# Water-based root exudates of Molinia caerulea (L.) Moench disrupt root nitrogen metabolism in Quercus petraea (Matt.) Liebl. seedlings with a fast negative effect on budburst

Larissa Adamik, Philippe Balandier, Jean-Stéphane Venisse, Marine

Fernandez, Philippe Malagoli

► To cite this version:

Larissa Adamik, Philippe Balandier, Jean-Stéphane Venisse, Marine Fernandez, Philippe Malagoli. Water-based root exudates of Molinia caerulea (L.) Moench disrupt root nitrogen metabolism in Quercus petraea (Matt.) Liebl. seedlings with a fast negative effect on budburst. Annals of Forest Science, 2025, 82 (1), pp.31-40. http://dx.doi.org/10.1186/s13595-025-01306-6

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# Water-based root exudates of *Molinia caerulea* (L.) Moench disrupt root nitrogen metabolism in *Quercus petraea* (Matt.) Liebl. seedlings with a fast negative effect on budburst

Larissa Adamik¹, Philippe Balandier², Jean-Stéphane Venisse¹, Marine Fernandez¹ and Philippe Malagoli¹*

**Abstract**

**Key message** *Molinia caerulea* (L.) Moench has been observed to significantly reduce budburst in *Quercus petraea* (Matt.) Liebl. seedlings through water-based *M. caerulea* root exudates. This suggests direct allelopathic effects between the two species. In terms of nutrient uptake, oak roots primarily take up nitrogen in the forms of ammonium and glycine. Interestingly, the application of root exudates from *M. caerulea* doubled the nitrate uptake in oak roots. Moreover, gene sets involved in nitrogen metabolism within oak roots exhibited strong deregulation when treated with *M. caerulea* root exudate, indicating that the interaction between these two plant species alters the nitrogen metabolism in the oak roots.

**Context** Oak regeneration encounters numerous impediments, including interactions with *Molinia caerulea* (L.) Moench, an understory grass species widespread in temperate forests. Besides competition for resources, our previous work suggested that the interaction between oak and *M. caerulea* may involve allelopathic processes.

**Aim** This study tested the hypothesis that *M. caerulea* affects the budburst dynamics, early growth of oak seedlings, and root N uptake transport and assimilation systems.

**Methods** Potted oak seedlings (*Quercus petraea* (Matt.) Liebl.) were watered with *M. caerulea* root exudates or water for 6 weeks during April and May 2021. The capacity of oak seedlings to take up nitrogen in its main molecular forms was characterized by an influx analysis of isotopically labeled (¹⁵N) nitrate, ammonium, and glycine on excised roots using the “teabag” technique. Concomitantly, targeted transcriptomics were carried out to monitor changes in gene expression related to nitrogen metabolism.

**Results** The treatment resulted in reduced budburst rates (–50%), together with an early reduction in height increment. Kinetics of N influx rates revealed that ammonium, and to a lesser degree glycine, were the predominant forms in which N was taken up by roots. Application of *M. caerulea* root exudate barely increased the ammonium influx rate and had no effect on the glycine influx rate. By contrast, the nitrate influx rate, despite its low values, doubled
after application of the exudate. Targeted transcriptomic analysis revealed a regulatory shift in gene sets associated
with key mechanisms underlying N uptake, assimilation, and long-distance transport in oak roots.

**Conclusion** Interactions between a perennial grass, *M. caerulea*, and a woody species, *Q. petraea*, do not only rely upon direct competition for resources. Allelopathic processes must be taken into consideration when designing regeneration operations.

**Keywords** Allelopathy, Phenology, Nitrogen, Root uptake, Assimilation, Transcriptome

# 1 Introduction

Natural oak regeneration has become difficult over the last three decades (Thomas et al. 2002), and oak planting remains a challenging alternative (Clark et al. 2016; Schuler and Robison, 2010).

Besides animal predation, seedbed properties, and turbance, well-documented negative plant-plant interactions have helped to explain failure in forest regeneration (Hoffmann and Haridasan 2008; Mallik 2003; Nilsson and Zackrisson 1992; Shimoda et al. 1994; Watt 1919). Several studies have highlighted the inhibitory action of understory plant species on tree regeneration (Mallik 2003). Although classical competition for resources among species is well documented, allelopathic interactions remain less understood. Physiological processes specifically targeted by allelochemicals are poorly identified. In temperate oak forests, *Molinia caerulea* (L.) Moench, a moor grass occurring on temporarily waterlogged acid soils, has been recognized as an important interfering species (Fernandez et al. 2022). Over 73% of biomass loss was recorded in *Quercus rubra* L. seedlings when growing with *M. caerulea* in non-fertilized systems (Timbal et al. 1990).

*M. caerulea* easily outcompetes temperate oak seedlings through its ability to quickly monopolize resources such as light, water, and nitrogen (Vernay et al. 2016, 2019). In forest environments, nitrogen (N) economy is a crucial factor for plant development (Fernandez et al. 2022). Vernay et al. (2016) observed limited N uptake by oak roots when oak was mixed-grown with *M. caerulea*. Findings from Fernandez et al. (2020, 2021) showed not only enhanced rhizodeposition from oak seedlings but also facilitated N transfer from oak to *M. caerulea*. Furthermore, *M. caerulea* is known to display an inhibitory allelopathic action on oak (Becker and Levy 1983; Fernandez et al. 2022; Weise 1960), although the responsible bioactive chemicals have not yet been identified, and thus their physiological targets remain unknown. Competition, facilitation, and allelopathy offer a clear advantage for *M. caerulea* and partly explain its dominance over oak seedlings.

Previous characterization of nitrogen flux dynamics between these two species suggested that allelopathic

action relied on decreased root N uptake in tree seedlings. Jabran et al. (2013) stated that allelopathic activity generally includes a decrease in plant root mineral uptake. This has been confirmed for nitrogen sources (Simon 2023; Warren and Adams 2007). Many allelochemicals are known to make nitrogen sources unavailable to the target plant. This may occur through actions on nitrification, ammonification, and N volatilization processes, by complexation with other elements in the soil (Zhang et al. 2021) or through inhibited nutrient root uptake, via reduced root growth and decreased nutrient transport system activity, and reduced nutrient use efficiency (Scavo et al. 2019). The non-negligible contribution of organic nitrogen to nutrition in tree species has gained greater recognition (Simon 2023). However, allelopathic action on organic nitrogen uptake has so far been scantily studied in forest ecosystems.

Regardless of the inorganic N forms, $NH_4^+$ is the primary cytosolic N substrate to be assimilated into numerous biomolecules via specific metabolism pathways. These are systemically reallocated in the plant through release in the vascular system. In this way, N content and its related metabolisms inside and/or outside plants are essential as regulatory signals and energy resources in the fine tuning of responses associated with plant adaptation to fluctuating environmental constraints, such as allelopathic stress.

At a physiological level, root nitrogen influx relies on activities of root transport systems specific to the chemical form of their substrates (Wang et al. 2012). Mineral transport takes place via two different transport systems depending on substrate concentrations in the soil solution: high affinity transport systems (HATS) function when substrate concentrations are low (typically 0.5–1 mM), while low affinity transport systems (LATS) take up substrate when their concentrations exceed 0.5–1 mM (Crawford and Glass 1998). At a molecular level, the root N uptake pathway relies on several $NO_3^-$ transporters. These include the nitrate transporter NRT1/PTR, NRT2 and NAR2 with the chloride channel (CLC), and the S-type anion channels SLACs and their homologs SLAHs. $NH_4^+$ uptake is regulated by the ammonium transporters (AMTs), and the
aqua-ammoniaporins, which are specific major intrinsic proteins (MIPs) or aquaporins (AQPs).

The present work sets out to characterize root N uptake and N assimilation in oak seedlings in response to the application of root exudates from *M. caerulea*, assumed to convey allochemicals (Fernandez et al. 2021). We hypothesized that allelopathic compounds would decrease the activity of N transport systems targeting root N transporters and alter N metabolism pathways during spring regrowth. Indeed, at this stage, tree seedling development is particularly dependent on N resource availability, a resource that is crucial for bud and shoot growth (Thitithanakul et al. 2012; Millard and Grelet 2010). We hypothesized that changes in N metabolism in oak root due to interaction with *M. caerulea* may result in changed phenology in oak shoots. Accordingly, we investigated the shoot's phenological response (budburst phenology and early growth). Functional responses were studied by monitoring root N influx using ¹⁵N labeling. This was extended by analyzing the expression of genes associated with N metabolism. We focused on key gene subfamilies involved in cellular uptake and N metabolism, encompassing enzymatic assimilation and intra- and intercellular fluxes of inorganic and organic N forms.

# 2 Materials and methods

## 2.1 Experimental site and plant material

All experiments were carried out at Clermont-Ferrand, France, 45° 77' 3" N, 3° 14' 3" E (394 m a.s.l.). *M. caerulea* was collected on January 27, 2021, from the French National Forest of Vierzon (47° 16' 4" N, 2° 07' 1" E). Plants were dug out along with the main parts of their root systems, and stored in plastic bags to maintain humidity during transport to the laboratory. On January 28, 2021, 60 tufts were individually potted in 10-L plastic pots. Concomitantly, 100 2-year-old *Q. petraea* seedlings from a tree nursery were potted (1 seedling per pot). The soil used for both species had a typical loam texture (sand 51%, silt 29%, clay 20%), with a pH<sub>water</sub> of 5.95. The soil C:N ratio (9.2) indicated active decomposition. The total nitrogen content was 2.1 g kg<sup>-1</sup>, indicating that N was not a limiting factor. Nitrogen content as ammonium was 32.1 mg kg<sup>-1</sup>, and as nitrate 123 mg kg<sup>-1</sup>. Phosphorus, potassium, and cation exchange capacities were not limiting.

## 2.2 Experimental design

From January 28 to April 07, 2021, plants were acclimated to pot environment before the onset of new growth, i.e., the beginning of the growing season. All plants were maintained under semi-natural conditions: the pots were left outdoors under natural light,

temperature, and precipitation. Plants were potted separately to avoid resource-based interactions. Manual weeding was done as needed. Irrigation began in March to avoid any water limitation.

When first budbursts occurred in 30% of the oaks, from April 07, 2021, *M. caerulea* roots were dug out every week and a root exudate was prepared (see Section 2.3). The *M. caerulea* plants then had an average fresh weight of 41.6 ± 3.9 g. A volume of 60 mL of their exudates was used to water 50 randomly selected oak seedlings. The remaining 50 seedlings received the same volume of distilled water free from *M. caerulea* compounds, constituting the control group. The same volume of exudates was added once a week for 6 weeks. Treatment duration was chosen according to Fernandez et al. (2020), who observed an effect of exudates a few weeks after treatment began. All plants' shoot growth and leaf budburst were assessed.

After 6 weeks of treatment (the week of May 17, 2021), oak plants were depotted and separated into shoots and roots. Soils containing root systems were coarsely sifted using a sieve of 2 mm mesh diameter and roots were collected. Roots were then sorted according to diameter. From five randomly selected individuals per modality, fine roots with a diameter less than 1 mm were selected for N influx analysis (see below). All fine roots were kept under a cool humid atmosphere to retain uptake capacity (Lucash et al. 2007) before carrying out influx measurements a few hours after harvest on the same day (see Section 2.6). For transcriptomic experiments, from the same individuals sampled for influx analysis, one root sample was taken per oak seedling. Accordingly, for each biological condition (control and treatment with *M. caerulea* exudate), five biological replicates were generated, each consisting of a mixture of six randomly selected seedlings. All were placed in liquid nitrogen and stored at -80 °C until use (see Section 2.9).

## 2.3 Collection of *M. caerulea* root exudates

*M. caerulea* roots keep their capacity to exudate secondary metabolites into their environment several days after the shoots have been removed (Lucash et al. 2007). Intact roots from freshly de-earthed plants were squeezed and placed in clean hermetic 1-L lunch boxes containing distilled water in an amount such that the final ratio of dry weight to water volume (DWV) of the solution was 2%. Boxes were placed in the dark at 20-25 °C for 48 h, the mean time before roots become inactive (Lucash et al. 2007). Water exudates from *M. caerulea* roots were applied to pots containing oak to convey *M. caerulea* allelochemicals to oak roots.
## 2.4 Budburst and early growth of oaks

First appearance of a leaf in at least one bud was recorded as budburst (BBCH 09). Relative budburst rate for a given time t was computed at the treatment group scale as follows:

labeled samples were cut. Manually into smaller pieces and transferred into 10 mL plastic tubes and dried for 72 h at 60 °C. An automated ball mill was used to obtain 3–4 mg of root powder, which was placed in pewter microcapsules. Isotope-ratio mass spectroscopy analysis of total N content and ¹⁵N abundance was carried out by

$$ \text{Relative budburst rate (t)} = \left[ \frac{\text{\%burst}(t) - \text{\%burst}(t - 1)}{\text{\%burst}(t - 1)} \right] \quad (1) $$

Budburst and growth of oak seedlings were monitored weekly, measuring maximal height of shoots during the treatment. Relative height increment (%) was calculated for each seedling to compare weekly changes:

INRAE SILVATECH (Silvatech, INRAE, 2018; Structural and functional analysis of tree and wood facility, https://doi.org/10.15454/1.5572400113627854E12). Analysis of nitrogen isotope composition d¹⁵N and nitrogen and

$$ \text{Relative height increment} = \left[ \frac{\text{height}(t) - \text{height}(t - 1)}{\text{height}(t - 1)} \right] \times 100 \quad (2) $$

## 2.5 Influx measurements for ¹⁵N-nitrate, ¹⁵N-ammonium, and ¹⁵N-glycine in Q. petraea seedling roots

Root N uptake kinetics in treated and non-treated oak seedlings were established based on ¹⁵N isotopic labeling of three different chemical forms, all contributing to N nutrition: mineral forms (¹⁵NO₃⁻ and ¹⁵NH₄⁺) and an organic form (amino acid ¹⁵N-glycine). Among amino acids, glycine was chosen because it is directly absorbed by trees (Warren and Adams 2007). Solutions with different nitrogen concentrations were prepared: 50 µM and 500 µM to assess high affinity transport systems (HATS) activity and 1500 µM for assessing low affinity transport systems (LATS) activity. For each combination of N form and concentration, the analysis was repeated using five different oak seedlings. First, 6 g of excised oak fine roots was placed in teabags and washed with 0.5 mM CaSO₄ solution for 30 min to rid roots of soil residues while maintaining membrane integrity (Boss and Mott 1980; Britto et al. 2000; Kronzucker et al. 1995a, 1995b). For acclimation, roots were then placed for 1 h in unlabeled solutions containing the test concentrations of N. Second, teabags containing roots were placed in a solution similar to the previous one except that N forms were ¹⁵N-labeled. Corresponding concentrations of ¹⁵N forms (K¹⁵NO₃, (¹⁵NH₄)₂SO₄, or ¹⁵N-glycine) were used. Roots were bathed in those solutions for 10 min (Kronzucker et al. 1995a, 1995b). Third, two washing steps were performed, both in N solutions identical to those used for acclimation. The first step lasted 5 s and freed roots of labeled N adsorbed on root surfaces. The second one lasted 2 min and removed apoplastic ¹⁵N. Timing was based on efflux studies to exclusively measure influx (Kronzucker et al. 1995a, 1995b, Britto et al. 2000). All solutions (pH adjusted to 6.5) were air-bubbled to ensure oxygenation and homogeneity of nutrient availability. Laboratory temperature averaged 20–25 °C. Finally,

carbon contents were performed by placing tin capsules containing dry material in an elemental analyzer (vario ISOTOPE cube, Elementar, Langenselbold, Germany) coupled, via a gas box interface, to a continuous flow isotope ratio mass spectrometer (Isoprime100, IRMS, Elementar UK, Cheadle, UK) available at SILVATECH. The sample was burned at 1025 °C in excess of oxygen. Then, the NOx were reduced using a quartz tube filled with copper heated at 650 °C. CO₂ was trapped at 35 °C by an adsorption column while N₂ passed through the thermal conductivity detector (TCD). Next, CO₂ was released from the adsorption column at 225 °C. Elementary gases were analyzed and detected by isotope ratio mass spectrometry, Isoprime100 IRMS (Cheadle, UK). Carbon and nitrogen contents were expressed in % of dry matter. d¹⁵N is expressed as delta values in ‰ relative to air N₂. The d¹⁵N uncertainty of measurements is 0.5‰ (2σ) in nitrogen for complex samples (i.e., not pure molecules).

## 2.6 Calculations of ¹⁵N influx rates in oak roots

Total N amounts ($N_{tot}$, [mg]) were calculated as follows:

$$ N_{tot} = \left( \frac{\%N_{tot} \times DW_{roots}}{100} \right) \quad (3) $$

where %N_{tot} is the nitrogen content (% of dry weight, DW) and DW_{roots} is DW (mg) of the roots.

The amount of ¹⁵N in the sample (¹⁵N_{amount}, [µg]) was given by the equation:

$$ ¹⁵N_{amount} = (IA_{sample} - IA_{reference}) \times N_{tot} \times 10 \quad (4) $$

using isotopic abundances of the labeled samples (IA_{sample}) and natural unlabeled ones (IA_{reference}). Isotopic abundance (IA) is the ratio of ¹⁵N over total N present in the sample. This can be written:
$$IA = {}^{15}\text{N}_{\text{amount}} \times 100/\text{N}_{\text{tot}} \quad (5)$$

Influx rates of nitrate, ammonium, and glycine ($I_{\text{Nit}}$, $I_{\text{Amm}}$, and $I_{\text{Gly}}$ respectively, [µmol h⁻¹ g (DW)⁻¹]) were given by the following equation:

(https://www.genscript.com/wolf-psort.html) (Horton et al. 2007), and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) (Chou and Shen 2010). Predicted subcellular localization and presence of transmembrane helices of Quercus rubra transporters are summarized in Appendix Table 1.

$$I = {}^{15}\text{N amount} \times (IA_{\text{labelling solution}}/100) \times (1/\text{MM}^{15}\text{N}) \times (60/d) \times (1/\text{DW}_{\text{roots}}) \quad (6)$$

$IA_{\text{labelling solution}}$ is the isotopic abundance of the nutrient solution enriched with ¹⁵N (atom-%=99%). MM¹⁵N is the molar mass of the labeling isotope (in this case 15 g/mol), and $d$ is the duration of the influx monitoring (10 min).

## 2.7 Identification of Q. petraea genes involved in N transport, assimilation, and vascular unloading

N-related metabolism was studied through the transmembrane uptake and assimilation processes of the two primary inorganic forms, nitrate ($NO_3^−$), and ammonium ($NH_4^+$). Finally, the release of nitrogenous metabolites (i.e., glutamine and asparagine) in the vascular system was assessed.

Putative Q. rubra genes (a species ontogenetically close to Q. petraea, as data were not available for Q. petreae, and specifically sought for this study) involved in the transmembrane diffusion and enzymatic assimilation of mineral nitrogen, followed by the release of nitrogenous metabolites into the vascular system, were retrieved from Phytozome (V13, https://phytozome-next.jgi.doe.gov/2023/11/28), using blast alignments (https://phytozome-next.jgi.doe.gov/blast-search) with known heterologous sequences from Arabidopsis (Heynh.) and Populus (L.), and basic searches by keywords. For each gene family, candidates were aligned to assign their potential subfamilies. Because our work focused on the general molecular responses developed by oak roots challenged by M. caerulea exudate, we prioritized the molecular investigation of genes that exhibited transcribed homologs in the NCBI EST libraries (https://www.ncbi.nlm.nih.gov/), or were identified as transcribed sequences in the Phytozome databases. A preliminary nomenclature was then attributed to each gene exhibiting transcribed homologs in the NCBI's public EST databases. The nomenclature is fully consistent with that traditionally assigned to each protein family and potential related subgroups. To unambiguously identify the transmembrane candidates, the helical transmembrane regions (TMHs) were predicted using the TMHMM-2.0 (www.cbs.dtu.dk/services/TMHMM/) (Sonnhammer et al. 1998). Likewise, the putative subcellular localizations were predicted using WoLF PSORT II

## 2.8 Gene expression analysis by real-time RT PCR

Approximately 150 mg of root material was removed from each harvested oak seedling (see Section 2.2), gently washed, immediately immersed in liquid nitrogen, and stored at -80 °C. Each sample was ground to a fine powder in liquid nitrogen. Total RNA was extracted with a CTAB extraction buffer (cetyltrimethylammonium bromide), as previously described in Lopez et al. (2012). The RNA concentration and purity were determined using a NanoDrop™ ND-1000 spectrophotometer (NanoDrop®, France), and RNA integrity was verified by 2% agar gel electrophoresis, staining with 10% (v/v) SYBR® Safe DNA gel stain (Invitrogen, Carlsbad, CA, USA). One microgram of total RNA extracted from each biological replicate was subsequently pooled to generate the five aforementioned biological replicates (see Section 2.2). First-strand cDNAs were synthesized from 2 µg of mixed total RNA, using SuperScript III (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions, and stored at -20 °C until use. Real-time quantitative PCR was carried out on an Applied Biosystems StepOnePlusTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The amplification reactions were performed in a 15 µL of final reaction mixture, including 1X of TakyonTM Rox SYBR® MasterMix dTTP Blue (Eurogentec, Liege Science Park, Belgium), 2 µL of 1:30 diluted cDNA, and 100 nM primers (Appendix Table 2). qPCR reactions were conducted according to the manufacturer's instructions, with the following conditions: pre-denaturing at 95 °C for 3 min, 35 cycles (95 °C for 10 s, 60 °C for 10 s, and 72 °C for 20 s), and 72 °C for 15 min. PCR reactions were ended by generating a dissociation curve of 60-95 °C, with an increment of 0.3 °C/15 s, to avoid primer dimers and nonspecific amplifications. Prior to qPCR with each primer pair, PCR conditions were determined by comparing threshold values in a dilution series (0, ×15, ×30, ×60) of a cDNA mix, generated with all RT products. For each gene, primer pairs were designed within consensus regions among homolog genes recovered from different oak species (NCBI), and the amplification efficiencies were calculated (Appendix Table 2). Transcript levels were
normalized to those of five *Quercus* encoding house-
keeping genes: *translation elongation factor 1-alpha
EF1α* (XP_050292676.1), *histone H4* (XM_024020482.1),
*polyubiquitin 10* (JAAMOV010000008.1), *serine/
threonine-protein phosphatase PP2A catalytic subunit*
(XM_031100507.1), and *ribosomal protein S13 mRNA*
(RPS13) (OW028771.1). These genes corresponded to the
best-suited reference genes (*p* < 0.001) from eight widely
used housekeeping genes. They were identified with the
software application BestKeeper (Pfaffl et al. 2004). The
BestKeeper Index was estimated and was used as a cali-
brator for calculating the transcript abundances. Each
referrer belongs to protein families involved in different
cell processes, to minimize the risk of co-regulations. The
relative expression of nitrogen-target genes was evalu-
ated by using the 2<sup>-ΔΔCt</sup> method (Livak and Schmitt-
gen 2001). For clarity, and especially for downregulated
genes, values were graphically normalized to 0 (signifying
no change in gene expression), where each unit on each
side of 0 corresponds to a twofold increase and a twofold
decrease, respectively. Overall constitutive transcript
steady-state levels were assessed by comparison of the
mean of the cycle threshold (Ct). The final results from
each statistical output were plotted after distribution and
assignation of a score value between 0 and 100, an arbi-
trary scoring range. A Ct of 20 was assigned an arbitrary
value of 100 (corresponding to the highest gene expres-
sion level). A Ct of 40 was assigned an arbitrary value of 0
(no expressed gene). All other Ct were then assigned val-
ues between 0 and 100, scaled based on their relative dis-
tributions. We emphasize that the complexity of studying
these large superfamilies (along with those presented
below) was overcome by retrieving all related mem-
bers from specialized public genomic resources, such as
NCBI and Phytozome. Subsequently, the genes whose
transcriptional expressions were monitored in our study
corresponded either to genes identified as potentially
transcribed (identifiable with the existence of transcribed
sequences integrating non-coding regions on both sides
of retrieved genes in Phytozome V13, and the presence of
their homologs from various oak EST libraries in NCBI),
or based on their ability to specifically transport nitrogen,
as observed with some AQPs. Regarding these latter, NH₃
diffusion is confirmed for the TIP2 and NIP1 subgroups,
which correspond to the tonoplast intrinsic protein and
the nodulin 26-like major intrinsic protein subfamilies,
respectively, and predicted for members of the X-intrin-
sic proteins (XIP) subfamily.

## 2.9 Statistical analyses

All tests were performed using R Studio software
(2023.09.1 + 494 "Desert Sunflower," using R edition
4.3.1). Differences in oak height increments and relative

budburst rates were analyzed with Wilcoxon's sum rank
test. For the N uptake assay, a root teabag represents an
experimental unit. The effect of the different factors (N
concentration, N form, treatment with *M. caerulea* root
exudates) and their interactions on the variable (influx)
were tested on the entire data set. Significance levels
were set to α=5%. Normality conditions were verified
with the Shapiro-Wilk test and QQ plots, and homosce-
dasticity with the Bartlett test. ANOVA was performed
and statistical groups were established with Tukey's hon-
estly significant difference test. Variation degree was cal-
culated for each factor combination. Gene expression
was analyzed with the Kruskal-Wallis test. The *p*-values
were adjusted according to the Benjamini-Hochberg
procedure (Benjamini and Hochberg 1995).

3 Results

## 3.1 Molinia caerulea root exudates reduced budburst and height growth in oak seedlings

The addition of *M. caerulea* root exudates caused a sig-
nificant decrease in the relative budburst rate (*p*=0.021,
Fig. 1A, Appendix Fig. 5A). While control plants pre-
sented a mean relative budburst rate of 31% on the whole
period, treated oaks averaged only about half of this
value. By the end of the experiment (6 weeks after start-
ing application of root exudate), 85% of control oaks and
62% of treated oaks displayed leaves.

The mean weekly relative height increment (RHI)
showed a significant negative response to treatment
(*p*=0.049) (Fig. 1B, Appendix Fig. 5). While control
plants displayed RHI of 1.58% ± 0.19 (SE), treated plants
showed values of 1.00% ± 0.16 (SE). Absolute height
values measured over time (Fig. 1, Appendix Table 3)
showed a significant negative treatment effect beginning
during the third week of the experience (*p*=0.004), when
control and treated plants measured 86.3 cm ± 1.72 (SE)
and 81.5 cm ± 2.33 (SE), respectively.

3.2 Kinetics of root N influx responded to treatment
with Molinia caerulea exudates

N influx rates responded to increasing substrate con-
centrations with a sharp increase from 50 to 500 µM N
concentration in the nutrient solution (p = 4.55 × 10⁻¹⁰),
followed by lower increase between 500 and 1500 µM in
most observations approaching a plateau that theoreti-
cally situates at 1500 µM. Under both conditions (control
and treated, Fig. 2), influx rate values varied according
to the tested N chemical forms (p = 2 × 10⁻¹⁶) as follows:
ammonium, glycine (tenfold decrease), and nitrate (40-
fold decrease). No significant effect of exudate applica-
tion was observed for either ammonium or glycine influx
rates after application of *M. caerulea* root exudates,
except at 500 µM NH₄⁺, when the influx for the treated
<figure><img src="image_5.png" /><figcaption>**Fig. 1** Weekly evolution of (A) budburst rate and (B) height increment of oak seedlings beginning at first *Molinia caerulea* exudate treatment. Treatment group consisted of oak seedling irrigated with *M. caerulea* exudates while control oaks received distilled water instead</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2** Influx rates of nitrate (A), glycine (B), and ammonium (C) in excised fine roots of oak seedlings in response to different concentrations of nitrogen sources. *p*-values indicate significance of differences between treatments at the same concentration (n.s. = *p*-value above 0.05). Lowercase letters indicate significance levels between concentrations for the same treatment modality</figcaption></figure>

exceeded the control (p = 0.004) (Fig. 2C). By contrast, applying exudates doubled nitrate influx rates compared to those measured in the roots of control plants, whatever the concentration (Fig. 2A).

### 3.3 The expression of genes related to nitrogen metabolism corroborated N influx rate pattern on application of *Molinia caerulea* root exudates

Transcriptomic analyses revealed a predominance of upregulated trends for NAR2, CLC, SLAH3, NIP1, TIP2, and XIP families (Fig. 3, Appendix Fig. 4). Expressions

were more contrasted for the NPF, NRT2, and AMP families under our biological conditions, with significant upregulations (NPF1, NPF3, NPF6, NIP1a, XIP2, AMT11b, AMT13, AMT32, and AMT33a), downregulations (NPF4a and -b, NPF5c, NPF7, NRT2a, AMT11a, AMT12, and AMT31b), and non-modulated genes (NPF5b, NRT2b, AMT2, AMT31a, and AMT33b). In the roots of *Q. petraea*, some transporter-related genes were not transcribed, such as NPF2a and -b, NPF5a, NIP1b, and XIP1.
<figure><img src="image_7.png" /><figcaption>**Fig. 3** Quantitative real-time RT-PCR of selected genes related to nitrogen transport, assimilation, and vascular release. Transcript abundances were recorded in root tissues of *Quercus petraea* seedlings at 6 weeks after treatment with *Molina caerulea* exudates. Color scales represent level of transcriptional expressions (green, up-expression; red, down-expression), and the transcript steady state level (white, extremely low transcript accumulation; dark blue, maximal transcript accumulation). Genes with no transcription are indicated by a gray square with a black cross. The significant differences were statistically analyzed between untreated and treated roots with *M. caerulea* exudates. Each biological condition comprised five biological replicates, with each replicate representing a pooled sample of six randomly selected seedlings (Kruskal–Wallis, adjusted *p*-value < 0.05). Complete statistical data, including the SE and *p* values, are detailed in Appendix Fig. 4. * *p*-value < 0.05, and ** *p*-value < 0.01; "ns" indicates no significant difference between treatments. Values corresponding to the "Relative transcript steady state level" are detailed in Appendix Table 2. AAP-EamA-LHT, amino acid transporter; AMT, ammonia transporter; ASNase, asparaginase; ASNS, asparagine synthetase; CLC-SLAC-SLAH, chloride channel; GDH, glutamate dehydrogenase; GOGAT, glutamate synthase; GS, glutamine synthase; NIP, nodulin 26-like intrinsic proteins; Nir, nitrite reductase; NR, nitrate reductase; NAR-NRT-NPF, nitrate transporter; TIP, tonoplast Intrinsic proteins, XIP, X-intrinsic protein</figcaption></figure>

Regarding genes involved in inorganic N assimila-
tion, the number of members from each family was rel-
atively restricted. The identification of genes encoding
proteins involved in the GS/GOGAT pathway includes
five glutamine synthase isoforms (three cytosolic iso-
forms with GS1, GS2, GS3; the chloroplastic GS isoform
GS4; and a pseudogene), and three glutamate synthase
(GOGAT) isoforms with two duplicated chloroplastic
NADH-dependent glutamate synthase isoforms (NADH-
GOGAT) and a ferredoxin-dependent amyloplastic
(Fd-GOGAT). Glutamine (Gln) metabolism is closely
connected to asparagine (Asn) metabolism, involving
asparagine synthases (9 members), and asparaginases (4

members). Additionally, seven glutamate dehydrogenases
were identified from the *Q. rubra* genome, with four
members (GDH1 to GDH4), for which transcript accu-
mulations were monitored in this work. Overall, tran-
script accumulations were significantly enhanced, except
for NADH-GOGAT, which was downregulated. Certain
members, namely GDH1, GDH2, Fd-GOGAT, GS1 and
-4, ASNSBb, and ASNaseb and -c, did not show signifi-
cant modulation in roots by *M. caerulea* exudates. No
GDH4 transcripts could be detected under our biological
conditions.

Lastly, three amino acid transporter families belong-
ing to the AAAP superfamily (AAPs, LHTs, and the
EamA-like transporter family, also known as AtSIAR1/
UnamiTs) were studied. These families include plasma
membrane transporters that transport neutral amino
acids, such as Gln and Asn. The diversity within these
families is high, resembling that of the NPF or MIP
superfamilies: AAPs with 83 putative members, 94 LHTs,
and 70 EamAs. We focused on some members whose
permease activity for Asn and/or Gln had been experi-
mentally demonstrated in orthologs. As observed above,
transcript accumulations were significantly enhanced,
except for LTH6e, AAP2, and EamAd, which were down-
regulated. LHT5 and LHT6f were either unaffected by
the treatment or not transcribed under our experimental
conditions, respectively.

4 Discussion

*M. caerulea* root exudates affected the whole spring phe-
nology of oak seedlings; budburst was reduced along with
early growth reduction. Contrarily to our hypothesis, N
uptake by root transport system was partly increased by
*M. caerulea* root exudates, while the transcriptomic anal-
ysis confirmed strong modifications of the expression of
genes associated with the whole N metabolism, indicat-
ing more indirect effects than expected.

## 4.1 Application of *M. caerulea* root exudates limited growth and budburst in oak seedlings

Exudates of *M. caerulea* roots were applied to investigate
their allelochemical properties on oak seedling phenol-
ogy and early growth. They affected oak height growth,
confirming a allelopathic action like previously reported
(Timbal et al. 1990). However, the effect on oak phenol-
ogy was unexpected. We observed that the first signifi-
cant differences on tree development appeared already
4 weeks after application of *M. caerulea* root exudates.
In previous studies, effects were observed after 6 weeks
(Fernandez et al. 2020). This suggests that allelopathic
action of *M. caerulea* occurs more rapidly than previously
demonstrated, pointing to a critical oak developmen-
tal period. Such responses might result from chemicals,
or induced pH variations possibly modifying availability
in soil anions and cations (Zambelli et al. 2025). Similar
observations of phenological effects mediated by plant-
plant allelopathy have been documented in other mod-
els, such as vanilla and beans exposed to allochemicals
(Adiputra et al. 2019; Amini et al. 2013). However, to our
knowledge, this is the first report of impeded tree phe-
nology mediated by allelopathy in forest ecosystems.

## 4.2 Reduced budburst of oak seedlings did not result from a decrease in N uptake

Ammonium was the preferred N form taken up in oak
seedling roots, followed by glycine, and lastly nitrate.

These results are in line with those observed in *Q. suber*
(Mata et al. 2000). According to Warren and Adams
(2007), plants prefer N forms that are predominantly
available in their natural habitats. Oaks interacting with
moor grass are mainly found on acidic soils supplying
nitrogen predominantly as ammonium (Koyama and
Kielland 2022; Ma et al. 2021). Glycine transport val-
ues, particularly when compared with nitrate transport,
underline the significant role of organic N sources in oak
seedling nutrition, even if it is ten times less than ammo-
nium. This role is emphasized in forest ecology (Farzadfar
et al. 2021) particularly during the non-growing sea-
son and the leaf expansion in deciduous trees (Koyama
and Kielland 2022; Ma et al. 2021). Despite knowledge
about the depressive effects of *M. caerulea* exudates on
oak morphological and phenological features, the find-
ings regarding nitrogen uptake rates were unexpected:
exudate treatment had no inhibitory effect on measured
influx rates. Instead, it significantly stimulated nitrate
uptake at all concentrations, increasing HATS and LATS
activity. For ammonium supply, a significant increase was
detected only at 500 µM, with higher values recorded
for treated plants. For other concentrations, no differ-
ences were measured, which jeopardizes conclusions on
ammonium HATS or LATS activity. No significant dif-
ference was found in measured glycine influx. Several
hypotheses may be suggested to explain these physiologi-
cal responses to root exudate application.

Possibly, the increase in nitrate, and to a lesser extent,
in ammonium influx for treated seedlings, might result
from an increase in N mineralization rate, leading to
higher nitrate and ammonium concentrations in the
soil after *M. caerulea* root exudate application (Chen
et al. 2009). Measurement of N mineralization processes
would be helpful to test this hypothesis. In this last study,
the allelopathic action exerted by the invasive species
*Mikania micrantha* (Kunth) led to increased soil nitrate
and ammonium concentrations, benefiting *M. micran-
tha*. Here, as no measurement of inorganic N was made
during the experiment in soil, induced soil nitrate and
ammonium concentrations resulting in higher N influx
rate may be hypothesized. However, transcriptomic
analyses may suggest other hypotheses, although we note
that the budburst reduction was not associated with a
decrease in N uptake in oak roots.

4.3 *M. caerulea* exudates modulated the expression
of genes encoding key enzymes in N metabolism

Increased nitrate influx may result from the observed
enhanced expression of genes encoding plasmalemma
transporters involved in HATS and LATS activities. By
contrast, the expression profiles of ammonium trans-
porters showed greater variability between members,
with many events of up- and downregulation. At a cellular level, the shift in transcription of genes coding for key enzymes in N metabolism after application of *M. caerulea* root exudates suggests an increase in the accumulation of glutamine, one of the key transport forms of N. The corresponding transporters are crucial for the uptake of inorganic N, and in our biological conditions, transcriptome analyses revealed a significant upregulation of most of them, whether putatively localized in the plasmalemma (NPF, NAR2, NIP1, CLC, SLAH, and XIP2), or tonoplast (NPF5 and -7, NRT2, TIP). These patterns echo those generally induced in some plant species grown under non-limiting N availability but challenged with various abiotic or biotic stresses, or during beneficial interaction with fungal ectomycorrhiza (ECM) or plant growth-promoting microorganisms (PGMs). In these situations, “stress tolerance” correlates with fluctuations in $NO_3^-$ and/or $NH_4^+$ availability, which in turn modulates transporter expression or activity (Calvo et al. 2019; Castro-Rodríguez et al. 2016; Ding et al. 2015; Selle et al. 2005). Transcriptional levels are not automatically correlated with those of their related proteins, but these modulated gene expressions indicate that the oak roots did respond to the presence of *M. caerulea* allelochemicals by modulating nitrate and ammonium uptakes and subcellular distributions. Expression of genes involved in the enzymatic assimilation of inorganic nitrogen is globally activated, resulting in the over-accumulation of Gln/Asn, a phenomenon accentuated by the inhibition of NADH-GOGAT expression. These gene expression profiles enhancing the Gln and Asn accumulations are consistent with previous studies, where N levels fluctuated during acclimatization of plants to various types of abiotic and biotic stress (Lea et al. 2007; Yin et al. 2022).

According to the upregulations of ASNases, which play a key role in Asp accumulation, we can conclude that oaks were stressed by root exposure to *M. caerulea* exudates. The expression of genes encoding ASNases, which play a key role in Asp accumulation, were significantly upregulated in roots challenged by *M. caerulea* exudates. Asparaginases are known to respond to various environmental cues, and influence the accumulation of Asn and its byproduct, Asp (Credali et al. 2013; Ivanov et al. 2012). Among the free cytosolic amino acids (AAs), L-aspartate (Asp) serves as a central building block in nitrogen and carbon metabolism, playing pivotal roles in various metabolic processes. Asp is therefore a biochemically active compound with pleiotropic functions, closely linked to stress phytohormones, which collectively drive the plant's responses to sudden stress and its acclimation to challenging environments (Han et al. 2021).

The transcript accumulation of AA transporters toward xylem vessels increased. Such changes decrease cellular

concentration of AAs, which are known to exert negative feedback on inorganic N influx rate, thereby activating nitrate influx rates through increased transcription of nitrate transporters in our experiment. Some down regulated expression trends were observed among these transporters, such as for AMP11a, AMP12, AMP31b, NPF4ab, NRT2a, LTH6e, AAP2, and EamAd. We are unable to propose specific functional interpretations for each transporter isoform. However, besides the fact that the role of transporters in the regulation of nitrogen homeostasis under allelopathic stress is likely complex and still largely unknown, the overall transcriptomic response highlighted a fine tuning of their availability, contributing to the complex regulation of N flows between various compartments (apoplasm, cytosol, and subsequently the morphoplasm, or vacuole). It is apparent that their activities are significantly influenced either by direct interaction with allelochemicals or indirectly through their phytotoxic effects on the molecular environment of the cell as a whole, as discussed below.

## 4.4 Which mechanisms might be involved in the allelochemical effects on oak seedling development?

Correlation between reduction in budburst and changes in N uptake, as well as transcriptomics in N metabolism, does not link them in a straightforward manner; however, it sheds light on several innovative hypotheses.

Allochemicals have an effect on signal transduction pathways, with several members exerting direct negative effects on membrane fluidity and integrity (Shearer et al. 2012). This is attributed to their detergent properties and their ability to induce imbalances in the antioxidant system, resulting in rapid production of reactive oxygen and nitrogen species (RONS), and the inhibition of the activity of various antioxidant enzymes at the contact area. The solubilization and peroxidation of phospholipids directly impact the membrane potential and fluidity, which ultimately affect primary functions such as signaling and transport. Identifying *M. caerulea* allelochemicals and testing their regulatory action (or “toxicity”) on various key transporters would be of great interest. In any case, the upregulation of the N metabolism can be observed in connection with the plausible oxidative stress elicited by the *M. caerulea* exudates. Interestingly, Gln metabolism is essential for maintaining cellular redox homeostasis by harnessing enhanced RONS levels through the biosynthesis of glutathione, a major antioxidant tripeptide (Glu–Cys–Gly) that deactivates peroxide-free radicals in cells (Amores-Sánchez and Medina 1999; Liao et al. 2022). Likewise, Gln is required to detoxify some toxic metabolites by conjugation (Cook 2019; Tao et al. 2022). Similarly, Asp and its derivative
compounds are among the key bioprotectants for reconfiguring the overall metabolic network to help plants respond to stress, recover, and acclimatize in the long term (Lehmann et al. 2009).

In the same line, the increase in N uptake potential of oak roots could be part of the adaptive response of oak seedlings to the stress induced by the understory species. However, in spring, to ensure budburst, stored N compounds are hydrolyzed and transported in the form of amino acids (asparagine and glutamine in most cases) toward growing buds. The decreased relative budburst rates of treated oaks therefore suggest that the reinforced conservation strategy also encompasses an inhibition of the remobilization of the stored N, or that oaks loose greater amounts of N than they take up during the winter despite a reinforced conservation strategy, potentially due to unfavorable facilitation of *M. caerulea* N supply (Fernandez et al. 2020).

## 5 Conclusion and perspectives

This molecular ecophysiology work is the first to report reduced budburst dynamics of oak seedlings caused by *M. caerulea* interference through water-based root exudates. At variance with an assumed decrease of root N uptake, uptake capacities of N sources were barely changed, and those of nitrate, and to a lesser degree ammonium, were even increased in treated seedlings. Moreover, *M. caerulea* root exudates induced a significant effect on overall nitrogen metabolism in oak roots suggested by the transcriptomic analysis. The specific ways in which allelochemicals affect the nitrogen metabolism are not yet elucidated, but our results suggest a general situation of stress caused by the *M. caerulea* root exudates. Our results point to a need to identify compounds with allelochemical properties acting on N metabolism, ultimately tree phenology.

The differential physiological responses derive from metabolic adjustments (biochemical and transcriptomic) linked to cellular nitrogen homeostasis, although further research will be required to elucidate the biological significance of the underlying metabolic and molecular mechanisms. Several primary diamide amino acids play a substantial role in the observed adjustments, such as Gln and Asn. Future studies will need to identify the key function of these diamides that may give protection against the emergence of these cellular disorders linked to allelopathic stress. More generally, they will need to address the importance of amino acid biosynthesis and transport processes in maintaining the physiological integrity and productivity of plants in situations of intra- and interspecific competition.

# Appendix

<table><caption>**Table 1** Predicted subcellular localization and presence of transmembrane helices of *Quercus rubra*'s transporters. Subcellular localization predictions were performed by using Wolf PSORT server (https://wolfpsort.hgc.jp/). Abbreviations of protein localization sites in the dataset are as follows: Plas, plasma membrane; ER, endoplasmic reticulum; Gol, Golgi apparatus; Pero, peroxisomes; Chlo, chloroplasts; Mito, mitochondria; Vac, vacuole; Cyto, cytosolic. The number mentioned besides localization roughly indicates the number of the nearest neighbors to the query which localizes to each site, but is adjusted to account for the possibility of dual localization. The strongest occurrences are highlighted in bold. Transmembrane helices predictions were performed by using the TMHMM-2.0 software (https://services.healt htech.dtu.dk/services/TMHMM-2.0/). The presence of transmembrane helices was notified « PM+ », without specifying the number of predicted TMHS, which requires dedicated molecular dynamics (MD) calculations</caption><thead><tr><th>QrAAP1</th><th>Plas: 9 (E.R.: 2, golg: 2, vac: 1)</th><th>PM+</th></tr></thead><tbody><tr><td>QrAAP2</td><td>Plas: 5 (vac: 4, golg: 3, cyto: 1, extr: 1)</td><td>PM+</td></tr><tr><td>QrAAP3a</td><td>Plas: 10 (E.R.: 2, extr: 1, vac: 1)</td><td>PM+</td></tr><tr><td>QrAAP3b</td><td>Plas: 10 (vac: 4)</td><td>PM+</td></tr><tr><td>QrAAP7</td><td>Plas: 8 (vac: 4, E.R.: 1, golg: 1)</td><td>PM+</td></tr><tr><td>QrLTH5</td><td>Plas: 6 (vac: 4, E.R.: 2, golg: 2)</td><td>PM+</td></tr><tr><td>QrLTH6a</td><td>Plas: 10 (golg: 2, vac: 1, E.R.: 1)</td><td>PM+</td></tr><tr><td>QrLTH6b</td><td>Plas: 10 (E.R.: 3, vac: 1)</td><td>PM+</td></tr><tr><td>QrLTH6c</td><td>Plas: 12 (vac: 1, E.R.: 1)</td><td>PM+</td></tr><tr><td>QrLTH6d</td><td>Plas: 10 (vac: 2, E.R.: 2)</td><td>PM+</td></tr><tr><td>QrLTH6e</td><td>Plas: 9 (vac: 3, E.R.: 2)</td><td>PM+</td></tr><tr><td>QrLTH6f</td><td>Plas: 14</td><td>PM+</td></tr><tr><td>QrLTH8</td><td>Plas: 10 (vac: 2, cyto: 1, E.R.: 1)</td><td>PM+</td></tr><tr><td>QrNIP1a</td><td>Plas: 11 (vac: 2, golg: 1)</td><td>PM+</td></tr><tr><td>QrNIP1b</td><td>Vac: 2 (plas: 2, E.R.: 1, golg: 1)</td><td>PM+</td></tr><tr><td>QrNIP2a</td><td>Vac: 13 (plas: 1)</td><td>PM+</td></tr><tr><td>QrNIP2b</td><td>Vac: 8 (plas: 6)</td><td>PM+</td></tr><tr><td>QrXIP1</td><td>Plas: 12 (plas: 12, vac: 1)</td><td>PM+</td></tr><tr><td>QrXIP2</td><td>Plas: 11 (plas: 11, golg: 2)</td><td>PM+</td></tr><tr><td>QrCLCa</td><td>Plas: 10 (vac: 2, E.R.: 2)</td><td>PM+</td></tr><tr><td>QrCLCc</td><td>Plas: 9 (vac: 3, golg: 2)</td><td>PM+</td></tr><tr><td>QrEamAa</td><td>Plas: 10 (E.R.: 3, chlo: 1)</td><td>PM+</td></tr><tr><td>QrEamAb</td><td>Plas: 10 (E.R.: 3, chlo: 1)</td><td>PM+</td></tr><tr><td>QrEamAc</td><td>Plas: 10 (E.R.: 4)</td><td>PM+</td></tr><tr><td>QrEamAd</td><td>Plas: 5 (vac: 3, E.R.: 3, golg: 2, cyto: 1)</td><td>PM+</td></tr><tr><td>QrNPF1</td><td>Plas: 10 (E.R.: 2, nucl: 1, vac: 1)</td><td>PM+</td></tr><tr><td>QrNPF2a</td><td>Plas: 8 (vac: 3, E.R.: 2, nucl: 1)</td><td>PM+</td></tr><tr><td>QrNPF2b</td><td>Plas: 9 (vac: 2, chlo: 1, E.R.: 1, golg: 1)</td><td>PM+</td></tr><tr><td>QrNPF3</td><td>plas: 10 (chlo: 1, nucl: 1, vacu: 1, pero: 1)</td><td>PM+</td></tr><tr><td>QrNPF4a</td><td>plas: 9 (vac: 3, E.R.: 2)</td><td>PM+</td></tr><tr><td>QrNPF4b</td><td>plas: 7 (vac: 3, E.R.: 2, cyto: 1, mito: 1)</td><td>PM+</td></tr><tr><td>QrNPF5a</td><td>plas: 13 (vac: 1)</td><td>PM+</td></tr><tr><td>QrNPF5b</td><td>plas: 14</td><td>PM+</td></tr><tr><td>QrNPF5c</td><td>plas: 12 (E.R.: 2)</td><td>PM+</td></tr><tr><td>QrNPF6</td><td>plas: 13 (vac: 1)</td><td>PM+</td></tr><tr><td>QrNPF7</td><td>plas: 11 (vac: 1, E.R.: 2)</td><td>PM+</td></tr><tr><td>QrNAR2a</td><td>plas: 10 (vac: 3, E.R.: 1)</td><td>PM+</td></tr><tr><td>QrNAR2b</td><td>plas: 8 (vac: 4, cyto: 1, golg: 1)</td><td>PM+</td></tr><tr><td>QrNAR2c</td><td>plas: 6 (vac: 5, cyto: 1, E.R.: 1, golg: 1)</td><td>PM+</td></tr><tr><td>QrNRT2a</td><td>plas: 12 (chlo: 1, E.R.: 1)</td><td>PM+</td></tr><tr><td>QrNRT2b</td><td>plas: 5 (E.R.: 3, vac: 2, chlo: 1, pero: 1, golg: 1)</td><td>PM+</td></tr><tr><td>QrSLAHa</td><td>plas: 5 (E.R.: 3, nucl: 2, cyto: 2, vac: 2)</td><td>PM+</td></tr></tbody></table>
<table><caption>**Table 2** Localization, sequence, PCR efficiency, and average Ct levels for primers used for qPCR amplification</caption><thead><tr><th>Loci/standard code</th><th>Primers pairs (5'—3')</th><th>PCR efficiency (E)</th><th>Average Ct level</th></tr></thead><tbody><tr><td colspan="4"><strong>Housekeeping genes</strong></td></tr><tr><td colspan="4"><strong>EF1α: Translation elongation factor 1-alpha</strong></td></tr><tr><td>QrEF1a</td><td>GAACGGAGATGCTGGGATGGTGA/TTTCTTTGC AGCGGACTTGGTGA</td><td>1.82</td><td>20.7</td></tr><tr><td colspan="4"><strong>TUB: Tubulin</strong></td></tr><tr><td>QrTUBa</td><td>CAATAACACAGCAGTAGCTGAGG/CAAGAGCAG CCAGATCTTCACG</td><td>1.97</td><td>23.62</td></tr><tr><td colspan="4"><strong>UBQ: Polyubiquitin 10</strong></td></tr><tr><td>QrUBQ10</td><td>CTGCGATTGCGTGGAGGAATGC/CATCTTCAAGCT GCTTGCCTGC</td><td>1.8</td><td>24.84</td></tr><tr><td colspan="4"><strong>PP2A: serine/threonine-protein phosphatase PP2A catalytic subunit</strong></td></tr><tr><td>QrPP2A</td><td>CAGGAGGTTCCACATGAAGGACC/GGGCTCTTG AAATCAGTGAGAGC</td><td>2.02</td><td>31.78</td></tr><tr><td colspan="4"><strong>RPS13: Ribosomal protein S13 mRNA</strong></td></tr><tr><td>QrRPS13</td><td>GTTAGGGCTTGCTCCTGAAATTCC/GTCTTCTTG TAGTAGCGAGCAAGG</td><td>1.92</td><td>23.45</td></tr><tr><td colspan="4"><strong>HIS: Histone H4 protein</strong></td></tr><tr><td>QrHIS4</td><td>CCCGCGATTCGTCGTTTGGCTCG/ACGACATCCATG GCCGTCACG</td><td>1.94</td><td>22.62</td></tr><tr><td colspan="4"><strong>Ammonium transporters</strong></td></tr><tr><td colspan="4"><strong>AMT: Ammonia transporter</strong></td></tr><tr><td>QrAMT1.1a</td><td>CCATAGCCGCCGCAGGAATCA/CCATCGGCGGAC CAGAACCCAGT</td><td>1.80</td><td>21.89</td></tr><tr><td>QrAMT1.1b</td><td>GGGAGAACCGCAGTCACAACCAC/GCAAACCCA CCAAGCAAACCATTA</td><td>2.08</td><td>29.83</td></tr><tr><td>QrAMT1.2</td><td>CTGCAGCTGGTGGCCTTTCTTAC/CCGGTGGGCTTA GGATAGTCTGTT</td><td>2.08</td><td>24.71</td></tr><tr><td>QrAMT1.3</td><td>AAGAGTGGGCCGGTTTGATGT/CTTGGCTGGTGT TGGGATAGG</td><td>2.04</td><td>23.04</td></tr><tr><td>QrAMT2</td><td>TGGGGCGGTGGGTTTCTTTAC/CCTGACCAACCC ATCCACAATAG</td><td>1.97</td><td>23.77</td></tr><tr><td>QrAMT3.1a</td><td>GAGAGAGAGAGTTTGGTTG/GATGTGCCGGATTGG TAAG</td><td>1.85</td><td>27.22</td></tr><tr><td>QrAMT3.1b</td><td>TGAAATGGGCTGCACTACCTGAA/GACACCCCA ATGGAACAAGAACC</td><td>2.09</td><td>24.57</td></tr><tr><td>QrAMT3.2</td><td>TGCATTGTGGGGTGATGGAG/TGCGCTACATGGCAA AACATC</td><td>2.21</td><td>30.58</td></tr><tr><td>QrAMT3.3a</td><td>CTCTCGAGCCTGTTCCTACCTGTC/GAGCTGCAT CATCCCCAATCTT</td><td>2.19</td><td>24.82</td></tr><tr><td>QrAMT3.3b</td><td>GGCTGGGCTGCACTGGTAATG/GAGGTATGGGTG AGCAAAGAGTCC</td><td>2.14</td><td>23.23</td></tr><tr><td colspan="4"><strong>NO3- transporteur</strong></td></tr><tr><td colspan="4"><strong>NPF: Nitrate and peptide transporter</strong></td></tr><tr><td>QrNPF1</td><td>GTTGATAACCTTACCTCAAGAGGG/GTACCAGTT ATTGCAACTCTTGTCC</td><td>1.95</td><td>29.86</td></tr><tr><td>QrNPF2a</td><td>GGCTTTCTCAAACTATCTAAGTGC/CTTCTCAGA CTGCTTTGTTAATTTCC</td><td>nd</td><td>0</td></tr><tr><td>QrNPF2b</td><td>CACCAGAACAAAAAGCAGGCCTG/CATCCACAT AAGGCTCATCTTCC</td><td>nd</td><td>0</td></tr><tr><td>QrNPF3</td><td>TTTACTGGCTACTCACATTGTTGC/ACCCCAACT GTGCCTAAAGCTGC</td><td>2.16</td><td>25.43</td></tr><tr><td>QrNPF4a</td><td>GAGCTCCTTATTGGTCTCTTTGG/GTGAAGCGATAA TGGCTATAGTCC</td><td>2.04</td><td>27.37</td></tr><tr><td>QrNPF4b</td><td>AGCTCACTCCTTGTTTCCCTCG/TGTGGGTGGTCT TGTCGATATGAC</td><td>2.13</td><td>28.99</td></tr></tbody></table>
<table><thead><tr><th>Loci/standard code</th><th>Primers pairs (5'—3')</th><th>PCR efficiency (E)</th><th>Average Ct level</th></tr></thead><tbody><tr><td>QrNPF5a</td><td>GGGGGTAAGAGCTGGATAGGC/CTAGAGGAGACT<br>TGTCCAAGGC</td><td>nd</td><td>0</td></tr><tr><td>QrNPF5b</td><td>TCACCGAGGATGGATTCTAAACG/TCCCTTCAATTC<br>TTCTGTAAGCAC</td><td>1.95</td><td>24.32</td></tr><tr><td>QrNPF5c</td><td>TAACCAAGAAGGGCGGACATGG/CCATTGAAGCTA<br>CACCTTAACATCC</td><td>1.91</td><td>24.44</td></tr><tr><td>QrNPF6</td><td>GGAACCCAGGCAAAGGACAGC/CCTATTTAACTA<br>ACCATGGAGAGG</td><td>1.86</td><td>27.85</td></tr><tr><td>QrNPF7</td><td>TATGAAGATATCAACTGAGGATCAC/ATGTCATCT<br>TCCTCATCTGCCTC</td><td>1.91</td><td>22.79</td></tr><tr><td colspan="4"><strong>NRT: NitRate transporter</strong></td></tr><tr><td>QrNRT2a</td><td>GATCCCGAGAAAGCCATTGAGG/AAGTTGACAATC<br>TTATTTGTCATAATTAC</td><td>1.94</td><td>33.31</td></tr><tr><td>QrNRT2b</td><td>GGCGAATGCGACAGAAGAAGAG/TAGCTAAACATG<br>TGCTGGTGAGG</td><td>1.76</td><td>22.62</td></tr><tr><td colspan="4"><strong>NAR: Nitrate Assimilation Related protein</strong></td></tr><tr><td>QrNAR2a</td><td>ATTGAGGAACATGATGGAGATTCG/CTAAAATGG<br>CACAAATTTCCATCC</td><td>1.78</td><td>30.49</td></tr><tr><td>QrNAR2b</td><td>GTTGAAAAGGAAGAAGAAGGTAGT/TCTTGGTCT<br>ACTATCAATTCTAC</td><td>2.27</td><td>24.54</td></tr><tr><td>QrNAR2c</td><td>AATGTTGAAAAGGGAGAAGAGAGC/TTCACCTTA<br>GTTCACCAATAATCTC</td><td>1.88</td><td>34.56</td></tr><tr><td colspan="4"><strong>SLAH: slow anion channel associated homologues</strong></td></tr><tr><td>QrSLAH3a</td><td>GAGAAGACTTGTCAAAGAGAAAAAGCC/CAGAAA<br>TTGTGACTATAGCTTCTG</td><td>2.03</td><td>27.58</td></tr><tr><td>QrSLAH3b</td><td>GGCTCTGTTTATAACAACTATCTGG/CACCTCTCT<br>CAGAGCTTGGAGG</td><td>1.82</td><td>25.42</td></tr><tr><td colspan="4"><strong>CLC: plasma membrane chloride channel</strong></td></tr><tr><td>QrCLCa</td><td>ATTGTACCTAAGTATGTAGCAGCC/GAAGACTCA<br>GTTCTCCTTTTCTCC</td><td>1.84</td><td>24.35</td></tr><tr><td>QrCLCc</td><td>TGTTGTACCAAAGAGTCAAGGG/ACCGACTATCAA<br>ACATTCAGTTGC</td><td>2.06</td><td>20.99</td></tr><tr><td colspan="4"><strong>Assimilation</strong></td></tr><tr><td colspan="4"><strong>NiR: Nitrite reductase</strong></td></tr><tr><td>QrNiR</td><td>AGATGTTTACAAGAAGGGTGTCC/GAAACCCAT<br>ACCATACCAAAACTATC</td><td>2.19</td><td>25.23</td></tr><tr><td colspan="4"><strong>NR: Nitrate reductase</strong></td></tr><tr><td>QrNR</td><td>CACAGAAAGTATTCTGCGAGAGC/CGCCTCACTTCT<br>AAAACTTGTTCC</td><td>1.92</td><td>25.11</td></tr><tr><td colspan="4"><strong>GS: Glutamine synthase</strong></td></tr><tr><td>QrGS1</td><td>GCGCCGTCTCACTGGCCGAC/CCTAAATGCAGG<br>TGGGTGTAGC</td><td>2.25</td><td>20.25</td></tr><tr><td>QrGS2</td><td>CAAAAMAGCCATTGAAAAGCTCRAGT/ACAGGT<br>TGAATAAATCTTCTTTATGG</td><td>1.99</td><td>33.52</td></tr><tr><td>QrGS3</td><td>GCAATTYTGAACCTGTCACTTCGC/GCTAGAGCT<br>TCAGCCTCCAGTG</td><td>2.08</td><td>26.77</td></tr><tr><td>QrGS4</td><td>ATAGTTGCTGCTGGCATTGATGG/CAGAACATAATC<br>AGCTTAATATCGG</td><td>2.16</td><td>27.62</td></tr><tr><td colspan="4"><strong>GS: Glutamine synthase</strong></td></tr><tr><td>QrfdGOGAT</td><td>CTGGGAGCAGTAAAGGTTCTGC/TTCAGTGCTGGT<br>CTCAGGGTAC</td><td>2.02</td><td>24.14</td></tr><tr><td>QrNadhGOGAT</td><td>CTGAAGGYCGGCAAGCTGCTGCA/GACGTCACC<br>TATGTCATTACAGTG</td><td>1.94</td><td>23.19</td></tr><tr><td colspan="4"><strong>GDH: Glutamate dehydrogenase</strong></td></tr><tr><td>QrGDH1</td><td>AGAGCCCTCAAAGCAGCTACTG/CAATAGTAATTG<br>TGCCTAATTCTTTGG</td><td>1.87</td><td>27.04</td></tr></tbody></table>
<table><thead><tr><th>Loci/standard code</th><th>Primers pairs (5'—3')</th><th>PCR efficiency (E)</th><th>Average Ct level</th></tr></thead><tbody><tr><td>QrGDH2</td><td>GAAAGTGAACAATGAGCTGAAAAC/GAATATGGT<br>TGAGAATCATTGGAGCC</td><td>2.13</td><td>23.76</td></tr><tr><td>QrGDH3</td><td>GTGGGATGAAGAGAAGGTAAATAGG/AAGGTTGAA<br>GATGAATSAGAAATGG</td><td>1.87</td><td>27.03</td></tr><tr><td>QrGDH4</td><td>TGAGGAGGGCTTTCCATGAAATC/GGATGATAACCT<br>YTGGATGCATC</td><td>nd</td><td>0</td></tr><tr><td colspan="4"><strong>ASNase: Asparaginase</strong></td></tr><tr><td>QrASNasea</td><td>GATATTTTGGAAACTCTATGGAACG/ATTCCGAAT<br>ATGAGCGAGGTTGC</td><td>1.90</td><td>25.39</td></tr><tr><td>QrASNaseb</td><td>TCATACGTGGGACCCTGGCTC/ACCCATCCTCAG<br>TAGCACAACC</td><td>2.08</td><td>20.88</td></tr><tr><td>QrASNasec</td><td>CAAAGGAGAAGTCACAATGTCTTAT/AAGAGTGTG<br>AGTGACTCACAGTG</td><td>2.10</td><td>28.73</td></tr><tr><td>QrASNased</td><td>AAAAGGAGAAGTTACAATGGCTTTG/GTCATTTGC<br>CAAATACTAAGTGTAAG</td><td>1.94</td><td>22.54</td></tr><tr><td colspan="4"><strong>ASNS: Asparagine synthetase</strong></td></tr><tr><td>QrASNSa</td><td>AGGATGCTTCTTCACATCGTCTG/CCCAATAATAAT<br>GACAGCTATGC</td><td>2.05</td><td>18.39</td></tr><tr><td>QrASNSb</td><td>GGGTGCATTTTCACGAATGGAAGT/AACACAGTT<br>TGATTACCAGCTTGC</td><td>1.97</td><td>28.80</td></tr><tr><td>QrASNSc</td><td>AGGATGTTTCTTCTCAACAGCAGT/ATACATTTTGTT<br>TGTCAAGTGTCC</td><td>2.06</td><td>18.49</td></tr><tr><td>QrASNSd</td><td>AGGGTGTATGTTCCACAGTGG/CAGCAGCTAAGC<br>TATTGAGAATCG</td><td>1.77</td><td>19.81</td></tr><tr><td>QrASNSe</td><td>AGCATGTTCCACAGTCAGAATGG/TCTGTGTCAGGA<br>ATTAATGTGAGC</td><td>2.18</td><td>21.15</td></tr><tr><td colspan="4"><strong>Aqua-ammoniaporines</strong></td></tr><tr><td colspan="4"><strong>NIP: Nodulin 26-like intrinsic proteins</strong></td></tr><tr><td>QrNIP1a</td><td>CGTGAGATCACGACGAGTGGC/TTTCGCTATAGTTAT<br>ATCCATAAAGG</td><td>1.84</td><td>23.58</td></tr><tr><td>QrNIP1b</td><td>CCGAGATCACCGAGCCTAAATC/CGGAGCAATCAC<br>TAAACAAGTTCC</td><td>nd</td><td>0</td></tr><tr><td colspan="4"><strong>TIP: Tonoplast intrinsic proteins</strong></td></tr><tr><td>QrTIP2a</td><td>ATTCGTCACAAATGGAGAGAGCG/CGGGTTCAT<br>CGACCCACCACTA</td><td>2.11</td><td>18.72</td></tr><tr><td>QrTIP2b</td><td>GCAGTCACTGGAGGCTTGGCAA/GTTCATTGATCC<br>ACCGGAGAATGG</td><td>1.67</td><td>19.12</td></tr><tr><td colspan="4"><strong>XIP: X-intrinsic proteins</strong></td></tr><tr><td>QrXIP1</td><td>GGTGCAAGGAACTTGACATAACG/AAGAATAGT<br>GGGATTATCATTTTCC</td><td>nd</td><td>0</td></tr><tr><td>QrXIP2</td><td>GGGCCTGCTATTGCATGTGTGGC/TTTCTTCACTTC<br>TTGTGAGCATGG</td><td>1.87</td><td>28.51</td></tr><tr><td colspan="4"><strong>N-organic diffusion—Amino acid permeases</strong></td></tr><tr><td colspan="4"><strong>AAP: amino acid permeases</strong></td></tr><tr><td>QrAAP1</td><td>CAAGTTCATCACAAGTGAGCATGC/GAAATTGAA<br>ACCAGGTCTTGAACG</td><td>2.48</td><td>23.34</td></tr><tr><td>QrAAP2</td><td>TTGGAGAACCATGTTCGTGATGTT/AGACAGCCA<br>GATAGTGTTCAAGC</td><td>2.62</td><td>25.30</td></tr><tr><td>QrAAP3a</td><td>TTGTAACAGCAGAATATGACATACC/CCATGGTTC<br>TCTCTTAGTAACTGG</td><td>1.96</td><td>29.53</td></tr><tr><td>QrAAP3b</td><td>ATGGAGGTCAATTTTTGTGGTTGC/AACTTCAAC<br>CTAATAGATGGCTGC</td><td>2.17</td><td>23.00</td></tr><tr><td>QrAAP7</td><td>TGGCTTTGTACATAATTTCTACACC/AATTACAGTAAT<br>GCCTCAACTCAG</td><td>1.84</td><td>22.85</td></tr></tbody></table>
<table><caption>**Table 3** Absolute height of oak seedlings throughout the experiment with *Molinia caerulea* exudate treatment (cm, means ± standard-error, *n* = 50)</caption><thead><tr><th>Loci/standard code</th><th>Primers pairs (5'—3')</th><th>PCR efficiency (E)</th><th>Average Ct level</th></tr></thead><tbody><tr><td colspan="4"><strong>EamA-like: Amino acid bi-directional transport (eq. MtN21/EamA-like/UMAMIT family)</strong></td></tr><tr><td>QrEamAa</td><td>AGCCATTGAAGAACCTGAAGCAG/CCACGTCCA CCATGCAACAATGC</td><td>1.95</td><td>22.70</td></tr><tr><td>QrEamAb</td><td>CAAGACGATGACAAAAGTGAACC/ATATTCAAA GCAGTTCCACTAGG</td><td>2.01</td><td>21.84</td></tr><tr><td>QrEamAc</td><td>TAAAAGCAAAGATTACAAGTCCTCG/TGTTGCAGA GGTGCATGGAACC</td><td>2.02</td><td>34.02</td></tr><tr><td>QrEamAd</td><td>ATACCAGATGTCATAAAGAGTACAC/CAATGCATC CATTTTGATAATTATGG</td><td>2.26</td><td>23.35</td></tr><tr><td colspan="4"><strong>LTH: lysine-histidine-like transporters</strong></td></tr><tr><td>QrLTH5</td><td>GGACGAGCCCTACGCCTCGC/ATAATAATTAAATCT AGTGACACAAAAG</td><td>1.97</td><td>33.14</td></tr><tr><td>QrLTH6a</td><td>CCAGGAATTGCACTCAGACTC/GTCATCAGGGAA ATGAGTTTAGCTT</td><td>1.78</td><td>34.71</td></tr><tr><td>QrLTH6b</td><td>CCAGGAATTGCACTCAGACTC/TCATCAGAAAAA TGAGTTCAGCCA</td><td>2.28</td><td>23.64</td></tr><tr><td>QrLTH6c</td><td>CTACAAGTACTCTGCGTTTCATTA/CCTTTATCTAAA AGGATTGCATTTGC</td><td>2.29</td><td>22.41</td></tr><tr><td>QrLTH6d</td><td>CCACAAGAATACTTCGGTTTACTG/TAACAGCAG CAGACTTCTCTTGG</td><td>2.09</td><td>22.15</td></tr><tr><td>QrLTH6e</td><td>GCCGACTAAGTTCCTTCGTTTCC/GGAACAAAGCAT TTTGTACCATCC</td><td>2.24</td><td>30.94</td></tr><tr><td>QrLTH6f</td><td>AAGTTCAAACCATCTTTTCATACTCC/CTATCATCT GATTCTGAACTAATGC</td><td>nd</td><td>0</td></tr><tr><td>QrLTH8</td><td>CTCTTCCAGTCACATTTGCTTACC/AACATTCGTTAT GCAGAACTCAGG</td><td>1.99</td><td>24.01</td></tr></tbody></table>

<table><thead><tr><th>Week after treatment</th><th>Control</th><th>Treated</th><th>p-value</th></tr></thead><tbody><tr><td>1</td><td>83.38 ± 13.81</td><td>79.38 ± 15.98</td><td>0.082</td></tr><tr><td>2</td><td>83.72 ± 13.67</td><td>79.88 ± 15.95</td><td>0.104</td></tr><tr><td>3</td><td>83.94 ± 13.83</td><td>80.12 ± 16.15</td><td>0.105</td></tr><tr><td>4</td><td>86.7 ± 14.05</td><td>81.5 ± 16.29</td><td>0.029</td></tr><tr><td>5</td><td>89.26 ± 14.46</td><td>82.68 ± 16.24</td><td>0.021</td></tr><tr><td>6</td><td>90.3 ± 14.32</td><td>83.14 ± 16.01</td><td>0.013</td></tr></tbody></table>
<figure><img src="image_8.png" /><figcaption>**Fig. 4** Quantitative real-time RT-PCR of selected genes related to nitrogen transport, assimilation, and vascular release. Transcript abundances of the genes were recorded by real-time qRT-PCR analyses, relative to those of the control, using the 2⁻^ΔΔCt^ method. The significant differences were statistically analyzed between untreated and treated roots with *Molinia* exudate. Each biological condition comprised five biological replicates, with each replicate representing a pooled sample of six randomly selected seedlings (Kruskal & Wallis, adjusted *p*-value < 0.05)</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 5** Means of weekly relative budburst rate (A) and weekly relative height increment (B) of oak seedlings throughout the experiment. Treatment group consisted of oak seedling irrigated with *Molinia caerulea* exudates while control oaks received distilled water instead. *p*-values are given above figures. Bars represent standard errors</figcaption></figure>

**Acknowledgements**

The authors thank SILVATECH (Silvatech, INRAE, 2018. Structural and functional analysis of tree and wood Facility, doi: https://doi.org/10.15454/1.55724 00113627854E12) from UMR 1434 SILVA, 1136 IAM, 1138 BEF and 4370 EA LERMAB, localized at the research center INRAE Grand-Est Nancy, for isotope ratio mass spectroscopy analyses. The SILVATECH facility is supported by the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01).

**Authors' contributions**

LA, PB, JSV, MF and PM have contributed to the conception and design of the work; LA, PB, JSV and PM have contributed to the acquisition of the data. LA and JSV analyzed data. LA, PB, JSV, MF and PM have contributed to interpretation of data; LA and JSV have drafted the work. MF, PB and PM have substantially revised it. PB and PM have supervised the project.

**Data availability**

Supplementary data is accessible at https://doi.org/10.57745/HEZHBC.

**Declarations**

**Competing interests**

The authors declare no competing interests.

**Author details**

¹University of Clermont Auvergne, Clermont-Ferrand, France. ²National Research Institute for Agriculture, Food and Environment, Paris, France.

Received: 31 January 2025 Accepted: 9 August 2025

Published online: 18 September 2025

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