Rooting Patterns and Aucubin Content in Plantago lanceolata

Abstract

Ribwort plantain (Plantago lanceolata L.) is expected to open up new crop cultivation perspectives in arable farming in order to significantly reduce nitrogen losses through leaching, N₂O emissions and to increase resilience under drought conditions. Therefore, this study investigated the properties of the roots as an alternative to grasses for forage production under drought conditions. Ten genotypes of ribwort plantain were compared under field conditions in northern Switzerland, focusing on the characteristics of the root system and the aucubin content in the leaves and roots. Aucubin, known for inhibiting the nitrification process in the soil, varied according to genotype and season. All tested genotypes showed higher aucubin content in leaves than in roots, which may indicate the greater importance of leaves in reducing soil nitrification. Dry matter yield and for the first time root characteristics, such as root length density (RLD) and specific root length (SRL), were evaluated at two different soil depths, and at different distances from the plants. The results showed that ribwort is well adapted to acquire water and nutrients in terms of rooting in topsoil. In addition, a second field experiment conducted in eastern Germany (Saxony) confirmed the plant’s ability to root deeply after one year of cultivation, reaching depths of up to even 1.88 m. The obtained results indicate the high capacity of ribwort plantain to take up water and nutrients also from deeper soil layers. To reduce N₂O emissions through the cultivation of ribwort plantain, the choice of genotype seems to be of great importance due to varying aucubin contents.

1. Introduction

Ribwort plantain belongs to the Plantaginaceae family and shows remarkable adaptability to different environmental conditions [1]. This perennial herbaceous plant [2] occurring worldwide has attracted attention for its potential role in sustainable agriculture due to its deep-rooted nature, which positively influences soil properties and facilitates efficient nutrient and water uptake [3]. Plantago lanceolata is also promising in mitigating the environmental impact of agriculture by reducing nitrous oxide (N₂O) emissions [4,5]. These above-average features ultimately also result in a reduction in NO₃⁻ [6] pollution of ground and surface water and a reduction in eutrophication [7]. This is due to the high capability of ribwort plantain for nitrate uptake and the release of nitrification inhibitors into the soil. This occurs through aucubin (an iridoid glycoside), which is stored in the leaves and roots and secreted directly into the soil by the roots [8].

So far, only a few studies have been published on the rooting patterns of P. lanceolata [3,9]. But there are still no data in the current literature on the specific characteristics of roots in the topsoil under field conditions and the maximum deep-rooting ability of ribwort plantain. It is important because roots play a key role in the uptake of water and nutrients by plants [10]. In addition, a higher rooting depth allows plants to access a larger volume of soil and increases their resistance to drought stress [11]. What is more, no studies have been carried out to compare the content of aucubin in the roots and leaves of different genotypes under arable field conditions. Scarcely a number of results have been published demonstrating the reduction of N₂O emissions by ribwort plantain under grassland conditions [4,5,12,13,14]. The plant is now widely used in pastures located in New Zealand, where varieties specifically adapted there to the climatic conditions were developed [15].

Two arable field experiments were designed to perform the analyses presented in this paper. A first field trial was conducted in Frick (northern Switzerland), where 10 genotypes of ribwort plantain originating from different countries were compared to evaluate the content of aucubin in leaves and roots. Also, the leaf yield and rooting patterns were characterized in the topsoil. In order to determine maximum rooting depth, a second site was used placed on a deeply rootable loess soil in Struppen (eastern Germany).

2. Materials and Methods

2.1. Experimental Sites

The first field experiment was set up on 18 June 2021 in a field belonging to the Research Institute of Organic Agriculture (FiBL) in Switzerland. The site (47°30′39.3″ N 8°01′23.3″ E) was located close to the village of Frick (Switzerland) at 348 m a.s.l. The mean annual precipitation and air temperature (2 m) were 1041 mm and 10.5 °C, on average, starting from 2006 to 2022. In the period of the trial, the average air temperature and total precipitation were 11.0 °C and 882 mm, respectively (weather station Frick). The experiment was conducted on clay loamy soil [16] and contained pH (H₂O) of 7.3, P (CAL): 3.4 mg kg⁻¹ and K (CAL): 6.3 mg kg⁻¹ with a stone content of the soil of around 17.5% (0.0–15 cm depth).

The field trial contained 40 plots (four replications) of 1.2 m² each in a randomized block design. Each plot had two 2 m rows and was 30 cm apart, which was recommended according to agricultural management for growing this crop [17]. The distance between rows containing different varieties in the plots was 70 cm. Seeds were sown by hand to a depth of 0.5 cm. Sowing density was 300 seeds per m². No fertilizers or plant protection products were applied during the experiment. It is worth emphasizing that the field was a non-permanent grassland before setting up the trial. Commercially available genotypes were acquired through purchase (

Table 1. Characteristics of genotypes used in the field experiment.

No.	Variety/Genotype Name	Origin of Seeds	Supplier	1000 Seed Weight [g]
1	Lot: 14981a	Switzerland	Sativa Rheinau AG (Rheinau, Switzerland)	2.92
2	Lot: 90245	Switzerland	Sativa Rheinau AG (Rheinau, Switzerland)	2.40
3	Lot: 21583a	Switzerland	Sativa Rheinau AG (Rheinau, Switzerland)	2.63
4	Genotype A	Poland	HerbFarm Edwin Lewczuk (Dawidy, Poland)	1.39
5	Genotype B	Poland	HerbFarm Edwin Lewczuk (Dawidy, Poland)	1.58
6	Genotype C	Poland	Agros S.C. (Okonek, Poland)	1.16
7	Libor	Czech Republic	PHARMASAAT Arznei- und Gewürzpflanzen Saatzucht GmbH (Artern, Germany)	2.57
8	BIO	Germany	PHARMASAAT Arznei- und Gewürzpflanzen Saatzucht GmbH (Artern, Germany)	2.35
9	Boston	New Zealand	Norwest Seed (Ashburton, New Zealand)	1.35
10	Tonic	New Zealand	PGG Wrightson Seeds Limited (Christchurch, New Zealand)	2.07

).

The second trial was established in the first week of August 2020 in Struppen, Germany; 50°55′38.3″ N 14°00′42.3″ E; 270 m a.s.l) on loess-derived soil. This site was chosen for the root analysis because of the presence of the ‘Libor’ variety, also part of the field experiment at Frick (Switzerland). In addition, the favorable soil conditions at Struppen allowed a detailed analysis of the underground parts of P. lanceolata in the deeper soil layers. Unfortunately, it was not possible to do at Frick. During the field experiment, the average air temperature and total precipitation were 11 °C and 589 mm, respectively (according to weather station Struppen). The soil contained 6.4 mg P per 100 g (CAL) and 11.5 mg K per 100 g (CAL) and was characterized by a pH (H₂O) of 6.8. The sowing density of cv. Libor was 300 seeds per m². The size of the plot was 7 m × 20 m.

2.2. Root Sampling and Measurements

• Location Frick (Switzerland)

Root samples were collected from 19 to 24 October 2021 during the flowering period using a single root auger (model: 05.01, producer: Eijkelkamp) following the soil coring method. Due to the presence of a large amount of stones, clay in the soil and equipment limitations, the first samples in each plot were collected from the upper part of the soil layer from five places (Figure 1) to a depth of 0.0–7.5 cm. Subsequently, another five samples were obtained at the same places but deeply on the plots at a depth of 7.5–15.0 cm. Each of them was 8 cm in diameter. Finally, 400 soil samples from 40 plots (10 plots × 4 replicates × 10 samples per plot), each with a cylinder volume of 377 cm³, were packed in plastic bags and immediately stored in a refrigerated chamber (0.1 °C) until manual root extraction.

Due to the high clay content of the soil, it was not possible to sieve soil samples containing roots, so the underground parts were manually extracted using metal tweezers. Each sample was next washed carefully with a jet of water and dried with a paper towel. Next, they were placed separately in plastic string bags and stored in the refrigerator (−18 °C) to avoid the loss of bioactive compounds. The extracted samples were defrosted, and the length of the root in each sample was analyzed using The Rhizo-II Root Biometrics Suite software (version V-2.5.0.4) [18] with a scanner (Toshiba, Tokyo, Japan, E-Studio 2010 AC). Each root sample was subsequently refrozen, lyophilized and its dry weight was determined. Root morphological parameters were estimated in each plot. The root length density (RLD, cm cm⁻³) value on a given volume of soil was defined. It refers to the concentration or abundance of roots in a given volume of soil. Therefore, it was a measure of their distribution over a specific area. A high root density in a plant directly influences its overall growth and productivity through efficient uptake of non-mobile soil resources such as potassium and phosphorus. In addition, specific root length (SRL, m g⁻¹) is based on the length of fresh roots and their freeze-dried weight. It is a measure of root morphology that takes into account root mass and length. This parameter characterizes the fineness of the root system, i.e., how much dry root mass the plant must produce to achieve one meter of root length. This determines the plant’s ability to extract resources from the soil, such as nutrients and water [19]. Moreover, root dry matter distribution (DM, g m⁻²) was also determined for individual genotypes. It refers to the distribution of total dry matter of the plant’s root system, and determines the uptake capacity of nutrients and water [20]. After root measurements were obtained, the final step was to grind the root dry matter (0.1 mm gradation) and store it in a dark, dry place until further chemical analyses.

2.3. Collection of Leaf Samples and Aucubin Measurements of Leaves and Roots—Frick (Switzerland)

Harvesting the aboveground plant parts of ribwort plantain at Frick (Switzerland) was carried out on 3 November 2021 and 17 May 2022. The process was performed by hand using hedge shears. In each plot containing two rows of 2 m length, 1.5 m of fresh leaf mass was cut from the center of each row. The cutting height was 3 cm above ground. The leaf yield from each plot was weighed, and 30 g of harvested leaves from each plot was placed in string bags, deep-frozen (−18 °C), freeze-dried and milled (0.01 mm gradation). For each variety, the dry matter (DM) to fresh matter (FM) ratio (in %) was also calculated.

Analyses for the aucubin content of P. lanceolata leaves were carried out on plants from the two harvests. For this purpose, 200 mg DM was weighed for each sample and transferred to a 15 mL falcon tube. Then, 10 mL of methanol was added to the plant material. The suspension was vigorously mixed for 3 h using a rotary laboratory mixer. Subsequently, 5 mL of extract was transferred into a 5 mL Eppendorf tube and centrifuged at 3000 rpm for 4 min. The extract prepared for analysis was transferred into a high-performance liquid chromatography (HPLC) vial. The same preparation procedure was carried out for the extract obtained from the DM of the roots, but the content of aucubin in the underground part was determined from the first harvest. Initially, 40 samples were obtained for the analysis of aucubin content in three replications containing the main root of the plant (root sampling site No. 3, depth 0.0–7.5 cm). Next, extracted lateral roots from all other depths (9 samples from each plot) were mixed to obtain a final 40 samples (one per plot) and analyzed in triplicate for aucubin content. HPLC was performed on a chromatographic system consisting of a degasser, a quaternary pump (LC-20AD), a column oven (CTO-20AC), a PDA detector (SPD-M20A) and a triple quadrupole mass spectrometer (LCMS-8030) (All Shimadzu, Kyoto, Japan). A ReproSil-Pur 120 C18-AQ column (150 × 3 mm, i.d., 3 μm, Dr. Maisch, Ammerbuch, Germany) equipped with a guard column (3.0 × 10 mm) was used for separation. The mobile phase consisted of water (A) and acetonitrile (B). A gradient of 1 to 15% B was applied for 10 min at a flow rate of 0.5 mL/min. Detection was performed at 200 nm as aucubin does not contain conjugated double bonds, and 5 μL was injected. Aucubin was eluted at 8.3 min. A total of 160 samples were analyzed in triplicate for the presence of aucubin content in the roots and leaves of P. lanceolata. For each analysis batch, a calibration curve (R² > 0.985) was prepared with a commercial sample of aucubin (Bioynth Ltd., Billingham, United Kingdom) and dissolved in methanol (0.014 to 0.365 mg mL⁻¹ aucubin).

• Location: Struppen (Germany)

Root measurements were carried out during the flowering period of ribwort plantain on 19.08.2021, one year after sowing. Measurements of the root length density (RLD) and rooting depth of P. lanceolata were conducted according to Böhm [21] using the profile wall method. RLD is a value representing the number of roots per unit volume of soil [cm cm⁻³]. Therefore, in order to test the parameters in a soil environment suitable for this type of examination, a 250 cm deep hole was dug out on the shorter side of the experimental field using an excavator. For an even surface, the profile wall was smoothed with a spade. The root profile was then exposed to a depth of 0.5 cm with pressurized water using a hand sprayer. The next step on the suitably prepared surface was to apply and fix a sheet of acrylic glass with a pre-attached plastic sheet of mesh foil with 4 cm × 4 cm grids. The size of the acrylic sheet on which the measurements were obtained was 100 cm wide and 200 cm deep. Points corresponding to a 0.5 cm of root length were marked by permanent marker, and value for each individual square was counted and recalculated for root length units. The root length density per soil volume was counted separately for each of the 25 squares per 4 cm soil depth by dividing the root length unit of each grid by 8 cm³ (4 cm × 4 cm × 0.5 cm). Standard deviation was calculated using 25 individual measurements per 4 cm soil depth.

2.4. Statistical Analyses

The statistical analysis was performed using SAS program version 9.3 (SAS Institute Inc., Cary, NC, USA, 2013). Normal distribution of the data was tested according to the Shapiro–Wilk test (univariate normal procedure). A one-factor analysis of variance (ANOVA) was performed for yield and secondary metabolite comparisons. Cultivar root sampling points were assessed using a two-factorial analysis of variance. For the multiple mean value comparison of balanced data, Tukey’s test was used. Significant differences between cultivars were indicated, with error probabilities (α) of <0.05, <0.01 and <0.001 and were marked by different letters in the figures.

3. Results

3.1. Total Leaf Dry Matter Yield

During the first harvest carried out on 3 November 2021, the highest average dry matter yield was found in two genotypes of Swiss origin (No. 2 and 3) and a variety of Czech origin (No. 7) 624, 587 and 575 g DM m⁻², respectively (Figure 2). The lowest dry matter yield was found in genotypes of New Zealand (No. 10) and Polish origin (No. 4 and 6; 443, 373 and 345 g DM m⁻², respectively). During the second harvest, 44% more dry matter, on average, was harvested from all tested genotypes compared to the first cut. The highest average dry matter yield was found, as in the first harvest, in variety 3 of Swiss origin (1292 g DM m⁻²), which was an increase of more than 108% compared to the first cut. As in the previous year (2021), genotype No. 2 and 7 had high dry matter yields of 1141 and 1166 g DM m⁻², respectively, an increase compared to the first harvest of 94 and 103%, respectively. In 2022, the lowest yields were found in the genotypes of Polish (No. 5 and 6) and New Zealand origin. The dry matter yields were 739, 539 and 403 g DM m⁻², respectively, resulting in an increase in dry matter of 57 and 56% and, in the case of the New Zealand variety, a lower DM yield of 9% (Figure 2). Statistically significant differences were found for harvest time (p = 0.0001) and genotype (p = 0.0001) but not for their interaction (p = 0.0625).

3.2. Morphometric Characteristics of the Roots of the Plantago lanceolata Genotypes Tested at Frick

The highest mean distribution of root dry mass of the tested genotypes was found directly under the plants (sampling position No. 3, Figure 3a). This amounted to 171 g m⁻² for the soil layer 0.0–7.5 cm deep and 34 g m⁻² for the bottom layer (7.5–15 cm; Figure 3). In both directions between and beyond the rows, the value decreased reaching similar values. Statistically significant differences were found according to sample positions in depths of 0.0–7.5 cm and 7.5–15.0 cm (p = 0.0001) and genotypes in depths of 7.5–15.0 cm (p = 0.0231). No statistically significant interactions (genotype × position) were found (p = 0.6809, p = 0.4007).

The mean root length density for all tested genotypes for the depth of 0.0–7.5 cm was 1.58 cm cm⁻³, while for the depth of 7.5 to 15 cm, it was 0.54 cm cm⁻³ (Figure 3b). The mean total root length of 0.0 to 7.5 cm for the tested genotypes was calculated to 1185 m m⁻² and at for 7.5 to 15 cm to 405 m m⁻². The highest mean total root length density per unit soil volume was determined directly under the plants (position No. 3) at a depth of 0.0–7.5 and 7.5–15 cm at the line of the row in the experimental plots (2.40 and 0.74 cm cm⁻³, respectively; Figure 3b). Statistically significant differences in root length density were found in relation to sampling position (p = 0.001) and genotype (p = 0.0269) but not for the interaction (p = 0.9379).

The maximum average specific root length (the finest roots) for the tested samples was found between the rows in the topsoil layer (0–7.5 cm) for samples No. 1, and 5 (outside the rows) and amounted to 107 and 92 m g⁻¹ (Figure 3c). The lowest average values of specific root length were recorded in the sample obtained from the center of the row, directly under the plants and for a depth of 0.0–7.5 cm, it was 13.7 m g⁻¹, and for a depth of 7.5–15.0 cm, it was 34.8 m g⁻¹. Differences in specific root length were significant in relation to sample position at depths of 0.0–7.5 cm (p = 0.0026) but not for genotype (p = 0.4928, p = 0.4652) or interaction (p = 0.9214, p = 0.9129).

3.3. Rooting Depth and Root Length Density of Tested Genotype at Struppen

The highest root length density on a loess soil was determined at a depth of up to 20 cm in the soil and had an average value of 1.44 cm cm⁻³ (Figure 4). The maximum value was obtained at a depth of 16 cm and was 1.52 cm cm⁻³. Subsequently, a decrease in root length density was observed in the soil profile and from 36 to 76 cm, it remained and ranged from 0.26 to 0.56 cm cm⁻³. With a slight increase to a 76 cm depth into the soil profile, the root length density decreased steadily. It could be noticed that P. lanceolata could root up deeply to be even 188 cm deep into the soil profile within one year (Figure 4). The total root length, up to 188 cm, was calculated in this case to be 1417 m m⁻².

3.4. Aucubin Content in Leaves and Roots at Frick

In the first year, the average value of aucubin in the tested genotypes was 15.7 mg g⁻¹ DM in leaves. The highest leaf aucubin content was in genotypes No. 1, 2 and 8 (18.6, 17.6 and 17.6 mg g⁻¹ DM, respectively), while the lowest was in genotypes No. 4, 6 and genotype No. 10, with values of 13.5, 13.4 and 12.6 mg g⁻¹ DM, respectively (Figure 5a). During the second harvest, a significant decrease in aucubin was observed in the shoot biomass, with an average of 4.6 mg g⁻¹ DM for the genotypes tested. Statistically significant differences were determined for harvest date (p = 0.0001) and an interaction genotype x harvest date (p = 0.0324) was found. The differences in aucubin content between the first and second dates were particularly large in genotype 1, 2, 3 and 8, while this was smaller in genotype 6 in comparison. No statistically significant differences were found between tested genotypes (p = 0.573).

Root samples from five sampling positions were collected at the same time at the first harvest of the aboveground parts of P. lanceolata. The first analyses of aucubin content in the underground parts were initially carried out only using plant material manually extracted from root sampling position 3 (Figure 1), located on the row line from the topsoil surface (depth of 0.0–7.5 cm). In this phase of the study, 40 root samples were tested for aucubin content (Figure 5b). The extracted material consisted of the main root, with a negligible amount of lateral roots. The samples differed slightly in proportion for the main root to lateral roots. The highest average aucubin content was recorded in these root samples of genotype No. 6 (5.7 mg g⁻¹ DM), 2 (4.4 mg g⁻¹ DM), 5 (4.4 mg g⁻¹ DM) and 4 (3.9 mg g⁻¹ DM). The lowest values were determined in genotype No. 1 (2.0 mg g⁻¹ DM), 7 (1.7 mg g⁻¹ DM) and 10 (1.7 mg g⁻¹ DM). No statistically significant differences were found between the analyzed genotypes. Further analyses of roots extracted from soil samples for aucubin content in individual genotypes were carried out using only lateral roots. However, no measurable amounts of aucubin were detected in any of these samples. There were also no statistically significant correlations found between the aucubin content of the leaves and roots (p = 0.3108).

4. Discussion

4.1. Leaf Yield

For arable field, there are not many scientific reports in the available literature describing the yield of ribwort plantain. Nevertheless, in the experiment without applying mineral fertilization, Kolodziej [22] received a maximum yield of 2.97 t DM/ha, which is much lower than the results presented in this paper. The significant differences we observed in dry matter yield due to genetic background confirm the high value of appropriate selection of genotypes for specific conditions.

4.2. Root System Characteristics

It has to be mentioned that in the experiment, the content of stones in the soil at Frick was relatively high, which may have had a negative effect on root development. Our results indicate that P. lanceolata could form an intermediate root length density compared to other crops. To better illustrate the obtained values, it can be compared, for example, with the root system of spring wheat, which has a value of 6.00 cm cm⁻³ under conditions of low water content to a depth of 20 cm in the soil profile. On the other hand, slightly lower values were recorded for pulses (chickpea, field pea, lentil) with 3.39 cm cm⁻³ and oilseeds (canola, flax, mustard) with 2.70 cm cm⁻³ [23]. Results on the root growth of ribwort plantain are currently only available from Rauber et. al. [9], who investigated ribwort plantain as an undersow in potatoes, whereby no distinction could be made between the roots of the potatoes and the ribwort plantain. However, these test results indicate similarly high root lengths and a root system of ribwort plantain extending up to 120 cm deep after 5 months of growth.

The currently available literature lacks any information related to the specific root length (SRL) of Plantago lanceolata grown under arable field conditions. Ribwort plantain, due to its nitrification-inhibiting properties in the soil and its speed and depth of rooting, is an alternative solution to grasses belonging to the order of panicles in combination with pasture species of legumes. Despite the importance of the indicator in question in the morphology of plant roots, there are limited bibliographic data for comparison with perennial grasses, especially under Central European conditions. Nevertheless, the SRL e.g., for bentgrass (Agrostis capillaris L.) for a depth of 20–30 cm is estimated at 155 m g⁻¹ and for comparison field sorrel (Rumex acetosa L.) was 24 m g⁻¹ [24], which is in the range found for Plantago lanceolata (Figure 3).

The obtained results in the study clearly indicate the very high rooting potential of ribwort plantain. After one year of their development, the roots of the ‘Libor’ variety were at a depth of almost 190 cm. Analysis of underground parts in the topsoil layer also showed an even higher root length density of the tested variety at Struppen (Figure 4) compared to the experiment conducted at Frick (Figure 3). This is most likely due to different soil conditions, more conducive to this type of root analysis. Therefore, the mentioned features of ribwort plantain make this plant attractive for agricultural practice [3] and may be competitive with some currently used plant species. For example, most grass species are successively becoming less and less drought tolerant due to limited possibilities of penetration of deeper soil layers by roots. Durand and Ghesquière [25], after analyzing obtained data through neutron probe measurements, determined the maximum water uptake capacity for Italian ryegrass (Lolium multiflorum Lam.) under field conditions 80 cm deep in the soil profile. Similar measurement results were obtained by Jacques [26] and Gibbs [27] where the deepest roots of annual ryegrass were also found at a depth of 80 cm. To make the best use of the properties of P. lanceolata, it would be beneficial to use it in agricultural fields in mixtures with plants with similar resistance to water shortages and binding atmospheric nitrogen, e.g., lucerne (Medicago sativa L.) [28], which is retained in the soil in its ammonium form (NH₄⁺) thanks to aucubin. In areas at risk of drought, farmers and pasture managers should use a combination of forage species that is more efficient than monocultures (e.g., Lolium perenne L.) to mitigate the effects of water shortages [29]. The use of P. lanceolata in pasture mixtures is currently popular in New Zealand due to its inhibition of soil N nitrification [30]. In addition, the presence of aucubin in the aboveground parts of the plant had a positive impact on the health of grazing animals [31].

4.3. Leaf and Root Aucubin Content

The leaf aucubin content during the first harvest carried out on 3 November 2021 was significantly different from that which was harvested during the second harvest on 17 May 2022. In the results presented by Tamura and Nishibe [32] obtained from a field experiment in Japan, a similar phenomenon was observed. In the experiment, two P. lanceolata genotypes Ceres Tonic and Grasslands Lancelot were compared in terms of bioactive compound content. In the tested varieties, the aucubin content increased in the leaves from late spring, reached a maximum value at the end of September and then decreased. In the cultivar Grasslands Lancelot, an aucubin level of approximately 2% in DM was recorded in the aboveground parts in the second half of June, while for the cultivar Ceres Tonic, it was determined to be around 1% DM. The highest value at the end of September for the Grasslands Lancelot variety was around 5% DM, and for the Ceres Tonic variety, it was just under 3% DM. Ribwort plantain genotypes tested for the purposes of aucubin content in the current study also had significantly lower levels of the tested plant parts during the spring harvest (minimum 0.3% DM, maximum 0.8% DM) compared to the autumn harvest (minimum 1.3% DM, minimum 1.9% DM). This confirms the hypothesis that aucubin variation in leaves undergoes seasonal changes [33,34]. The wide differences in the results for aucubin content in the leaves between Tamura and Nishibe [32] and the experiment conducted at Frick (Switzerland) are most likely due to environmental conditions. Further research carried out by Miehe-Steier et al. [35] in a greenhouse concluded that phenotypic plasticity responding to environmental changes could be responsible for the variation in the aucubin content of the aboveground parts of ribwort plantain. The authors obtained aucubin level results ranging from 1.6% of DM to just over 1.8% of DM under high light conditions (14 h of light per day). The 12% difference in the content of the test ingredient was due to nutrient application. In the experiment carried out at Frick, no application of any fertilizer was performed, so it is possible that this also had an impact on the reduced amount of aucubin measured during the second harvest. The production of secondary metabolites, given the examples of other species, can also be affected by drought [36,37]. In addition, the bioactive compound synthesis rate in the shoots may also be influenced by water availability, temperature fluctuations [38] and the presence of pests [39], although these were not observed during the field experiment in Frick. In the present study, the tested genotypes in Bowers et al. [38] were characterized by different contents of aucubin at harvest. The three-way analysis of variance (ANOVA) performed by the authors clearly indicated significant statistical differences in terms of genotype, date, site and genotype × site and genotype × date interactions. Tamura and Nishibe [32] also found statistically significant differences due to the sampling date of aboveground parts of the ribwort plantain. This confirms the hypothesis that leaves differ in aucubin content due to their age. Additionally, Klockars et al. [40] also observed a significant positive correlation between leaf age and total iridoid glycoside concentration. The aucubin level increased from being undetectable in older leaves to 9% DM in the newest leaves. In this study, the same amounts of green matter were collected from each plot for aucubin analyses, although they may have differed slightly in terms of the proportion of younger and older leaves. This probably resulted in an increased standard deviation between the tested samples (Figure 5).

As with the leaves, the aucubin content of the roots is influenced, among other factors, by the plant’s access to light. For aucubin analyses, root samples of the tested genotypes were collected in parallel with the first harvest. In an experiment under controlled environmental conditions, Miehe-Steier et al. [35] showed the effect of light availability on the aucubin content in the belowground parts of ribwort plantain. The aucubin content in the roots under good light conditions was seven times higher (2.1% DM) than under poor light conditions (0.3% DM), and the hypothesis of an effect of nutrient availability on the aucubin content in the roots of P. lanceolata was ruled out. Values obtained from Frick showed the average aucubin content in the tested genotypes at the level of 0.4% DM. The obtained value is comparable to the results shared by the cited author under low light conditions. The low aucubin content of the tested component in the belowground parts of the plants could be caused by the soil factors and also the availability of water during the experiment that was carried out. For the analyses of the extracted roots, the first samples were obtained from the line of the row and contained varying amounts of lateral roots and the main root (0.0–7.5 cm deep into the soil profile). This could have influenced the final absence of statistical correlation between the analyzed samples. Moreover, for the purposes of further research, lateral roots were analyzed for aucubin content, and no measurable value was detected. Currently, there is no information in the literature on the various contents of aucubin in the different root parts of ribwort plantain; thus, further studies are required to better describe the main root and lateral roots in terms of its content.

5. Conclusions

The results presented indicate that P. lanceolata is well adapted to water-deficit conditions through its rooting properties almost 190 cm deep in the soil profile after one year of cultivation. Furthermore, based on the values characterizing roots (RLD and SRL), it can be concluded that ribwort plantain has a good ability to take up immobile and mobile nutrients from the soil. Major differences in aucubin content between leaves and roots are related to the higher value of the aboveground parts of this plant, which is especially essential for inhibiting the nitrification process in the soil. To reduce N₂O emissions through the cultivation of ribwort plantain, the choice of genotype seems to be of great importance due to varying aucubin contents. It can be concluded that ribwort plantain is a crop species, that due to its above properties, can have multifunctional applications in agriculture, improving overall productivity and reducing N losses of agricultural systems.