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Age-related changes in protein metabolism of beech (Fagus sylvatica L.) seeds during alleviation of dormancy and in the early stage of germination
Plant Physiology and Biochemistry 94 (2015) 114e121
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Research article
Age-related changes in protein metabolism of beech (Fagus sylvatica L.) seeds during alleviation of dormancy and in the early stage of germination Ewelina Ratajczak, Ewa M. Kalemba, Stanislawa Pukacka* rnik, Poland Institute of Dendrology, Polish Academy of Sciences, 62-035 Ko
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2015 Received in revised form 1 June 2015 Accepted 2 June 2015 Available online 4 June 2015
The long-term storage of seeds generally reduces their viability and vigour. The aim of this work was to evaluate the effect of long-term storage on beech (Fagus sylvatica L.) seeds at optimal conditions, over 9 years, on the total and soluble protein levels and activity of proteolytic enzymes, including endopeptidases, carboxypeptidases and aminopeptidases, as well as free amino acid levels and protein synthesis, in dry seeds, after imbibition and during cold stratification leading to dormancy release and germination. The same analyses were conducted in parallel on seeds gathered from the same tree in the running growing season and stored under the same conditions for only 3 months. The results showed that germination capacity decreased from 100% in freshly harvested seeds to 75% in seeds stored for 9 years. The levels of total and soluble proteins were highest in freshly harvested seeds and decreased significantly during storage, these proportions were retained during cold stratification and germination of seeds. Significant differences between freshly harvested and stored seeds were observed in the activities of proteolytic enzymes, including endopeptidases, aminopeptidases and carboxypeptidases, and in the levels of free amino acids. The neosynthesis of proteins during dormancy release and in the early stage of seed germination was significantly weaker in stored seeds. These results confirm the importance of protein metabolism for seed viability and the consequences of its reduction during seed ageing. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Amino acids Aminopeptidases Carboxypeptidases Endopeptidases Germination Protein synthesis Seed ageing
1. Introduction Seeds of many plant species are characterized by different life spans after maturation and harvesting that depend on their genetic and physiological storage potential as well as by deteriorative processes that occur before or during storage and are affected by environmental factors (Arc et al., 2011). The life span of seeds may also be associated with various metabolic and biophysical conditions (Bailly, 2004; Ballesteros and Walters, 2011). During the storage of seeds, germinability and vigour may be lost over the course of ageing (Bailly, 2004; Çakmak et al., 2010). Seed ageing is accompanied by a progressive loss of vigour and germination capacity even when seeds are stored under optimal temperature, humidity and oxygen levels (Pukacka and Ratajczak, 2007). Seed longevity is a multigenic character (Arc et al., 2011; Rajjou et al.,
rnik, * Corresponding author. Insitute of Dendrology, PAS, Parkowa 5, 62-035 Ko Poland. E-mail address: [email protected] (S. Pukacka). http://dx.doi.org/10.1016/j.plaphy.2015.06.003 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
2012), and reactive oxygen species (ROS) and free radicals are considered the most important factors that determine seed ageing (Bailly, 2004; Rajjou et al., 2012; Ratajczak et al., 2015). ROS prompt many oxidative injuries to macromolecular components of cells (ElMaarouf-Bouteau et al., 2011; Kranner et al., 2011), of which the most affected are proteins (Davies, 2005). Protein oxidation (i.e., carbonylation) can affect the activities of enzymes and increase the susceptibility of proteins to proteolysis (Rajjou et al., 2012; Kalemba and Pukacka, 2014). The loss of seed vigour induced by ageing is thus the result of protein changes in dry seeds during storage, the regulation of protein synthesis and turnover, and posttranslational modifications (PTMs), as well as a reduction in translational activity during germination (Rajjou et al., 2012). PTMs, including carbonylation, S-nitrosylation and nitration, and glycosylation (Murthy et al., 2003), can play important roles in the loss of vigour and viability (Arc et al., 2011; Rajjou et al., 2012). Ranges of 10 to 20 C for temperature and of 7.8e11.5 % for moisture level have been established as optimal conditions for the successful storage of beech seeds (Pukacka et al., 2003). Despite the provision of optimal storage conditions, beech seeds
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lose their viability after 5e10 years of storage (Suszka et al., 1996). Beech seeds are typically classified as intermediate or sub-orthodox because they are characterized by a greater sensitivity to drying and storage conditions compared to orthodox seeds (Pukacka and Ratajczak, 2007). The germination capacity of beech seeds stored in optimal conditions for up to 10 years was strongly positively correlated with soluble protein level, whereas a strong negative correlation was found between germination capacity and hydrogen peroxide, superoxide anion radical and lipid hydroperoxide (LHPO) levels (Pukacka and Ratajczak, 2007). A clear negative correlation was demonstrated between the protein carbonylation level and the germination capacity of beech seeds stored long-term (Kalemba and Pukacka, 2014). Ratajczak et al. (2015) established that the loss of beech seed germinability is related to ROS accumulation in the root apical meristem, and DNA fragmentation occurred primarily in embryonic axes of seeds stored for 5 years or longer. The aforementioned results were reported for dry seeds. The germination of beech seeds requires upwards of 10e12 weeks of cold stratification at 3 C in an imbibed state. The stratification treatment releases dormancy and promotes germination (Weitbrecht et al., 2011). During this period, many processes occur in seeds, among them the upregulation of transcripts encoding proteins involved in protein synthesis and hormone metabolism, as well as DNA and RNA synthesis (Nonogaki, 2014). The transition between seed dormancy and germination involves the reactivation of metabolism and the mobilization of reserve substances (Müntz et al., 2001). During the development of beech seeds, storage proteins are sequestered in protein storage vacuoles (PVS), which are transformed to protein bodies at the end of maturation and drying. Storage proteins consist of 11S globulins (legumin type) and 7S globulins (vicilin type), of which albumins constitute 14.6%, globulins 77.4%, prolamins 1.7%, and glutelins 6.3% (Collada et al., 1988, 1993). However, there are no reports describing the mobilization of protein reserves during dormancy release and germination in beech seeds. Published data indicate that endopeptidases, including cysteine, serine, aspartic proteases and metalloproteases, are responsible for the degradation of storage proteins (Kim et al., 2011; Tan-Wilson and Wilson, 2012). Proteomic studies of dormancy release in beech seeds have indicated that among the more quantitatively changeable proteins, up to 33% are connected with protein metabolism, with roles in synthesis, destination, folding and degradation (Pawlowski, 2007). Moreover, the majority of identified carbonylated proteins in longterm stored beech seeds were also associated with protein metabolism: synthesis, folding, and degradation (Kalemba and Pukacka, 2014). Based on the above data from the literature, we wanted to investigate the effect of long-term storage for 9 years on beech (Fagus sylvatica L.) seeds. We characterized changes in total and soluble protein level, proteolytic activity, free amino acid levels and neosynthesis of proteins in dry seeds and during subsequent steps of the germination process: imbibition, dormancy release, and radicle protrusion. These results were compared with those obtained for seeds stored in the same conditions for only 3 months after gathering. 2. Materials and methods 2.1. Plant material rnik Arboretum in Beech seedlots were collected in the Ko western Poland from a single tree during two different growing seasons. Shed seeds were transferred to a laboratory where they were cleaned, separated from empty seeds and dried in a special room maintained at 18 C, 60e70 % RH, to 8e9 % WC. Afterwards,
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seeds were kept in tightly sealed containers at e 10 C until use. The experiments were performed on seeds stored for 9 years and seeds collected in the current growing season and stored for 3 months (0 years) prior to experiments in containers marked as “non-stored.” Suitable portions of each seed lot designated for analyses were subjected to cold stratification at 3 C, in a similar manner as for germination tests, in separate boxes, where moisture was maintained at a stable level. Analyses of protein level, proteolytic activity, free amino acid levels and protein biosynthesis activity were performed on the embryonic axes (Eas) and cotyledons (Cots) of seeds in the dry state, after imbibition and 3, 6, or 9 weeks of cold stratification as well as in germinated seeds (with embryonic axes protruded to approximately 1 cm). 2.2. Germination test After hydration at 100% RH for 24 h to prevent imbibitional injury, samples of 4 50 seeds from each seed lot were prepared for germination at 3 C between moist paper towels in separate boxes. Cold stratification of seeds was conducted for 20e26 weeks. Each seed was scored as it germinated when the radicle protruded to 5 mm. Germination counts were made every week, and germinated seeds were removed (ISTA, 1999). Decayed seeds were also removed. 2.3. Total protein extraction and determination The samples of Eas and Cots were ground to a powder in liquid nitrogen. Then, 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). Twenty mg fat-free powder (dried under nitrogen gas) was extracted in 0.5 ml of 60 mM TriseHCl buffer (pH 6.8) containing 10% (v/v) glycerol and 2% (w/v) SDS (Stone and Gifford, 1997) at 90 C for 1 h. The mixture was centrifuged at 10,000 g for 15 min, and clear supernatant was used for total protein determination according to Markwell et al. (1981) using the FolinCiocalteu reagent and bovine serum albumin (BSA) as the standard. Protein concentration is expressed in mg per gram dry mass of tissue. 2.4. Soluble protein extraction and determination The samples of Eas and Cots were homogenized in 2 ml of 100 mM TriseHCl buffer (pH 7.5) containing 0.4 M sucrose, 10 mM KCl, 1 mM EDTA, 1 mM PMSF, 0.6% (w/v) PVPP and 50 mM 2mercaptoethanol in a cold mortar and pestle. The homogenate was centrifuged at 10 000 g for 15 min. The supernatant was used for determination of soluble protein level according to Bradford (1976), using BSA as the standard. Protein concentration is expressed in mg per gram dry mass of tissue. 2.5. Extraction of enzyme crude extract The samples of Eas and Cots were homogenized in buffer A (20 mM sodium phosphate (pH 7.0) containing 80 mM NaCl and 10 mM 2-mercaptoethanol) for the determination of aminopeptidase and carboxypeptidase activity and in buffer B (100 mM sodium acetate (pH 5.0) containing 10 mM 2-mercaptoethanol) for the determination of endopeptidase activity, according to Isola and Franzoni (1993). Homogenates were filtered through four layers of gauze and centrifuged at 1000 g for 10 min. The supernatants were desalted through a Sephadex G-25 column before enzymatic assay. All steps were performed at 0e4 C.
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1
2.6. Assay of enzyme activity
expressed as cpm min
Activities of proteolytic enzymes were measured as described by Isola and Franzoni (1993). Endopeptidase activity was essayed in the reaction mixture containing in 0.75 ml of final volume: 0.2 M sodium acetate buffer (pH 5.0), 1 mM 2-mercaptoethanol, 1 mg of gelatine and 100 ml of enzyme extract. The mixture was incubated at 40 C for 60 min, and the reaction was stopped by the addition 0.5 ml of 20% TCA. The precipitated proteins were removed by centrifugation, and in the supernatant, free amino acid content was determined by the ninhydrin method (Rosen, 1957) using leucine as the standard. The extinction was measured at 570 nm using UV-2501 PC spectrophotometer (Shimadzu, Kyoto, Japan). Endopeptidase activities are expressed as mmol mg 1 protein h 1. Carboxypeptidase activity was assayed using aN-benzoyl-DLarginine-nitroanilide (BAPNA) as the substrate. The reaction mixture contained 30 ml BAPNA (6 mM in dimethylformamide), 1 ml 10 mM Na-phosphate buffer (pH 6.8) and 250 ml of enzyme extract. The absorbance of the liberated p-nitroaniline was measured at 405 nm, and carboxypeptidase activities are expressed in mmol mg 1 protein h 1 based on p-nitroaniline standard curve. Aminopeptidase activity was assayed by the release of pnitroaniline from the substrate L-leucine-p-nitroanilide (LPNA). The reaction mixture contained 0.25 ml LPNA (6 mM in 25% methanol), 1 ml 10 mM Na-phosphate buffer (pH 6.5) and 150e200 ml of enzyme extract. The absorbance of liberated p-nitroaniline was measured at 405 nm. The activity of aminopeptidase is expressed as mmol mg 1 protein h 1 based on p-nitroaniline standard curve.
2.9. Statistical analysis
Three typical phases of water uptake were demonstrated, predominantly in the Eas of beech seeds, during imbibition, dormancy release and germination (Fig. 1A). Imbibition of dry beech seeds caused a remarkable increase to approximately 55% WC in Eas and approximately 35% WC in Cots (Phase I, Imbibition). In Eas, the WC remained relatively unchanged (55e60 % WC) up to the 9th week of stratification (Phase II, Stable water content). Then, the WC increased to approximately 85% in germinating seeds, indicating a second phase of water uptake (Phase III, Radicle protrusion). In Cots, Phase II was reported between the 3rd week of stratification and seed germination. The viability of the beech seeds was assessed on the basis of their ability to germinate. Nonestored seeds were characterized by high germination capacity (100%) and high vigour, but the germination capacity of seeds stored for 9 years declined to 75%, and their vigour was worse than that of younger seeds (Fig. 1B).
2.7. Amino acid determination
3.2. Proteins level
The samples of Eas and Cots were homogenized in 5 ml of 80% (v/v) ethanol. Homogenates were heated in a water bath at 70 C for 15 min and then centrifuged at 3500 g for 10 min. The supernatant was separated, and the pellet was extracted with ethanol twice. Combined extracts were evaporated under the vacuum to the known volume. Samples of 5 ml were combined with 1 ml of chloroform to remove pigments and fats. Free amino acids were measured in the water phase by the ninhydrin method according to Rosen (1957), using leucine as the standard. Extinction was measured at 570 nm.
In the Eas and Cots of seeds stored for 9 years, the total (Fig. 2A and B) and soluble protein levels (Fig. 2C and D) were significantly lower in dry imbibed seeds at all stages of stratification and in germinating seeds compared to non-stored seeds.
2.8. Protein synthesis activity Protein synthesis activity was measured by L (U 14C)-leucine incorporation into protein fractions of Eas and Cots of beech seeds according to Kalpana and Rao (1997), with some modifications. Samples of 20 Eas or 5 Cots were placed in 1 ml or 3 ml, respectively, of incubation medium containing 0.1 M potassium phosphate (pH 6.0) buffer with 10 mg ml 1 sucrose, 40 mg ml 1 of streptomycin and 1 mCi ml 1 L (U 14C)-leucine. The samples were vacuum infiltrated and incubated for 18 h at 30 C. After incubation, the samples were separated from the buffer, washed with deionized water, and then surface dried on paper towel. Next, samples were homogenized on an ice bath in 0.01 M Tris buffer (pH 7.5), containing 0.2 mg ml 1 12C L-leucine. The homogenate was filtered through glass wool, and protein was precipitated using 2% (w/v) TCA and centrifuged at 20 000 g for 15 min. The precipitate was dissolved in 0.5 ml (Eas) or 1 ml (Cots) of 2.0 N ammonium hydroxide. From these solutions, samples of 50 ml were transferred to rings of Whattman 3 filter paper and dried at 60 C for 10 min. The incorporation of L (U 14C)-leucine into proteins was measured on a Beckman liquid scintillation counter using an Ultima Gold (Perkin Elmer) scintillator system. The protein synthesis activity is
g
FW.
The data are presented as the means ± standard deviation of four replicates. The significant differences between non-stored and stored seeds were tested using an analysis of variance (ANOVA). Levels of significance are indicated as *P < 0.05 and **P < 0.01. 3. Results 3.1. Water uptake and seed germination
3.3. Peptidases activity In non-stored seeds, endopeptidase activity significantly increased in Eas and Cots during cold stratification beginning in the 3rd week and reached a maximum at the 9th week, then decreased to the level of onset in germinating seeds (Fig. 3A and B). At the 9th week of cold stratification the activity of endopeptidase in nonstored seeds, was 13 and 5 fold higher in Eas and Cots respectively than in stored ones. In stored seeds, the activity of endopeptidases only slightly increased at the 3rd week of cold stratification, and in germinating seeds, a distinct increase in activity of these enzymes was noted in Eas and Cots. Carboxypeptidase activity in Eas was significantly increased in stored seeds during imbibition and was significantly higher (six times) than in non-stored seeds for the duration of stratification (Fig. 3C). In the Cots of stored seeds, carboxypeptidase activity was significantly higher during imbibition (nine times) and during the first steps of germination (almost two times) compared to nonstored seeds. In the Cots of non-stored seeds, carboxypeptidase activity increased significantly only in germinated seeds (Fig. 3D). During imbibition, the activity of aminopeptidases increased significantly in the Eas and Cots of stored seeds (thirteen and five times respectively), while in non-stored seeds, the increase appeared in Eas and Cots at the 3rd week of cold stratification and was maintained to the end of stratification and decreased at the first step of germination (Fig. 3E and F). In the Eas and Cots of germinating seeds, the activity of aminopeptidases in stored seeds was significantly higher (fourteen and two times) than in non-
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Fig. 1. Water uptake (A) in embryonic axes (Eas) and cotyledons (Cots) and germination capacity (B) of beech seeds during cold stratification at 3 C, after storage for 0 and 9 years at 10 C. D, dry seeds; G, germinated seeds. Data are means of four replicates ± SD.
Fig. 2. The total (A, B) and soluble (C, D) protein level in embryonic axes and cotyledons of dry (D), imbibed (I), at 3rd, 6th, 9th week of cold stratification at 3 C and germinated (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
stored seeds. 3.4. Free amino acids level Interesting differences between stored and non-stored seeds were evident in the levels of free amino acids (Fig. 4A and B). In the Eas of dry, imbibed seeds and at the 3rd week of cold stratification, the level of free amino acids was low in both groups of seeds. In non-stored seeds at the 6th week, a 2-fold increase was reported in the free amino acid level. Then, the free amino acid level decreased and in germinating seeds reached a level identical to that in dry and imbibed seeds. In stored seeds, the maximum level of free amino acids occurred later, at the 9th week of cold stratification, while in
germinated seeds, it was significantly higher compared to nonstored seeds. In the Cots of non-stored seeds, the change in the free amino acid level was similar to that in Eas. However, in the Cots of stored seeds, the level of free amino acids was significantly higher in dry, imbibed seeds and in all steps of cold stratification as well as in germinated seeds, compared to non-stored seeds. Marked differences in the levels of free amino acids were noted in dry (D) and imbibed seeds (I). 3.5. Protein synthesis The activity of protein synthesis was measured after imbibition and at each week of cold stratification for stored and non-stored
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Fig. 3. The activity of endopeptidases (A, B) carboxypeptidases (C, D) and aminopeptidases (E, F) in embryonic axes and cotyledons of dry (D), imbibed (I), at 3rd, 6th, 9th week of cold stratification at 3 C and germinated (G) (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
seeds (Fig. 5A and B). In general, protein synthesis activity, measured in g FW of tissue, was almost ten times higher in Eas than in Cots. The lowest protein synthesis intensity in Eas was reported in imbibed (term 0 weeks) seeds (Fig. 5A). Protein synthesis increased over the next 3 weeks, reached its highest level at the 3rd week, and then decreased at the 4th week of stratification. The increase in translational activity at the 3rd week of stratification was markedly larger in nonestored seeds. In these seeds, the next notable increase in protein synthesis activity appeared at the 9th week of stratification, and then, the activity of protein synthesis decreased in germinating seeds. In contrast, the translational activity in stored seeds did not change at the 4th or 9th week of stratification and in germinating seeds. Changes in protein synthesis in Cots exhibited a different pattern. Similar high levels of
protein synthesis activity had already occurred in imbibed nonestored and stored seeds (Fig. 5B). Protein synthesis then decreased in both seedlots. In the Cots of nonestored and stored seeds, protein synthesis activity was almost stable beginning at the 4th week and lasting until 9th week of stratification but was markedly lower in stored seeds compared to nonestored seeds. In germinated seeds, the activity of protein synthesis decreased proportionally in both types of seeds but remained significantly higher in nonestored seeds. 4. Discussion In the present study, beech seeds retained relatively high viability (75%) after storage for nine years. However, their vigour
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Fig. 4. Free amino acid levels in embryonic axes (A) and cotyledons (B) of dry (D), imbibed (I), at 3rd, 6th, 9th week of cold stratification at 3 C and germinated (G) (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
Fig. 5. De novo protein synthesis in embryonic axes (A) and cotyledons (B) in imbibed (Im), during 9 weeks of cold stratification at 3 C and germinated (G) (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
was worse than that of non-stored seeds (Fig. 1B). Seed viability is dependent on the environmental conditions during development and preparation for storage after harvest (Suszka et al., 1996). Previous studies have revealed that beech seeds stored for 7 years can possess up to 66% germination capacity (Pukacka and Ratajczak, 2007), while seeds stored for 8 years can possess up to 82% germination capacity (Kalemba and Pukacka, 2014), depending on the seed lot. During storage, total and soluble protein levels decreased in Eas and Cots significantly (Fig. 2AeD). Although the seeds were collected from the same tree, the significant differences in the levels of the total and soluble proteins in stored and non-stored seeds may be due to the weather conditions experienced by a given year's crop of seeds. However, similar trends were observed for other beech seedlots used in the works of Pukacka and Ratajczak (2007) and Kalemba and Pukacka (2008, 2014) and in work studying artificial ageing of seeds of different plant species (Kalpana and Rao, 1997; Xin et al., 2011). In seeds where germination requires several weeks of cold stratification, proteolysis can begin at the imbibition of seeds and last until radicle protrusion and seedling development (Bewley, 1997). In general, the level of protein is the resultant of both proteolysis and protein synthesis activities. In Dicotyledones, protein bodies, in addition to containing storage whereas aminopeptidases are localized in the cytoplasm (Müntz et al., 2001; Tan-Wilson and Wilson, 2012). Nothing is known about the location of proteolytic enzymes along the embryonic axes and in the cotyledons of beech
seeds; however, judging by the composition of the storage proteins, it can be assumed that beech seeds are typical of dicots. Our results clearly show that long-term storage of beech seeds changes the activities of proteolytic enzymes. The activity of endopeptidases in these seeds is several times lower than in non-stored seeds (Fig. 3A and B). In stored seeds, the activity of endopeptidases increased at the beginning of seed germination. The weaker and delayed endopeptidase activity in stored seeds could be the cause of depleted vigour and viability. Kiyosaki et al. (2009) and Prabucka et al. (2013) demonstrated that gibberellic acid (GA3) affects endopeptidase synthesis in wheat seeds during germination. It is possible that in beech seeds, where breaking dormancy and beginning germination is under the control of GA (Mortensen and Eriksen, 2004), endopeptidase activity is also connected with this hormone. However, the hormonal control of storage protein mobilization in dicots has not yet been resolved (Tan-Wilson and Wilson, 2012). Carboxypeptidases cooperate with endopeptidases to increase the number of peptides with free C-termini. The higher activity of carboxypeptidases in the Eas and Cots of stored beech seeds may be the result of the ROS-affected breakdown of storage proteins because ROS are known to accumulate in aged beech seeds (Pukacka and Ratajczak, 2007; Ratajczak et al., 2015). The final products of endopeptidases and carboxypeptidases are free amino acids and small oligopeptides, which are degraded to amino acids by aminopeptidases (Müntz, 1996; Tan-Wilson and Wilson, 2012). An age-related effect on aminopeptidase activity has been demonstrated in Cots, where weaker activity of these enzymes in
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stored seeds until the 9th week of cold stratification has been observed. Considering that the breakdown of storage proteins begins with endopeptidase activity (Tan-Wilson and Wilson, 2012), it can be suggested that beech seeds characterized with 100% germination capacity (i.e., non-stored seeds), the mobilization of proteinous reserves begins at the 3rd week of cold stratification and is further intensified up to the 9th week in both Eas and Cots (Fig. 3A and B). The increase in carboxypeptidase (Fig. 3C and D) and aminopeptidase (Fig. 3E and F) activity in imbibed stored seeds coincides with first water uptake (Fig. 1A). High proteolytic activity at this particular time might be related to the removal of oxidatively injured proteins in stored seeds, because phase I of water uptake is considered to be a moment of initiation of repair mechanisms for membranes and proteins (Weitbrecht et al., 2011). Aged seeds exhibited significantly higher proteolytic activity in germinating seeds (Fig. 3) when compared to non-stored seeds, whereas differences in soluble and total protein level between stored and nonstored germinating seeds are similar to the initial differences in dry seeds (Fig. 2). It seems that the completion of dormancy release and the germination program in beech seeds requires intense endopeptidase activity, probably in protein bodies, between the 3rd and 9th weeks of stratification (Fig. 3A and B) as well as intense de novo protein synthesis, particularly at the 3rd and 9th weeks of stratification (Fig. 5) because stored seeds lack this phenomenon. In general, protein synthesis activity is significantly higher in nonstored seeds compared to stored seeds. It is possible that the first peak of protein synthesis in beech seeds is the result of the translational activity of stored mRNA. The work of Rajjou et al. (2012) and Kimura and Nambara (2010) revealed that proteins and mRNA pools stored in dry seeds play an important role in the germination process. De novo protein synthesis from stored mRNA allows cells to repair or replace important proteins altered during storage to reinitiate metabolism during germination. On the other hand, Chibani et al. (2006) demonstrated that after successful dormancy, released seeds acquire the capacity to reprogram the pattern of protein biosynthesis during imbibition, allowing the completion of germination. It can be assumed that perturbing protein synthesis and proteolysis does not allow completion of the dormancy release program in stored seeds and that therefore, no more than 75% of them germinated (Fig. 1B). During the later stages of cold stratification, de novo synthesis of proteolytic enzymes, particularly endopeptidases and aminopeptidases (Galland et al., 2014), is also possible (Tan-Wilson and Wilson, 2012). Lowered protein neosynthesis in stored seeds at the 9th week of stratification (Fig. 5) coincides with the greatest decrease in endopeptidase activity (Fig. 3A and B) compared to non-stored seeds. In this light, it can be hypothesized that the age-related impairment of neosynthesis of endopeptidases at later stages of stratification might contribute to a decrease in the germination capacity of stored beech seeds. The aminopeptidase activity in the Eas of stored seeds seems to be more affected by water than seed age, because the only reported differences in enzymatic activity occurring in imbibed and germinating seeds co-occurs with the first and the second documented water uptake (Fig. 1A). It is clear that the free amino acid levels in the Eas and Cots of stored and non-stored seeds during cold stratification and the early stage of germination are related to the neosynthesis of proteins in these organs, particularly in the Eas of non-stored seeds between the 3rd and 9th week of stratification, where careful coordination is visible between these two processes (Fig. 4A and 5A). Larger amounts of available free amino acids in the Cots of stored seeds (Fig. 4B) might be the effect of inefficient translation in this organ (Fig. 5B) and may indicate greater protein degradation by the 26S proteasome in the cytoplasm.
5. Conclusion Decreases in seed viability are considered to be age related. Beech seeds stored for only 3 months maintained 100% germination capacity, whereas seeds stored for 9 years maintained 75% germination capacity under identical dormancy alleviation and germination conditions. These results suggest that lower protein levels and perturbations in proteolysis and protein synthesis are important age-related factors that contribute to seed viability. Seeds stored for 9 years contained less protein in the dry state and continuously during dormancy alleviation, as well as during germination. The largely decreased activity of endopeptidases in seeds stored for 9 years, particularly at the 3rd-9th week of cold stratification, might be the cause of affected storage protein mobilization. Moreover, despite of the abundance of free amino acids, the weakened de novo synthesis of proteins, possibly also endopeptidases, at the end of the stratification period contributes to the disruption of the dormancy release program or to the inaccurate initiation of the germination process in stored seeds. Contributions SP designed the experiments; ER conducted all experiments; ER, EMK, SP analyzed the data; SP wrote the manuscript; EMK, SP created figures; ER, EMK, SP revised and edited the manuscript. Acknowledgements This study was supported by research funds of the Polish Ministry of Science and Higher Education. The authors thank dr Renata Rucinska from the Isotope Laboratory, Adam Mickiewicz University in Poznan, for the help with assay of protein synthesis. References Arc, E., Galland, M., Cueff, G., Godin, B., Lounifi, I., Job, D., Rajjou, L., 2011. Reboot the system thanks to protein post-translational modifications and proteome diversity: how quiescent seeds restart their metabolism to prepare seedling establishment. Proteomics 11, 1606e1618. Bailly, C., 2004. Reactive oxygen species and antioxidant in seed biology. Seed Sci. Res. 14, 93e107. Ballesteros, D., Walters, C., 2011. Detailed characterization of mechanical properties and molecular mobility within dry seed glasses: relevance to the physiology of dry biological systems. Plant J. 68, 607e619. Bewley, J.D., 1997. Seed germination and dormancy. Plant Cell 9, 1055e1066. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein edye binding. Anal. Biochem. 72, 248e254. Çakmak, T., Atici, O., Agar, G., Sunar, S., 2010. Natural aging-related biochemical changes in alfalfa (Medicago sativa L.) seeds stored for 42 years. Int. Res. J. Plant Sci. 1, 001e006. Collada, C., Caballero, R.G., Casado, R., Aragoncillo, C., 1988. Different types of major storage seed proteins in Fagaceae species. J. Exp. Bot. 39, 1751e1758. Collada, C., Aragoncillo, P., Aragoncillo, C., 1993. Development of protein bodies in cotyledons of Fagus sylvatica. Physiol. Plant 89, 354e359. Chibani, K., Ali-Rachedi, S., Job, C., Job, D., Jullien, M., Grappin, P., 2006. Proteomic analysis of seed dormancy in Arabidopsis. Plant Physiol. 142, 1493e1510. Davies, M.J., 2005. The oxidative environment and protein damage. Biochim. Biophys. Acta 1703, 93e109. El-Maarouf-Bouteau, H., Mazury, C., Corbineau, F., Bailly, C., 2011. DNA alteration and programmed cell death during ageing of sunflower seed. J. Exp. Bot. 62, 5003e5011. Galland, M., Huguet, R., Arc, E., Cueff, G., Job, D., Rajjou, L., 2014. Dynamic proteomic emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination. Mol. Cell. Proteomics 13, 253e268. Isola, M.C., Franzoni, L., 1993. Studies on the proteolytic activities of potato tuber. Plant Physiol. Biochem. 31, 169e174. ISTA, 1999. International rules for seed testing. Rules 1999 (Suppl.). Seed Sci. Technol. 27, 1e133. Kalemba, E.M., Pukacka, S., 2008. Changes in late embryogenesis abundant proteins and small heat shock protein during storage of beech (Fagus sylvatica L.) seeds. Env. Exp. Bot. 63, 274e280. Kalemba, E.M., Pukacka, S., 2014. Carbonylated proteins accumulated as vitality decreases during long-term storage of beech (Fagus sylvatica L.) seeds. Trees 28,
E. Ratajczak et al. / Plant Physiology and Biochemistry 94 (2015) 114e121 503e515. Kalpana, R., Rao, K.V.M., 1997. Protein metabolism of seeds of pigeonpea (Cajanus cajan (L.) Millsp.) cultivars during accelerated ageing. Seed Sci. Technol. 25, 271e279. Kim, H.T., Choi, U.-K., Ryu, H.S., Lee, S.J., Kwon, O.-S., 2011. Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth. Biochim. Biophys. Acta 1814, 1178e1187. Kimura, M., Nambara, E., 2010. Stored and neosynthesized mRNA in Arabidopsis seeds: effects of cycloheximide and controlled deterioration treatment on the resumption of transcription during imbibition. Plant Mol. Biol. 73, 119e129. Kiyosaki, T., Asakura, T., Matsumoto, I., Tamura, T., 2009. Wheat cysteine proteases triticain a, b and g exhibit mutually distinct responses to gibberellin in germinating seeds. J. Plant Physiol. 166, 101e106. Kranner, I., Chen, H., Pritchard, H., Pearce, S., Birti c, S., 2011. Inter-nucleosomal DNA fragmentation and loss of RNA integrity during seed ageing. Plant Growth Reg. 63, 63e72. Markwell, M.A., Haas, S.M., Tolbert, N.E., Bieber, L.L., 1981. Protein determination in membrane and lipoprotein samples: manual and automated procedures. Meth. Enzym 72, 296e303. Mortensen, L.C., Eriksen, E.N., 2004. The effect of gibberellic acid, paclobutrazol and etephon on the germination of Fagus sylvatica and Picea sitchensis seeds exposed to varying duration of moist chilling. Seed Sci. Technol. 32, 21e33. Müntz, K., 1996. Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds. J. Exp. Bot. 47, 605e622. Müntz, K., Belozersky, M.A., Dunaevsky, Y.E., Schlereth, A., Tiedemann, J., 2001. Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth. J. Exp. Bot. 52, 1741e1752. Murthy, U.M.N., Kumar, P.P., Sun, W.Q., 2003. Mechanisms of seed ageing under different storage conditions for Vigna radiate (L.) Wilczek: lipid peroxidation, sugar hydrolysis. J. Exp. Bot. 54, 1057e1067. Nonogaki, H., 2014. Seed dormancy and germination-emerging mechanisms and
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new hypotheses. Front. Plant Sci. 5, 233. Pawlowski, T.A., 2007. Proteomics of European beech (Fagus sylvatica L.) seed dormancy breaking: influence of abscisic and gibberellic acids. Proteomics 7, 2246e2257. Prabucka, B., Drzymala, A., Grabowska, A., 2013. Molecular cloning and expression analysis of the main gliadin-degrading cysteine endopeptidase EP8 from triticale. J. Cereal Sci. 58, 284e289. jkiewicz, E., 2003. Water and Pukacka, S., Hoffmann, S.K., Goslar, J., Pukacki, P.M., Wo lipid relations in beech (Fagus sylvatica L.) seeds and its effect on storage behaviour. Bioch. Biophys. Acta 1621, 48e56. Pukacka, S., Ratajczak, E., 2007. Age-related biochemical changes during storage of beech (Fagus sylvatica L.) seeds. Seed Sci. Res. 17, 45e53. Rajjou, L., Duval, M., Gallardo, K., Catusse, J., Bally, J., Job, C., Job, D., 2012. Seed germination and vigor. Ann. Rev. Plant Biol. 63, 507e533. Ratajczak, E., Małecka, A., Bagniewska-Zadworna, A., Kalemba, E.M., 2015. The production, localization and spreading of reactive oxygen species contributes to the low vitality of long-term stored common beech (Fagus sylvatica L.) seeds. J. Plant Physiol. 174, 147e156. Rosen, H., 1957. A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67, 10e15. Stone, S.L., Gifford, D.J., 1997. Structural and biochemical changes in loblolly pine (Pinus taeda L.) seeds during germination and early-seedling growth. I. Storage protein reserves. Int. J. Plant Sci. 158, 727e737. Suszka, B., Muller, C., Bonnet-Masimbert, M., 1996. Seeds of Forest Broadleaves: from Harvest to Sowing. INRA, Paris. Tan-Wilson, A.L., Wilson, K.A., 2012. Mobilization of seed protein reserves. Physiol. Plant 145, 140e153. Weitbrecht, K., Müller, K., Leubner-Metzger, G., 2011. First off the mark: early seed germination. J. Exp. Bot. 62, 3289e3309. Xin, X., Lin, X.H., Zhou, Y.C., Chen, X.L., Liu, X., Lu, X.X., 2011. Proteome analysis of maize seeds: the effect of artificial ageing. Physiol. Plant 143, 126e138.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Age-related changes in protein metabolism of beech (Fagus sylvatica L.) seeds during alleviation of dormancy and in the early stage of germination Ewelina Ratajczak, Ewa M. Kalemba, Stanislawa Pukacka* rnik, Poland Institute of Dendrology, Polish Academy of Sciences, 62-035 Ko
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 March 2015 Received in revised form 1 June 2015 Accepted 2 June 2015 Available online 4 June 2015
The long-term storage of seeds generally reduces their viability and vigour. The aim of this work was to evaluate the effect of long-term storage on beech (Fagus sylvatica L.) seeds at optimal conditions, over 9 years, on the total and soluble protein levels and activity of proteolytic enzymes, including endopeptidases, carboxypeptidases and aminopeptidases, as well as free amino acid levels and protein synthesis, in dry seeds, after imbibition and during cold stratification leading to dormancy release and germination. The same analyses were conducted in parallel on seeds gathered from the same tree in the running growing season and stored under the same conditions for only 3 months. The results showed that germination capacity decreased from 100% in freshly harvested seeds to 75% in seeds stored for 9 years. The levels of total and soluble proteins were highest in freshly harvested seeds and decreased significantly during storage, these proportions were retained during cold stratification and germination of seeds. Significant differences between freshly harvested and stored seeds were observed in the activities of proteolytic enzymes, including endopeptidases, aminopeptidases and carboxypeptidases, and in the levels of free amino acids. The neosynthesis of proteins during dormancy release and in the early stage of seed germination was significantly weaker in stored seeds. These results confirm the importance of protein metabolism for seed viability and the consequences of its reduction during seed ageing. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Amino acids Aminopeptidases Carboxypeptidases Endopeptidases Germination Protein synthesis Seed ageing
1. Introduction Seeds of many plant species are characterized by different life spans after maturation and harvesting that depend on their genetic and physiological storage potential as well as by deteriorative processes that occur before or during storage and are affected by environmental factors (Arc et al., 2011). The life span of seeds may also be associated with various metabolic and biophysical conditions (Bailly, 2004; Ballesteros and Walters, 2011). During the storage of seeds, germinability and vigour may be lost over the course of ageing (Bailly, 2004; Çakmak et al., 2010). Seed ageing is accompanied by a progressive loss of vigour and germination capacity even when seeds are stored under optimal temperature, humidity and oxygen levels (Pukacka and Ratajczak, 2007). Seed longevity is a multigenic character (Arc et al., 2011; Rajjou et al.,
rnik, * Corresponding author. Insitute of Dendrology, PAS, Parkowa 5, 62-035 Ko Poland. E-mail address: [email protected] (S. Pukacka). http://dx.doi.org/10.1016/j.plaphy.2015.06.003 0981-9428/© 2015 Elsevier Masson SAS. All rights reserved.
2012), and reactive oxygen species (ROS) and free radicals are considered the most important factors that determine seed ageing (Bailly, 2004; Rajjou et al., 2012; Ratajczak et al., 2015). ROS prompt many oxidative injuries to macromolecular components of cells (ElMaarouf-Bouteau et al., 2011; Kranner et al., 2011), of which the most affected are proteins (Davies, 2005). Protein oxidation (i.e., carbonylation) can affect the activities of enzymes and increase the susceptibility of proteins to proteolysis (Rajjou et al., 2012; Kalemba and Pukacka, 2014). The loss of seed vigour induced by ageing is thus the result of protein changes in dry seeds during storage, the regulation of protein synthesis and turnover, and posttranslational modifications (PTMs), as well as a reduction in translational activity during germination (Rajjou et al., 2012). PTMs, including carbonylation, S-nitrosylation and nitration, and glycosylation (Murthy et al., 2003), can play important roles in the loss of vigour and viability (Arc et al., 2011; Rajjou et al., 2012). Ranges of 10 to 20 C for temperature and of 7.8e11.5 % for moisture level have been established as optimal conditions for the successful storage of beech seeds (Pukacka et al., 2003). Despite the provision of optimal storage conditions, beech seeds
E. Ratajczak et al. / Plant Physiology and Biochemistry 94 (2015) 114e121
lose their viability after 5e10 years of storage (Suszka et al., 1996). Beech seeds are typically classified as intermediate or sub-orthodox because they are characterized by a greater sensitivity to drying and storage conditions compared to orthodox seeds (Pukacka and Ratajczak, 2007). The germination capacity of beech seeds stored in optimal conditions for up to 10 years was strongly positively correlated with soluble protein level, whereas a strong negative correlation was found between germination capacity and hydrogen peroxide, superoxide anion radical and lipid hydroperoxide (LHPO) levels (Pukacka and Ratajczak, 2007). A clear negative correlation was demonstrated between the protein carbonylation level and the germination capacity of beech seeds stored long-term (Kalemba and Pukacka, 2014). Ratajczak et al. (2015) established that the loss of beech seed germinability is related to ROS accumulation in the root apical meristem, and DNA fragmentation occurred primarily in embryonic axes of seeds stored for 5 years or longer. The aforementioned results were reported for dry seeds. The germination of beech seeds requires upwards of 10e12 weeks of cold stratification at 3 C in an imbibed state. The stratification treatment releases dormancy and promotes germination (Weitbrecht et al., 2011). During this period, many processes occur in seeds, among them the upregulation of transcripts encoding proteins involved in protein synthesis and hormone metabolism, as well as DNA and RNA synthesis (Nonogaki, 2014). The transition between seed dormancy and germination involves the reactivation of metabolism and the mobilization of reserve substances (Müntz et al., 2001). During the development of beech seeds, storage proteins are sequestered in protein storage vacuoles (PVS), which are transformed to protein bodies at the end of maturation and drying. Storage proteins consist of 11S globulins (legumin type) and 7S globulins (vicilin type), of which albumins constitute 14.6%, globulins 77.4%, prolamins 1.7%, and glutelins 6.3% (Collada et al., 1988, 1993). However, there are no reports describing the mobilization of protein reserves during dormancy release and germination in beech seeds. Published data indicate that endopeptidases, including cysteine, serine, aspartic proteases and metalloproteases, are responsible for the degradation of storage proteins (Kim et al., 2011; Tan-Wilson and Wilson, 2012). Proteomic studies of dormancy release in beech seeds have indicated that among the more quantitatively changeable proteins, up to 33% are connected with protein metabolism, with roles in synthesis, destination, folding and degradation (Pawlowski, 2007). Moreover, the majority of identified carbonylated proteins in longterm stored beech seeds were also associated with protein metabolism: synthesis, folding, and degradation (Kalemba and Pukacka, 2014). Based on the above data from the literature, we wanted to investigate the effect of long-term storage for 9 years on beech (Fagus sylvatica L.) seeds. We characterized changes in total and soluble protein level, proteolytic activity, free amino acid levels and neosynthesis of proteins in dry seeds and during subsequent steps of the germination process: imbibition, dormancy release, and radicle protrusion. These results were compared with those obtained for seeds stored in the same conditions for only 3 months after gathering. 2. Materials and methods 2.1. Plant material rnik Arboretum in Beech seedlots were collected in the Ko western Poland from a single tree during two different growing seasons. Shed seeds were transferred to a laboratory where they were cleaned, separated from empty seeds and dried in a special room maintained at 18 C, 60e70 % RH, to 8e9 % WC. Afterwards,
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seeds were kept in tightly sealed containers at e 10 C until use. The experiments were performed on seeds stored for 9 years and seeds collected in the current growing season and stored for 3 months (0 years) prior to experiments in containers marked as “non-stored.” Suitable portions of each seed lot designated for analyses were subjected to cold stratification at 3 C, in a similar manner as for germination tests, in separate boxes, where moisture was maintained at a stable level. Analyses of protein level, proteolytic activity, free amino acid levels and protein biosynthesis activity were performed on the embryonic axes (Eas) and cotyledons (Cots) of seeds in the dry state, after imbibition and 3, 6, or 9 weeks of cold stratification as well as in germinated seeds (with embryonic axes protruded to approximately 1 cm). 2.2. Germination test After hydration at 100% RH for 24 h to prevent imbibitional injury, samples of 4 50 seeds from each seed lot were prepared for germination at 3 C between moist paper towels in separate boxes. Cold stratification of seeds was conducted for 20e26 weeks. Each seed was scored as it germinated when the radicle protruded to 5 mm. Germination counts were made every week, and germinated seeds were removed (ISTA, 1999). Decayed seeds were also removed. 2.3. Total protein extraction and determination The samples of Eas and Cots were ground to a powder in liquid nitrogen. Then, 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). Twenty mg fat-free powder (dried under nitrogen gas) was extracted in 0.5 ml of 60 mM TriseHCl buffer (pH 6.8) containing 10% (v/v) glycerol and 2% (w/v) SDS (Stone and Gifford, 1997) at 90 C for 1 h. The mixture was centrifuged at 10,000 g for 15 min, and clear supernatant was used for total protein determination according to Markwell et al. (1981) using the FolinCiocalteu reagent and bovine serum albumin (BSA) as the standard. Protein concentration is expressed in mg per gram dry mass of tissue. 2.4. Soluble protein extraction and determination The samples of Eas and Cots were homogenized in 2 ml of 100 mM TriseHCl buffer (pH 7.5) containing 0.4 M sucrose, 10 mM KCl, 1 mM EDTA, 1 mM PMSF, 0.6% (w/v) PVPP and 50 mM 2mercaptoethanol in a cold mortar and pestle. The homogenate was centrifuged at 10 000 g for 15 min. The supernatant was used for determination of soluble protein level according to Bradford (1976), using BSA as the standard. Protein concentration is expressed in mg per gram dry mass of tissue. 2.5. Extraction of enzyme crude extract The samples of Eas and Cots were homogenized in buffer A (20 mM sodium phosphate (pH 7.0) containing 80 mM NaCl and 10 mM 2-mercaptoethanol) for the determination of aminopeptidase and carboxypeptidase activity and in buffer B (100 mM sodium acetate (pH 5.0) containing 10 mM 2-mercaptoethanol) for the determination of endopeptidase activity, according to Isola and Franzoni (1993). Homogenates were filtered through four layers of gauze and centrifuged at 1000 g for 10 min. The supernatants were desalted through a Sephadex G-25 column before enzymatic assay. All steps were performed at 0e4 C.
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1
2.6. Assay of enzyme activity
expressed as cpm min
Activities of proteolytic enzymes were measured as described by Isola and Franzoni (1993). Endopeptidase activity was essayed in the reaction mixture containing in 0.75 ml of final volume: 0.2 M sodium acetate buffer (pH 5.0), 1 mM 2-mercaptoethanol, 1 mg of gelatine and 100 ml of enzyme extract. The mixture was incubated at 40 C for 60 min, and the reaction was stopped by the addition 0.5 ml of 20% TCA. The precipitated proteins were removed by centrifugation, and in the supernatant, free amino acid content was determined by the ninhydrin method (Rosen, 1957) using leucine as the standard. The extinction was measured at 570 nm using UV-2501 PC spectrophotometer (Shimadzu, Kyoto, Japan). Endopeptidase activities are expressed as mmol mg 1 protein h 1. Carboxypeptidase activity was assayed using aN-benzoyl-DLarginine-nitroanilide (BAPNA) as the substrate. The reaction mixture contained 30 ml BAPNA (6 mM in dimethylformamide), 1 ml 10 mM Na-phosphate buffer (pH 6.8) and 250 ml of enzyme extract. The absorbance of the liberated p-nitroaniline was measured at 405 nm, and carboxypeptidase activities are expressed in mmol mg 1 protein h 1 based on p-nitroaniline standard curve. Aminopeptidase activity was assayed by the release of pnitroaniline from the substrate L-leucine-p-nitroanilide (LPNA). The reaction mixture contained 0.25 ml LPNA (6 mM in 25% methanol), 1 ml 10 mM Na-phosphate buffer (pH 6.5) and 150e200 ml of enzyme extract. The absorbance of liberated p-nitroaniline was measured at 405 nm. The activity of aminopeptidase is expressed as mmol mg 1 protein h 1 based on p-nitroaniline standard curve.
2.9. Statistical analysis
Three typical phases of water uptake were demonstrated, predominantly in the Eas of beech seeds, during imbibition, dormancy release and germination (Fig. 1A). Imbibition of dry beech seeds caused a remarkable increase to approximately 55% WC in Eas and approximately 35% WC in Cots (Phase I, Imbibition). In Eas, the WC remained relatively unchanged (55e60 % WC) up to the 9th week of stratification (Phase II, Stable water content). Then, the WC increased to approximately 85% in germinating seeds, indicating a second phase of water uptake (Phase III, Radicle protrusion). In Cots, Phase II was reported between the 3rd week of stratification and seed germination. The viability of the beech seeds was assessed on the basis of their ability to germinate. Nonestored seeds were characterized by high germination capacity (100%) and high vigour, but the germination capacity of seeds stored for 9 years declined to 75%, and their vigour was worse than that of younger seeds (Fig. 1B).
2.7. Amino acid determination
3.2. Proteins level
The samples of Eas and Cots were homogenized in 5 ml of 80% (v/v) ethanol. Homogenates were heated in a water bath at 70 C for 15 min and then centrifuged at 3500 g for 10 min. The supernatant was separated, and the pellet was extracted with ethanol twice. Combined extracts were evaporated under the vacuum to the known volume. Samples of 5 ml were combined with 1 ml of chloroform to remove pigments and fats. Free amino acids were measured in the water phase by the ninhydrin method according to Rosen (1957), using leucine as the standard. Extinction was measured at 570 nm.
In the Eas and Cots of seeds stored for 9 years, the total (Fig. 2A and B) and soluble protein levels (Fig. 2C and D) were significantly lower in dry imbibed seeds at all stages of stratification and in germinating seeds compared to non-stored seeds.
2.8. Protein synthesis activity Protein synthesis activity was measured by L (U 14C)-leucine incorporation into protein fractions of Eas and Cots of beech seeds according to Kalpana and Rao (1997), with some modifications. Samples of 20 Eas or 5 Cots were placed in 1 ml or 3 ml, respectively, of incubation medium containing 0.1 M potassium phosphate (pH 6.0) buffer with 10 mg ml 1 sucrose, 40 mg ml 1 of streptomycin and 1 mCi ml 1 L (U 14C)-leucine. The samples were vacuum infiltrated and incubated for 18 h at 30 C. After incubation, the samples were separated from the buffer, washed with deionized water, and then surface dried on paper towel. Next, samples were homogenized on an ice bath in 0.01 M Tris buffer (pH 7.5), containing 0.2 mg ml 1 12C L-leucine. The homogenate was filtered through glass wool, and protein was precipitated using 2% (w/v) TCA and centrifuged at 20 000 g for 15 min. The precipitate was dissolved in 0.5 ml (Eas) or 1 ml (Cots) of 2.0 N ammonium hydroxide. From these solutions, samples of 50 ml were transferred to rings of Whattman 3 filter paper and dried at 60 C for 10 min. The incorporation of L (U 14C)-leucine into proteins was measured on a Beckman liquid scintillation counter using an Ultima Gold (Perkin Elmer) scintillator system. The protein synthesis activity is
g
FW.
The data are presented as the means ± standard deviation of four replicates. The significant differences between non-stored and stored seeds were tested using an analysis of variance (ANOVA). Levels of significance are indicated as *P < 0.05 and **P < 0.01. 3. Results 3.1. Water uptake and seed germination
3.3. Peptidases activity In non-stored seeds, endopeptidase activity significantly increased in Eas and Cots during cold stratification beginning in the 3rd week and reached a maximum at the 9th week, then decreased to the level of onset in germinating seeds (Fig. 3A and B). At the 9th week of cold stratification the activity of endopeptidase in nonstored seeds, was 13 and 5 fold higher in Eas and Cots respectively than in stored ones. In stored seeds, the activity of endopeptidases only slightly increased at the 3rd week of cold stratification, and in germinating seeds, a distinct increase in activity of these enzymes was noted in Eas and Cots. Carboxypeptidase activity in Eas was significantly increased in stored seeds during imbibition and was significantly higher (six times) than in non-stored seeds for the duration of stratification (Fig. 3C). In the Cots of stored seeds, carboxypeptidase activity was significantly higher during imbibition (nine times) and during the first steps of germination (almost two times) compared to nonstored seeds. In the Cots of non-stored seeds, carboxypeptidase activity increased significantly only in germinated seeds (Fig. 3D). During imbibition, the activity of aminopeptidases increased significantly in the Eas and Cots of stored seeds (thirteen and five times respectively), while in non-stored seeds, the increase appeared in Eas and Cots at the 3rd week of cold stratification and was maintained to the end of stratification and decreased at the first step of germination (Fig. 3E and F). In the Eas and Cots of germinating seeds, the activity of aminopeptidases in stored seeds was significantly higher (fourteen and two times) than in non-
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Fig. 1. Water uptake (A) in embryonic axes (Eas) and cotyledons (Cots) and germination capacity (B) of beech seeds during cold stratification at 3 C, after storage for 0 and 9 years at 10 C. D, dry seeds; G, germinated seeds. Data are means of four replicates ± SD.
Fig. 2. The total (A, B) and soluble (C, D) protein level in embryonic axes and cotyledons of dry (D), imbibed (I), at 3rd, 6th, 9th week of cold stratification at 3 C and germinated (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
stored seeds. 3.4. Free amino acids level Interesting differences between stored and non-stored seeds were evident in the levels of free amino acids (Fig. 4A and B). In the Eas of dry, imbibed seeds and at the 3rd week of cold stratification, the level of free amino acids was low in both groups of seeds. In non-stored seeds at the 6th week, a 2-fold increase was reported in the free amino acid level. Then, the free amino acid level decreased and in germinating seeds reached a level identical to that in dry and imbibed seeds. In stored seeds, the maximum level of free amino acids occurred later, at the 9th week of cold stratification, while in
germinated seeds, it was significantly higher compared to nonstored seeds. In the Cots of non-stored seeds, the change in the free amino acid level was similar to that in Eas. However, in the Cots of stored seeds, the level of free amino acids was significantly higher in dry, imbibed seeds and in all steps of cold stratification as well as in germinated seeds, compared to non-stored seeds. Marked differences in the levels of free amino acids were noted in dry (D) and imbibed seeds (I). 3.5. Protein synthesis The activity of protein synthesis was measured after imbibition and at each week of cold stratification for stored and non-stored
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Fig. 3. The activity of endopeptidases (A, B) carboxypeptidases (C, D) and aminopeptidases (E, F) in embryonic axes and cotyledons of dry (D), imbibed (I), at 3rd, 6th, 9th week of cold stratification at 3 C and germinated (G) (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
seeds (Fig. 5A and B). In general, protein synthesis activity, measured in g FW of tissue, was almost ten times higher in Eas than in Cots. The lowest protein synthesis intensity in Eas was reported in imbibed (term 0 weeks) seeds (Fig. 5A). Protein synthesis increased over the next 3 weeks, reached its highest level at the 3rd week, and then decreased at the 4th week of stratification. The increase in translational activity at the 3rd week of stratification was markedly larger in nonestored seeds. In these seeds, the next notable increase in protein synthesis activity appeared at the 9th week of stratification, and then, the activity of protein synthesis decreased in germinating seeds. In contrast, the translational activity in stored seeds did not change at the 4th or 9th week of stratification and in germinating seeds. Changes in protein synthesis in Cots exhibited a different pattern. Similar high levels of
protein synthesis activity had already occurred in imbibed nonestored and stored seeds (Fig. 5B). Protein synthesis then decreased in both seedlots. In the Cots of nonestored and stored seeds, protein synthesis activity was almost stable beginning at the 4th week and lasting until 9th week of stratification but was markedly lower in stored seeds compared to nonestored seeds. In germinated seeds, the activity of protein synthesis decreased proportionally in both types of seeds but remained significantly higher in nonestored seeds. 4. Discussion In the present study, beech seeds retained relatively high viability (75%) after storage for nine years. However, their vigour
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Fig. 4. Free amino acid levels in embryonic axes (A) and cotyledons (B) of dry (D), imbibed (I), at 3rd, 6th, 9th week of cold stratification at 3 C and germinated (G) (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
Fig. 5. De novo protein synthesis in embryonic axes (A) and cotyledons (B) in imbibed (Im), during 9 weeks of cold stratification at 3 C and germinated (G) (with embryonic axes protruded to ca 1 cm) of beech seeds, after storage for 0 and 9 years at 10 C. Data are means of four replicates ± SD.
was worse than that of non-stored seeds (Fig. 1B). Seed viability is dependent on the environmental conditions during development and preparation for storage after harvest (Suszka et al., 1996). Previous studies have revealed that beech seeds stored for 7 years can possess up to 66% germination capacity (Pukacka and Ratajczak, 2007), while seeds stored for 8 years can possess up to 82% germination capacity (Kalemba and Pukacka, 2014), depending on the seed lot. During storage, total and soluble protein levels decreased in Eas and Cots significantly (Fig. 2AeD). Although the seeds were collected from the same tree, the significant differences in the levels of the total and soluble proteins in stored and non-stored seeds may be due to the weather conditions experienced by a given year's crop of seeds. However, similar trends were observed for other beech seedlots used in the works of Pukacka and Ratajczak (2007) and Kalemba and Pukacka (2008, 2014) and in work studying artificial ageing of seeds of different plant species (Kalpana and Rao, 1997; Xin et al., 2011). In seeds where germination requires several weeks of cold stratification, proteolysis can begin at the imbibition of seeds and last until radicle protrusion and seedling development (Bewley, 1997). In general, the level of protein is the resultant of both proteolysis and protein synthesis activities. In Dicotyledones, protein bodies, in addition to containing storage whereas aminopeptidases are localized in the cytoplasm (Müntz et al., 2001; Tan-Wilson and Wilson, 2012). Nothing is known about the location of proteolytic enzymes along the embryonic axes and in the cotyledons of beech
seeds; however, judging by the composition of the storage proteins, it can be assumed that beech seeds are typical of dicots. Our results clearly show that long-term storage of beech seeds changes the activities of proteolytic enzymes. The activity of endopeptidases in these seeds is several times lower than in non-stored seeds (Fig. 3A and B). In stored seeds, the activity of endopeptidases increased at the beginning of seed germination. The weaker and delayed endopeptidase activity in stored seeds could be the cause of depleted vigour and viability. Kiyosaki et al. (2009) and Prabucka et al. (2013) demonstrated that gibberellic acid (GA3) affects endopeptidase synthesis in wheat seeds during germination. It is possible that in beech seeds, where breaking dormancy and beginning germination is under the control of GA (Mortensen and Eriksen, 2004), endopeptidase activity is also connected with this hormone. However, the hormonal control of storage protein mobilization in dicots has not yet been resolved (Tan-Wilson and Wilson, 2012). Carboxypeptidases cooperate with endopeptidases to increase the number of peptides with free C-termini. The higher activity of carboxypeptidases in the Eas and Cots of stored beech seeds may be the result of the ROS-affected breakdown of storage proteins because ROS are known to accumulate in aged beech seeds (Pukacka and Ratajczak, 2007; Ratajczak et al., 2015). The final products of endopeptidases and carboxypeptidases are free amino acids and small oligopeptides, which are degraded to amino acids by aminopeptidases (Müntz, 1996; Tan-Wilson and Wilson, 2012). An age-related effect on aminopeptidase activity has been demonstrated in Cots, where weaker activity of these enzymes in
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stored seeds until the 9th week of cold stratification has been observed. Considering that the breakdown of storage proteins begins with endopeptidase activity (Tan-Wilson and Wilson, 2012), it can be suggested that beech seeds characterized with 100% germination capacity (i.e., non-stored seeds), the mobilization of proteinous reserves begins at the 3rd week of cold stratification and is further intensified up to the 9th week in both Eas and Cots (Fig. 3A and B). The increase in carboxypeptidase (Fig. 3C and D) and aminopeptidase (Fig. 3E and F) activity in imbibed stored seeds coincides with first water uptake (Fig. 1A). High proteolytic activity at this particular time might be related to the removal of oxidatively injured proteins in stored seeds, because phase I of water uptake is considered to be a moment of initiation of repair mechanisms for membranes and proteins (Weitbrecht et al., 2011). Aged seeds exhibited significantly higher proteolytic activity in germinating seeds (Fig. 3) when compared to non-stored seeds, whereas differences in soluble and total protein level between stored and nonstored germinating seeds are similar to the initial differences in dry seeds (Fig. 2). It seems that the completion of dormancy release and the germination program in beech seeds requires intense endopeptidase activity, probably in protein bodies, between the 3rd and 9th weeks of stratification (Fig. 3A and B) as well as intense de novo protein synthesis, particularly at the 3rd and 9th weeks of stratification (Fig. 5) because stored seeds lack this phenomenon. In general, protein synthesis activity is significantly higher in nonstored seeds compared to stored seeds. It is possible that the first peak of protein synthesis in beech seeds is the result of the translational activity of stored mRNA. The work of Rajjou et al. (2012) and Kimura and Nambara (2010) revealed that proteins and mRNA pools stored in dry seeds play an important role in the germination process. De novo protein synthesis from stored mRNA allows cells to repair or replace important proteins altered during storage to reinitiate metabolism during germination. On the other hand, Chibani et al. (2006) demonstrated that after successful dormancy, released seeds acquire the capacity to reprogram the pattern of protein biosynthesis during imbibition, allowing the completion of germination. It can be assumed that perturbing protein synthesis and proteolysis does not allow completion of the dormancy release program in stored seeds and that therefore, no more than 75% of them germinated (Fig. 1B). During the later stages of cold stratification, de novo synthesis of proteolytic enzymes, particularly endopeptidases and aminopeptidases (Galland et al., 2014), is also possible (Tan-Wilson and Wilson, 2012). Lowered protein neosynthesis in stored seeds at the 9th week of stratification (Fig. 5) coincides with the greatest decrease in endopeptidase activity (Fig. 3A and B) compared to non-stored seeds. In this light, it can be hypothesized that the age-related impairment of neosynthesis of endopeptidases at later stages of stratification might contribute to a decrease in the germination capacity of stored beech seeds. The aminopeptidase activity in the Eas of stored seeds seems to be more affected by water than seed age, because the only reported differences in enzymatic activity occurring in imbibed and germinating seeds co-occurs with the first and the second documented water uptake (Fig. 1A). It is clear that the free amino acid levels in the Eas and Cots of stored and non-stored seeds during cold stratification and the early stage of germination are related to the neosynthesis of proteins in these organs, particularly in the Eas of non-stored seeds between the 3rd and 9th week of stratification, where careful coordination is visible between these two processes (Fig. 4A and 5A). Larger amounts of available free amino acids in the Cots of stored seeds (Fig. 4B) might be the effect of inefficient translation in this organ (Fig. 5B) and may indicate greater protein degradation by the 26S proteasome in the cytoplasm.
5. Conclusion Decreases in seed viability are considered to be age related. Beech seeds stored for only 3 months maintained 100% germination capacity, whereas seeds stored for 9 years maintained 75% germination capacity under identical dormancy alleviation and germination conditions. These results suggest that lower protein levels and perturbations in proteolysis and protein synthesis are important age-related factors that contribute to seed viability. Seeds stored for 9 years contained less protein in the dry state and continuously during dormancy alleviation, as well as during germination. The largely decreased activity of endopeptidases in seeds stored for 9 years, particularly at the 3rd-9th week of cold stratification, might be the cause of affected storage protein mobilization. Moreover, despite of the abundance of free amino acids, the weakened de novo synthesis of proteins, possibly also endopeptidases, at the end of the stratification period contributes to the disruption of the dormancy release program or to the inaccurate initiation of the germination process in stored seeds. Contributions SP designed the experiments; ER conducted all experiments; ER, EMK, SP analyzed the data; SP wrote the manuscript; EMK, SP created figures; ER, EMK, SP revised and edited the manuscript. Acknowledgements This study was supported by research funds of the Polish Ministry of Science and Higher Education. The authors thank dr Renata Rucinska from the Isotope Laboratory, Adam Mickiewicz University in Poznan, for the help with assay of protein synthesis. References Arc, E., Galland, M., Cueff, G., Godin, B., Lounifi, I., Job, D., Rajjou, L., 2011. Reboot the system thanks to protein post-translational modifications and proteome diversity: how quiescent seeds restart their metabolism to prepare seedling establishment. Proteomics 11, 1606e1618. Bailly, C., 2004. Reactive oxygen species and antioxidant in seed biology. Seed Sci. Res. 14, 93e107. Ballesteros, D., Walters, C., 2011. Detailed characterization of mechanical properties and molecular mobility within dry seed glasses: relevance to the physiology of dry biological systems. Plant J. 68, 607e619. Bewley, J.D., 1997. Seed germination and dormancy. Plant Cell 9, 1055e1066. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein edye binding. Anal. Biochem. 72, 248e254. Çakmak, T., Atici, O., Agar, G., Sunar, S., 2010. Natural aging-related biochemical changes in alfalfa (Medicago sativa L.) seeds stored for 42 years. Int. Res. J. Plant Sci. 1, 001e006. Collada, C., Caballero, R.G., Casado, R., Aragoncillo, C., 1988. Different types of major storage seed proteins in Fagaceae species. J. Exp. Bot. 39, 1751e1758. Collada, C., Aragoncillo, P., Aragoncillo, C., 1993. Development of protein bodies in cotyledons of Fagus sylvatica. Physiol. Plant 89, 354e359. Chibani, K., Ali-Rachedi, S., Job, C., Job, D., Jullien, M., Grappin, P., 2006. Proteomic analysis of seed dormancy in Arabidopsis. Plant Physiol. 142, 1493e1510. Davies, M.J., 2005. The oxidative environment and protein damage. Biochim. Biophys. Acta 1703, 93e109. El-Maarouf-Bouteau, H., Mazury, C., Corbineau, F., Bailly, C., 2011. DNA alteration and programmed cell death during ageing of sunflower seed. J. Exp. Bot. 62, 5003e5011. Galland, M., Huguet, R., Arc, E., Cueff, G., Job, D., Rajjou, L., 2014. Dynamic proteomic emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination. Mol. Cell. Proteomics 13, 253e268. Isola, M.C., Franzoni, L., 1993. Studies on the proteolytic activities of potato tuber. Plant Physiol. Biochem. 31, 169e174. ISTA, 1999. International rules for seed testing. Rules 1999 (Suppl.). Seed Sci. Technol. 27, 1e133. Kalemba, E.M., Pukacka, S., 2008. Changes in late embryogenesis abundant proteins and small heat shock protein during storage of beech (Fagus sylvatica L.) seeds. Env. Exp. Bot. 63, 274e280. Kalemba, E.M., Pukacka, S., 2014. Carbonylated proteins accumulated as vitality decreases during long-term storage of beech (Fagus sylvatica L.) seeds. Trees 28,
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