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Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech (Fagus sylvatica L.) seeds
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Environmental and Experimental Botany 63 (2008) 274–280
Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech (Fagus sylvatica L.) seeds Ewa M. Kalemba ∗ , Stanisława Pukacka 1 Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 K´ornik, Poland Received 3 April 2007; received in revised form 30 November 2007; accepted 23 December 2007
Abstract Beech seed physiology, including the effect of stress proteins like late embryogenesis abundant (LEA) and small heat shock proteins (sHSP) on viability during storage, is not fully understood. Four lots of beech (Fagus sylvatica L.) seeds have been stored for 1, 4, 6 and 8 years at −10 ◦ C and 8–9% moisture content (MC). Under these conditions, the germination capacity ranges from 81.5% to 100% in the youngest seeds. However, the seeds decrease in vigour with prolonged time of storage. Dehydrins and dehydrin-like proteins were identified both in cotyledons and embryonic axes of the dry stored seeds. In general, decreased contents of LEA proteins as well as reduced content of total soluble protein were detected during prolonged storage. The contents of soluble proteins in embryonic axes and nearly all detected dehydrins and dehydrin-like proteins were correlated with germination capacity. Moreover a sHSP with molecular mass of approximately 22 kDa was identified. The largest content of this protein was observed in the oldest seeds, especially in embryonic axes. The proteins identified may play a protective role during water deficit and storage. © 2008 Elsevier B.V. All rights reserved. Keywords: Beech; Seed; Storage; LEA; Dehydrin; sHSP
1. Introduction Beech (Fagus sylvatica L.) is a large shade tree with a slow growth rate. Wild type beech is native in almost all of Europe, where it creates Fagetum or grows in mixed forests with oak, hornbeam and pine. Beech produces seeds in large intervals from 5 to 10 years, beginning from more or less the 50th year of its life. So there is a need to store the seeds in seedbanks as a source for human-induced activities of seeding or planting. The optimum storage conditions have been established for the whole seeds: 7.8–11.5% moisture content (MC) and temperature −10 to −20 ◦ C (Le´on-Lobos and Ellis, 2002). Additionally, these optimal storage conditions were confirmed through investigations of glass formation in embryonic axes of beech seeds (Pukacka et al., 2003). Due to their reduced longevity during storage, beech seeds are classified to the intermediate category (Bonner, 1990; Gosling, 1991; Le´on-Lobos and Ellis, 2002), in which seeds can tolerate some desiccation, but cannot survive dehydration below those in equilibrium with about 40–50% rel-
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ative humidity (Ellis et al., 1990). During storage, the activity of the detoxifying system is not sufficient to remove reactive oxygen species (ROS). ROS initiate lipid peroxidation and membrane damage, which can lead to loss of viability of seeds (Hendry, 1993; McDonald, 1999; Bailly, 2004). In seeds, the glassy state assures cell stability in times of dehydration and all reactions become slowed (Buitink et al., 1999). Intracellular glass can prevent crystallization of chemical compounds in spite of their high concentration, membrane fusion and protein conformational changes. The glassy state is significant for the storage longevity of seeds (Buitink et al., 1999). In woody plant seeds, the presence of the glassy state was determined in embryonic axes of beech (Pukacka et al., 2003). Late embryogenesis abundant (LEA) proteins, together with oligosaccharides and possibly small heat shock proteins (sHSPs), participate in glass formation and stabilization. LEA proteins increase the glass transition temperature and the average strength of hydrogen bonding of the amorphous matrix (Wolkers et al., 2001). LEA proteins are produced in many plant organs during plant development and under stress conditions. LEAs are heatstable and hydrophilic proteins usually classified into three or six groups, based on sequence similarity and properties (Bray, 1993; Cuming, 1999; Wise, 2003). The widespread and widely analysed LEA proteins belong to group 2, also called the D-
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11 family (Dure et al., 1989) or dehydrins (Close, 1997). The K, S and Y segments are characteristic of dehydrins (Close, 1996). The S segment is a serine-rich motif and the Y segment is the conserved sequence DEYGNP. Only the K segment, containing the consensus amino acids sequence EKKGIMDKIKELPG, is present in all types of dehydrins (Close, 1997). Based on the presence and combination of these segments, dehydrins are classified into the Yn SK2 , Kn , Kn S, SKn and Y2 Kn classes. Dehydrins are synthesized during seed development as an element of the embryogenesis program, when their accumulation is related to the acquisition of desiccation tolerance (Xu et al., 1996; Close, 1997). Their expression can be induced in seeds by drought and abscisic acid (Chandler and Robertson, 1994). Dehydrins or dehydrin-related proteins were detected in the orthodox woody plant seeds of Acer platanoides L. and many recalcitrant seeds of Castanospermum australe L., Clausena lansium (Lour.), Quercus robur L., Castanea sativa L., Aesculus hippocastanum L., Acer pseudoplatanus L. and Acer saccharinum L. (Finch-Savage et al., 1994). By binding to and stabilizing denatured proteins, maintenance of lipid and membrane structure are the main roles of dehydrins in cells under stress conditions (Rorat, 2006). In plants, sHSPs are a numerous and diverse group of proteins that are localized to the cytosol, nucleus, plastids, endoplasmic reticulum and mitochondria (Vierling, 1991; Sun et al., 2002). sHSP are synthesized during embryogenesis, germination, pollen production and fruit maturation (Sun et al., 2002). Their expression can be induced by oxidative stress, cold stress, heavy metals, ozone, UV and ␥ radiation (Waters et al., 1996; Sun et al., 2002). This suggests that sHSP can be associated with the general mechanism of cellular response to abiotic stress, not only heat shock. sHSPs might participate in glassy state formation (Wehmeyer and Vierling, 2000). They can attach to denatured proteins, prevent their aggregation (Waters et al., 1996; Lee et al., 1997; Halsbeck et al., 2005), stabilize their conformation and also assist in protein folding, oligomer formation and intracellular transport (Hendrick and Hartl, 1995). This study reports the presence of LEA proteins and a sHSP in beech seeds during storage. We have used seeds stored at optimal conditions for prolonged time. The study of stress proteins changes may help to understand the seed physiology during storage. The aim of this study was to investigate whether LEA proteins and sHSP are correlated with a decrease of germination capacity of beech seeds during storage through 8 years. A potential role of those proteins in maintenance of beech seed viability during prolonged storage is discussed. 2. Materials and methods
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2.2. Germination test The germination test was performed on four samples of 50 seeds each. Seeds were hydrated at 100% relative humidity for 24 h. Fully imbibed seeds were placed between moist rolled paper towels in separate boxes and incubated at 3 ◦ C. Stratification was conducted for 20–26 weeks. A seed was scored as germinated when the radicle protruded 5 mm. Germination counts were made every week and germinated seeds were removed (ISTA, 1999). All seeds that had not germinated after 26 weeks were considered dead. 2.3. Protein extraction Embryonic axes and cotyledons were separated and ground to a powder in liquid nitrogen. After the powder was incubated in hexane (1:10, w:v) at −20 ◦ C for 24 h to remove fat and centrifuged (20,000 × g at 4 ◦ C for 20 min). To obtain soluble proteins, dried (under nitrogen gas) and fatfree powder was homogenized at 4 ◦ C in 1:2 (w:v) extraction buffer containing 20 mM Tris–HCl, pH 7.5, 5% glycerol, 10 mM -mercaptoethanol, 7 l of protease inhibitor cocktail (Sigma–Aldrich, Poland, P-9599) for each 0.2 g plant tissue and polyvinylpolypyrrolidone (Sigma–Aldrich, Poland) and then centrifuged (20,000 × g at 4 ◦ C for 20 min). To obtain heatstable proteins, the supernatant was boiled 10 min, cooled on ice 15 min and centrifuged as above. Protein concentration was measured according to Bradford (1976). The level of heat-stable proteins was expressed as a percentage of heat-stable to soluble proteins. Proteins were resolved by 15–17% SDS-PAGE (Laemmli, 1970) and stained with Coomassie brilliant blue R250 (Sigma–Aldrich, Poland). 2.4. Western blotting Seven to twenty-five micrograms of protein extract, calculated based on the calibration curve data, was loaded onto a gel (Marian et al., 2003). Fractioned proteins were transferred onto polyvinylidene fluoride membrane (ImmobilonTM -P, Millipore) at 350 mA for 1 h, blocked and incubated with primary antibody raised against the dehydrin consensus K segment (Close et al., 1993) and Arabidopsis Hsp 17.4 (Wehmeyer et al., 1996), biotinylated secondary antibodies (Sigma–Aldrich, Poland), streptavidin-conjugated alkaline phosphatase (Sigma–Aldrich, Poland) and visualized by reaction with alkaline phosphate substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma–Aldrich, Poland). Primary antibodies were raised in rabbit and secondary antibody was developed in goat (anti-rabbit IgG).
2.1. Plant material 2.5. Densitometry analysis Beech (F. sylvatica L.) seeds were obtained from the Forest Gene Banks in Bialogard and Kostrzyca. They originated from nearby forest districts in NW Poland. Four lots of beechnuts were used in the experiments. All of the seeds had been stored at −10 ◦ C and 8–9% moisture content for 1, 4, 6 and 8 years.
The densitometry data was prepared in the BIO 1D++ (Vilber Lourmat) program using the Vilber Lourmat apparatus. Densitometry in image analysis is based on the digitalization of the image in pixels whose intensity is coded on a scale of 256 levels of grey. The density of a spot is calculated from its volume
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Fig. 1. The germination test of stored beech seeds. 100%, 92%, 81.5% and 85.5% germination capacity were observed in seeds stored for 1, 4, 6 and 8 years, respectively.
Fig. 2. Soluble proteins in cotyledons and embryonic axes of beech seeds stored for prolonged time. Data indicated with the same letter are not statistically significant.
(V), which is a sum of all (3D) intensities (I) and presented in 1 × 10−3 units obtained from V = ni I, and the number of pixels inside the area of the spot.
Table 1 The percentage of soluble proteins that are heat-stable in stored beech seeds
2.6. Statistical analysis Data are presented as means ± standard deviation of three replicates. The statistical differences between seed viability after different storage periods and the particular parameters were tested using a correlation coefficient analysis. The significance amongst the means of components (between-group component and within-group component) was verified in F-test at P < 0.05. 3. Results Both the percentage of germination and an estimation of seed vigour were measured to indicate the seed physiological quality. In the analysed seed lots, germination capacity ranged from 81.5% to 100%. Values of 100%, 92%, 81.5% and 85.5% germination were observed in seeds stored for 1, 4, 6 and 8 years, respectively (Fig. 1). The germination test illustrates a diverse vigour of seeds during storage. Seeds that had been stored for 1 year started to germinate in the 13th week of stratification and ended in week 17 with 100% success, indicating high vigour. In other seed lots, seeds started to germinate earlier (11–12th week of starification) but finished later (18–19th week) indicating decreased vigour. Lengthening the time of storage resulted in a slightly decreased ability to germinate. However after 4, 6 and 8 years of storage, the seeds maintained over 81% germination.
Time of storage (years)
1 4 6 8
Embryonic axes
Cotyledons
%
±
%
±
45.3 47.5 48.1 60.7
11.9 9.1 9.6 8.9
44.8 44.8 48.3 51.1
12.1 6.8 9.6 5.2
Since lipids and proteins are the main storage compounds in beech seeds, the protein profile of soluble and heat-stable fractions in cotyledons and embryonic axes were measured. Significant differences in the soluble proteins were observed only in embryonic axes of seeds stored for 6 years (Fig. 2), characterized by the lowest germination capacity (Fig. 1), and in cotyledons of seeds stored for 8 years (Fig. 2). During storage, a general decreasing trend of some proteins was observed, particularly in the fraction of soluble proteins because the ratio of heatstable to soluble proteins was higher in older seeds (Table 1). The relationship between the contents of soluble proteins from embryonic axes and germination capacity is significant. The correlation coefficient equals 0.89, indicating moderately strong relationship between the variables (Fig. 7). Antisera raised against the K segment sequence TGEKKGIMDKIKEKLPGQH were used to identify the LEA proteins (Close et al., 1993). Dehydrins and dehydrin-like proteins were detected both in cotyledons and embryonic axes (Fig. 3). Two proteins with molecular masses of approximately
Fig. 3. LEA proteins identified in stored beech seeds: 1 and 2, cotyledons; 3 and 4, embryonic axes, where 1 and 3 are soluble proteins; 2 and 4 are heat-stable proteins. Equal amounts of 10 g of protein extracts were loaded onto the gel. Molecular masses of proteins were identified according to Prestained Protein Molecular Weight Marker (Fermentas, Poland).
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Fig. 4. Densitometry analyses of LEAs and LEA-like proteins. Protein bands of heat-stable fraction (26 and 44 kDa) and soluble fraction (35 and 40 kDa) were analysed. Data indicated with the same letter are not statistically significant.
26 and 44 kDa were heat-stable and present both in cotyledons and embryonic axes. Quantitative analyses showed that expression of these proteins was higher in embryonic axes than in cotyledons. The level of the 26 kDa protein decreased with longer storage time (Fig. 4). After 8 years of storage, it was reduced twofold in embryonic axes. Content of the 26 kDa protein could be extremely low in the cotyledons of seeds with decreased viability, since it was detected in seeds with 41% of germination (data not shown). However, this correlation was not observed in embryonic axes. Two proteins with molecular masses of approximately 35 and 40 kDa were soluble and were present only in embryonic axes. As storage time increased, lower contents of these proteins were detected. For 44 kDa dehydrin no clear descending trend was observed. Through the 8 years of storage, its level remained relatively high. The contents of almost all detected dehydrins and dehydrin-like proteins were positively correlated with germination capacity. In particular, germination capacity correlated with the 26 kDa protein in embryonic axes and in cotyledons, and with 35, 40 and the 44 kDa protein in cotyledons (Fig. 7). These relationships were moderately strong. sHSPs were identified in beech seeds using antisera against sHSP 17.4, a class I sHSP from Arabidopsis thaliana (Wehmeyer et al., 1996). A protein with a molecular mass of approximately 22 kDa was detected both in cotyledons and embryonic axes. The embryonic axes possessed high content of this protein (Fig. 5). In addition quantitative analyses confirmed that in embryonic axes the content of the 22 kDa protein increased together with prolonged time of storage (Fig. 6). However, the statistical analyses showed that there is a moderately strong negative correlation between the germination capacity and 22 kDa protein content in both seed tissues (Fig. 7).
Fig. 5. sHSP (22 kDa) in stored beech seeds. Numbers 1, 2, 3 and 4 refer to seeds stored for 8, 6, 4 and 1 year, respectively. Equal amounts of 10 g of protein extracts were loaded onto the gel.
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Fig. 6. Densitometry analyses of 22 kDa sHSP that was detected in embryonic axes of beech seeds. Data indicated with the same letter are not statistically significant.
4. Discussion Vigour is defined as the sum of all seed properties that determine the activity and seed performance during germination and seedling emergence (Perry, 1978) and can be read from the shape of germination curve (Bailly et al., 1998). In particular, genetic, mechanical, pathological and intrinsic factors, preharvest, maturational effect, temperature, moisture conditions and storage environment influence seed vigour (Heydecker, 1972). Seed vigour is one of the most important factors that determine plant growth, yield and potency to tolerate environmental stress. Eighty-five percent of germination capacity in seeds stored for 8 years (Fig. 1) indicates that this lot of seeds was in very good condition and came from a good cropping year. Higher germination percentage observed for 8 years stored seeds compared to 6 years stored seeds can be a result of seed quality. Data from Forest Gene Bank Kostrzyca show that similar differences in viability were observed between these two seed lots just after harvest and after 1 year of storage (Gugała, 2002). However, the most important factors for viability and longevity of seeds in storage are temperature and seed moisture content. In general, lowering the temperature and moisture content to an optimum the longer the period of viability. Well-established and preserved storage conditions resulted in the capability to store viable intermediate category seeds for a prolonged time. Statistically significant decreased contents of soluble proteins (Fig. 2) possibly are caused by protease activity, oxidation and degradation related to ROS and free radicals activity or could be a result of Amadori and Maillard reactions (Murthy and Sun, 2000). Some proteases are present only in embryonic axes and their activities are not associated with the mobilization of the accumulated storage materials (Cerc´os et al., 2003). Besides, in embryonic axes, the moisture content is higher than in cotyledons and thus all of the reactions can be accelerated (Pukacka et al., 2003). The K segment is characteristic of all dehydrins that belong to the LEA-2 class (Close, 1997). The K segment might be organized in an amphipathic helix, which may play an important role in the protection of cellular molecules and organelles and the presence of proteins with such a domain may intensify the
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Fig. 7. Correlation coefficient (r) between germination capacity and soluble proteins in embryonic axes, LEA protein 26 kDa in embryonic axes and cotyledons, LEA-like 35 kDa protein, LEA-like 40 kDa protein, LEA protein 44 kDa in cotyledons and sHSP 22 kDa in embryonic axes and cotyledons.
protection mechanism in embryonic axes during water deficit. During storage, the moisture content in seeds is only 8–9%, so the presence of water is limited. The polar groups of LEA amino acids participate in preferential hydration. During water deficit, they can replace water in the cell. The roles of these proteins as protectors of cell structures during drought and desiccation are evident (Hoekstra et al., 2001). In embryonic axes strongest correlation between heat-stable dehydrins and germination capacity was computed for the 26 kDa protein (Fig. 7). Possibly the protein participates in glassy state formation. The protein is not detectable during seed maturation and germination (data not published). Moreover, the reaction with dehydrin antibodies might suggest that the 35 and 40 kDa proteins have a LEA-type
domain in their sequence. Atypical LEA-like proteins were identified in plants, i.e. the dormancy-related peroxiredoxin PER1 of the gene Per1 in barley (Hordeum vulgare L.), which plays a protective role in seeds (Stacy et al., 1999). These two proteins also correlate with seed viability. Compared to cotyledons, embryonic axes have more LEA-type proteins and these proteins are present in larger contents. Therefore, embryonic axes seem to be an organ that is preferentially protected during storage. sHSPs act as protective proteins (Forreiter et al., 1997; L¨ow et al., 2000). Their universal protective role and possible participation in glassy state formation (Hsp17.4 Arabidopsis) have been proposed (Wehmeyer and Vierling, 2000). During storage, the temperature was constant (−10 ◦ C). Therefore, the increase of
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sHSP protein is not directly related with heat shock or changes in temperature. It might be associated with the ageing of seeds and oxidative stress. It was shown that an increased production of ROS and lipid peroxides is observed during the storage of beech seeds (Pukacka and Ratajczak, 2007). The expression of some cytosolic (Sun et al., 2001) and mitochondrial sHSPs (Banzet et al., 1998) can be induced by oxidative stress. sHSPs that possess a methionine-rich amphipathic ␣-helix are crucial for the prevention of the effects of oxidative stress (H¨arndahl et al., 1999; Lee and Vierling, 2000). Moreover, Qshsp10.4-CI class-CI sHSP, obtained from Quercus suber L. cork, accumulates in cork and other oxidatively stressed tissues (Jofre et al., 2003). Therefore, the oxidative stress and damaging processes that occur in ageing may induce the expression of the 22 kDa sHSP in beech seeds. sHSPs can decrease the intracellular level of ROS in a glutathione (GSH)-dependent way and therefore enhance the survival of cells exposed to oxidative stress (Arrigo, 1998). Moreover, sHSPs can induce an increase in the cellular level of reduced form of GSH (Preville et al., 1999). Increased GSH content was observed in 5- and 7-year-old beech seeds compared to 2-year-old seeds (Pukacka and Ratajczak, 2007). sHSP in the soluble protein fraction, which decreased contents in ageing seeds, are chaperones that are possibly especially protected, so the increase in their content must be a result of degradation of other soluble proteins rather than de novo synthesis. Newly synthesized proteins are observed in fully hydrated seeds (Kalpana and Madhava Rao, 1997). In dry tissue, where the glassy state is formed, the viscosity of the cytoplasm is so high that the mobility of molecules is limited and reactions are slowed down. Therefore, the repairing mechanisms can be less effective. However, additional information is still needed to determine the role of this sHSP in seeds during storage. What is more the protein band (Fig. 5) could be a suite of proteins. In Arabidopsis thaliana seeds two sHSP with molecular masses 17.4 and 17.6 kDa were detected (Wehmeyer et al., 1996). Thus the establishment of exact number of sHSP in beech seeds is also needed. Acknowledgements The antisera raised against the K segment were kindly supplied by T.J. Close, Department of Botany and Plant Sciences, University of California, Riverside, CA, USA. The antisera raised against the Arabidopsis anti-Hsp17.4 were kindly supplied by E. Vierling, Department of Biochemistry & Molecular Biophysics, University of Arizona, Tucson. This research was supported by The Ministry of Science and Information (Poland)—grant no. 2PO6L02927. References Arrigo, A.P., 1998. Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. Biol. Chem. 379 (1), 19–26. Bailly, C., 2004. Active oxygen species and antioxidants in seed biology. Seed Sci. Res. 14, 93–107. Bailly, C., Benamar, A., Corbineau, F., Cˆome, D., 1998. Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds. Physiol. Plant. 104, 646–652.
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Changes in late embryogenesis abundant proteins and a small heat shock protein during storage of beech (Fagus sylvatica L.) seeds Ewa M. Kalemba ∗ , Stanisława Pukacka 1 Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 K´ornik, Poland Received 3 April 2007; received in revised form 30 November 2007; accepted 23 December 2007
Abstract Beech seed physiology, including the effect of stress proteins like late embryogenesis abundant (LEA) and small heat shock proteins (sHSP) on viability during storage, is not fully understood. Four lots of beech (Fagus sylvatica L.) seeds have been stored for 1, 4, 6 and 8 years at −10 ◦ C and 8–9% moisture content (MC). Under these conditions, the germination capacity ranges from 81.5% to 100% in the youngest seeds. However, the seeds decrease in vigour with prolonged time of storage. Dehydrins and dehydrin-like proteins were identified both in cotyledons and embryonic axes of the dry stored seeds. In general, decreased contents of LEA proteins as well as reduced content of total soluble protein were detected during prolonged storage. The contents of soluble proteins in embryonic axes and nearly all detected dehydrins and dehydrin-like proteins were correlated with germination capacity. Moreover a sHSP with molecular mass of approximately 22 kDa was identified. The largest content of this protein was observed in the oldest seeds, especially in embryonic axes. The proteins identified may play a protective role during water deficit and storage. © 2008 Elsevier B.V. All rights reserved. Keywords: Beech; Seed; Storage; LEA; Dehydrin; sHSP
1. Introduction Beech (Fagus sylvatica L.) is a large shade tree with a slow growth rate. Wild type beech is native in almost all of Europe, where it creates Fagetum or grows in mixed forests with oak, hornbeam and pine. Beech produces seeds in large intervals from 5 to 10 years, beginning from more or less the 50th year of its life. So there is a need to store the seeds in seedbanks as a source for human-induced activities of seeding or planting. The optimum storage conditions have been established for the whole seeds: 7.8–11.5% moisture content (MC) and temperature −10 to −20 ◦ C (Le´on-Lobos and Ellis, 2002). Additionally, these optimal storage conditions were confirmed through investigations of glass formation in embryonic axes of beech seeds (Pukacka et al., 2003). Due to their reduced longevity during storage, beech seeds are classified to the intermediate category (Bonner, 1990; Gosling, 1991; Le´on-Lobos and Ellis, 2002), in which seeds can tolerate some desiccation, but cannot survive dehydration below those in equilibrium with about 40–50% rel-
∗ 1
Corresponding author. Tel.: +48 6181 70033; fax: +48 6181 70166. E-mail address: [email protected] (E.M. Kalemba). Tel.: +48 618170033; fax: +48 618170166.
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ative humidity (Ellis et al., 1990). During storage, the activity of the detoxifying system is not sufficient to remove reactive oxygen species (ROS). ROS initiate lipid peroxidation and membrane damage, which can lead to loss of viability of seeds (Hendry, 1993; McDonald, 1999; Bailly, 2004). In seeds, the glassy state assures cell stability in times of dehydration and all reactions become slowed (Buitink et al., 1999). Intracellular glass can prevent crystallization of chemical compounds in spite of their high concentration, membrane fusion and protein conformational changes. The glassy state is significant for the storage longevity of seeds (Buitink et al., 1999). In woody plant seeds, the presence of the glassy state was determined in embryonic axes of beech (Pukacka et al., 2003). Late embryogenesis abundant (LEA) proteins, together with oligosaccharides and possibly small heat shock proteins (sHSPs), participate in glass formation and stabilization. LEA proteins increase the glass transition temperature and the average strength of hydrogen bonding of the amorphous matrix (Wolkers et al., 2001). LEA proteins are produced in many plant organs during plant development and under stress conditions. LEAs are heatstable and hydrophilic proteins usually classified into three or six groups, based on sequence similarity and properties (Bray, 1993; Cuming, 1999; Wise, 2003). The widespread and widely analysed LEA proteins belong to group 2, also called the D-
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11 family (Dure et al., 1989) or dehydrins (Close, 1997). The K, S and Y segments are characteristic of dehydrins (Close, 1996). The S segment is a serine-rich motif and the Y segment is the conserved sequence DEYGNP. Only the K segment, containing the consensus amino acids sequence EKKGIMDKIKELPG, is present in all types of dehydrins (Close, 1997). Based on the presence and combination of these segments, dehydrins are classified into the Yn SK2 , Kn , Kn S, SKn and Y2 Kn classes. Dehydrins are synthesized during seed development as an element of the embryogenesis program, when their accumulation is related to the acquisition of desiccation tolerance (Xu et al., 1996; Close, 1997). Their expression can be induced in seeds by drought and abscisic acid (Chandler and Robertson, 1994). Dehydrins or dehydrin-related proteins were detected in the orthodox woody plant seeds of Acer platanoides L. and many recalcitrant seeds of Castanospermum australe L., Clausena lansium (Lour.), Quercus robur L., Castanea sativa L., Aesculus hippocastanum L., Acer pseudoplatanus L. and Acer saccharinum L. (Finch-Savage et al., 1994). By binding to and stabilizing denatured proteins, maintenance of lipid and membrane structure are the main roles of dehydrins in cells under stress conditions (Rorat, 2006). In plants, sHSPs are a numerous and diverse group of proteins that are localized to the cytosol, nucleus, plastids, endoplasmic reticulum and mitochondria (Vierling, 1991; Sun et al., 2002). sHSP are synthesized during embryogenesis, germination, pollen production and fruit maturation (Sun et al., 2002). Their expression can be induced by oxidative stress, cold stress, heavy metals, ozone, UV and ␥ radiation (Waters et al., 1996; Sun et al., 2002). This suggests that sHSP can be associated with the general mechanism of cellular response to abiotic stress, not only heat shock. sHSPs might participate in glassy state formation (Wehmeyer and Vierling, 2000). They can attach to denatured proteins, prevent their aggregation (Waters et al., 1996; Lee et al., 1997; Halsbeck et al., 2005), stabilize their conformation and also assist in protein folding, oligomer formation and intracellular transport (Hendrick and Hartl, 1995). This study reports the presence of LEA proteins and a sHSP in beech seeds during storage. We have used seeds stored at optimal conditions for prolonged time. The study of stress proteins changes may help to understand the seed physiology during storage. The aim of this study was to investigate whether LEA proteins and sHSP are correlated with a decrease of germination capacity of beech seeds during storage through 8 years. A potential role of those proteins in maintenance of beech seed viability during prolonged storage is discussed. 2. Materials and methods
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2.2. Germination test The germination test was performed on four samples of 50 seeds each. Seeds were hydrated at 100% relative humidity for 24 h. Fully imbibed seeds were placed between moist rolled paper towels in separate boxes and incubated at 3 ◦ C. Stratification was conducted for 20–26 weeks. A seed was scored as germinated when the radicle protruded 5 mm. Germination counts were made every week and germinated seeds were removed (ISTA, 1999). All seeds that had not germinated after 26 weeks were considered dead. 2.3. Protein extraction Embryonic axes and cotyledons were separated and ground to a powder in liquid nitrogen. After the powder was incubated in hexane (1:10, w:v) at −20 ◦ C for 24 h to remove fat and centrifuged (20,000 × g at 4 ◦ C for 20 min). To obtain soluble proteins, dried (under nitrogen gas) and fatfree powder was homogenized at 4 ◦ C in 1:2 (w:v) extraction buffer containing 20 mM Tris–HCl, pH 7.5, 5% glycerol, 10 mM -mercaptoethanol, 7 l of protease inhibitor cocktail (Sigma–Aldrich, Poland, P-9599) for each 0.2 g plant tissue and polyvinylpolypyrrolidone (Sigma–Aldrich, Poland) and then centrifuged (20,000 × g at 4 ◦ C for 20 min). To obtain heatstable proteins, the supernatant was boiled 10 min, cooled on ice 15 min and centrifuged as above. Protein concentration was measured according to Bradford (1976). The level of heat-stable proteins was expressed as a percentage of heat-stable to soluble proteins. Proteins were resolved by 15–17% SDS-PAGE (Laemmli, 1970) and stained with Coomassie brilliant blue R250 (Sigma–Aldrich, Poland). 2.4. Western blotting Seven to twenty-five micrograms of protein extract, calculated based on the calibration curve data, was loaded onto a gel (Marian et al., 2003). Fractioned proteins were transferred onto polyvinylidene fluoride membrane (ImmobilonTM -P, Millipore) at 350 mA for 1 h, blocked and incubated with primary antibody raised against the dehydrin consensus K segment (Close et al., 1993) and Arabidopsis Hsp 17.4 (Wehmeyer et al., 1996), biotinylated secondary antibodies (Sigma–Aldrich, Poland), streptavidin-conjugated alkaline phosphatase (Sigma–Aldrich, Poland) and visualized by reaction with alkaline phosphate substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma–Aldrich, Poland). Primary antibodies were raised in rabbit and secondary antibody was developed in goat (anti-rabbit IgG).
2.1. Plant material 2.5. Densitometry analysis Beech (F. sylvatica L.) seeds were obtained from the Forest Gene Banks in Bialogard and Kostrzyca. They originated from nearby forest districts in NW Poland. Four lots of beechnuts were used in the experiments. All of the seeds had been stored at −10 ◦ C and 8–9% moisture content for 1, 4, 6 and 8 years.
The densitometry data was prepared in the BIO 1D++ (Vilber Lourmat) program using the Vilber Lourmat apparatus. Densitometry in image analysis is based on the digitalization of the image in pixels whose intensity is coded on a scale of 256 levels of grey. The density of a spot is calculated from its volume
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Fig. 1. The germination test of stored beech seeds. 100%, 92%, 81.5% and 85.5% germination capacity were observed in seeds stored for 1, 4, 6 and 8 years, respectively.
Fig. 2. Soluble proteins in cotyledons and embryonic axes of beech seeds stored for prolonged time. Data indicated with the same letter are not statistically significant.
(V), which is a sum of all (3D) intensities (I) and presented in 1 × 10−3 units obtained from V = ni I, and the number of pixels inside the area of the spot.
Table 1 The percentage of soluble proteins that are heat-stable in stored beech seeds
2.6. Statistical analysis Data are presented as means ± standard deviation of three replicates. The statistical differences between seed viability after different storage periods and the particular parameters were tested using a correlation coefficient analysis. The significance amongst the means of components (between-group component and within-group component) was verified in F-test at P < 0.05. 3. Results Both the percentage of germination and an estimation of seed vigour were measured to indicate the seed physiological quality. In the analysed seed lots, germination capacity ranged from 81.5% to 100%. Values of 100%, 92%, 81.5% and 85.5% germination were observed in seeds stored for 1, 4, 6 and 8 years, respectively (Fig. 1). The germination test illustrates a diverse vigour of seeds during storage. Seeds that had been stored for 1 year started to germinate in the 13th week of stratification and ended in week 17 with 100% success, indicating high vigour. In other seed lots, seeds started to germinate earlier (11–12th week of starification) but finished later (18–19th week) indicating decreased vigour. Lengthening the time of storage resulted in a slightly decreased ability to germinate. However after 4, 6 and 8 years of storage, the seeds maintained over 81% germination.
Time of storage (years)
1 4 6 8
Embryonic axes
Cotyledons
%
±
%
±
45.3 47.5 48.1 60.7
11.9 9.1 9.6 8.9
44.8 44.8 48.3 51.1
12.1 6.8 9.6 5.2
Since lipids and proteins are the main storage compounds in beech seeds, the protein profile of soluble and heat-stable fractions in cotyledons and embryonic axes were measured. Significant differences in the soluble proteins were observed only in embryonic axes of seeds stored for 6 years (Fig. 2), characterized by the lowest germination capacity (Fig. 1), and in cotyledons of seeds stored for 8 years (Fig. 2). During storage, a general decreasing trend of some proteins was observed, particularly in the fraction of soluble proteins because the ratio of heatstable to soluble proteins was higher in older seeds (Table 1). The relationship between the contents of soluble proteins from embryonic axes and germination capacity is significant. The correlation coefficient equals 0.89, indicating moderately strong relationship between the variables (Fig. 7). Antisera raised against the K segment sequence TGEKKGIMDKIKEKLPGQH were used to identify the LEA proteins (Close et al., 1993). Dehydrins and dehydrin-like proteins were detected both in cotyledons and embryonic axes (Fig. 3). Two proteins with molecular masses of approximately
Fig. 3. LEA proteins identified in stored beech seeds: 1 and 2, cotyledons; 3 and 4, embryonic axes, where 1 and 3 are soluble proteins; 2 and 4 are heat-stable proteins. Equal amounts of 10 g of protein extracts were loaded onto the gel. Molecular masses of proteins were identified according to Prestained Protein Molecular Weight Marker (Fermentas, Poland).
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Fig. 4. Densitometry analyses of LEAs and LEA-like proteins. Protein bands of heat-stable fraction (26 and 44 kDa) and soluble fraction (35 and 40 kDa) were analysed. Data indicated with the same letter are not statistically significant.
26 and 44 kDa were heat-stable and present both in cotyledons and embryonic axes. Quantitative analyses showed that expression of these proteins was higher in embryonic axes than in cotyledons. The level of the 26 kDa protein decreased with longer storage time (Fig. 4). After 8 years of storage, it was reduced twofold in embryonic axes. Content of the 26 kDa protein could be extremely low in the cotyledons of seeds with decreased viability, since it was detected in seeds with 41% of germination (data not shown). However, this correlation was not observed in embryonic axes. Two proteins with molecular masses of approximately 35 and 40 kDa were soluble and were present only in embryonic axes. As storage time increased, lower contents of these proteins were detected. For 44 kDa dehydrin no clear descending trend was observed. Through the 8 years of storage, its level remained relatively high. The contents of almost all detected dehydrins and dehydrin-like proteins were positively correlated with germination capacity. In particular, germination capacity correlated with the 26 kDa protein in embryonic axes and in cotyledons, and with 35, 40 and the 44 kDa protein in cotyledons (Fig. 7). These relationships were moderately strong. sHSPs were identified in beech seeds using antisera against sHSP 17.4, a class I sHSP from Arabidopsis thaliana (Wehmeyer et al., 1996). A protein with a molecular mass of approximately 22 kDa was detected both in cotyledons and embryonic axes. The embryonic axes possessed high content of this protein (Fig. 5). In addition quantitative analyses confirmed that in embryonic axes the content of the 22 kDa protein increased together with prolonged time of storage (Fig. 6). However, the statistical analyses showed that there is a moderately strong negative correlation between the germination capacity and 22 kDa protein content in both seed tissues (Fig. 7).
Fig. 5. sHSP (22 kDa) in stored beech seeds. Numbers 1, 2, 3 and 4 refer to seeds stored for 8, 6, 4 and 1 year, respectively. Equal amounts of 10 g of protein extracts were loaded onto the gel.
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Fig. 6. Densitometry analyses of 22 kDa sHSP that was detected in embryonic axes of beech seeds. Data indicated with the same letter are not statistically significant.
4. Discussion Vigour is defined as the sum of all seed properties that determine the activity and seed performance during germination and seedling emergence (Perry, 1978) and can be read from the shape of germination curve (Bailly et al., 1998). In particular, genetic, mechanical, pathological and intrinsic factors, preharvest, maturational effect, temperature, moisture conditions and storage environment influence seed vigour (Heydecker, 1972). Seed vigour is one of the most important factors that determine plant growth, yield and potency to tolerate environmental stress. Eighty-five percent of germination capacity in seeds stored for 8 years (Fig. 1) indicates that this lot of seeds was in very good condition and came from a good cropping year. Higher germination percentage observed for 8 years stored seeds compared to 6 years stored seeds can be a result of seed quality. Data from Forest Gene Bank Kostrzyca show that similar differences in viability were observed between these two seed lots just after harvest and after 1 year of storage (Gugała, 2002). However, the most important factors for viability and longevity of seeds in storage are temperature and seed moisture content. In general, lowering the temperature and moisture content to an optimum the longer the period of viability. Well-established and preserved storage conditions resulted in the capability to store viable intermediate category seeds for a prolonged time. Statistically significant decreased contents of soluble proteins (Fig. 2) possibly are caused by protease activity, oxidation and degradation related to ROS and free radicals activity or could be a result of Amadori and Maillard reactions (Murthy and Sun, 2000). Some proteases are present only in embryonic axes and their activities are not associated with the mobilization of the accumulated storage materials (Cerc´os et al., 2003). Besides, in embryonic axes, the moisture content is higher than in cotyledons and thus all of the reactions can be accelerated (Pukacka et al., 2003). The K segment is characteristic of all dehydrins that belong to the LEA-2 class (Close, 1997). The K segment might be organized in an amphipathic helix, which may play an important role in the protection of cellular molecules and organelles and the presence of proteins with such a domain may intensify the
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Fig. 7. Correlation coefficient (r) between germination capacity and soluble proteins in embryonic axes, LEA protein 26 kDa in embryonic axes and cotyledons, LEA-like 35 kDa protein, LEA-like 40 kDa protein, LEA protein 44 kDa in cotyledons and sHSP 22 kDa in embryonic axes and cotyledons.
protection mechanism in embryonic axes during water deficit. During storage, the moisture content in seeds is only 8–9%, so the presence of water is limited. The polar groups of LEA amino acids participate in preferential hydration. During water deficit, they can replace water in the cell. The roles of these proteins as protectors of cell structures during drought and desiccation are evident (Hoekstra et al., 2001). In embryonic axes strongest correlation between heat-stable dehydrins and germination capacity was computed for the 26 kDa protein (Fig. 7). Possibly the protein participates in glassy state formation. The protein is not detectable during seed maturation and germination (data not published). Moreover, the reaction with dehydrin antibodies might suggest that the 35 and 40 kDa proteins have a LEA-type
domain in their sequence. Atypical LEA-like proteins were identified in plants, i.e. the dormancy-related peroxiredoxin PER1 of the gene Per1 in barley (Hordeum vulgare L.), which plays a protective role in seeds (Stacy et al., 1999). These two proteins also correlate with seed viability. Compared to cotyledons, embryonic axes have more LEA-type proteins and these proteins are present in larger contents. Therefore, embryonic axes seem to be an organ that is preferentially protected during storage. sHSPs act as protective proteins (Forreiter et al., 1997; L¨ow et al., 2000). Their universal protective role and possible participation in glassy state formation (Hsp17.4 Arabidopsis) have been proposed (Wehmeyer and Vierling, 2000). During storage, the temperature was constant (−10 ◦ C). Therefore, the increase of
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sHSP protein is not directly related with heat shock or changes in temperature. It might be associated with the ageing of seeds and oxidative stress. It was shown that an increased production of ROS and lipid peroxides is observed during the storage of beech seeds (Pukacka and Ratajczak, 2007). The expression of some cytosolic (Sun et al., 2001) and mitochondrial sHSPs (Banzet et al., 1998) can be induced by oxidative stress. sHSPs that possess a methionine-rich amphipathic ␣-helix are crucial for the prevention of the effects of oxidative stress (H¨arndahl et al., 1999; Lee and Vierling, 2000). Moreover, Qshsp10.4-CI class-CI sHSP, obtained from Quercus suber L. cork, accumulates in cork and other oxidatively stressed tissues (Jofre et al., 2003). Therefore, the oxidative stress and damaging processes that occur in ageing may induce the expression of the 22 kDa sHSP in beech seeds. sHSPs can decrease the intracellular level of ROS in a glutathione (GSH)-dependent way and therefore enhance the survival of cells exposed to oxidative stress (Arrigo, 1998). Moreover, sHSPs can induce an increase in the cellular level of reduced form of GSH (Preville et al., 1999). Increased GSH content was observed in 5- and 7-year-old beech seeds compared to 2-year-old seeds (Pukacka and Ratajczak, 2007). sHSP in the soluble protein fraction, which decreased contents in ageing seeds, are chaperones that are possibly especially protected, so the increase in their content must be a result of degradation of other soluble proteins rather than de novo synthesis. Newly synthesized proteins are observed in fully hydrated seeds (Kalpana and Madhava Rao, 1997). In dry tissue, where the glassy state is formed, the viscosity of the cytoplasm is so high that the mobility of molecules is limited and reactions are slowed down. Therefore, the repairing mechanisms can be less effective. However, additional information is still needed to determine the role of this sHSP in seeds during storage. What is more the protein band (Fig. 5) could be a suite of proteins. In Arabidopsis thaliana seeds two sHSP with molecular masses 17.4 and 17.6 kDa were detected (Wehmeyer et al., 1996). Thus the establishment of exact number of sHSP in beech seeds is also needed. Acknowledgements The antisera raised against the K segment were kindly supplied by T.J. Close, Department of Botany and Plant Sciences, University of California, Riverside, CA, USA. The antisera raised against the Arabidopsis anti-Hsp17.4 were kindly supplied by E. Vierling, Department of Biochemistry & Molecular Biophysics, University of Arizona, Tucson. This research was supported by The Ministry of Science and Information (Poland)—grant no. 2PO6L02927. References Arrigo, A.P., 1998. Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death. Biol. Chem. 379 (1), 19–26. Bailly, C., 2004. Active oxygen species and antioxidants in seed biology. Seed Sci. Res. 14, 93–107. Bailly, C., Benamar, A., Corbineau, F., Cˆome, D., 1998. Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds. Physiol. Plant. 104, 646–652.
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