The First Evidence of Gibberellic Acid’s Ability to Modulate Target Species’ Sensitivity to Honeysuckle (Lonicera maackii) Allelochemicals

Abstract

Invasive species employ competitive strategies such as releasing allelopathic chemicals into the environment that negatively impact native species. Decomposing Amur honeysuckle (Lonicera maackii) leaves leach various allelopathic phenolics into the soil, decreasing the vigor of several native species. Notable differences in the net negative impacts of L. maackii metabolites on target species were argued to depend on soil properties, the microbiome, the proximity to the allelochemical source, the allelochemical concentration, or environmental conditions. This study is the first to address the role of target species’ metabolic properties in determining their net sensitivity to allelopathic inhibition by L. maackii. Gibberellic acid (GA₃) is a critical regulator of seed germination and early development. We hypothesized that GA₃ levels might affect the target sensitivity to allelopathic inhibitors and evaluated differences in the response of a standard (control, Rbr), a GA₃-overproducing (ein), and a GA₃-deficient (ros) Brassica rapa variety to L. maackii allelochemicals. Our results demonstrate that high GA₃ concentrations substantially alleviate the inhibitory effects of L. maackii allelochemicals. A better understanding of the importance of target species’ metabolic properties in their responses to allelochemicals will contribute to developing novel invasive species control and biodiversity conservation protocols and may contribute to applications in agriculture.

1. Introduction

Invasive plant species are significant threats to biodiversity, negatively impacting native species, altering the community structure and dynamics, and affecting the ecosystem health and functions, regionally and globally [1,2,3,4]. A better understanding of the eco-physiological and molecular impacts that the invasives exert on native species and communities is essential for cost-effective and successful invasive species management, native species conservation, restoration, and agricultural weed management practices [5,6,7,8,9,10,11,12,13,14].

Amur honeysuckle (Lonicera maackii (Rupr.) Herder), a deciduous, highly successful invasive shrub, was introduced to the U.S. in the 18th century from Asia for its ornamental value [15,16]. In Asia, extracts from Lonicera spp. have traditionally been used as medicinal supplements to combat colds and influenza [17,18,19] and have been exploited for their anti-inflammatory [20,21], antioxidant, and anti-mutagenic properties [22,23,24].

L. maackii was a species recommended by the U.S. Soil Conservation Service for soil erosion control and habitat restoration. Its use for such applications likely contributed to the rapid spread and broad expansion of its habitat. Its invasive success was not recognized until the 1950s [15,25]. Today, L. maackii aggressively invades the deciduous forest-populated eastern and mid-North American regions. Infestations have been reported and cataloged from North Dakota to Texas in the South and in Massachusetts and Georgia in the North and Southeast [16,25,26]. The invasive success and widespread distribution of this species prompted intense scientific interest over the past decades, with studies revealing its complex, multitrophic impacts on native populations, communities, landscapes, and ecosystems. The complexity and varying impacts of L. maackii across ecosystems at multiple ecological scales make this species of high interest in serving as a model of invasion impacts [16]. The recent sequencing of the L. maackii genome [27] further emphasizes the increasing scientific interest in this species and the molecular traits underlying its high invasive success.

L. maackii’s widespread invasive ability is underpinned by a suite of invasive traits [28,29,30,31,32]. These include rapid growth and phenotypic plasticity [31,33,34,35,36,37,38,39], phenological differences from native species in the invaded habitat, resistance to environmental stresses [37,40,41,42], and long-range seed dispersal facilitated by birds, deer, or others [29,30,43,44,45]. L. maackii also synthesizes a variety of secondary metabolites, some of which act as herbivore deterrents or toxins [46,47,48,49,50].

Some of the metabolites synthesized in the roots, leaves, and fruits of L. maackii have also been reported to exert negative allelopathic activity, suppressing the germination, growth, survival, or reproduction of native plant species without auto-toxic impacts, possibly further enhancing L. maackii’s competitive ability [51,52,53,54,55,56,57]. Cipollini et al. (2008) [47] attributed these adverse effects to phenolic molecules and their derivatives isolated from L. maackii tissue extracts.

Studies found L. maackii fruit extracts to suppress or delay the germination of grass species (e.g., tall fescue, Festuca arundinacea, and Kentucky bluegrass, Poa pratensis), garden coreopsis (Coreopsis lanceolata), or a dwarf impatiens hybrid (Impatiens walleriana) [55]. On the other hand, leaf extracts only affected the latter negatively [55]. The negative impacts of L. maackii leaf extracts were also documented in woody species, with Trisel (1997) and Gorchov and Trisel (2003) showing a reduced germination of green ash (Fraxinus pennsylvanica) and sugar maple (Acer saccharum) [58,59]. The adverse allelopathic effects are not limited to L. maackii. Leaf extracts of another species, L. japonica (Japanese honeysuckle), reduced the growth of loblolly and shortleaf pine (Pinus taeda and P. echinata), an effect ascribed to the secondary phenolic metabolites identified from the leaf tissue [60]. Furthermore, L. maackii root and shoot extracts reduced the germination of several midwestern species’ seeds, such as jewelweed (Impatiens capensis Meerb.), tall thimbleweed (Anemone virginiana L.), and the model Arabidopsis thaliana [47,51,52,53,54,55,56,57]. Other studies reported no apparent allelopathic impacts of L. maackii on native herbaceous species [61]. Cipollini and Dorning (2008) also attributed confounding effects to L. maackii leaf extracts, reporting a decrease in the survival and a delay in the flowering of A. thaliana; however, this was associated with the increased seed production and leaf size at maturity [52]. These observations highlight a potentially complex phytochemical interaction between L. maackii and other species. It is also feasible for the described interactions between L. maackii and other species to be affected by the targets’ endogenous characteristics and, possibly, biotic or environmental factors, which may enhance or attenuate the allelopathic effects in a context-dependent manner [62]. Recent work also emphasizes that environmental pressures, such as stress conditions, may lower L. maackii performance; however, this is likely to be offset by the benefits gained in plant–plant competition by an increasing allelopathic potential [63].

Despite a good understanding of L. maackii’s impacts on ecosystems [6,16,30,33,52,59,61,64,65,66,67,68,69,70], the complex and potentially context-dependent direct chemical interactions between L. maackii and target plant species are not understood.

In the current work, we sought to evaluate the potentially fundamental role of the seed germination stimulator gibberellic acid (GA₃) [71,72,73,74,75,76,77,78,79] in determining target sensitivity to L. maackii allelochemicals. We used a model system that allowed for the comparative evaluation of the impacts of L. maackii leaf metabolites on the germination and growth of control, GA₃-overproducing, and GA₃-deficient Brassica rapa varieties [80,81,82].

A better understanding of the physiological and biochemical controls modulating the sensitivity of target species to allelopathic influences is expected to contribute to developing innovative invasive species management approaches and novel applications in agriculture and weed management under current and future climatic conditions.

2. Results

2.1. Lonicera maackii Leaf Extracts Inhibit Brassica rapa Seed Germination

L. maackii extracts, prepared from late-season harvested leaves, significantly decreased B. rapa seed germination rates in a range from 20 to 95% compared to controls (Standard variety, Rbr, Wisconsin Fast Plants^®) in Petri dish assays in a concentration range of 0.5–2 g (leaf tissue) mL⁻¹. The effects were concentration-dependent, with lower germination rates associated with higher concentrations of the applied extract. All control B. rapa seeds, treated with sterile distilled water, germinated within 24 h. The germination rates of seeds treated with 0.5, 0.75, 1, and 1.5 g (leaf tissue) mL⁻¹ L. maackii extracts were reduced by 20, 50, 85, and 95%, respectively. Treatments with 2 g (leaf tissue) mL⁻¹ extracts fully inhibited B. rapa seed germination (Figure 1).

L. maackii extracts of a 1 g (leaf tissue) mL⁻¹ concentration, prepared from mature leaves in different moments of the season, from early spring to late fall, had varying effects on B. rapa var. Rbr germination. Early-season leaves (harvested between March and June) did not inhibit the germination of B. rapa seeds. Leaves harvested later, from mid-summer to late fall, had a progressively increasing negative impact on Rbr seed germination. Germination rates declined by 10, 20, 50, 85, and 95% in response to L. maackii extracts prepared from leaves harvested monthly between July and November, respectively (Figure 2).

2.2. Gibberellic Acid Influences Brassica rapa’s Sensitivity to Lonicera maackii Allelopathic Effects

The germination success in seeds treated with L. maackii extracts varied substantially in the function of the endogenous gibberellin production capacity of the tested B. rapa varieties. Significant differences were observed between the response of the GA₃-overproducing, tall (ein, elongated internode) variant, the gibberellin-deficient, rosette-dwarf (ros) B. rapa variant, and the standard, Rbr variety, producing physiological levels of gibberellins (Figure 3A and Figure 4A). The control seeds of all three varieties, treated with sterile water, germinated within 72 h after imbibition, with the seeds of the Rbr and ein varieties germinating after 24 h. The ros variety control germination rate was 92, 95, and 100% after 24, 48, and 72 h, respectively. After twenty-four hours of imbibition with 1 g (leaf tissue) mL⁻¹ L. maackii extracts prepared from leaves harvested in October, Rbr and ein seed germination significantly decreased in both varieties by over 85%. After 48 h, 55% of the L. maackii-treated Rbr and 75% of the ein seeds germinated, while after 72 h, 80% of the Rbr and 95% of the ein seeds germinated. Imbibition with the L. maackii leaf extract completely inhibited the germination of the gibberellin-deficient ros seeds; no seedlings germinated within 72 h. The statistical analysis revealed a significant impact of the applied L. maackii leaf extract on the germination success of all varieties when compared to the corresponding controls; this effect was particularly enhanced at the beginning of the treatment after 24 and 48 h. While L. maackii leaf extracts negatively affected the germination success of all varieties, delaying the germination of both Rbr and ein varieties, ein seeds showed a lower sensitivity to L. maackii-derived inhibitors than Rbr. In contrast, the germination of the ros seeds was fully inhibited by the L. maackii leaf extract (Figure 3A and Figure 4A). Further statistical evaluation of these data with two-way ANOVA analysis of variance also supports that the variety responses to L. maackii allelochemicals are significantly different. However, these differences become less accentuated over time (Figure 3A inset), except for the ros variety, whose seeds did not germinate within our observation window.

The response of B. rapa varieties revealed a similar trend when L. maackii extracts were administered in conjunction with a supplemental exogenous 100 µmol GA₃ solution (Figure 3B and Figure 4B). The controls of all three varieties, treated with sterile water supplemented with exogenous GA₃, germinated after 24 h, including the GA₃-deficient dwarf variety. After 24 h, 75 and 65% of Rbr and ein seeds imbibed with the combination of GA₃ + L. maackii leaf extracts germinated. Supplementation with exogenous GA₃ enhanced the germination rates of L. maackii-treated Rbr and ein seeds and, after 48 h, ros seeds as well, as compared to the rates recorded without the supplement (Figure 3A and Figure 4A). Exogenous GA₃ enhanced the germination of Rbr and ein seeds treated with the L. maackii extract after 24 h (Figure 3B), as compared to the Rbr and ein seed germination rates without exogenous GA₃ (Figure 3A). At later timepoints, a further stimulation of Rbr and ein was detected in the presence of exogenous GA₃ (Figure 3A,B). After 48 h, 90 and 95% of Rbr and ein seeds germinated, respectively. The complete germination of the Rbr and ein seeds treated with GA₃ + L. maackii leaf extract was recorded by the 72 h timepoint.

Finally, in the presence of exogenous GA_3, the seeds of the rosette-dwarf, gibberellin-deficient variety were also able to partially overcome the harmful effects of L. maackii inhibitors, and, albeit with a delay compared to the other varieties and controls, 80% of the ros seeds germinated within 72 h. Two-way ANOVA analysis of variance revealed that the variety dependence of the response of B. rapa seeds to L. maackii allelochemicals is still significant in the presence of exogenous GA₃; however, the differences are attenuated on a longer timescale (Figure 3B and inset).

Within the first 72 h after exposure to L. maackii leaf extracts, we detected significant differences in the growth and development of seedlings with differing capacities to produce GA₃. Similar differences were recorded when seedlings were treated with exogenous GA₃ in conjunction with the L. maackii extract (Figure 5). The growth pattern of the control Rbr, ein, and ros varieties, treated with sterile water, showed the typical response expected from varieties synthesizing physiological amounts of GA₃, overproducing and showing deficiencies in GA₃ synthesis. The ein variety seedlings reached 5 cm in length, the Rbr seedlings reached 4.5 cm, and the ros seedlings reached 1.3 cm. Imbibition with L. maackii extracts significantly suppressed the growth of both Rbr and ein varieties, with the average seedling length not exceeding 0.5 cm for either variety. Ros seedlings did not germinate within the timeframe of our observation window; therefore, their length was considered 0 cm.

Exogenous GA₃ supplementation benefitted, in particular, the Rbr and ros seedlings under control conditions in sterile water supplemented with GA₃. The average seedling length was 5.7, 5.45, and 1.95 cm in the Rbr, ein, and ros varieties. While the exogenous GA₃ did not fully compensate for the inhibitory effects of L. maackii leaf extracts, the seedling lengths recorded 72 h after the exposure to the combination of L. maackii extract + GA₃ were more extensive for each variety compared to those of the same variety exposed to L. maackii extracts alone. Rbr, ein, and ros seedlings reached an average length of 1.5, 1.4, and 0.4 cm when treated with L. maackii, extracts supplemented with exogenous GA₃. Two-way ANOVA was used to test for differences in the growth of control and L. maackii extract-treated (with or without exogenous GA₃) B. rapa varieties. Analyses revealed a significant, variety-dependent growth response to the L. maackii leaf extract treatment and supported significant differences between the control and L. maackii extract-treated samples both in the presence and absence of exogenous GA₃ (Figure 5 inset).

2.3. Apigenin, Luteolin, and Their Combination Exert Varying Effects on the Germination and Growth of B. rapa Seeds

Apigenin (API) and luteolin (LUT) have been previously identified from aqueous extracts of L. maackii leaves as dominant flavones with potential allelochemical properties [47]. In seed assays, 50 µg mL⁻¹ solutions of API, LUT, and their combination triggered various responses in the germination and development of B. rapa var. Rbr (Figure 6 and Figure 7). The allelochemical concentration for this test was selected to represent concentrations likely occurring under field conditions due to L. maackii leaf decomposition at the end of the growing season in the soil [47]. API stimulated the germination of Rbr seeds, especially in the early stages of the treatment, with higher germination rates observed, particularly after 24 and 36 h, when compared to the controls. After 48 h, this effect was lost, with all of the control and API-treated seeds germinating.

On the other hand, API had a significant suppressive impact on the seedlings’ growth compared to untreated controls. The average API-treated seedling length was 1.5 cm compared to untreated controls, whose average size reached 4.7 cm within 48 h. LUT suppressed germination and growth, although its effects on growth were weaker than those of API. Only about 60% of LUT-treated seeds germinated after 48 h, and the seedling size averaged 2.8 cm. The combination of API + LUT had a strong inhibitory effect on germination, similar to that of LUT, inhibiting germination by 35%.

Moreover, it had a highly significant suppressive impact on growth compared to API or LUT alone. The average seedling lengths did not exceed 0.7 cm when treated with API + LUT. The representative photographs of seedlings in Figure 7 also indicate that while API, LUT, and their combination suppress seedling growth, combining the two flavones affects root and hypocotyl development the strongest.

3. Discussion

In the Midwestern U.S., L. maackii has been recognized to have an aggressive invasive success and devastating multitrophic impacts on ecosystems, rapidly invading forests and disturbed landscapes across N. America [15,25,26]. Its invasive success relies on several competitive mechanisms, which make this species a good candidate for an invasive model [16]. One of the numerous strategies employed by invasive plant species is the synthesis of secondary metabolites with allelopathic properties. When released into the environment, these chemicals may influence their neighbor’s germination, growth, development, reproduction, and overall survival [8,83,84,85,86,87,88,89,90,91,92]. Allelopathic invasives, including L. maackii, have raised numerous concerns for species conservation and restoration, with further challenges posed by a warming climate. Climate change is likely to enhance the competitive relevance of allelochemistry in the biological invasion strategies of this species [63]. Therefore, a better understanding of these interactions is pivotal for biodiversity conservation, landscape and weed management, as well as potential applications in agriculture and forestry.

Lonicera spp. synthesize and release various bioactive phytochemicals from their live or degrading root, shoot, leaf, and fruit tissues [16,17,51,52,53,54,55,56,57,60,61,62]. These metabolites may play essential roles in plant defense against abiotic stressors, such as high-intensity and ultraviolet radiation [47,51], and protection against herbivores [46,47,48,49,50]. It is likely that their early recognized medicinal properties may also be attributed to their specialized phytochemistry [17,18,19,20,21,22,23,24]. Nevertheless, L. maackii phytochemicals may also be released into the soil through roots or leaf litter decomposition, negatively affecting the germination, growth, and development of native species [47,51].

Notably, most studies evaluated the phytochemical composition and allelopathic impacts of L. maackii in extracts prepared in organic solvents [47,85,93], with few exceptions [51,54,55,56,94]. However, in realistic field settings, the metabolites that are most likely effective in inhibiting other plant species must be water-soluble. In our study, we used a passive extraction method mimicking the progressive, gradual leaching of water-soluble phytochemicals from dense L. maackii leaf litter into the soil water at the end of its growing season. Leachates derived from late-season leaf litter inhibited the germination of B. rapa seeds in a concentration-dependent manner, with the more concentrated extracts exerting the highest inhibitory effects and a 2 g (leaf tissue) mL⁻¹ concentration being lethal to the tested seeds (Figure 1). Even though the net impact of allelochemical products may also be affected by other factors [53,54,95,96,97,98,99,100,101], possibly attenuating their effects, they may substantially alter germination success and plant development [47,51,52,53,54,55,56,57,94]. Concentrations similar to those used in this study were detected in the soil for various phenolics [99,100,101,102].

These data are in good agreement with earlier descriptions of the inhibitory impacts of L. maackii leaf extracts on a variety of other species, including Arabidopsis spp., green ash, sugar maple, or an impatiens hybrid [47,51,52,53,54,55,56,57,58,59,60,61], and provide additional support for the L. maackii leaf litter inhibitory effects on yet another species. We chose B. rapa, a member of the Brassicaceae family, as the model species for our studies, as its wild varieties are widespread across the U.S., including areas affected by L. maackii infestations [26]. Moreover, B. rapa is sensitive to water-soluble allelochemicals released from various other allelopathic species [103,104,105,106,107,108,109].

Ontogeny and seasonal variations in environmental conditions control the synthesis of constitutive secondary metabolites in plant tissues [110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127]. Age-dependent and seasonal variations in the synthesis of terpenoids, phenolics, and alkaloids in leaf tissue have been commonly described and frequently attributed to, among other factors, the availability of metabolic precursors for their synthesis, deriving from primary metabolism [115,116,117,118,120,121,122,123,124,125,126,127]. The duration of L. maackii’s life cycle and period of photosynthetic competency exceeds that of the native vegetation in the invaded regions. L. maackii defoliation was recorded very late in the fall, sometimes retaining leaves until early in the following spring [16,40]. We hypothesized that leaf ontogeny also affects the synthesis and accumulation of metabolites with allelopathic properties in L. maackii. Extracts prepared from L. maackii leaves at non-lethal concentrations (1 g (leaf tissue) mL⁻¹, Figure 1) inhibited B. rapa germination most significantly, and increasingly, only when prepared from tissue harvested in the late season, from September to November (Figure 2). Such seasonal variation in the inhibitory potential of L. maackii leaf extracts was not unexpected. Our data also support an earlier assessment provided by Cipollini and coworkers on specific variations in the synthesis of some phenolic metabolites in L. maackii leaves in summer versus fall months [47]. Some of those metabolites are also hypothesized to be responsible for the species’ allelopathic impacts. Like most other secondary metabolites, phenolics are versatile molecules and play various roles in plants [47]. Cipollini and coworkers ascribed the differential accumulation of select phenolics from among the 13 metabolites identified from L. maackii leaf extracts to their potentially varied functions [47]. They suggested that synthesizing phenolics that protect leaves from ultraviolet radiation-induced damages may peak during the summer months. Other metabolites, which are deterrents for herbivore activity, may be synthesized to best fit the herbivore activity cycle during each season [47].

Nevertheless, Cipollini and coworkers [47] did not address the net inhibitory potential of whole aqueous leaf extracts, as the current work does. One of the novelties in our results is the evidence that the strength of L. maackii leaf extracts in inhibiting B. rapa germination increases during the fall. We suggest the progressive inhibitory capacity of seasonal L. maackii extracts to be a proxy for the variations in the biosynthesis of allelopathic inhibitors in this species’ leaves. It is also likely that leaf litter forming in the fall breaks down during the wet season, leaching into soil water, with expected inhibitory effects exerted in early spring, when conditions become favorable for seed germination.

Today, allelochemicals are frequently argued to serve as “novel chemical weapons” against naïve native species that have not yet been exposed to them. Consequently, native species may be expected to have a higher sensitivity to allelochemical inhibition than species cohabiting the environment from which the invasive originated [8,83,128]. Several metabolic candidates were isolated from Lonicera spp., such as phenolics, flavones, flavonoids, their glucoside derivatives, phenolic acids, and iridoids as likely allelopathic inhibitors [47,58,59,60,129]. Nevertheless, the net allelopathic impacts varied substantially in studies using different plant species and variable controlled or field conditions [47,51,52,53,54,55,56,57,58,59,60,61,62]. When considering the context-dependency of allelopathic effects, under field conditions, soil properties, the microbiome, the proximity to the allelochemical source, or other environmental conditions were suggested to affect the net negative impact of L. maackii metabolites on target species [16,51,52,62,98,99,130].

Surprisingly, however, to date, no study has considered the possibility that the confounding effects reported earlier regarding L. maackii’s net impact on different species might also be influenced by endogenous recipient-dependent factors.

Allelochemicals affect various physiological and metabolic processes in plants. These include alterations in the cellular ultrastructure; the inhibition of cell division and elongation; imbalances in the antioxidant system; increases in cell membrane permeability; and alterations in enzyme activities, respiration, photosynthesis, water and nutrient uptakes, and plant growth regulation (reviewed by [89]). A target species’ sensitivity to allelochemical-induced effects may also depend on their metabolic properties. Phenolics and their derivatives are some of plant tissues’ most prevalent and extensively studied allelochemical groups [131,132,133,134,135,136,137]. Phenolics were also suggested to be the predominant allelochemical molecules in L. maackii leaves [16,47,58,59,60,138].

Substantial evidence supports that some plant species can counteract allelopathic phenolics through direct counter-allelopathic defense or other metabolic strategies. For example, Weir and coworkers showed that oxalate synthesized and excreted by Gaillardia grandiflora and Lupinus sericeus could neutralize catechin, an allelopathic substance released by Centaurea maculosa [139]. Exogenous oxalate could also protect Arabidopsis spp. from catechin-induced allelopathy [139]. Species such as Lolium perenne, Trifolium repens, Rumex aquaticus, and others were stimulated by concentrated allelochemical solutions, making use of them as fertilizers [84,85,140,141]. Legumes and species with larger seeds are speculated to be more likely to resist allelopathy due to their abundant nutrition and energy, which can be invested into resistance [142]. Garlic root allelochemicals altered the tomato seedling transcriptome, oxidant–antioxidant, and growth-regulating phytohormone balance, inducing resistance [143].

Phytohormones are critical regulators of plants’ vegetative and reproductive cycles. Environmental factors affect the transition from dormancy to germination competency in seeds. However, germination is tightly regulated by the balance between two phytohormones. Abscisic acid (ABA) sustains dormancy, while gibberellic acid (GA₃) is required in high amounts, along with low ABA/GA₃ ratios, to alleviate dormancy in order to permit germination [71,73,79]. GA₃ stimulates seed germination, enhancing imbibition, weakening the seed coat, and activating hydrolytic enzymes. Germination involves bidirectional interactions between the embryo and the endosperm. The endosperm senses the appropriate conditions for germination, hence regulating the embryo’s growth. The embryo controls amylase release and endosperm degradation. Amylases break down the storage nutrients required for the nutrition and development of the embryo [72,73,74,75,76,77,78,79]. GA₃ also stimulates the expression of genes responsible for embryonic cell expansion in the early stages of germination and seedling development [77].

The biosynthesis of GA₃ was previously shown to be significantly inhibited by phenolic allelochemicals [137,143]. Cheng and coworkers also suggested that the allelochemical-induced alteration in phytohormone levels might increase plant resistance [143].

Here, we demonstrated that a standard (Rbr) B. rapa variety is sensitive to L. maackii aqueous extracts, supporting earlier sensitivity studies by Cipollini and colleagues [47]. These extracts inhibited B. rapa seed germination in a concentration-dependent manner (Figure 1), also controlled by L. maackii leaf ontogeny (Figure 2). In addition to the standard variety, using other B. rapa varieties with capacities to synthesize GA₃ differing from that of the Rbr, we also tested the hypothesis that elevated endogenous GA₃ levels may provide resistance against L. maackii allelochemicals.

We assessed the germination success and early-stage growth and development of a GA₃-overproducing (ein) and a GA₃-deficient (ros) B. rapa variety [81,82] treated with concentrations of L. maackii leaf extracts that are non-lethal to the Rbr variety (1 g (leaf tissue) mL⁻¹, Figure 1). Compared to the sensitivity of the Rbr, seeds of the ein variety were less sensitive to inhibition. At the same time, ros proved to be the most inhibited by L. maackii allelopathic metabolites, with no germination recorded up to 72 h after treatment. By that time, most seeds of the other varieties and all of the water-treated controls germinated (Figure 3A and Figure 4A). In addition, exogenous GA₃, in physiologically relevant concentrations (100 µmol, [144]), further decreased the sensitivity of all three tested B. rapa varieties to L. maackii inhibitors. The exogenous GA₃ supplement was sufficient to substantially compensate for the diminished endogenous GA₃ synthesis in the ros variety, with 78% of ros seeds exposed to L. maackii allelochemicals germinating within 72 h (Figure 3B and Figure 4B). We also noted a similar trend in the early seedling growth, with both elevated endo- and exogenous GA₃ compensating for the inhibitory effects of L. maackii extracts (Figure 5). The highly significant differences in the sensitivity of B. rapa varieties and the impact of exogenous hormone supplements (Figure 3 and Figure 5, and insets) suggest that GA₃ plays a substantial role in determining a species’ sensitivity and response to L. maackii allelochemicals. The observed differences between the growth and development of seedlings could be a consequence of the direct effects of GA₃ on seed germination, and not a direct effect of GA₃ on growth. We stipulate that the earlier-reported differences in the sensitivity of various species to L. maackii allelopathic inhibition [47,51,52,53,54,55,56,57,58,59,61,62] may, at least in part, be attributable to potential differences in the GA₃ synthetic ability of the species. However, further studies are needed to ascertain these differences in various native species cohabiting with L. maackii to identify candidates with the highest capacity for resistance.

To our knowledge, the current work is the first to examine the role of a target species’ metabolic properties in determining their net sensitivity to allelopathic inhibition by L. maackii.

Earlier, Cipollini and coworkers [47] identified 13 phenolic metabolites from degrading L. maackii leaves. They suggested that API, LUT, and their glycosidic derivatives are likely allelopathic candidates. Flavones and flavonoids are phenolic metabolites commonly synthesized by many plant species, with demonstrated bioactivity [145]. The molecules isolated from L. maackii by Cipollini and colleagues [47] were also detected in other species such as Cistus ladanifer and Matricaria chamomilla. These species are also allelopathic [102,110,146]. From the metabolites analyzed by Cipollini and coworkers [47], we tested the effects of API, LUT, and their combination on B. rapa germination success and development. We focused on these two flavones, as the concentration of their glycosidic derivatives was reported to be low during the fall [47]. The chemical properties and the solubility of both API and LUT have been amply characterized in a variety of solvents, including water [147,148,149,150]. The water solubility of API is as low as 183 µg mL⁻¹ [147,148], while LUT’s is 387.5 µg mL⁻¹ at 25 °C [149,150]. Compared to their solubility in other solvents, API and LUT’s solubility in water is low [147,148,149,150]. The poor solubility of these substances implies that the bioactive concentrations of API and LUT cannot exceed 183 µg mL⁻¹ and 387.5 µg mL⁻¹, respectively, under field conditions and in the soil. These values also reflect the likely maximum extractable amounts of API and LUT from leaves in an aqueous extract, under ideal conditions. However, under field conditions, it is likely that the water-soluble API and LUT amounts released from degrading leaves are significantly lower, considering that leaf degradation occurs at relatively low temperatures in the fall, which may further decrease API and LUT solubility in the aqueous environment [147,148,149,150]. Thus, they are likely to be present in fully aqueous extracts at low concentrations compared to the amount extractable using organic solvents, in which their solubility is higher [47,146]. However, these flavones can still accumulate in the soil at bioactive concentrations that are relevant for allelopathic interactions [102].

In this study, we treated standard B. rapa seeds with 50 µg mL⁻¹ API, LUT, and a 1:1 ratio combination of the two. These concentrations are lower than those used in prior studies [47] and represent a concentration range that plants are more likely to be exposed to under field conditions. We found that the two metabolites have different effects on B. rapa. Surprisingly, API stimulated B. rapa seed germination. Other studies also observed similar stimulating effects triggered by different members of the phenylpropanoid family, the group of metabolites to which API belongs. For example, Bi et al. (2017) [151] documented that sinapic acid stimulates the germination of wild-type Arabidopsis seeds in a concentration-dependent manner in the range of 0.1 to 1 mM.

On the other hand, API significantly inhibited seedling growth. LUT repressed both germination and growth. The combination of apigenin and luteolin decreased germination to a similar extent as LUT alone. However, the combination had a much stronger effect on seedling growth than either of the two flavones alone, suggesting that the simultaneous occurrence of multiple metabolites in degrading leaves may result in enhanced allelopathic effects (Figure 6). In addition, stereomicroscopic examination revealed aberrant morphology and deficiency in the root development of seedlings treated with API and LUT combined (Figure 7).

From the obtained data, we conclude that the net allelopathic impacts of degrading L. maackii leaves on B. rapa are controlled by complex chemistry and the combined effects of multiple metabolites. In particular, the described effects of aqueous L. maackii extracts are similar to those triggered by the combination of API and LUT (Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7), supporting earlier assumptions made by Cipollini and colleagues [47]. Nevertheless, we do not exclude the possibility of different metabolites also playing a role in the net allelopathic effects of L. maackii, on which our study did not focus.

To better understand the mechanisms at the basis of our observations, future work will test for GA₃ recovery effects on the allelopathic inhibition by API, LUT, and their combination, will test for potential synergistic or antagonistic effects of the API + LUT combination, and will be complemented with a comparative analysis of responses to API, LUT and API + LUT of the varieties differing in their ability to synthesize GA₃.

In conclusion, this study demonstrated that the net allelopathic potential of L. maackii leaves is defined by complex chemistry and the interactive effects of multiple metabolites on the target species. For the first time, we showed that the target’s metabolic properties might also influence the net allelopathic impacts of invasives, in addition to environmental factors and the proximity to the source. In particular, high GA₃ concentrations may substantially alleviate the inhibitory effects of L. maackii allelochemicals. The mechanism responsible for this effect will be further investigated in the future. A better understanding of the direct impacts of allelochemicals on target species will contribute to developing novel invasive species control and biodiversity conservation protocols. In addition, this knowledge may also contribute to additional applications in other fields—for example, to support applications employing allelopathic species in agriculture [11,89,92,152,153,154], in water recycling [155], and as substitutes for synthetic herbicides [136,156].

4. Materials and Methods

4.1. Plant Material and Growth Conditions

4.1.1. Lonicera maackii Leaf Collection and Extraction

L. maackii (Amur honeysuckle) leaves were collected from honeysuckle-infested forest stands in the Otoe Creek Natural Area on the Missouri Western State University campus (GPS coordinates: 39.7597° N, 94.7845° W) on the third week of each month between March and November 2020 and in 2021, throughout the growing season. Species identification was provided by the Missouri Department of Conservation’s Northwest Regional Office, located on the Missouri Western State University campus. The harvested leaves were rinsed three times with sterile distilled water to remove surface contaminants, blotted, frozen, and stored at −20 °C in a freezer (Frigidaire, Charlotte, NC, USA) until processing.

Aqueous extracts were prepared from whole leaves, modifying a method previously used by Dorning and Cipollini [51]. The leaves were incubated and shaken without disruption in sterile distilled water at a concentration of 2 g (leaf tissue) mL⁻¹ at room temperature for 72 h. The extract stock was decanted and filtered using a Pall Acrodisc^® sterile syringe filter with Supor^® membrane, with a 0.2 µm pore-size and 32 mm OD (Pall Corp., Port Washington, NY, USA). The filtered extracts were stored at 4 °C until use in a walk-in cooler unit (Amerikooler QC061277FBSC, Hialeah, FL, USA).

For germination and seedling development tests, the stock extract of 2 g (leaf tissue) mL⁻¹ was diluted with sterile distilled water to obtain concentrations of 0; 0.5; 0.75; 1; 1.5; and 2 g (leaf tissue) mL⁻¹.

4.1.2. Brassica rapa Varieties and Growth Conditions

The effects of L. maackii leaf extracts and their chemical components on seed germination and seedling growth were tested on Wisconsin Fast Plants^® Brassica rapa varieties in Petri dish seed assays. Wisconsin Fast Plants^® Brassica rapa subsp. dichotoma [80] seed stocks were obtained from the University of Wisconsin-Madison, WI, USA and stored at 4 °C until testing in a walk-in cooler.

Standard Wisconsin Fast Plants^® seeds (Rbr) were used as controls. The Standard stock characterizes the typical morphology and growth patterns for Fast Plants^® [81,82].

The Rosette-Dwarf variety (ros) is a Fast Plants^® stock homozygous recessive (ros/ros) for a gene that causes deficiency in the synthesis of the growth hormone gibberellic acid (GA₃). Dwarf plants produce 4–10 times less GA₃ than Standard (Rbr) plants, resulting in stems that do not elongate after seedlings emerge [81,82].

The Tall Plant (Elongated Internode, ein) variety is a Fast Plants^® stock homozygous recessive (ein/ein) for a gene causing the over-production of phytohormones in the gibberellin family, resulting in overly elongated stems and internodes. Tall plants produce up to 12 times more GA₃ than Standard (Rbr) Wisconsin Fast Plants^®. This stock develops tall plants [81,82].

For each seed assay, 20 Brassica rapa seeds of either variety were placed on wet filter paper in 15 × 100 mm plastic Petri dishes. A 5 mL solution of the selected composition and concentration was added to each Petri dish. The plates were sealed with parafilm to minimize evaporation. The dishes were placed on racks of a climate-controlled growth chamber (Percival, AR36L2, Perry, IA, USA). The plated seeds were kept under a day/night temperature cycle of 27/25 °C and a cycle of 16 h days with 250 µmol m⁻² s⁻¹ intensity irradiation and 8 h of dark for the duration of each assay.

Three independent replicates (N = 3) were performed for each treatment and seed variety for all assays, with 60 individual seeds of either type examined under each condition (n = 60).

4.2. Germination and Seedling Development Bioassays

4.2.1. Concentration Dependence and Seasonal Variation in the Effects of Lonicera maackii Extracts on Brassica Seeds

The concentration dependence of the effects of L. maackii leaf extracts on Standard (Rbr) control seeds was tested within the 0–2 g (leaf tissue) mL⁻¹ concentration range. The extracts were prepared from the stock harvested in October 2020. Rbr seed germination rates were recorded at each applied concentration as the percent fraction of water-treated control seeds after 24 h.

Seasonal variations in the inhibitory potential of honeysuckle extracts prepared from leaves harvested throughout the 2020 and 2021 growth seasons, from March to November, were assessed in Petri dish bioassays. Such variations were expected to serve as a proxy for the dynamic synthesis and accumulation of inhibitors in the leaf tissue throughout the plant’s life cycle.

Rbr seeds were treated for 24 h with 1 g (leaf tissue) mL⁻¹ extracts of leaves harvested on the third week of each month during the honeysuckle life cycle, combining leaves harvested in the same months of 2020 and 2021. The choice of the extract concentration for this assay was based on preliminary data indicating that, at this concentration, extracts prepared from leaves harvested in October may inhibit seed germination by over 80%, but no lethal effects have been observed in Rbr seeds yet.

4.2.2. The Impact of the Plant Growth Hormones, Gibberellins, on Brassica Seed Responses to Lonicera maackii Leaf Extracts

The Standard (Rbr), Rosette-Dwarf (ros), and Tall (ein) Brassica rapa Fast Plant^® varieties differ in their endogenous synthesis and accumulation of gibberellins [80,81,82]. To compare their sensitivity to the L. maackii leaf extracts, the seeds of each variety were treated with 1 g (leaf tissue) mL⁻¹ L. maackii extract for 72 h in Petri dishes, in parallel with the water-treated controls for each variety.

For assessing the potential impact of exogenous gibberellin supplementation on Brassica seed responses, Petri dishes with Rbr, ros, and ein seeds, treated with L. maackii extract, were also supplemented with 100 µM sterile aqueous solution of GA₃ (Millipore Sigma, Merck KgaA, Darmstadt, Germany). Each seed variety’s control was treated with sterile distilled water instead of the L. maackii extract.

Germination and seedling growth were measured at 24 h intervals for up to 72 h. Germination rates were expressed as a percent (%) fraction of the water-treated controls’ germination under each condition for each variety. The seedling length was measured in centimeters (cm).

4.2.3. Apigenin, Luteolin, and Their Combined Effects on Brassica Seed Germination and Early Seedling Development

To assess the individual or combined effects of these flavones on the germination and early-stage development of Rbr Brassica seedlings, seeds were treated with 50 µg mL⁻¹ solutions of API, LUT, and the combination of 50 µg mL⁻¹ API + 50 µg mL⁻¹ LUT in 0.05% DMSO (Millipore Sigma, Merck KgaA, Darmstadt, Germany). The seed response to the flavones was assayed in Petri dishes, with the treatments lasting up to 48 h. Control seeds were treated with sterile distilled water. Germination rates were expressed as the % change over time, and seedling lengths were established as the average lengths of all germinated seedlings at the time of the measurements for each variety and treatment. Germination and seedling development were assessed at a frequency of 12 h for the 48 h duration of the test.

Representative images of the developing control and treated (API, LUT, or API + LUT combination) Rbr Brassica seedlings were photographed under the lowest magnifying power (1.5×) of a Labomed Luxeo 4Z Stereomicroscope (Labo America Inc., Fremont, CA, USA) fitted with an AmScope CA-CAN-SLRIII adapter (United Scope, Llc., Los Angeles, CA, USA) for a Canon EOS Rebel T7 DSLR Camera (Canon Inc., Melville, NY, USA).

4.3. Statistical Analysis

All statistical analyses were performed using the Statistical Package for the Social Sciences software, version 20 (SPSS Inc., Chicago, IL, USA). Data were expressed as the means ± standard deviation (SD) of three independent replicates (N = 3, n = 60 seeds total for each treatment and condition). Student’s t-test was used to compare the differences in Brassica seed responses to varying honeysuckle extract concentrations and to analyze the seasonal variation in the L. maackii extracts’ negative allelopathic efficiency. Students’ test was also used to assess the significance of different variety seed germination and growth changes in response to L. maackii-induced inhibition in the presence and absence of exogenous GA₃ compared to the corresponding controls. The same test was used to determine the significance of the impact of flavones or their combination on Rbr seed germination and seedling growth after 48 h, as compared to the controls. Analysis of variance (two-way ANOVA) was used to test for differences in the germination and growth of the used B. rapa varieties treated with L. maackii extracts without or in the presence of exogenous GA₃ over time. Graphing was performed with Microsoft^® Excel^® 365 MSO, Version 2210 (Microsoft Corporation, Redmond, WA, USA).