Germinating seeds and seedlings are exposed at critical stages of plant development to various abiotic stresses that pose serious risks to the plants growing near the soil surface. The stresses include drought, salinity and cold [
18]. Seeds reaching maturity in a highly dehydrated state exhibit dramatic changes in metabolism [
19]. Upon imbibition, dry seeds rapidly consume oxygen, an element required for oxidative phosphorylation that provides energy for intensified metabolic processes. Oxidative phosphorylation and hydrolysis of storage compounds generate reactive oxygen species (ROS), which, if insufficiently scavenged, cause extensive structural and functional damage in cells [
20]. Exposure of sensitive seedlings to chilling reduces the number of germinated seeds, decreases root and shoot growth and increases leakage of solutions of low molecular weight metabolites from cells due to a loss of membrane integrity [
21]. Cold also plays a key role in breaking seed dormancy, as it may induce GA biosynthesis during early phases of germination [
22]. Each plant species has a specific optimal temperature range for germination. Cold may also induce synthesis of cold-shock domain proteins or transcription factors affecting seed germination [
23,
24]. Narrow-leaf lupine is not a cold-sensitive species and it is sown in early spring. Our study demonstrates that low temperature (7 °C) significantly extends the time necessary for germination. As per our experience (data not shown), the temperature below 7 °C limits narrow-leaf lupine seed germination in the dishes, while 13 °C is optimal for seed germination and growth at early developmental stage of this plant species. In the present study, we found significant differences in low-temperature germination ability among the lines/cultivars of narrow-leaf lupine. Similarly, Nordborg and Bergelson [
25] observed considerable differences in low-temperature germination times of 35 ecotypes of
Arabidopsis thaliana.
In our investigation most narrow-leaf lupine lines/cultivars did not germinate at 7 °C, even six days after sowing. In the weakly germinating group, on average 2.5 times fewer seeds germinated at the lower than at the higher temperature. In the groups well (II) and very well (III) germinating under cold, this ratio for the seeds germinating at 13 °C and 7 °C was much lower. At this stage of the research, it is difficult to pinpoint the reason for variable germination ability at low temperatures. Genetic background seems to be the major factor controlling germination in a specific climate. We have not found any correlations between the origin of the studied lines/cultivars and their ability to germinate at low temperature. Cultivars from the same country demonstrated both weak and strong germination ability at 7 °C. The seeds of all three groups showed greater efflux of ions from cells under cold as compared with 13 °C. Kaur et al. [
26] noticed a decrease in the germination vigour of
Cicer arietinum seeds accompanied by greater EL and a decrease in seed dehydrogenase activity in cold conditions. In our experiment, the seed germination vigour of weakly and well germinating groups (I and II) depended on EL, which may indicate a degree of plasma membrane permeability. The weakly germinating lines/cultivars experienced the greatest ion leakage from seed cells at low temperature, while the group of very well germinating lines/cultivars (group III) showed the lowest ion efflux; this feature thus seems to be a major determinant of low-temperature germination ability. Cell membrane conditions affect all metabolic processes, and, especially in germinating seeds, oxidative phosphorylation. According to Leopold and Musgrave [
27], a decrease in TTC reduction suggested a loss of mitochondrial stability. The relationship between germination vigour at low temperature and dehydrogenase activity may be explained by the necessity of providing energy for de novo synthesis of many compounds and hydrolysis of storage compounds in the endosperm. In our experiment, seed germination vigour correlated with dehydrogenase activity only in the weakly germinating cultivars. In addition, the seeds in group I had lower amylolytic activity than those in groups II and III. Weak germination observed under cold conditions seemed to depend on such factors as cell membrane permeability, activity of dehydrogenases and amylolytic activity. In contrast, the vigour of very well germinating seeds depended only on amylolytic activity.
One of the aims of our research was to improve seed germination of narrow-leaf lupine at low temperature via application of smoke water as well as pre-sowing 3-h hydropriming at 20 °C. The results indicate that the hydropriming significantly increased germination vigour of most studied lines/cultivars. Water uptake at room temperature was rapid during the first three hours, and subsequent transfer of seeds to low temperature did not cause any major damage to the structural membranes. It is difficult to tell whether in such a short time any rearrangement of cytoplasmic membranes occurred, which in nature is a typical physiological response of plants to changes in the temperature and degree of the cytoplasm hydration. This question requires molecular analysis of the changes in cell membrane structure taking place during the initial three hours of imbibition. According to Bewley [
2] membranes return to a more stable configuration shortly after rehydration. Based on our observations, we conclude that the initial hydration of seeds protected their cell membranes against cold-induced damage as manifested by a decrease in ion leakage from seed cells. Similar results were obtained by Dubert and Filek [
5], who reported that 100% of hydroprimed soybean seeds germinated at 5 °C without showing any structural or functional disorders. They postulated that seed imbibition at low temperature requires a quick reconstruction of the cell membrane of dry seeds into a new structure characteristic of germinating seeds. This reconstruction requires an activation of appropriate enzymatic processes that run too slowly at low temperature, which in turn causes damage to the cell membranes. Contrary to that, hydropriming at room temperature allows for harmonizing the swelling of cells with the processes of remodeling of their membranes. Smoke water may mobilise seed metabolism via activation of dehydrogenases engaged in glycolysis and the Krebs cycle. Karrikins contained in SW were confirmed to enhance germination of approximately 1200 plant species worldwide [
15]. However, it should be noticed that most studies on the stimulating action of SW on seed germination focused on dormancy break. Although the molecular mechanism underlying this response remains unknown, some investigations demonstrated a key role of GAs and NO
2 in smoke-induced seed germination [
12,
28]. Seeds of many plant species originating from various world regions show a positive response to the compounds of smoke. Apart from typical post-fire flora, responsive species include crop plants such as
Lactuca sativa,
Zea mays,
Apium graveolens and
Avena fatua [
29].
Most studies investigating the role of antagonistic hormones, such as GAs and ABA, were performed on dormant seeds [
30]. One of the aims of our research was to find out whether the content of ABA and GAs determine the germination ability of non-dormant seeds of narrow-leaf lupine under cold. Both hormones occur in the seeds in active and non-active forms, for example conjugated in the case of ABA, or hydroxylated in the case of GA
1 to GA
8. According to Halińska and Lewak [
31], the equivalence between changes in active and non-active GAs suggests that the latter are also involved in the control of physiological levels of active gibberellins. In our study, we determined the levels of both forms of ABA and GAs, and the results indicated that only active forms of ABA determined the germination ability of non-dormant seeds of narrow-leaf lupine. In our experiment, such active forms as GA
1, GA
3, GA
4, GA
5, GA
6 as well as non-active GA
8 were quantified. The most common active gibberellin forms are GA
1, GA
3 and GA
4. Universal occurrence of GA
1 and GA
4 in plants suggests that they are functionally active forms and co-occur with their biosynthetic precursors and metabolites, which are often present at much higher concentrations than the hormones themselves [
32,
33]. Our analysis showed that the seeds of narrow-leaf lupine contained the largest amounts of GA
8 as compared with individual free gibberellins. We found differences in gibberellin accumulation between lines/cultivars varying in terms of germination ability at cold. This was particularly visible for GA
8 accumulation pattern. Generally, the groups of well and very well germinating seeds showed higher GA
8 levels than the group of weakly germinating ones. According to Ross et al. [
34], seeds approaching maturity often contain high levels of inactive GA, ensuring against concentrations of bioactive GAs in mature seed that could provoke premature germination and abnormal seedling growth. It seems that the capability of maintaining high rate of GA metabolism is more important for germination efficiency than total GAs accumulation. Our opinion could be confirmed by positive correlation between GA
1 and GA
8 as well as negative correlation between GA
4 and GA
8 content. Gibberellin GA
8 is a direct metabolite of GA
1 deactivation, while GA
4 is a part of metabolic pathway parallel to GA
1 [
33,
35]. On the other hand, GA
4 bioactivity is greater than GA
1 [
36,
37], thus, they mutually affect their accumulation as well as metabolism rate. Analysing the role of individual gibberellins in germination only on the basis on their accumulation in seeds is not easy due to their very complicated metabolic pathway providing many possibilities of synthesis and degradation as well as conversion of one compound into another. For example GA
5 may be converted into GA
6 or GA
3 [
33]. Statistical analysis did not reveal any correlation between either GA
8 or total GA level in the seeds and their germination vigour at both studied temperatures. Seed germination seemed to be affected rather by reciprocal equilibrium of the gibberellins than by the content of individual GAs alone. Chien et al. [
37] reported that the combination of GA
4 and GA
7 had a greater effect on the germination of dormant
Taxus mairei seeds than GA
4 alone. A prevailing view is that seed germination is controlled by ABA and GAs ratio [
30]. In our study, the ratio of active forms of gibberellins to free abscisic acid had no effect on the germination ability. Similarly to GAs, only the levels of free ABA correlated with lupine seed germination vigour. The stimulating effect of 3-h hydropriming on germination vigour was caused not only by a decrease of cell membrane permeability but also by a reduction in ABA content in the imbibed seeds. Smoke water and hydropriming were more effective in ABA reduction than in GAs increase. Contrary to our results, Chiwocha et al. [
38] reported that some studies on dormant
Arabidopsis seeds demonstrated no effect of KAR
1 on endogenous ABA and GAs content. It seems that this effect is species specific. It is worth mentioning that SW contains many various compounds exerting positive and negative impact on seed germination, so the effect of a single compound could not be so extensive compared to SW [
16]. Also our other research on the effects of butenolide, i.e., 2(5
H)-furanone on lupine seed germination (not yet published) demonstrated its less intensive impact than SW. Although the level of ABA seems to play a key role in seed germination, a decrease in the endogenous ABA does not always correlate with germination [
39] or is not sufficient to break dormancy in some dormant seeds [
40]. Humplik et al. [
41] showed that ABA was essential for hypocotyl elongation and that appropriate control of the endogenous level of ABA was required in order to drive the growth of etiolated seedlings.
Taking into account positive effects of SW and hydropriming on seed germination at low temperature, we hope that they could be used also in the field cultivation of crop plants. Both pre-sowing treatments do not change surface properties of seeds which can still be sown using standard seed drills.