AppliChem, Germany) in 0.2 M Tris–HCl, pH 7.4, with addition of TBS buffer (0.9% NaCl, 0.02% sodium azide, and 0.2% Tween20) was used as a blocking solution. Membranes were incubated in this solution for 2 h at room temperature on the shaker or overnight in the refrigerator. Thereaf ter, membranes were rinsed with TB buffer (the same buffer devoid of NaCl) and treated with the antibody. Commercial rabbit antidehydrin polyclonal anti body (Stessgen, United States) was used after its dilu tion (1 : 1000). The initial solution of primary anti body (10 μl) was added to 10 ml of TBS containing 0.6% of dry defatted milk, and the mixture was incu bated for 4 h at room temperature or 18 h on the shaker in the refrigerator. Thereafter, the solution of primary antibody was removed, and membrane was washed with 50 ml of TBS containing 0.6% of dry defatted milk (washing buffer) three times (10 min each) on the shaker. The primary antibody bound to the antigen on membrane was detected using the secondary antirab bit antibody conjugated with horseradish peroxidase (Medganal, Russia). Commercial serum 0.2 ml in vol ume was diluted with 2 ml of double distilled water The solution obtained (20 μl) was added to 10 ml of buffer for antibodies (final dilution was 1 : 5000). Membrane was incubated in the solution of secondary antibody for 1.5 h at room temperature on the shaker. Thereafter, the solution of antibody was removed, and membrane was washed three times (10 min each) with
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50 ml of buffer for antibodies. Then, peroxidase was detected on membrane. For detection of peroxidase on membrane treated with the secondary antibody, its substrate tetramethyl benzydine (TMB) was used. TMB reaction with the enzyme gives a blue insoluble product. Washed mem brane was placed in a suitable vessel with protein side upward, and 2 ml of TMB solution was poured onto its surface. In 10–15 min stained bands appeared, and staining intensity did not change afterward. Excess TMB was removed, and membrane was dried in air and then stored, photographed, and scanned.
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Dehydrin number and molecular weights The analysis of polypeptide composition of pro teins from horse chestnut seeds, which we have per formed earlier [8, 11] showed that the bulk of proteins (more than 80%) of axial organs during deep dor mancy was represented by soluble proteins (in the postmitochondrial supernatant), whereas only 12– 15% of total cell protein are present in the cell struc tures (excluding nucleus). Cytosolic proteins differ significantly in their sensitivity to heat treatment. Heating at 75°С for 5–10 min coagulated about 60% of cell homogenate proteins. These proteins are heat sensitive. About 30% of protein remained in the solu tion after heating; they are heatstable proteins. For the period of stratification, the proportion of heatsensitive proteins increased steadily but only slightly (from 55 to 70%). Approximate calculation of heatstable and heatsensitive proteins in the cell extract showed that the level of heatsensitive proteins did not substantially change during stratification but increased markedly during radicle emergence and axial organ growth, whereas the level of heatstable proteins reduced strongly and continued to decrease until their almost complete disappearance during ger mination. It is well known that dehydrins are extremely toler ant to heat denaturing [1]. In horse chestnut seeds, heatstable proteins comprised about 30% of soluble cytosolic proteins in axial organs and more than 80% in cotyledons [8, 11]. They had characteristic polypeptide composition (Fig. 1a) and comprised mainly three or two groups of very abundant polypep tides with mol wts of about 50, 25 and below 16 kD in axes and about 25 and below 16 kD in cotyledons. According to scarce published data concerning dehy drins in horse chestnut seeds, these proteins represent the group of lowmolecularweight, watersoluble, heatstable proteins with mol wts of 12, 14, and 18 kD [7, 17]. In this connection, in our initial attempts to detect dehydrins in horse chestnut seeds, we paid especial attention to the group of lowmolecular weight polypeptides in the fraction of heatstable cyto solic proteins. After separation of heatstable proteins RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 1. Electrophoregrams of heatstable proteins (a) and detection of dehydrin among heatstable proteins (b) from various parts of horse chestnut seed embryos. (1) Axial organs; (2) cotyledon petioles; (3) cotyledon parenchyma.
of axial organs (or cotyledons and cotyledon petioles) by SDSPAGE, only half of the separating gel plate comprising proteins with mol wts below 25 kD was treated with antibody and transferred to nitrocellulose membrane. Gel staining after protein transfer to membrane showed that transfer of lowmolecularweight proteins was always complete. However, transferred proteins did not crossreact with antidehydrin antibody. A great number of heatstable protein preparations were isolated from different embryo parts (axes, coty ledon petioles, and cotyledons) of mature horse chest nut seeds, and in all cases dehydrins were not detected among lowmolecular weight polypeptides. Thus, the results of our study differ from published data [7, 17]. The analysis of proteins with mol wts of 25 kD and higher, which were positioned in the upper part of the separating gel and were not analyzed in the above experiments, showed that, almost always, their trans fer to membrane was not complete. Nevertheless, all polypeptides detected in the stained gel were present on the membrane. After membrane treatment with antidehydrin antibody, it was found that, like low No. 6
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Fig. 2. Detection of dehydrins and ubiquitin among heatstable proteins of horse chestnut seeds. (a) Treatment with antidehydrin antibody; (b) treatment with antiubiquitin antibody. (1) Axial organs; (2) cotyledon petioles; (3) ubiquitin.
molecularweight polypeptides, a dominating ~25kD polypeptide did not crossreact with the antibody. At the same time, a heatstable polypeptide with a mol wt of about 50 kD gave a clear and very strong signal (Fig. 1b). Thus, only a single dehydrinlike protein with a mol wt slightly higher than 50 kD was detected in heatstable proteins of horse chestnut seeds. Dehydrin Tissue and Intracellular Localization in Horse Chestnut Embryos To elucidate whether tissue specificity in dehydrin distribution in horse chestnut embryos exists, heat stable proteins isolated from axes, cotyledons, and cotyledon petioles were analyzed by immunoblotting. The 50kD polypeptide was the only one detected in the heatstable fraction and it was present in all embryo tissues tested. It should be noted that the brightness of the band of 50kD dehydrin in samples with similar protein con tent differed in different tissues. It was most bright in the proteins of axial organs (Fig. 1b, 1) and the least bricht in cotyledon proteins (Fig. 1b, 3). The results obtained indicate that there is no tissue specificity in dehydrin distribution and that the relative content of 50kD dehydrin differed in heatstable proteins of dif ferent embryo tissues.
Dehydrins and Ubiquitin When membranes were treated with antibody agaist ubiquitin (Fig. 2), they detected the same pro tein (50 kD), which crossreacted with antidehydrin antibody. This means that just dehydrinlike protein of horse chestnut seeds may be bound with ubiquitin. No other polypeptides interacted with antiubiquitin anti body. When ubiquitin protein was electrophoresed, its position in the gel and signal at immunoblotting were found in the zone of polypeptides with a mol wt of 8 kD (Fig. 2b), which corresponds to the mol wt of ubiquitin. This fact seems very interesting because just 50kD dehydrin disappeared firstly during horse chestnut seed germination. Dehydrins in Horse Chestnut Seeds during Dormancy and Germination To appraise the behavior of dehydrins in dormant seeds during stratification and in germinating seeds, heatstable proteins were isolated from embryos of mature freshly fallen seeds before stratification and the seeds subjected to stratification for 4, 8, 14, 18, and 19 weeks in moist sand at 5°C, and also from seeds during radicle emersion. Heatstable proteins were also iso lated from excised axes germinated in vitro for 3 days at 28°C. 50kD dehydrin was present in embryos dur ing the entire period of dormancy but was hardly detected after in vivo radicle emersion. No other dehydrins were found in embryos during this period.
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Fig. 3. Dehydrins in axes excised from horse chestnut seeds after 3 days of growing at 28°C. The results of two independent experiments (I and II) are presented. (1) Before incubation; (2) after incubation on water (control); (3) after incubation on 10 µM ABA; (4) after incubation on 50 µg/ml cycloheximide; (5) after incubation on 0.06 M glucose; (6) after incubation on glucose solution + ABA.
During excised axis germination in vitro for 3 days at 28°C, axes manifested growth activity and corre sponded to axes from seeds germinated in vivo at a moment of radicle emersion [10]. 50kD dehydrin was absent from such axes (Fig. 3, 2). When in vitro germi nation was performed in the presence of ABA (Fig. 3, 3), cycloheximide (Fig. 3, 4), or αamanitin, which inhibited germination of isolated axes, 50kD dehy drin was detected as a bright band. Germination in the presence of 0.06 M glucose, which activated axis growth, dehydrin disappeared (Fig. 3, 5). It is likely that germination of recalcitrant seeds is accompanied by dehydrin disappearance, similarly as germination of orthodox seeds. It is worth mentioning that dehydrins were not detected in immature yet unfallen seeds. Are All Dehydrins HeatStable Proteins? Since horse chestnut seeds contain along with heatstable proteins numerous heatsensitive proteins, it was of importance to elucidate whether dehydrin like proteins are present among the latter. Nobody analyzed this protein fraction for the presence of dehydrinlike proteins. In the fraction of heatsensi tive proteins of horse chestnut seeds, we detected a component with a mol wt of 80 kD (Fig. 4, 2), which crossreacted with antidehydrin antibody, i.e., was immunologically revealed as a dehydrinlike protein. The analysis of different subcellular protein fractions of axial organs (Fig. 4), cotyledons, and cotyledon petioles showed that both 50kD dehydrin and 80kD dehydrinlike protein could be detected in the total homogenate protein (Fig. 4, 1). 50kD dehydrin was absent from heatsensitive cytosolic proteins and gave a weak band in the proteins of the pellet after homoge RUSSIAN JOURNAL OF PLANT PHYSIOLOGY
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Fig. 4. Dehydrins and dehydrinlike proteins in horse chestnut seeds. (1) Cell homogenate; (2) heatsensitive cytosolic proteins; (3) heatstable cytosolic proteins; (4) total cytosolic pro teins (20000 g supernatant); (5) proteins of cell structures (20000 g pellet).
nate centrifugation at 20000 g (Figs. 4, 5). It might be that the presence of 50kD dehydrin in the fraction of cell structures (the 20000 g pellet) is explained by con tamination of this fraction by cytosolic proteins, among which heatstable proteins and 50kD dehy drin are present. It is not excluded a possibility of the presence in this fraction of mitochondrial dehydrins [4], although we did not analyzed such a possibility experimentally. DISCUSSION The molecular weight of the single dehydrin we detected in the horse chestnut seeds was slightly above 50 kD. At the same time, other researchers reported other values [7, 17], e.g., 12, 14, and 18 kD or 30–55 kD. So far it is difficult to explain such differences in dehy drin sizes. It is not excluded that this is related to some specific feature of horse chestnut plants or their popu lations in different countries. It seems more likely that these differences are related to the influence of differ ent growth conditions. Thus, seeds collected in differ ent years differed in the size of dehydrins: 12, 14, and 18 kD in seeds collected in 1992; 23, 30, and 35–55 kD in seeds collected in 1994; and 14 kD in seeds col lected in 1993 [7, 17]. During stratification, there were no substantial changes in the content of 50kD dehydrin. However, this protein disappeared rapidly during seed germina tion. Since a small heat shock protein ubiquitin plays an important role in cell protein degradation, it was of interest to elucidate whether there is any connection between dehydrin, which should to disappear during seed germination, and ubiquitin, which marks specifi cally proteins destined for degradation. It turned out that 50kD dehydrin crossreacted with antiubiquitin No. 6
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antibody. This means that dehydrin ubiquitination might provide for dehydrin rapid disappearance after radicle protrusion. Earlier we have established that embryo axes of dormant seeds are not in the dormant state and could germinate in vitro (72 h on water at 28°С) in each period of stratification of dormant seeds [10]. Like during seed germination in vivo, 50kD dehydrin was not detected in such axes germinated in vitro. As we have demonstrated earlier [10], treatment of excised axes with ABA (10–5 M), cycloheximide, or αaman itin suppressed their germination; and under these conditions of suppressed growth, 50kD dehydrin remained in the axes and was easily detected by immu noblotting. The mechanism of ABA inhibitory action on excised axis germination remains unknown. It is also unknown whether ABA induces 50kD dehydrin synthesis in axes or simply prevents its degradation, resulting in its presence in axes on the level detected before germination. However, at the comparison of ABAtreated axes with those treated with cyclohexim ide, or αamanitin, the substantially stronger signal may be noted in the case of ABA treatment. This may indicate indirectly on the induction of dehydrin syn thesis at germination inhibition by ABA. It seems important that one of axis heatsensitive polypeptides crossreacted with the antidehydrin antibody but differed from heatstable dehydrin by a higher molecular weight (about 80 kD). This is the first indication on the possible presence of dehydrinlike proteins among heatsensitive polypeptides of horse chestnut seeds. In general, it was shown for the first time that, in recalcitrant horse chestnut seeds, dehydrins are repre sented a homogenous and relatively highmolecular weight (about 50 kD) component. Dehydrins com prise a small part of heatstable proteins. The func tions of other heatstable proteins accumulating in horse chestnut recalcitrant seeds in great amounts remain unclear. ACKNOWLEDGMENTS This work was partially supported by the Russian Foundation for Basic Research (project no. 0304 48156a) and by the Presidium of Russian Academy of Sciences (program Molecular and Cell Biology). REFERENCES 1. Close, T.J., Dehydrins: Emergence of a Biochemical Role of a Family of Plant Dehydration Proteins, Phys iol. Plant., 1996, vol. 97, pp. 795–803. 2. Close, T.J., Dehydrins: A Commonalty in the Response of Plants to Dehydration and Low Temperature, Phys iol. Plant., 1997, vol. 100, pp. 291–296.
3. Shakirova, F.M. and Allagulova, Ch.R., Dehydrins in Plants, Vestn. Bashkir. Gos. Univ., 2001, no. 2 (1), pp. 175–178. 4. Borovskii, G.B., Stupnikova, I.V., Antipina, A.I., Vladimirova, S.V., and Voinikov, V.K., Accumulation of DehydrinLike Proteins in the Mitochondria of Cere als in Response to Cold, Freezing, Drought and ABA Treatment, BMC Plant Biol., 2002, vol. 2, pp. 5–11. 5. Allagulova, Ch.R., Gimalov, F.R., Shakirova, F.M., and Vakhitov, V.A., Dehydrins in Plants: Structure and Esti mated Functions, Biokhimiya, 2003, vol. 68, pp. 1157– 1165. 6. Rorat, T., Dehydrins – Tissue Location, Structure and Function, Cell Mol. Biol. Lett., 2006, vol. 11, pp. 536– 556. 7. Farrant, J.M., Pammenter, N.W., Berjak, P., Farn sworth, E.J., and Vertucci, C.W., Presence of Dehy drinLike Proteins and Level of Abscisic Acid in Recal citrant (Desiccation Sensitive) Seeds May Be Related to Habitat, Seed Sci. Res., 1996, vol. 6, pp. 175–182. 8. Gumilevskaya, N.A., Azarkovich, M.I., Komarova, M.E., and Obroucheva, N.V., Proteins of Axial Organs of Dormant and Germinating Horse Chestnut Seeds: 1. General Characterization, Russ. J. Plant Physiol., 2001, vol. 48, pp. 1–11. 9. Gumilevskaya, N.A., Azarkovich, M.I., Lityagina, S.V., and Obroucheva, N.V., Proteins of Axial Organs of Dormant and Germinating Horse Chestnut Seeds: 2. ProteinSynthesizing Capacity of Embryo Axes, Russ. J. Plant Physiol., 2003, vol. 50, pp. 460–469. 10. Gumilevskaya, N.A. and Azarkovich, M.I., Growth Capacity of Embryo Axes Excised from Dormant and Germinating Horse Chestnut Seeds and Their Response to Exogenous Abscisic Acid, Russ. J. Plant Physiol., 2004, vol. 51, pp. 75–85. 11. Azarkovich, M.I. and Gumilevskaya, N.A., Proteins of Cotyledons of Mature Horse Chestnut Seeds, Russ. J. Plant Physiol., 2006, vol. 53, pp. 629–637. 12. Gumilevskaya, N.A. and Azarkovich, M.I., Physiolog ical and Biochemical Characteristics of Recalcitrant Seeds (Review), Prikl. Biokhim. Mikrobiol., 2007, vol. 43, pp. 366–375. 13. Skazhennik, M.A., Gumilevskaya, N.A., Kuvaeva, E.B., and Kretovich, V.L., Electrophoretic Analysis of Com ponents in Total Proteins from Pea Seed Cotyledons, Prikl. Biokhim. Mikrobiol., 1981, vol. 17, pp. 918–926. 14. Towbin, H., Staehlin, T., and Gordon, J., Electro phoretic Transfer of Proteins from Polyacrylamide Gel to Nitrocellulose Sheets: Procedure and Some Applica tions, Proc. Natl. Acad. Sci. USA, 1979, vol. 76, pp. 4350–4354. 15. Harlow, E. and Lane, D., Immunoblotting, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory, 1988, pp. 471–510. 16. Protein Electrophoresis. Application Guide, Hoefer Sci entific Instruments, USA, 1994. 17. FinchSavage, W.E., Pramanik, S.K., and Bewly, J.D., The Expression of DehydrinProteins in Desiccation Sensitive (Recalcitrant) Seeds of Temperate Trees, Planta, 1994, vol. 193, pp. 478–485.
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