4.1. Vigour and Viability
Temperature and air humidity are considered the main factors affecting seed viability [
24]. Field bean (
Vicia faba var.
minor) seeds that were dried and stored for 30 years at −14 °C germinated at 91% and 98% in cultivars Dino and Nadwiślański, respectively. Seed storage at room temperature resulted in a complete loss of germination capability (
Table 1). Our studies thus confirm that low temperature storage of seeds favours the preservation of seed viability. Similar observations were made by Pradhan and Badola [
25]. Lee et al. [
26] compared viability of seeds belonging to selected species in the Fabaceae family and found that it decreased by 15% in pea and soybean seeds as a result of a 10-year storage at +4 °C. Walters et al. [
27] reported various degrees of viability loss in seeds of Fabaceae (47 species) stored at −18 °C. For instance, within the
Trifolium genus
Triffolium campestre did not lose viability after 44-year storage, whereas
T. caudatum after 38 years of storage had the germination capacity of just 2%. In
Vicia sp. seeds stored for 42 years the germination percentage decreased by just 20%.
An important prerequisite for preservation of high seed quality is to keep the air relative humidity and seed water content at a low level [
9]. Mira et al. [
28], however, maintain that seed water content does not affect seed viability if only the storage temperature is below +30 °C. In our experiments seed water content was 8.8% at the start of the experiments and in Nadwiślański after 30 years of storage at −14 °C and +20 °C it shifted to 10.6% and 6.7%, respectively. Similarly, in Dino, after the same period of storage at −14 °C and +20 °C the seed water content changed to 8.6% and 6.5%, respectively (
Table 1). In both cultivars (Dino and Nadwiślański), therefore, seeds stored at room temperature had a lower water content than those stored at −14 °C. Pérez-García et al. [
29] have also found that changes in seed water content within the range 2–15% do not affect seed lifespan under the conditions of low temperature. Considering these data, we assumed that the air relative humidity and seed water content did not affect field bean (
Vicia faba var.
minor) seed lifespan in our experiments.
4.2. Seed Coat Characteristics
Phenolic compounds affect the plant metabolic activity and antioxidative properties of plant derived foods [
30,
31]. They also contribute to the colour of plant products [
32]. In synchronous fluorescence method the intensity of fluorescence is measured as a function of the emission and excitation wavelengths with difference step of Δλ or Δν. Good resolution and multiplying of absorption and emission intensities (in comparison to the conventional studies of emission spectra) are the major advantages of this method [
33,
34]. The Stokes shifts were published corresponding to the best Δλ for observations of synchronous spectra of various phenols. Chlorogenic acid in a methanol solution, for instance, has a single fluorescence maximum 337 nm for Δλ = 110 nm [
35]. Sergiel et al. [
36] studied several phenolics dissolved in methanol and gave the wavelength ranges and Δ
λ values suitable for the analyses of these compounds (
Table 6).
Considering the data given in
Table 6 and the obtained results, we can assume that the seed coats of field bean (
Vicia faba var.
minor) may contain all of phenolic metabolites mentioned there. To gain a better insight into the changes occurring in seed coats during prolonged storage, the total content of phenols, non-tannin phenolics, total tannins, and proanthocyanidins were measured. The obtained results show clearly that in seeds damaged by inadequate storage conditions the contents of all the above-mentioned compounds decreases, by up to 95% (
Table 2). On the other hand, the synchronous fluorescence spectra show increased levels of fluorescent metabolites in seeds stored at +20 °C (increased band intensity,
Figure 3). We should remember that phenols in spite of their wide structural diversity share the propensity for becoming oxidized [
37]. Nasar-Abbas et al. [
19] demonstrated, that in bean a 12-month storage in the atmosphere of oxygen results in accelerated seed darkening, whereas the atmosphere of nitrogen delays this change. A condensed tannin contributes to the darkening of pinto bean seed coats and this process occurs more rapidly in a cultivar with a higher initial content of proanthocyanidin compared to the one with a low content of this metabolite [
38]. Seeds of pinto bean contain the main monomer of the flavonol, kaempferol, and three flavonols—kaempferol 3-
O-glucoside, kaempferol 3-
O-glucosylxylose, and kaempferol 3-
O-acetylglucoside. Seed ageing resulted in a decrease of kaempferol content by nearly a half and it did not change significantly in those seeds that did not change their colour. In seed coats procyanidins are the main polymers with the degree of polymerisation above 10. Seed ageing decreased the level of these polymers and increased the level of low molecular weight procyanidins. However, our results do not show the occurrence of free kaempferol in field bean (
Vicia faba var.
minor) seeds (
Figure 3) (the extract spectra do not contain the bands corresponding to kaempferol). The most significant features of phenolics are their ability to undergo oxidation and to polymerise. The decreased contents of total phenols and non-tannin phenols may probably result from dimerization of polyphenols leading to the formation of insoluble high molecular weight polymers or products of their oxidation. Two reasons of the browning of lentil seeds were described: polymerisation of phenols [
39] and oxidation of phenols [
40]. Our results, the observation of increased fluorescence in methanol extracts from field bean (
Vicia faba var.
minor) seeds stored at +20 °C, thus indicate that oxidized forms of phenols or condensed phenols can be formed in seed coats of such inadequately stored seeds.
4.3. Characteristics of Protein
Although there have been numerous reports on field bean (
Vicia faba var.
minor) seed storage (e.g., [
41,
42]) and the storage proteins of
Vicia faba (e.g., [
43,
44,
45]), there have been few reports on the relation between seed storage proteins and the storability of grain legume seeds. Mbofung et al. [
46] did not notice any correlation between total protein content and vigour and viability of soybean seeds; however, Dobiesz and Piotrowicz-Cieślak [
47] indicate that in yellow lupin there is a correlation between the levels of conglutins γ and δ and seed biological quality. Simiarly, Sathish et al. [
48] described a close relation of some proteins from black gram seeds to seed viability and vigour.
Shaban [
49] found that various factors may cause both quantitative and qualitative changes in seed storage proteins. Proteins make a large part of legume seed dry mass—from approximately 20% in pea and bean to 38–40% in soybean and lupin. Field bean (
Vicia faba var.
minor) seeds used in our experiments contained proteins at the level of up to 26.8% of their dry mass (
Table 1) which corroborates the data provided by Księżak et al. [
50]. Considering the differences in their solubility, seed proteins are divided into four groups: albumins (soluble in water and buffered solutions with neutral acidity), globulins (soluble in salt solutions), prolamins (soluble in alcohols), and glutelins (soluble in acids and alkali) [
51]. In our experiments seed storage proteins were divided into the following fractions: albumin, vicilin, and legumin, based on solubility controlled by pH and salt concentration, following the method of Rubio et al. [
19]. Although the designations of these fractions suggest large differences in molecular weights of these proteins, their electrophorograms contain diverse peptides with size ranges partially overlapping. This may be explained by the fact, well established that seed storage proteins have an oligomeric structure and electrophoresis reveals their subunits [
52,
53]. Non-germinating seeds were distinguished in our work by decreased intensity of electrophoretic bands and, with some proteins, by their complete disappearance (
Figure 5,
Figure 6,
Figure 7 and
Figure 8). The largest protein differences related to seed storage regime were observed within the albumin fraction. Polypeptides with molecular masses 45.2 and 20.4 kDa were not detected in nongerminating seeds. Similarly, within the legumin fraction of Nadwiślański there was seed protein bands corresponding to molecular masses 56.6 and 32 kDa that were not observed in the deteriorated, non-germinating seeds. The relationship between seed vigour/viability and seed proteins was also reported by Mbofung et al. [
46] and Sathish et al. [
48]. The disappearance of protein bands from ageing lupin seeds was noted by Dobiesz et al. [
22]. Rajjou et al. [
6] and Sathish et al. [
48] suggested that the disappearance of some protein bands results from protein degradation in stored seeds. Gao et al. [
54] found that the content of seed storage proteins may be the main determinant of seed viability. 2-D electrophoresis of pre-purified fractions enabled us to identify proteins, the occurrence of which was most clearly related to the seed storage regime.
The use of 2-D electrophoresis to study seed proteins can be a challenge. Storage proteins usually greatly prevail in the separations and mask the members of other groups and functional categories. Seeds of Nadwiślański and Dino, stored at −14 °C were characterised by 47 and 54 spots, respectively, within the albumin fraction (
Figure 3). The numbers of albumin spots obtained with seeds stored at +20 °C were lower by 8 and 24 in Nadwiślański and Dino, respectively. The albumins occurring in large quantities in seeds of many crops, e.g., soybean, sunflower, mustard, or Brazil nut, where they fulfil the storage functions [
55]. Albumins of some plants have already been subjected to in depth analyses, e.g., in common castor (
Ricinus communis)—RicC1 and RicC3, common sunflower (
Helianthus annuus)—SFAs (sunflower albumins), soybean (
Glycine max)—AL1 and AL3 proteins, or garden pea (
Pisum sativum)—PA2 proteins. Albumin-like storage proteins are even accumulated by some ferns; for instance, matteucin protein occurs in
Matteuccia struthiopteris spores [
51]. In this paper among 11 identified proteins belonging to the albumin fraction and related to seed storage regime, seven turned out to be lectins (
Table 3). Their abundance decreased in seeds stored at room temperature (spots 1, 2, 3, 4, 8, 9, and 11) (
Figure 8) or they totally disappeared from such seeds (spots 1, 2, and 4 in Dino). Lectins can have antimycotic properties; however, they are not able to bind the glycoconjugates in fungal cell membranes or penetrate the fungal cells due to impermeable fungal cell walls. Their action may, therefore, be limited to binding the carbohydrates on fungal cell wall surfaces, and thus hindering the synthesis or deposition of chitin [
56]. The decrease in seed lectin content is likely, therefore, to promote seed infection by fungi.
Superoxide dismutase (Cu–Zn) was identified on 2-D proteomic maps as spot number 10 (
Figure 8). The content of superoxide dismutase protein was higher in Nadwiślański seeds stored at +20 °C compared to seeds stored at −14 °C. A similar pattern, however regarding enzyme activity, not enzymatic protein quantity, was observed in
Jatropha curcas L. seeds, where the degree of seed deterioration correlated with an increase in superoxide dismutase activity after 15 months storage [
57].
On the other hand, Sahua et al. [
58] suggests that expression of a specific superoxide dismutase isoenzyme is positively correlated with seed viability. It should be emphasized that plant superoxide dismutases can be classified according to their different cofactors, Cu–Zn, Fe, or Mn. Cu–Zn superoxide dismutase can be found in the cytosol, chloroplasts, and peroxisomes, while Fe superoxide dismutase is mainly found in chloroplasts, to a lesser extent in peroxisomes and apoplasts, and Mn superoxide dismutase is mainly found in mitochondria [
59]. Thus, the determination of overall superoxide dismutase activity does not take into consideration the occurrence of several isoforms of this enzyme with potentially different activation patterns.
Globulins are even more abundant in plants than the albumins. They include, e.g., vicilin, convicilin, and legumin of
Vicia faba and
Pisum sativum, conglycynin and glycynin of
Glycine max, or phaseolin of
Phaseolus vulgaris [
60]. Storage proteins of
V. faba include mainly the globulins and can be classified into two groups with different sedimentation constants: legumins and vicilins. The 2-D electrophoretic maps obtained for the legumin fraction contained 82 and 75 spots in the case of seeds of Nadwiślański and Dino, respectively, stored at −14 °C. In seeds stored at +20 °C, 67 and 63 spots were obtained in Nadwiślański and Dino, respectively (
Figure 8). Identification of the legumin revealed 6 legumins (spots 16, 17, 18, 19, 20, and 22), a convicilin (spot 12) and five vicilins (spots 13, 14, 15, 21, and 23). The vicilin fraction produced 93 and 72 protein spots in the case of Nadwiślański and Dino seeds, respectively, stored at −14 °C. The vicilin fraction was separated into 65 and 66 spots in the case of seeds of the same cultivars stored at +20 °C (
Figure 8). The changes in vicilin-related protein contents were observed in ageing maize (
Zea mays L.) seeds [
61], and the rapid decline of these proteins correlated with decreasing germination rate and seedling growth rate.