Effect of light, gibberellic acid and nitrogen source on germination of eight taxa from dissapearing European temperate forest, Potentillo albae-Quercetum

Little is known about how light affects seed germination and revegetation of species of thermophilous oak forest. To reveal this relationship effects of white, red, far-red irradiations and dark incubation on germination of eight Potentillo albae-Quercetum taxa were examined. Attempts were also made to evaluate the influence of gibberellic acid and different nitrogen sources on the germination characteristics. Interaction between light and nitrogen was also studied. Freshly matured seeds of all taxa germinated very poorly, indicating presence of primary dormancy. Germination rates increased after wet-stratification treatment and were low in darkness. The highest concentration of the nitrogenous solutions that resulted in high germination level was 10 mM, whereas higher concentrations had a negative effect. Nitrate had the strongest influence which can be proved by a ‘gap detection’ mechanism for gaps in the vegetation. Far-red and red irradiation showed antagonistic effect on seed germination. There was a decrease in germination when far-red was followed by red and an improvement when red was followed by far-red treatment. Under red light, gibberellic acid enhanced germination of positively photoblastic taxa. It was concluded that light factor, associated with vegetation gaps, was the most important signal stimulating germination of the studied taxa.


Results
Effect of Different Quality of Light. Seed germination was greatly affected by light quality (Table 2).
Exposure to white light significantly (Tukey's test, P < 0.05) increased germination compared to the dark control for all species (Table 3). The white light effect was greatest for the seeds of Serratula tinctoria and Stachys officinalis which germinated poorly (<16%) in the dark but high germination rate (>96%) was observed in the light. The seeds of other six taxa germinated between 23-43% in dark. R light fully replaced the effects of white light in seed germination for four (Aquilera vulgaris, Calamintha acinos, Digitalis grandiflora, Festuca amethystina) taxa. For Calamintha acinos, Digitalis grandiflora and Lychnis viscaria no difference in germination between light and darkness conditions were observed. Seeds of Calamintha acinos, Digitalis grandiflora and Festuca amethystina were highly responsive to R light. Seed germination depended upon the kind of treatment applied at the end. The effect of R light was reversed by FR light and vice versa. Seeds of all taxa imbibed in FR light and then exposed to R light had increased germination compared to those not exposed to R light, but this increase was much smaller than that for seeds imbibed in darkness followed by R light. This indicates insensitivity of the seeds to FR light following an initial exposure to R light.
The PGI values varied between 0.44 and 0.88 (Table 4). Six of the eight studied taxa (rated 6 or 7 for light requirements) occured as adults in large gaps, had a seed mass <1.5 mg, and required light for germination (PGI > 0.56). The most shade-tolerant species tested, Melica nutans (Ellenberg's value of 4), had a seed mass 1.59 mg and germinated better in light than in dark (PGI = 0.44).

Effect of Different Type and Concentrations of Nitrogen Source and Chilling. Germination was
generally very low in the seeds incubated immediately after harvesting. Maximum promotion of germination of all taxa was obtained with the concentration of 10 mM NH 4 NO 3 after 16 wk chilling. Calamintha acinos, Festuca amethystina, Lychnis viscaria and Melica nutans presented the highest germination. A considerable enhancement of germination was also obtained at 1 mM. Germination of all taxa was again shown to be promoted by KNO 3 (NH 4 NO 3 being slightly more effective than KNO 3 ). NH 4 Cl had less effect than both NH 4 NO 3 and KNO 3 ( Table 5).
In the absence of exogenous N or chilling, only 0-8% of seed germinated. Chilling and addition of either nitrate (NO 3 − ) and ammonium (NH 4 + ) significantly enhanced germination. There was also significant interaction between the influence of NO 3 − or NH4 + and chilling on germination. In the absence of chilling and after 4 weeks of chilling, germination increased with growing NO 3 and NH 4 + concentration. Overall, chilled seeds germinated better than non-chilled ones. Chilling stimulated germination in seven of the eight taxa. N level, chilling period and their interaction significantly affected germination (Table 6).
SciEntiFic RepoRts | 7: 13924 | DOI:10.1038/s41598-017-13101-z Effect of Nitrogen Source Type and Light. Nitrogen added as nitrate ions (KNO 3 ), was statistically more effective than that from ammonium ions (added as NH 4 Cl). In addition, germination in the combined presence of the two ions, added as NH 4 NO 3 solution, was statistically higher than with either one of them (Table 3).
Although both light and exogenous nitrogen alone resulted in statistically significant promotion of germination, the combined presence of these factors was the most inductive. Two-way interactions of N form and light were significant ( Table 7). Effect of GA 3 . GA 3 promoted dark germination of all species. Seeds of all examined taxa given R light irradiation and then imbibed in 100 ppm GA 3 germinated just as well as dark-treated seeds kept in water. Exposure of imbibed seeds to 48 h FR before the application of GA 3 prevented germination (Table 8).  Table 2. Germination percentages of seeds exposed to different light treatments. 24-h dark imbibied seeds were exposed for 20 days to 10 min of either light conditions. Germination was observed in dark chamber at 23 °C.
Values are mean ± SD (n = 4). Different lower-case letters indicate significant diffences by Tukey's test with Bonferroni correction in the germination percentages among different light conditions.   Table 4. Photo-requirement germination index (PGI) and average seed mass for taxa included in the study. *The mean seed mass of each taxa was determined by weighing 100 air-dried seeds.

Discussion
Seeds of all studies species sown soon after harvest showed very low germination which indicates presence of primary dormancy. Furthermore, germination rates of all taxa increased after wet-stratification treatment. In nature such a mechanism effectively delays germination until spring. It is consistent with the previous study on the forest species in a temperate region 20 .
It was shown in this study that germination can be induced by short R irradiation applied after 24 h of RF light inhibition. Promotion of seed germination caused by R light was reported for a great number of plant species and is a phenomenon well established in literature [21][22][23] . For example, germination of Ruellia tuberose L. 24 and Asteracantha longifolia (L.) Nees 25 seeds can be promoted by R light treatment. This promotion can be reversed by a subsequent exposure to FR. Described inhibitory effect of FR expired after 17-20 h of R light treatment. All physiological actions that can be altered by changing R/FR ratio (including germination) are considered as processes controled by phytochrome. Our study showed that Lv and Dg were weakly inhibited by FR while level of inhibition for Av, Ca, Fa, Mn, St and So was moderate. Similar results were obtained for two of these species (Lv and Av) after field studies 23 . It may indicate that phytochrome-mediated regulation of Av, Ca, Fa, Mn, St and So seed germination is tuned to maximalize chance for fast population establishment by germination of banked seeds. It is worth mentioning that germination of some forest woody species (e.g. Abies alba L., Betula pubescens Ehrh., P. strobus L. and P. sylvestris L.) is also sensitive to light quality: R light stimulates germination while FR light inhibits this process 26,27 . Elimination of specimens with canopy that strongly affects light spectrum may therefore lead to competition beetwen herbaceous and woody species. It also suggests that many herbs occuring in Potentillo albae-Quercetum are able to win competition for resouces due to efficient phytohormone regulation of germination.   Phytochromes are well known to mediate light-promoted germination; they are also known to increase the amount of bioactive gibberellins in seeds 28 . Our results showed that exogenously applied gibberellins promoted dark germination of the studied species. Prolonged FR light pretreatment caused permanent loss of sensitivity to GA 3 . However, it was stated that continuous FR light irradiation delayed gibberellin mediated promotion of germination but did not prevent germination permanently 29 . It was also indicated that different kinds of processes are involved in the biochemical control of germination 30 . This was consistent with studies on Lactuca sativa L. 31 .
Nitrates which naturally occurr in soil, can substitute for the light requirement in some cases 32,33 . In this study, four taxa: Ca, Mn, St and So are connected with poor-soil environments and their Ellenberg's nitrogen index is 3 (Table 1). Up to now little was known about the effects of nitrate concentration combined with light quality on germination. Our results show that germination of these species was enhanced by the lowest concentration (in the range 1-10 mM) of any of the nitrogenous solutions. High concentrations of nitrogen compounds (25-50 mM) resulted in lower germination percentage than controls. This could be related to their ability to colonize soils with low nitrogen concentration. Similar results for seeds soaked in nitrogen solutions as those used in our study have been shown for 10 species from shrubby woodlands in central-western Spain 34 . However, the optimal conditions for seedling growth might not correspond to those for seed germination and seedling survival 10,35 . It is interesting to note that the critical nitrate concentration range for germination induction, observed in laboratory experiment, is spectacularly close to that encountered in natural ecosystems 36 .
Seeds of all examined species need light exposure to complete germination. Only Ca, Mn and Lv germinated >30% in the dark. Based on the white light germination characteristics, the data indicate that many of these species may be able to form a large persistent seed bank. This response, previously shown for many small-seeded temperate species 37,38 , can be seen as an evolutionary adaptive mechanism that prevents seed germination under shaded conditions 10 , as well as under excessively deep soil layers 39 .
The distinct light requirements for seed germination of the studied taxa could be a major factor hampering its natural regenerations. Strong requirements of light suggest germination preference for large vegetation gaps. For forest canopy of temperate forests, areas under canopy openings were found to have higher light intensities as well as higher air and soil temperatures than the surrounding closed forest 40,41 . Therefore, to improve the natural regeneration of Potentillo albae-Quercetum, disturbance (e.g., thinning) should be applied to allow more light reaching the understory layer.
A general correlation between seed weight and light requirement for germination has been suggested 37,38,42 . It was found that in temperate forest seed dry mass of 1.5 mg was an approximate cutoff between herbaceous species that are light-dependent and light-independent 39 . Our experiments indicated that seed germination of all 8 studied taxa of Potentillo albae-Quercetum was promoted by light. A light requirement for germination was stronger in smaller than in larger seeded species of Potentillo albae-Quercetum. PGI decreased with increasing seed mass. Such relationship was reported previously for example for Campanulaceae 42 and for herbaceous species of northern temperate deciduous forests 43 .
In recent years fast advancing changes in some types of heliophilous oak and oak-pine forests, where there are the best habitat conditions for the studied species, have been observed. The gradual invasion of C. avellana and C. betulus shrubs is connected with a considerable deterioration of light conditions in the ground layer which results from the closure of the canopy 1 . This variation in vegetation density creates new conditions with altered R/RF ratios of irradiance. Thus, germination of any seed falling under plant canopy (e.g. cover shrubs) could be predominantly inhibited by R/RF ratio which would also contribute to the formation of persistent seed banks of these species 7,8,11 . Germination of any seed falling under a plant canopy may be inhibited by exposure to FR and by lack of R light. Removal of C. avellana and C. betulus results in greater irradiance of R light than under intact canopies. It could be hypothesized that seedling emergence of the studied taxa may be increased after canopy removal as a result of increased germination of seeds exposed to white and R light. Moreover, the germination of most seeds of the studied taxa occurs in early spring before leaf expansion. The subsequent reduction in light transmittance after leaf expansion would be disadvantageous to growth of seedlings of helio-and thermophilous species.
In many habitats the establishment of seedlings depends on the presence of sites that are clear of vegetation, namely vegetation gaps 12 . Environmental conditions in vegetation gaps often differ considerably, depending on type and size of the gaps, as compared to those of the intact forest 44 . For example, it was found that vine maple gaps, compared with closed canopy of conifer forest, had significantly higher pH values and higher concentrations of Ca, Mg and K in the forest floor 45 . A tendency for lower C/N ratios and higher total N concentrations in the surface mineral soil was also observed. Germination in response to elevated NO 3 − concentrtion in our study can be interpreted as gap formation or as gap-detection mechanisms 10,37 . Some authors stated that nitrates could be a useful indicator of small scale disturbances in forests, since rise in the soil nitrate level can even be observed in single tree-fall gaps 46 . Seedling that rapidly establish in gaps have an advantage over plants that germinate later, when there is a greater competition for resources 10 . The results of this study have important implications for Potentillo albae-Quercetum restoration programmes. The distinct light requirements for seed germination of the species of Potentillo albae-Quercetum phytocoenoses could be the most important factor hampering its natural regeneration. In the phytocoenoses of Potentillo albae-Quercetum, emergence of light-demanding woody species from the seed bank is triggered by disturbance when gaps in litter or plant canopy expose seeds to light or higher R/FR ratio. Therefore, to improve the natural regeneration of phytocoenoses in thermophilous oak forests, disturbances (e.g., thinning) should be applied to allow more light reaching the understorey layer. Moreover, cold stratification breaks dormancy and promotes germination of selected species occurng in temperate forest and this suggests that the examined taxa are specialized to germinate in spring before leaf expansion of canopy. It leads to the conclusion that for many of these species revegetation is strictly connected with seed bank establishment and efficient detection of light condition change. The study site is located in the transition zone between the temperate oceanic (Atlantic) climate in the west and the moderate continental climate in the east. Mean annual temperature (2000-2010) is 8.8 °C; mean annual precipitation is 570.1 mm, with a maximum in June-July; the average length of the growing season is 210-220 days 47 .
Seed material was collected in the study region between May and October 2015 depending on the time of ripening. In order to obtain representative seed samples of the local populations, mature seeds of each taxa were collected from at least 15 individuals of one single large population and mixed before use. In the laboratory, the seeds were removed from fruits, dried at room temperature and stored in paper bags with relative humidity 40-60% in the dark at laboratory temperature (23 °C) for a maximum of two weeks. By selecting eight species we intended to include typical species of termophilous oak forest occuring under oak tree stand with different seed weights and dispersal strategies, representing a broad variety of families and life forms.

General Germination Procedures.
All germination tests were done in sterile plastic Petri dishes (9 cm diameter) lined with two layers of filter paper (Whatman no. 1) moistened with 3 ml of distilled water (pH 6) or the tested solutions. The Petri dishes were sealed with parafilm to minimize the loss of water. Each experiment consisted of four replicate Petri dishes of 25 seeds for each of the eight species and each treatment. Germination was recorded daily for 20 days. Germinated seeds were removed from the Petri dishes. A seed was considered to have germinated when a radicle had emerged. All manipulations in the dark treatments were done under dim green safe light. Germination percentages were calculated on the basis of the number of viable seeds; dead seeds, which were identified based on their softness and brownish embryo colour, were excluded. Number of dead seeds did not differ statistically among groups and treatments (P < 0.05). Then some seeds were stratified in a refrigerator at 5 °C for a 16 weeks to fully break dormancy. This temperature is a standard for testing cold stratification and simulated average winter (5 °C) temperature condition. Non-chilled seeds were stored at 23 °C. The temperature of 23 °C appeared most suitable for optimal germination for herbaceous species from the temperate region 14,48,49 . In addition, this thermoperiod represents the mean daily maximum and minimum monthly temperatures at the Lodz Weather Station during June and July, when most seeds germinate in natural habitat. Fluorescent and incandescent sources of light were used. Light was filtered through 3 mm thick plexiglas filters (locally manufactured).
Effect of Different Quality of Light. This experiment was performed using the seeds after 16 weeks of stratification at 5 °C prior to germination. Four light treatments plus dark control were used. First, the cold stratified seeds were imbibed in darkness (dishes covered with two layers of aluminium foil) for 12 h on filter paper moistened with 5 ml distilled water. Next, the seeds were exposed to different light treatments: (a) white light provided by a single fluorescent lamp (60 W); (b) R light for 10 min per day obtained by filtering the white light of 100 W incandescent bulb through red plexiglas; (c) FR light for 10 min per day obtained by filtering the white light of 100 W incandescent bulb through blue and red plexiglas; (d) 10 min of R, followed by 10 min of FR per day; (e) 10 min of FR, followed by 10 min of R per day. Dark controls were conducted in absolute darkness. The bulbs were placed 30 cm above the level of the seeds. Germination was observed in a dark chamber at 23 °C for 20 days.
For each taxa, an index of light requirement for germination (photo-requirement germination index; PGI) was derived: PGI = 1 − (FGD/FGL) where FGD is the percentage of germination in the dark and FGL is the percentage of germination in the light. Therefore if all of the seeds germinated in the dark as well as in the light during one day, PGI index would be 0; a value of 1 indicates germination only occurring in the light 37 .

Effect of Different Type and Concentrations of Nitrogen Source and Chilling. Chilled (16 weeks)
and non-chilled seeds were treated with the following nitrogen (N) concentrations: 0, 1, 10, 25, 50 mM N, applied either as KNO 3 , NH 4 Cl or NH 4 NO 3 . Each seed lot was moistened with the appropriate N solution and placed in the dark for the chilling treatment. For the non-chilling treatment, seeds were treated in a similar manner, but chilling (5 °C) for 24 h was substituted for room temperature (23 °C). Seeds were then returned to Petri dishes and incubated in the dark. Germinated seeds were counted under dim green safe light.
Effect of Nitrogen Source Type and Light. 10 mM sources of N were applied as KNO 3 , NH 4 Cl or NH 4 NO 3 solutions. All taxa were tested under two light conditions (complete darkness and exposure to daylight). Each seed lot was moistened with the appropriate N solution and placed in the dark for the chilling treatment. Next, seeds were transferred to 23 °C and wrapped in aluminium foil for dark treatment or exposed to light for 12 h. Germinated seeds were counted under dim green safe light or in the daylight, respectively. Effect of GA 3 . Seeds were first imbibed in 5 ml of distilled water under R or RF light for 48 h. Then the seeds were imbibed either in distilled water or GA 3 solution (100 ppm) in the dark for 24 h. Procedures with imbibed seeds were carried out under a dim green lamp at 23 °C. The seeds were incubated at 23 °C in continuous darkness.