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Article

Plantago Species Show Germination Improvement as a Function of Nitrate and Temperature

by
António Teixeira
1,2,*,
Pietro P. M. Iannetta
3,* and
Peter E. Toorop
1
1
Royal Botanic Gardens Kew, Millenium Seed Wakehurst Place, Ardingly, West Sussex RH17 6TN, UK
2
Earth and Environmental Sciences Department, University of Pavia, Via S. Epifanio 14, 27100 Pavia, Italy
3
Ecological Sciences, James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
*
Authors to whom correspondence should be addressed.
Nitrogen 2024, 5(3), 790-807; https://doi.org/10.3390/nitrogen5030052
Submission received: 9 August 2024 / Revised: 11 September 2024 / Accepted: 18 September 2024 / Published: 20 September 2024
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Abstract

:
At the optimum temperature, which is the ideal range in which seeds germinate most efficiently, seed germination may be lower than expected under favorable conditions, and this is indicative of seed dormancy. Also, germination may be enhanced by additional and interacting factors, such as nitrate and light. However, little is known about the interplay between temperature, nitrate, and seed germination. Using seeds from 22 accessions of four Plantago species that occupy distinct pedoclimates, we applied a factorial experimental design to assess the relationship between exogenously applied nitrate (KNO3) and temperature on germination in a Petri dish experiment. The data explore the relationship between seed germination, temperatures, and seed- and maternal-source soil N content as either nitrite (NO2−), nitrate (NO3−), or ammonium (NH4+). The interpretation also considered the total N and C contents of seeds, and the soil of the maternal plant (of the test seed) sources. Significant interspecific effects of nitrate and temperature on seed germination were observed. The capacity of nitrate to enhance final germination may be diminished substantially at supra-optimum temperatures, e.g., P. lagopus germination at 15 °C was 7% lower than that seen for water-only treatment. In contrast, at sub-optimum and alternating temperatures, nitrate enhanced final germination differentially across the species tested. This suggests a shift to enhanced germination at lower temperatures in the presence of sufficient soil nitrate, facilitating seedling establishment earlier in the growing season. The seeds of some Plantago species showed increased germination as a function of nitrate and temperature, particularly those of P. lagopus. The findings indicate that species (and genotype) responses correlated with the prevailing temperature and rainfall patterns of the locality; such local adaptation would ensure that seed germination and establishment occur during a period when environmental conditions are optimal.

1. Introduction

In the soil seed bank, seed dormancy is adjusted continuously in response to the sensing of environmental signals, such as light, mineral nitrogen (N) forms, and water potential. These environmental factors interact with the seeds’ physiological capacity, such that a low seed dormancy state is attained when the most suitable conditions for germination are present [1]. Among such non-dormant seeds, variation in the state of germination may still be observed. The germination of non-dormant seeds is completed when appropriate endogenous nutritional conditions, and especially N forms, are present [2,3].
Temperature is also a critical abiotic factor controlling seed germination. The effect of temperature on seed germination varies between species and intraspecific variants, as determined by environmental gradients [4,5,6,7,8]. The optimal temperatures for the total germination of Mediterranean species are typically within the range of 5–15 °C, with germination percentages decreasing at higher temperatures [8,9,10,11]. The stimulation of seed germination by alternating temperatures is also common in species from arid zones [12,13,14]. It has been reported that species producing small seeds require alternating temperatures and light as an adaptation for germination close to the soil surface [13]. Altogether, there is an abundance of evidence regarding the important role of temperature in controlling seed germination and seedling establishment.
Mineral N forms can act to reduce seed dormancy and promote seed germination in a wide range of species [15,16,17,18]. In Arabidopsis, nitrate is thought to advance the degradation of ABA during imbibition [19], and in Sisymbrium officinale, Toorop (2015) demonstrated that testa rupture is controlled by nitrate, likely through increased embryo growth potential, and associated with dormancy release [20]. However, such studies did not assess other N forms that also stimulate seed germination—whether those N-forms were in the seeds or maternal (soil) environment. Some evidence for differential mineral N preference is available for a small range of species, suggesting a role in re-colonization and making this an ecologically relevant mechanism [21].
However, in the field, seeds must operate under complex and variable environmental conditions. Soil organic matter (SOM) mineralization is an important process in which carbon (C) and nutrients are transformed into CO2 and plant-available forms of nitrogen (N), phosphorus (P), and sulfur (S) [22]. Carbon mineralization is tightly coupled to the release of mineral N, P, and S, and can be driven by microbial requirements for C and nutrients for their maintenance, growth, and the production of extracellular metabolites, including enzymes [23,24,25,26].
It has been reported that soils with lower C:N ratios (i.e., with a smaller pool of C to support microbial growth) can favor longer seed persistence [27,28]. In contrast, [29] showed that soil organic N levels have a significant negative impact on seed longevity in the soil seed bank, which may be explained by the likelihood that N promotes seed germination (i.e., removal from the soil seedbank) [30], and/or that N enhances the activity of microbes, including their capacity as seed pathogens [31]. Seed exogenous nitrite, nitrate, and ammonium ions (e.g., 10−1, 10−2, and 10−3 M) have also been described to break dormancy and stimulate germination in the seeds of various species, including Capsella bursa-pastoris Medik. [32], Oryza sativa L. [33,34], and Avena fatua L. [35]. A recent study on the soil seedbank of acid grasslands in the UK showed that atmospheric N deposition was responsible for decreased seed abundance and species richness [36]. Such long-term observations may have been due to soil-N x temperature interactions, negatively impacting the seed dormancy state and capacity to sense when environmental conditions are most suitable for germination. The effect of N addition (15 kg N ha−1 year−1) to soil on seed germination rates and the composition of nine native plant species exhibited no or poor emergence in field plots, which was species-specific [37].
The germination of plant species shows different reactions to N type and dose [21], but limited information is available regarding how N form preference influences the effect of temperature on seed germination (e.g., [21]). A deeper understanding of the effect of mineral N, temperature, and their interaction on seed performance can provide important insight into the fundamental mechanisms governing seed–soil interactions, and ultimately plant fitness. However, N–temperature interactions have not been studied extensively for wild plant species, and especially with respect to a ‘system-level’ approach where the pedoclimate and the N status of seed and soil are also considered. Knowing the detailed N status of the soil environment for the maternal (i.e., seed) source with respect to the germination characteristics of the progeny may provide useful and applicable perspectives, for example, when selecting species for the seed-based restoration or conservation of specific niches.
Given that the above-discussed variables may interact to promote seed germination, in the present study, we tested the following hypotheses: (1) the application of exogenous nitrate improves seed germination differently between species and incubation temperature; (2) endogenous seed and maternal soil N forms and carbon positively contribute to seed germination; and (3) the positive effects of edaphic factors differ between pedoclimate regions.
To address these hypotheses, four ruderal species of Plantago (P. albicans L., P. coronopus L., P. lagopus L., and P. lanceolata L.) were selected as they occupy different, but overlapping, pedoclimates and are frequently used for seed-based conservation and restoration efforts. Plantago’s significance lies not only in its role as a main genus for grassland restoration, but also as a commercially cultivated wild crop. This dual utility highlights its potential to contribute to both ecological conservation endeavors and commercial cultivation, rendering it a versatile and valuable resource. In addition, they are common native grassland species in the Iberian Peninsula, yielding many small seeds for scientific study and are typically well-adapted to arid and low-nutrient soil conditions [38,39,40]. The existing literature also suggests that these species are likely responsive to different N types, doses, and temperatures [41,42,43,44].

2. Materials and Methods

2.1. Plant Material

Seeds from four Plantago species (P. albicans, P. coronopus, P. lagopus, and P. lanceolata) were collected in 2015, totaling 22 accessions from three Iberian Peninsula pedoclimatic regions: LUS—Lusitanian, MDS—Mediterranean South, and MDN—Mediterranean North (Figure 1, Table S1) [45]. For each species, mature seeds were sampled (accessions) at their natural time for seed dispersal. Upon collection, the seeds of each accession were cleaned and kept in a controlled environment (15 °C, 15% RH) until the germination experiments were conducted over the next six months. For each accession, three subsamples of soil were also collected (0–10 cm depth) after removing any crop residue from the soil’s surface. Each soil sample was placed in a zip-lock bag and stored at 4 °C.

2.2. Seed Germination Experiments

Preliminary experiments showed that 10 mM of KNO3 solution (1, 10, and 30 mM used) was the best concentration to increase the germination percentage. The laboratory germination experiments were performed within the six months after collection in temperature-controlled incubators (LMS, Sevenoaks, UK) following a factorial design with two factors (temperature and substrate) and with two levels each: (a) constant temperatures in 10 mM KNO3 solution (potassium nitrate, hereafter referred to as nitrate); (b) constant temperatures in water; (c) alternating temperatures in nitrate; and (d) alternating temperatures in water. The germination temperature was set to six levels: daily constant regimes of 5, 10, and 15 °C and daily alternating regimes of 10/0, 15/5, and 20/10 °C with a 12 h light/12 h dark photoperiod; darkness was synchronized with the low temperature for alternating regimes.
Seeds were sown on 9 cm Petri dishes holding two layers of filter paper (Whatman® grade 1, Sigma Aldrich, Gillingham, UK) and 4 mL of Milli-Q® water (Merck Millipore, Watford, UK) or the same volume of 10 mM nitrate solution (# P8394 ≥ 99.0%, Sigma Aldrich, Gillingham, UK) and placed in closed plastic bags to prevent evaporation, and Milli-Q water added when necessary. One replicate dish with 50 seeds was used per experimental treatment, and three replicate experiments were performed independently. Completion of germination was scored daily for seeds with protruded radicles >1 mm. At the end of the experiments, non-germinated seeds were cut open and classified visually as either: ‘apparently viable’, ‘empty’, or ‘fungus-infected’. Final germination percentages (FGPs) were calculated and further statistical analyses (as described below) were undertaken using viable seed (germinated plus remaining viable) data only [46,47].

2.3. Seeds and Soil Carbon and Nitrogen Content

Triplicate sub-samples of the seeds from the 22 accessions and respective soil samples were oven-dried (three days, 105 °C) and ball-milled for three minutes (radial oscillation frequency 20 s−1, Retsch MM200, Haan, Germany) to a fine powder, and 2 mg was added to tin containers for combustion and processing with a CE440™ Elemental Analyser according to the manufacturer’s recommendations (Exeter Analytical, Coventry, UK).

2.4. Quantification of Mineral-N Forms in Seeds and Soil

The concentrations of nitrite, nitrate, and ammonium in seed and soil samples were quantified using colorimetric methods. One hundred milligrams of dry seed powder (DW) or five hundred milligrams of soil powder (DW) were added to one or five milliliters (respectively) of milli-Q water and vortexed vigorously. The homogenates were centrifuged at 18,000× g for 30 min and NO3− and NO2− quantification were performed using the Roche NITRITE/NITRATE colorimetric method kit (# 11 746 081 001, Sigma Aldrich, Gillingham, UK) according to the manufacturer’s recommendations and with reagent volumes scaled down 5× to allow the use of the method in a 96-well plate format (Nunc-Immuno 96 MicroWell). Ammonium quantification was performed according to Hernandez-Lopez and Vargas-Albores (2003).

2.5. Statistical Analysis

All statistical analyses were performed in R (R Core Team 2021), except for the accumulated germination curve fitting using the Boltzmann equation (OriginLab 2016 software, Northampton, MA, USA). The effects of imbibition in water and nitrate solution at constant and alternating temperatures on germination were visualized with violin plots and the differences were analyzed using generalized linear mixed models (GLMMs) (Package lme4) [48] with binomial error distribution and logit link with germination proportions as response variables. Also, the species, imbibition, incubation conditions (constant temperatures in 10 mM KNO3 solution; constant temperatures in water; alternating temperatures in 10 mM KNO3; and alternating temperatures in water), and their interactions were set as fixed-effects independent variables (and factors) and the region and provenance altitude were set as random effects. The model-estimated probability of change factor was calculated by exp(log-odds).
To evaluate the effect of each temperature in both water- and KNO3-solution-imbibed seeds, germination data were analyzed for each species using generalized linear models (GLMs) with binomial error distribution and logit link function to identify significant effects of N on germination at alternating (10/0, 15/5, and 20/10 °C) and constant temperatures (5, 10, and 15 °C). Hence, two models were fitted separately for each species’ accessions: (a) nitrate versus water at constant temperature and (b) nitrate versus water at alternating temperatures. In each case, a full factorial model was fitted first, including germination temperature, imbibition condition, and their interaction. Subsequently, the model was simplified by the stepwise deletion of non-informative model parameters until a minimum adequate model for each case was selected.
Additionally, germination variation was assessed with the delta variation for each incubation condition and graphically represented in a heat-map cluster. To test the effect of seed and soil mineral N and carbon contents (NO2−, NO3−, NH4+, total C, N, and the C:N ratio) in the FGP of the three temperatures used in each incubation condition, a factor analysis of mixed data (FAMD) was used to reduce the dimensionality. FAMD (Package FactoMineR Version: 1.36) was performed for each condition with species (but not pedoclimatic region) as the categorical variable, and germination percentages as numerical variables. The FAMD second dimensions were then used in generalized linear models (GLMs) with Gaussian distribution and an identity link was performed to test the effect of seed and soil mineral N and carbon contents. Subsequently, the Akaike information criterion (AIC) was used for the step-wise deletion of non-informative parameters to simplify the models until a minimum adequate model for each case was selected. Significant variables in each model were visualized with the function type = “pred” (Package sjPlot, Version: 2.8.11). The model estimates’ probability of change was calculated by exp(log-odds)-1) × 100).
New GLMs were used to test the effect of the above seed and soil mineral N and carbon on individual species germination. As above, the Akaike information criterion (AIC) was used for the step-wise deletion of non-informative parameters to simplify the models until a minimum adequate model for each case was selected.

2.6. Base Temperatures and Thermal Times

For each experimental condition, the germination rate was inferred from the reciprocal of the time until 50% germination (t50), estimated through a sigmoidal curve fit of the cumulative germination for each dish using the Boltzmann equation. With the range of constant temperatures and the mean of the alternating temperatures of each replicate experiment per accession, the reciprocal values of t50 (GR50) were regressed with a linear model to estimate the base temperature at which the germination rate was equal to zero (Tb). The thermal time (θ50) was estimated as the reciprocal of the coefficient, ascertaining a sub-optimum temperature range. Tb and θ50 variations were assessed with the delta variation for each treatment condition. GLMs were used to test the effects of seed and soil mineral N and carbon contents (NO2−, NO3−, NH4+, total-C, total-N, and the C:N ratio) on Tb and θ50. As above, the Akaike information criterion (AIC) was used for the step-wise deletion of non-informative parameters to simplify the models until a minimum adequate model for each case was selected and the results each model were visualized with the sjmisc package (Version: 2.8.11).

3. Results

3.1. Germination in Nitrate vs. Water under Two Temperature Regimes

The germination profile of seeds collected in three distinct pedoclimatic regions (LUS, MDS, and MDN) of the Iberian Peninsula showed significant variation between species (Figure 2A). P. albicans and P. lanceolata revealed low FGP in several accessions, while P. lagopus showed a germination profile ranging from low to high FGP. Most accessions of P. coronopus showed a high FGP (Figure 2B).
Compared with water, the imbibition in nitrate solution showed an increase in the germination of all species accessions (Figure 2C). This increase was particularly evident in P. lagopus, where the accessions with low germination ceased to be observed, and P. coronopus, with most of the accessions exhibiting near 100% germination (Figure 2C). The GLMM analysis indicated log-odds for P. coronopus and P. lagopus of 1.92 and 1.08, respectively, corresponding to the probability of germination increasing by factors of 6.95 and 2.94, respectively, compared with the P. albicans. In contrast, the P. lanceolata germination probability decreased by a factor of 0.31 (Figure 2A).
Violin plots showed that water-imbibed seeds had higher germination percentages at constant temperatures than at alternating temperatures in accessions characterized by low germination (Figure 2D), which was confirmed by the GLMM model with a factor of 1.54. However, it was observed that the species interaction with incubation treatment had a factor of 0.55 for P. coronopus and P. lagopus, indicating a decrease in these two species under constant temperatures compared with alternating temperatures, and contrary to P. lanceolata, which showed an increase (Figure 2A). When incubated in nitrate solution, a strong increase was observed in germination in the accessions previously characterized by low germination under both alternating and constant temperatures (Figure 2E). The GLMM output of species interactions with the treatment regime (species x KNO3), indicated positive odds for all species (an increase by a factor of up to 6.88) (Figure 2A). The violin plots showed the germination improvement of nitrate-imbibed seeds of accessions from LUS and MDS pedoclimatic regions, which was less evident for the MDN region (Figure 2F,G).
The generalized linear models (GLM) estimating germination at each temperature showed a high correlation with observed germination values (Figure S1A). At constant temperatures, compared with the reference temperature (10 °C), the results showed that, in the presence of nitrate, a temperature of 15 °C increased germination in P. albicans, P. coronopus, and P. lanceolata, while germination in P. lagopus showed a significant decrease (Figure S1B, Table S2A). GLM A (nitrate solution versus water at constant temperatures) showed a negative effect on the FGP of all species incubated at 5 °C (Table S2A), indicating that the increase observed was reduced by nitrate, compared with water only and the reference temperature of 10 °C (Figure S1B). This indicates that nitrate did not reduce the effect of less-than-optimal seed temperature. In contrast, and compared with the control (water), the GLM showed that nitrate has a positive interaction with temperature at 5 °C in P. lanceolata, since the FGP decrease from 10 to 5 °C was lower for this species (Figure S1; Table S2A).
Nitrate also increased the FGP of seeds incubated at alternating temperatures, particularly for seeds of P. lagopus (Figure S1). Nitrate interacted negatively with alternating temperatures in the germination of the other three species: at 20/10 in P. albicans and P. lanceolata, and at 10/0 in P. coronopus (Table S2B). On the other hand, in P. albicans and P. lanceolata, seed germination showed a negative interaction with temperature at 10/0 °C. This indicated that, when nitrate-imbibed, FGP decreased in colder environments (from 15/5 °C to 10/0 °C) in P. albicans and P. lanceolata species, which did not occur in water-imbibed seeds of P. lagopus and P. coronopus (Table S2B).
The delta variation in germination treatments showed a distinct species pattern, with most of the accessions of P. coronopus, P. lagopus, and P. lanceolata clustering by species (Figure 3). Seeds incubated in nitrate solution at constant temperatures (ΔKNO3:H2O), showed a stronger positive effect on germination compared with those at alternating temperatures (Δalter.:const.), with P. lagopus and P. coronopus exhibiting the best response. Seeds incubated in nitrate with alternating temperatures (Δalter.:const) revealed negative variations, particularly P. lanceolata at 10/5 and 15/10 °C (Figure 3).

3.2. Factor Analysis of Mixed Data Reveals Species-Specific Nitrate Response

The first dimension of the FAMD explained approximately half of the germination variance (up to 57%) for constant and alternating temperatures when seeds were imbibed in nitrate or water, separating the species from lower to higher germination accessions. The FAMD second dimension accounted for up to 20% of the germination variance under the above-mentioned conditions and showed that the accessions tentatively clustered by biogeographical region, with species from the LUS pedoclimatic region mainly positioned in the negative second dimension when imbibed in water at alternating temperatures and KNO3 under both temperature regimens (Figure 4 and Figure S2).

3.3. Comparative Analysis

Comparative analysis was performed to assess the relationship between the soil and seed mineral N and carbon (C) contents and FGP. Considering data from the four species, we found that the seed nitrite content was significantly higher than the nitrate content (up to 190-fold more than that found for nitrate), and was occasionally not detected in some accessions (Table S1). The seed total N content was significantly higher in P. coronopus than in the other species (46% dry mass; Figure 5A).
Using the reduced dimensionality of the FAMD’s second dimensions (Figure 4) as the dependent variable, the GLMs showed that the probabilities of germination in water-imbibed seeds at constant temperatures were increased by the soil total C (1.3%), seed nitrite (NO2−) (15%), and ammonium (NH4+) (17%), and decreased by the soil nitrite and seed total-C content (Figure 5A). The germination probabilities of seeds imbibed in KNO3 solution at constant temperatures were increased by soil total-N, soil C:N ratio, seed nitrite, and seed ammonium (25%) content and decreased by the seed nitrate (NO3+) and seed C:N ratio (Figure 5B; Table S3). The germination probabilities of water-imbibed seeds at alternating temperatures were increased by the soil C:N ratio and the seed nitrite and ammonium (25%) contents, and decreased by the seed total-N, C:N ratio, and nitrate (Figure 6A). The increased seed germination when imbibed in KNO3 solution was correlated with the soil C:N ratio, and seed nitrite and ammonium (27%) contents, while decreased germination was correlated with the seed total N, C:N ratio, and nitrate contents (Figure 6B, Table S3).
To elucidate more detail, further GLM comparative analysis assessed the relationship between seed and soil traits, and the temperature x germination response by each species (Table S4). The seed C:N ratio showed only positive correlations with P. albicans germination at 5, 10, and 10/0 °C in water-imbibed seeds and at 5 °C in KNO3-solution-imbibed seeds, contrasting with the total carbon, which showed negative correlations. The seed endogenous nitrate, nitrite, and ammonium increased the probabilities of germination for this species, except NO3+ at 15 °C in seeds imbibed in water (Table S4). For P. coronopus the probabilities of germination increased with the seed C:N ratio and total-N, and decreased with total-C, but only at 20/10 °C for both water- and KNO3-imbibed seeds. The seed total C:N ratio showed a strong negative effect on the germination of P. lagopus seeds imbibed on water at both constant and alternating temperatures, which was less negative in KNO3 solution-imbibed seeds. In contrast, at alternating temperatures, the seed total-C content increased the probabilities of P. lagopus percentage seed germination, except in KNO3 solution-imbibed seeds at 5 °C. Ammonium also positively contributed to P. lagopus germination at 5 °C and negatively contributed at 20/10 °C in water-imbibed seeds (Table S2). The GLMs also revealed a positive effect of endogenous nitrite content on P. lanceolata germination at 10 and 10/0 °C. In contrast, the seed total N and C:N ratio showed negative effects on P. lanceolata germination at 20/10 °C in water-imbibed seeds and total C showed the same effect at 10/0 °C in KNO3-solution-imbibed seeds (Table S4).
As noted above, P. coronopus seeds possessed the highest seed total N content, even when the soil total N content was low, which was indicative of its high capacity to accumulate N and its efficiency in translocating this N for reproduction. The soil total N content was up to 74-fold lower than that found in seeds, and GLM models showed positive estimates for nitrogen on P. albicans seed germination at 20/10 °C in KNO3-imbibed seeds (Table S5). This exception aside, the soil total N content revealed a strong and negative effect on P. coronopus germination at 15/5 °C when KNO3-imbibed, and P. lagopus germination at several temperatures, whether water-imbibed or nitrate-imbibed. Soil nitrite showed negative effects on P. lagopus germination at 5 °C when water-imbibed and P. lanceolata seeds when KNO3-imbibed at 20/10 °C. Soil nitrate showed negative effects under most germination conditions of P. coronopus, while the other N forms had no significant effect. As the soil nitrate levels increased, so did the probability of P. lagopus germination under several experimental conditions, contrary to that observed in P. lanceolata. Soil ammonium showed a positive effect on P. albicans FGP at 5, 10, and 20/10 °C in water-imbibed seeds, and a negative effect on P. lanceolata FGP at 20/10 °C in nitrate-imbibed seeds (Table S5). These results indicate that the positive correlations of soil N forms with the FGP improvement depended on the applied temperature regime.

3.4. Base Temperature and Thermal Time Variation

The differences in the base temperatures for seed germination in nitrate versus water incubation and alternating versus constant temperatures were tested by GLM analysis, showing positive coefficient effects of seed endogenous nitrate on germination base temperature (Tb) for water-imbibed seeds at constant temperatures, while seed endogenous ammonium showed negative effects (Figure 7A). At the same temperatures but imbibed with KNO3, source soil and seeds with high nitrite levels showed positive effects on Tb germination, while seed endogenous ammonium showed negative effects (Figure 7B).
Soil nitrate, total N, C:N ratio, and seed total C showed negative effects on Tb germination in water-imbibed seeds at alternating temperatures (Figure 7C), while at the same temperatures but imbibed in KNO3, the soil total N and seed endogenous ammonium showed positive effects, contrary to the levels of soil total C (Figure 7D). Considering all traits, soil nitrite had the strongest influence on the increase in the Tb under constant temperatures, and the decrease in Tb under alternating temperatures.
Soil nitrite and endogenous seed nitrate showed negative effects on the thermal times (θ50) of water-imbibed seeds at constant temperatures (Figure 7E), with soil N form also showing strong negative estimates on the germination θ50 of KNO3-imbibed seeds at the same temperatures (Figure 7F). At alternating temperatures, endogenous ammonium showed negative effects on the germination θ50 of water-imbibed seeds while the C:N ratio showed a positive effect (Figure 7G), and a significant effect was observed in KNO3-imbibed seeds at the same temperatures (Figure 7H). Considering all traits, soil nitrite has the strongest influence in decreasing θ50 for both water- and KNO3-imbibed seeds, while the seed C:N ratio increased θ50, indicating that these variables also influenced the time taken to complete germination of P. lanceolata.

4. Discussion

4.1. The Studied Species Showed Variations in Individual Requirements for Germination

Temperature and nitrogen (and light) availability are the most critical environmental factors that have profound impacts on seed dormancy and germination (reviewed by [18]). Our factorial study showed inter-species effects of nitrate and temperature on the FGP of seeds collected from accessions occupying different pedoclimates. Although many studies have already described the positive effect of exogenous nitrate on the seed germination of different species when dormancy is present [15,16,17,18,20,49,50,51], few studies have been undertaken on the effect of exogenous nitrate in germination over a range of temperatures [52,53,54,55].
We observed that the application of nitrate improved the germination under both alternating and constant temperatures, though the enhancement was more effective at constant temperatures, but not at all temperatures, or for all species (Figure 2, Figure 3, and Figure S1). Previous studies have also reported a strong stimulation of dormancy release in many species under alternating temperatures regimes [56,57,58]. Our results demonstrate that the capacity of nitrate to enhance FGP varies not only between species, but also with the temperature of germination. Despite the imbalanced number of species collected in each region, the results showed a germination response pattern according to the collection environment of the maternal sources (Figure 4), which correlated with regional observations for N forms and C quantified in the soil samples (Figure S3A). It was also observed that the addition of nitrate was more effective in P. lagopus, while in P. lanceolata, effectiveness was confined to low temperatures. The negative interaction of these two species (and P. albicans) with endogenous nitrate at higher and constant germination temperatures suggested that, at supra-optimum temperatures, nitrate addition possibly causes the thermoinhibition of Plantago seed germination at constant temperatures. Indeed, a slight decrease in FGP in P. coronopus, P. lagopus, and P. lanceolata was observed (Figure S1B, Table S2). At alternating temperatures, our results agree with a report for Arabidopsis [59], where the percentage seed germination response to nitrate increased from 5–20 °C.
Our results also showed a negative effect of nitrate on seeds under alternating temperature regimes (P. albicans and P. lanceolata at 20/10 °C), suggesting a temperature-dependent nitrate response for at least some of the Plantago species tested, favoring germination and seedling establishment in the field at temperatures that are lower than optimal in the presence of elevated soil nitrate contents. This result was confirmed by the FGP increase in accessions from the LUS pedoclimatic region (with lower temperatures), where a stronger germination recovery was observed when seeds were incubated in KNO3 solution (Figure 2G).

4.2. Germination Response to Soil N Forms and C Content Suggested a Transgenerational Effect

Seed dormancy is an adaptive trait [60] that displays strong plasticity in response to abiotic factors, such as the pedoclimate (i.e., biogeographical location), and seasonal conditions [61,62]. Furthermore, seed dormancy release depends on several factors that vary with site, e.g., chemical properties of soils, including the nutrient content, organic matter, pH, water-holding capacity, and nitrate content [28]. Our results showed a clear inter-species variation in seed N forms and C content (Figure S3B) when the soils of maternal (seed) sources substantially differed between pedoclimatic regions, but not between maternal sources (Figure S3A).
The relationships observed between FGP with seed and soil mineral N and carbon content suggest a regional influence, either for positive or negative contributions, to seed germination, independent of the incubation temperature regimen (Figure 5 and Figure 6). The strong positive correlation between the seed total N content and FGP of P. albicans at low temperatures (Table S4) suggests that, for this species, FGP may be dependent on the seed total N content. Conversely, P. coronopus exhibited a positive association with the seed total N content, but only at high temperatures. Either total N or specific ion forms appeared as species-specific capacities that reflected selection pressure which helps to ensure sufficient seed N for germination in low-soil-N environments (Figure 5A). Nitrate fertilizer application to Arabidopsis (col0) seeds showed that the seed nitrate concentration was correlated with higher FGP [63]. A recent study on riparian and dryland community species showed that seeds from wetland species germinated rapidly and in high percentages, regardless of nitrate treatments, whereas dryland species had unique responses [64], which may explain the variability in the effects that we observed for the different species.
Contrary to what was observed for seeds, the soil total N content was low and up to 74-fold lower than the seed total N content. Contrary to the other species in this study, soil nitrate positively contributed to the P. lagopus FGP at both constant and alternating temperatures (Table S5). In addition, since the majority of soil total-N (>98%) is bound within organic matter, it is not directly plant-available [65]. A previous study into germination in soils treated with NH4+ (1 mM), NO3− (1 mM), and NH4NO3 (0.5 mM) reported no effects on seed germination, and the authors explained that the lack of effect was due to the absence of seed dormancy [66]. However, an explanation based solely on a lack of dormancy may be an oversimplification, since (1) the soils on which the tested populations grow are not nitrogen-rich, (2) overfertilization poses a risk to germination in a maternal effect manner, and (3) direct toxicity as ammonia nitrogen is greater at the germination stage than nitrate-nitrogen. The negative effects of soil N forms, observed here for P. lagopus FGP, reveal that this species response is sensitive to N form, contrary to P. albicans (Table S5).
It has been suggested that a nitrite concentration above 80 ppm could be toxic to plants, resulting in stunted roots, restriction of nutrient absorption, and causing chlorosis [67,68]. The soil nitrate amounts observed in our samples were much lower than the toxic quantities reported. In fact, the values of nitrate observed in the soil samples were within the same range as those in other reports [69,70], although several samples were below the detection limit, and we could not exclude a possible interference with our model accuracy. A study into the effects of soil urea fertilizer on seed germination (Triticum aestivum, L. (wheat); Secale cereale, L. (rye); Hordeum vulgare L. (barley); and Zea mays, L. (maize)) indicated an adverse effect of N (as urea) on seed germination, and that this was due to ammonia formed through the hydrolysis of urea by soil urease and impurities, such as biuret or nitrite formed by the nitrification of urea N [71]. Although the soil components could not directly influence the germination of seeds in the present study, they can have an indirect effect by influencing the growth and physiology of the maternal plant. The transgenerational effects of draught, heat, light, or herbivory are well-documented in the literature [72,73,74], contrary to the effect of nitrogen forms. A study on the N enrichment of maternal conditions in two consecutive years showed contradictory, but significant, effects on the offspring biomass of Artemisia frigida. The offspring seeds of this species also showed an increase in germination rate, contrary to Stipa krylovii, which showed a decrease in germination [75]. The same study showed that the maternal addition of P decreased the offspring biomass of S. kryloii and increased the offspring biomass of A. frigida. The negative effects of N forms on germination observed in the present study may result from transgenerational effects.
The base temperature for germination has been found to change with dormancy status, and physiological dormancy release was described in terms of Tb reduction, gradually allowing germination to occur at progressively lower temperatures [76,77]. Contrary, a study on Lippia javanica, L. seeds treated with GA3 and KNO3 showed higher Tb values [55]. Our results demonstrated that the soil total N, C:N ratio, and nitrate and seed endogenous total C and ammonium generally facilitated a decrease in the germination base temperature, which ultimately may contribute to the increase in FGP (Figure 7). At the same time, θ50 increased with seed C:N ratio in H2O-imbibed seeds at alternating temperatures, which may indicate a longer germination time under natural conditions (Figure 7). Therefore, the base temperature and thermal time appear to contribute to the germination and establishment in the field in a complex response to soil conditions.

5. Conclusions

Overall, the results obtained allow for the assessment of the effect of exogenously applied N as KNO3 on seed germination for four ruderal species of Plantago, across six levels of temperature, in relation to the seed total C and N contents, and maternal source soil. Nitrate treatments diminished the germination of seeds at supra-optimum constant temperatures. In contrast, at sub-optimum and alternating temperatures, nitrate enhanced final germination differentially across the species tested. This suggests a shift to germination at lower temperatures in the presence of sufficient soil nitrate levels, encouraging the germination of seeds, and promoting seedling establishment and growth earlier in the growing season.
There is evidence that elevated ambient temperatures can accelerate the decline in seed viability and compromise the bet-hedging strategies of species, especially in dryland regions [78]. Consequently, a lack of precipitation (due to climate change) in such regions will determine plant recruitment, with lower levels of success and net losses to seed bank population densities. Models assessing global warming impacts on Mediterranean soil seed banks demonstrate a trend toward an increase in plant-devoid areas under drought conditions, and a decrease in the seed bank, especially for short-lived species. This may result in further positive feedback effects, which would accelerate vegetation loss [79]. The dual role of Plantago as a key target genus for grassland restoration and a commercially cultivated wild crop underscores its great potential in simultaneously contributing to ecological conservation efforts and meeting commercial demands.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nitrogen5030052/s1, Table S1. Metadata of plant species seeds collected at the time of seed dispersal for each region and soil samples of each location; N forms and C content in both seeds and soil samples; the total mean of content and the ratio seeds/soil content; Table S2. Minimal adequate generalized linear models (binomial error, logit link) fitted to the germination results (alternating versus constant temperatures in water; alternating versus constant temperatures in nitrate solution (10 mM); nitrate solution versus water at constant temperature; and nitrate solution versus water at alternating temperatures) of each species. The minimal adequate models were obtained by a stepwise deletion of non-significant parameters, starting from a full factorial model; Table S3. Minimal adequate generalized linear models of testing the effect of soil and seed nitrogen forms and carbon on species germination (based on second axis of the FAMD); Table S4. GLM comparative analysis assessing the relationship between seed traits, and the temperature vs. germination response by each species in water and KNO3 imbibition; Table S5. GLM comparative analysis assessing the relationship between soil traits and the temperature vs. germination response by each species in water and KNO3 imbibition; Figure S1. Percentage of seed germination of 22 accessions of four Plantago species from different pedoclimates. (A) Linear regression between estimated and observed germination percentage; (B) estimated percentage of germination at constant and alternating temperatures under two imbibition conditions (water and 10 mM of nitrate). Data estimates were acquired using GLM (binomial distribution, logit link); the species (and number of accessions) are: P. albicans (5); P. coronopus (5); P. lagopus (5); and P. lanceolata (7). Data estimates were acquired using GLM (binomial distribution, logit link). Colored bars show means across treatments for each temperature regime, and error bars denote 95% binomial confidence interval. Results are grouped per treatment (H2O = water imbibition; and KNO3 = nitrate imbibition (10 mM)); Figure S2. Plot loadings of the factor analysis of mixed data (FAMD) presented in Figure 4; Figure S3. Factor Analysis of mixed data (FAMD) of nitrogen (N) forms and carbon (C) content for: (A) soil and (B) seed samples. Colored/shadowed areas indicate clustering of accessions by pedoclimate regions for soil samples and by species for seed samples.

Author Contributions

Conceptualization, A.T., P.P.M.I. and P.E.T.; Formal analysis, A.T. and P.P.M.I.; Investigation, A.T.; Resources, and P.E.T.; Data curation, A.T.; Writing—original draft, A.T.; Writing—review & editing, A.T., P.P.M.I. and P.E.T.; Project administration, P.P.M.I. and P.E.T.; Funding acquisition, P.P.M.I. All authors have read and agreed to the published version of the manuscript.

Funding

The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/under REA Grant Agreement number 607785. The Royal Botanic Gardens, Kew, receives grant-in-aid from DEFRA. The James Hutton Institute is supported by the Rural & Environment Science & Analytical Services (RESAS), a Division of the Scottish Government. This research was supported by the European Union’s Horizon 2020 Research and Innovation Action (RIA) project; ‘Realising Dynamic Value Chains for Underutilised Crops’ (RADIANT; www.radiantproject.eu), Grant Agreement number 101000622.

Data Availability Statement

The data presented in this study are available in the Supplementary Files of the present manuscript.

Acknowledgments

We are grateful to Candido Galvez Ramirez (Semillas Silvestres) and Álvaro Bueno (Jardín Botánico Atlántico) for their valuable assistance in choosing the seed collection sites. We also thank Matías Hernández González, Stephanie Frischie (Semillas Silvestres), and Eduardo Fernández Pascual (Royal Botanic Gardens, Kew) for their help in seed collection.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Map illustrating the Iberian Peninsula’s pedoclimatic regions (adapted from Metzger, Marc J., 2018), where the seed accessions were collected. Each species is represented by a colored circle.
Figure 1. Map illustrating the Iberian Peninsula’s pedoclimatic regions (adapted from Metzger, Marc J., 2018), where the seed accessions were collected. Each species is represented by a colored circle.
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Figure 2. Seed germination of the 22 accessions of four Plantago species from three different pedoclimates of the Iberian Peninsula. (A) Generalized linear mixed model (GLMM) of seed incubation temperatures and imbibition conditions on germination percentage by species. (B) Violin plots showing percentage germination of water−imbibed seeds; (C) potassium nitrate solution−imbibed seeds (10 mM KNO3); (D) water−imbibed seeds at alternating and constant temperatures; (E) potassium nitrate solution−imbibed seeds at alternating and constant temperatures; (F) water−imbibed seeds by pedoclimate region; (G) nitrate solution−imbibed seeds by pedoclimate region. The species (and number of accessions) used were: P. albicans (5); P. coronopus (5); P. lagopus (5); and P. lanceolata (7). Asterisks indicate significant statistical differences: * p ≤ 0.05; *** p ≤ 0.001.
Figure 2. Seed germination of the 22 accessions of four Plantago species from three different pedoclimates of the Iberian Peninsula. (A) Generalized linear mixed model (GLMM) of seed incubation temperatures and imbibition conditions on germination percentage by species. (B) Violin plots showing percentage germination of water−imbibed seeds; (C) potassium nitrate solution−imbibed seeds (10 mM KNO3); (D) water−imbibed seeds at alternating and constant temperatures; (E) potassium nitrate solution−imbibed seeds at alternating and constant temperatures; (F) water−imbibed seeds by pedoclimate region; (G) nitrate solution−imbibed seeds by pedoclimate region. The species (and number of accessions) used were: P. albicans (5); P. coronopus (5); P. lagopus (5); and P. lanceolata (7). Asterisks indicate significant statistical differences: * p ≤ 0.05; *** p ≤ 0.001.
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Figure 3. Heatmap cluster diagram of the delta variation (Δ) for the percentage of seed germination between constant (const.) and alternating (alter.) temperature incubations in nitrate solution and water (ΔKNO3: H2O); or the reciprocal percentage germination incubations (Δalter.:const). Means are the average of three replicates Δ%.
Figure 3. Heatmap cluster diagram of the delta variation (Δ) for the percentage of seed germination between constant (const.) and alternating (alter.) temperature incubations in nitrate solution and water (ΔKNO3: H2O); or the reciprocal percentage germination incubations (Δalter.:const). Means are the average of three replicates Δ%.
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Figure 4. Factor analysis of mixed data (FAMD) of the final percentage germination of seeds from 22 accessions of Plantago albicans, P. coronopus, P. lagopus, and P. lanceolata (n = 5, 5, 5, and 7, respectively). (A) Water imbibition at constant temperature; (B) water imbibition at alternating temperatures; (C) nitrate solution imbibition (10 mM) at constant temperature; (D) nitrate solution imbibition (10 mM) at alternating temperatures. Each colored ellipse highlights clustering of accessions of the same species, and labels represent the collection region of each accession, where: LUS—Lusitanian; MDS—Mediterranean South; MDN—Mediterranean North. All tests were performed with the means of data calculated from the average of three experimental replicates.
Figure 4. Factor analysis of mixed data (FAMD) of the final percentage germination of seeds from 22 accessions of Plantago albicans, P. coronopus, P. lagopus, and P. lanceolata (n = 5, 5, 5, and 7, respectively). (A) Water imbibition at constant temperature; (B) water imbibition at alternating temperatures; (C) nitrate solution imbibition (10 mM) at constant temperature; (D) nitrate solution imbibition (10 mM) at alternating temperatures. Each colored ellipse highlights clustering of accessions of the same species, and labels represent the collection region of each accession, where: LUS—Lusitanian; MDS—Mediterranean South; MDN—Mediterranean North. All tests were performed with the means of data calculated from the average of three experimental replicates.
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Figure 5. Generalized linear models testing the effect of soil nitrogen forms and carbon on species germination (based on the second axis of the FAMD). (A) Water imbibition and (B) KNO3 solution imbibition at constant temperatures. All tests were performed with three replicates for each accession.
Figure 5. Generalized linear models testing the effect of soil nitrogen forms and carbon on species germination (based on the second axis of the FAMD). (A) Water imbibition and (B) KNO3 solution imbibition at constant temperatures. All tests were performed with three replicates for each accession.
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Figure 6. Generalized linear models testing the effects of soil nitrogen forms and carbon on species germination (based on second axis of the FAMD). (A) Water imbibition and (B) KNO3 solution imbibition at alternating temperatures. All tests were performed with three replicates for each accession.
Figure 6. Generalized linear models testing the effects of soil nitrogen forms and carbon on species germination (based on second axis of the FAMD). (A) Water imbibition and (B) KNO3 solution imbibition at alternating temperatures. All tests were performed with three replicates for each accession.
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Figure 7. Generalized linear models testing the effect of soil and seed nitrogen forms and carbon on species Tb and θ50 of seeds imbibed in H2O and KNO3 solution (10 mM), incubated at constant and alternating temperatures. All tests were performed with three replicates for each accession. Asterisks indicate significant statistical differences: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 7. Generalized linear models testing the effect of soil and seed nitrogen forms and carbon on species Tb and θ50 of seeds imbibed in H2O and KNO3 solution (10 mM), incubated at constant and alternating temperatures. All tests were performed with three replicates for each accession. Asterisks indicate significant statistical differences: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
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MDPI and ACS Style

Teixeira, A.; Iannetta, P.P.M.; Toorop, P.E. Plantago Species Show Germination Improvement as a Function of Nitrate and Temperature. Nitrogen 2024, 5, 790-807. https://doi.org/10.3390/nitrogen5030052

AMA Style

Teixeira A, Iannetta PPM, Toorop PE. Plantago Species Show Germination Improvement as a Function of Nitrate and Temperature. Nitrogen. 2024; 5(3):790-807. https://doi.org/10.3390/nitrogen5030052

Chicago/Turabian Style

Teixeira, António, Pietro P. M. Iannetta, and Peter E. Toorop. 2024. "Plantago Species Show Germination Improvement as a Function of Nitrate and Temperature" Nitrogen 5, no. 3: 790-807. https://doi.org/10.3390/nitrogen5030052

APA Style

Teixeira, A., Iannetta, P. P. M., & Toorop, P. E. (2024). Plantago Species Show Germination Improvement as a Function of Nitrate and Temperature. Nitrogen, 5(3), 790-807. https://doi.org/10.3390/nitrogen5030052

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