Legumes and cereals are the main sources of proteins in human diets. Legumes are important foods for people with specific nutritional preferences, like vegans, vegetarians, and hyperlipidemia patients, who must observe lipid restriction (Arozarena et al., 2001). In addition to the nutritive value, legume seed proteins affect food structure and stability (Makri et al., 2005). First of all, however, they are vital components of seeds, affecting their vigor and potential for producing healthy seedlings. The germplasm of the most promising varieties is deposited in gene banks and subjected to long term storage. With the passage of time, adverse changes in seed macromolecules and intracellular systems accumulate and eventually they lead to the loss of seed viability. Diverse biochemical and physiological disturbances accumulate in seed and the rate of this deterioration is determined by the storage temperature and relative humidity (RH) (Priestley, 1986; Ratajczak et al., 2015). There are several hypotheses about the mechanics of seed aging. The main one has been proposed by Harman (1992). He assumed that the basic source of aging, in various organisms, not only plant seeds, is the accumulation of cell damage caused by reactive oxygen species (ROS) (Harman, 1992; Ratajczak et al., 2015). The accumulation of ROS in seeds results in damage to DNA, lipid, and proteins. Severe protein damage caused by oxidative stress may lead to a reduction in seed vigor and viability (Ratajczak et al., 2015; Barreto and Garcia, 2017).

Proteomic analysis of seeds should thus provide an insight into the effects of storage conditions on seed quality and longevity. Analysis of proteins extracted from the seeds of legume family is rather daunting. The main limitations of proteomic analysis are associated with the great diversity of proteins in terms of physico-chemical properties and significant differences in their amounts (Martínez-Maqueda et al., 2013). The vast majority of the isolated proteins are storage proteins. Proteins playing metabolic, structural, regulatory or defensive roles are represented less frequently and are often masked by storage proteins (Magni et al., 2007).

Analysis of the seed proteome may be more efficient when modern methods for fractioning and purification are applied (Berghout et al., 2015). These methods are based on cell fracture followed by protein solubilisation and/or precipitation in order to obtain the desired protein fraction. Of paramount significance is the minimisation of the effects of other agents (e.g., phenolic compounds) on the electrophoresis result (Martínez-Maqueda et al., 2013). The fractioning process requires that large quantities of water, energy and reagents be used. The disadvantages of this method also include significant losses of material, particularly during degreasing and drying (Berghout et al., 2015).

In the seeds of lupins, the main storage proteins are conglutins which, based on the electrophoretic mobility, were divided into four families, i.e., α, β, γ, and δ conglutins. δ Conglutin content of white lupin mature seeds was estimated to a level of 10–12%; however, the latest studies suggest a lower content of approximately 3–4% (Duranti et al., 2008).

This discrepancy most probably results from inaccurate purification of fractions during earlier studies. δ Conglutin is a monomer with a low molecular weight (Salmanowicz and Przybylska, 1994), comprising two subunits with weights of 4 and 9 kDa (Duranti et al., 2008). δ Conglutins are water soluble and include enzymatic proteins and inhibitors (Klupšaitė and Juodeikienė, 2015). Therefore, in a 2D electrophoretic image, they are often masked by the more abundant proteins (mainly γ conglutins). β Conglutin found in cotyledons accounts for 44–45% of all storage proteins, and is thus the most abundant protein present in the seeds of all lupin species. This protein is a tetramer whose monomers are comprised of polypeptides with a molecular weight of 16–70 kDa (Duranti et al., 2008), resulting in a complex pattern in a 2D electrophoretic image. γ Conglutin is a protein specific for lupin seeds and accounts for approximately 4–5% of the total protein of mature seeds. γ Conglutin is a hexamer whose every monomer is composed of two heterogeneous subunits with molecular weights of 17 and 29 kDa. However, the heterogeneity of γ conglutin subunits is not as clear as it is in the case of β conglutin subunits (Duranti et al., 2008). β Conglutin and γ conglutin are characterized by minimum solubility at the pH value of four to five. The manipulation of protein solubility, and the application of appropriate techniques using hydrodynamic properties of proteins, may help obtain the desired protein fractions (Klupšaitė and Juodeikienė, 2015; Muranyi et al., 2016). However, it is not always possible to obtain a uniform fraction, which is due to the fact that many proteins exhibit intermediate solubility (Nadal et al., 2011). γ Conglutin at neutral pH is readily soluble in water and in salt solutions (Duranti et al., 2008).

Decreased contents of soluble carbohydrates, biogenic amines and proteins was detected in yellow lupin (Lupinus luteus L.) seeds subjected to 29 years storage at room temperature (Dobiesz et al., 2017). The physiological role of the proteins correlating with seed vigor is pretty straightforward in some cases, for instance in free radical scavenging proteins. However, it was demonstrated that seed storability can be strongly affected by conglutins too. These proteins are considered storage molecules, and should undergo proteolysis at the onset of seed germination. It was interesting, therefore, to find out if the significance of conglutins in determining seed vigor and viability is a characteristic of most members of this protein group or just a few specific molecules. The objective of this paper was to fractionate white lupin seed conglutins into β, γ, and δ to obtain possibly precise 2-D electrophoretic separations and verify the hypothesis that most or some of the identified proteins could be considered as key molecules affecting seed vigor and vitality.

The study material comprised seeds of white lupin (L. albus L.), Hetman variety. Seeds were stored in linen sacks, inside hermetically sealed glass ‘twist’ jars at -14°C and 50% (RH) and under laboratory conditions [temperature about +20°C and 44% (RH) for 26 years]. Hair hygrometer [model TR415 (C.P. Poland)] was used to measure the air RH. At the beginning of the experiment (in 1989) seeds contained 8.8% of water. The water content of the seeds after 26 years of storage (in 2015) was found to be 7.0% (storage at -14°C) and 4.8% (storage at +20°C). Water content was determined with the gravimetric method and expressed in relation to seed dry-weight (5 g samples were used for each replication of these determinations).

The vigor and viability of white lupin seeds (International Seed Testing Association [ISTA], 1999) were measured. Seed germination was carried out in an incubator with a constant temperature of +20°C. One hundred seeds of white lupin were placed evenly on a moist germination paper. The germination results were recorded as a percent of seed with embryonic roots emerging within 7 days of incubation. Subsequently, the length of stems and roots was measured and the fresh and dry weight of the seedlings was determined. In order to determine the electrical conductivity of seed leachates, the seed surfaces were sterilized by immersing them in 1% sodium hypochlorite for 2 min; the seeds were then washed in distilled water for 3–4 min. Subsequently, a seed sample (100 seeds) was soaked in milliQ water (250 ml) at 20°C for 24 h. The electroconductivity of seed leachate was measured with a conductometer (model HI 2315, Hanna Instrument). The measurements were conducted in triplicates for each batch of seeds. The electroconductivity was related to seed fresh weight and expressed in μS cm^-1 g^-1.

Storage conditions such as the temperature and humidity affect the viability of seeds (Parrish and Leopold, 1978; Jatoi et al., 2001; Mohammadi et al., 2011). White lupin seeds, despite the long period of storage (26 years) under the conditions of lowered temperature, retained viability at a level of 86.3% (Table 1). This observation corroborates previous studies suggesting that low seed storage temperature has a positive effect on seed storability (Cortelazzo et al., 2005; Sastry et al., 2007; Balešević-Tubić et al., 2010; Pradhan and Badola, 2012). Seeds of lupin are considered orthodox, i.e., they tolerate well drying to a water content of 5–10% dry weight (Piotrowicz-Cieślak et al., 2008). Therefore, they retain the ability to germinate for a long time. It has been established that orthodox seeds best retain their viability when stored at a temperature below 0°C and RH of approx. 5–8% (Sastry et al., 2007; Shaban, 2013). Walters et al. (2005) reported that the viability of the seeds of narrow-leafed lupin (L. angustifolius L.) dropped by 50% after 41 years of storage at -18°C. Piotrowicz-Cieślak et al. (2008) established that the viability of yellow lupin seeds after 20 years of storage at -14 and 0°C was similar and remained at a level of 99 and 96%, while the viability of the seeds stored at +20°C dropped to only 2%. The results obtained by Piotrowicz-Cieślak et al. (2008) confirm that a temperature of 0°C was sufficient for maintaining the viability of lupin seeds at a high level. According to Elias and Copeland (1994), a temperature of 5°C contributes to the slowing down of metabolic processes that cause deterioration of seed material. Lee et al. (2013), while comparing the viability of seeds of selected species of the Fabaceae family, noted an approx. 15% drop in the viability of pea (Pisum sativum L.) and soybean (Glycine max Merr.) seeds after 10 years of storage at +4°C. It was found in the same study that the most durable seeds are those of Arachis hypogaea, Vigna angularis var. angularis, and Phaseolus vulgaris, whose viability decreased by only 5%. On the other hand, in a study by Walters et al. (2005), the best storability was exhibited by pea seeds. After long-term storage at -18°C, a drop in the viability by 25% was noted in pea seeds (after 64 years of storage). As regards pea seeds, an above-zero storage temperature is not sufficient for them to retain their maximum durability.

Maintaining the RH of the air and water content of seeds at a low level is also significant for retaining a high quality of seed material (Priestley, 1986; Ratajczak et al., 2015). However, according to Mira et al. (2015), during storage at a temperature lower than +30°C, a reduction in the amount of water in seeds has no effect on their storability. The water content of the seeds at the beginning of the experiment was 8.8%, while after 26 years of storage at -14 and +20°C it was, respectively, 7.0 and 4.8%. Ellis and Hong (2006) concluded in their study that changes in water content at a level of 2–15% during the storage of seeds under the conditions of a lowered temperature had no effect on the longevity of seeds of plants of the Fabaceae family. Similar conclusions were reached by Pérez-García et al. (2007) in a study on the seed longevity of plants of the Brassicaceae family. Given these results, in our experiment we excluded the RH of the air and water content of seeds as a factor affecting the durability of white lupin seeds.

According to Tabe et al. (2002) and Shaban (2013), the quantity and quality of stored proteins may vary depending on the impact of various factors. In seeds stored at room temperature, the contents of γ, δ, and β conglutins decreased. A similar relationship was observed by Cortelazzo et al. (2005) in the seeds of French beans (P. vulgaris L.), Piotrowicz-Cieślak et al. (2008) in yellow lupin and Mbofung et al. (2013) in soybean. A direct relationship between seed viability and vigor and seed storage proteins was confirmed in a study by Machado et al. (2001), Mbofung et al. (2013), and Sathish et al. (2015). The changes in structural proteins of non-germinating seeds of white lupin are indicated by a reduction in the intensity of protein spots and the fading of the band with a molecular weight of 16.4 kDa (band 11, fractions γ and δ) (Figures 1A,B). A similar fading of bands in aging bean seeds was observed by Machado et al. (2001). Along with a gradual loss of seed viability, the authors observed a gradual disappearance of polypeptide bands in the seed protein profile. According to Rajjou et al. (2008) and Sathish et al. (2015), the fading of polypeptide bands indicates post-translational modifications and protein degradation occurring during seed storage. According to Gao et al. (2016), the content of storage proteins is the main determinant of seed longevity. This statement is supported by the fact that the biggest differences between seeds with high and low durability are seen in their storage proteins. Furthermore, it was shown that the degradation of storage proteins during seed storage leads to disturbances in the supply of amino acids necessary for the synthesis of new proteins required during germination and seedling formation (Sathish et al., 2015).

The application of two-dimensional electrophoresis for study of seed proteins is a challenge. A 2D-electrophorogram generated for seed proteins is often dominated by the main storage proteins (particularly β conglutins) which mask other fractions and functional categories. Therefore, in our experiment we decided to carry out a preliminary fractionation of white lupin seed proteins before separating them by electrophoresis. In a study of narrow-leafed lupin seed proteome, published by Islam et al. (2012), the 2D-electrophorogram comprised mostly of polypeptides similar to β conglutin. On the other hand, in gels with yellow lupin seed proteins 205 β conglutin spots were found among a total of 341 seed protein spots (Ogura et al., 2014). The performance of preliminary protein fractionation proceeding the 2D electrophoresis in our experiments enabled us to identify proteins that change during seed storage.

The identified proteins (Table 3 and Figure 2) were storage proteins: β conglutins (belonging to 7S globulins or vicilin-like proteins), γ conglutins (belonging to 7S globulins or vicilin-like proteins), δ conglutins (belonging to 2S albumins), and proteins responsible for bioenergetic metabolism, i.e., glyceraldehydes-3-phosphate dehydrogenase (GAPDH). In our experiment, the amount of all polypeptides identified as γ and δ conglutins decreased in the seeds stored at room temperature (Figures 2, 4). As regards the polypeptides identified as β conglutins, nineteen occurred in higher amounts in seeds stored at -14° C, while two (spots 16 and 18) dominated in seeds stored at 20°C. Changes in contents of vicilin-like proteins were demonstrated in aging seeds of maize (Zea mays L.) (Xin et al., 2011) and rice (Oryza sativa L.) (Gao et al., 2016). Xin et al. (2011), while observing the rapid disappearance of vicilin-like proteins, concluded that the level of these proteins is correlated with the rate of germination and seedling growth. It is suggested that vicilin-like proteins, in addition to the role of nutrient reservoir, may serve additional functions. The disappearance of 2S albumins was observed in the seeds of Arabidopsis (Nguyen et al., 2015). Mature seeds of Arabidopsis, devoid of this fraction, were characterized by a reduced total protein content and were more susceptible to aging. Stability of γ conglutin during seed germination and its resistance to proteolysis make the storage function of this protein rather doubtful. It is exuded from seeds under stress conditions and during germination (Scarafoni et al., 2013). Due to the high homology of γ conglutin to glycoside hydrolase, it was suggested that this protein may participate in cell defense reactions. It was demonstrated by Scarafoni et al. (2010) γ conglutin from white lupin does not inhibit the cell wall degrading enzymes. Ribeiro et al. (2014) classify γ, β conglutins and albumins as family II of legume lectins. Lectins bind carbohydrates and are involved in plant defense from insects and pathogens, interactions with symbionts and regulation of cell adhesion. Moreover, these proteins probably participate in the signal transduction pathways, induce hypersensitivity reaction and strengthen the cell wall and cell membrane (Ribeiro et al., 2014; De Oliveira Dias et al., 2015). Jimenez-Lopez et al. (2016) have found that the β conglutins facilitate the oxidative burst, while at the same time they prevent its extinguishing and induction of apoptosis. The protective function of δ conglutins is also suggested by their similarity to the alpha-amylase inhibitor of cereal plants (Duranti et al., 2008). Nguyen et al. (2015) have shown that the decay of seed albumins resulted in decreased longevity of seeds and their greater sensitivity to oxidation. These authors suggested that albumins play a buffering role and provide protection from oxidative stress for other proteins necessary at the later stages of seed germination and seedling growth. Our experiments confirmed that γ, δ and β conglutins deteriorate parallel to the deterioration of seed vigor and longevity. This was demonstrated by one- and two-dimensional electrophoresis of the extracted protein fractions.

MD performed molecular experiments, AP-C supervised the research design and wrote the manuscript. All authors discussed the results and commented on the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.