The interplay between drought and P scarcity shapes resilience to stress in Quercus spp. by modulating metabolomic profiles

The interplay between drought and P scarcity shapes resilience to stress in Quercus spp. by modulating metabolomic profiles | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The interplay between drought and P scarcity shapes resilience to stress in Quercus spp. by modulating metabolomic profiles Morena Rolando, Paola Ganugi, Leilei Zhang, Francesca Secchi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6420617/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Plant and Soil → Version 1 posted 5 You are reading this latest preprint version Abstract Background and Aims Plant resilience to drought conditions is modulated by physiological and metabolic responses, including the accumulation of antioxidant metabolites. These mechanisms can be in influenced by nutrient availability, especially phosphorus (P). In European mixed forests, native species with high resource demands often compete with more efficient alien species, whose advantages under abiotic stress can enhance their invasiveness. This study investigates how drought and P availability affect the resilience of two Quercus species differing in stress tolerance and resource use efficiency. Methods We conducted a mesocosm experiment in which the native Q. robur (EO) and alien Q. rubra (RO) were grown under well-watered or drought conditions, with or without P supplementation. Leaf gas exchange and stem water potential were measured, leaves and soil were chemically characterized, and metabolite biosynthesis was investigated using an omics approach. Results During drought, the metabolism of P-supplied EO shifted toward the accumulation of phenylpropanoids, flavonoids, saikosaponins, mannitol, and long-chain fatty acids, which are compounds known for their antioxidant, osmoprotectant, and membrane-stabilizing functions. Conversely, RO plants displayed a more conservative metabolic profile, with limited changes in response to P supply. Under P deficiency, RO accumulated secondary metabolites such as phenylpropanoids and alkaloids, highlighting its ability to withstand with combined nutrient and water stress. Conclusions Our results reveal contrasting adaptive strategies: EO is more susceptible to drought stress, but its resilience can be modulated by P availability, whereas RO maintains more stable physiological and metabolic functions, consistent with higher nutrient-use efficiency. drought abiotic stress phosphorous Quercus spp. metabolism resilience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ecosystems increasingly face more frequent and prolonged extreme conditions due to climate change (IPCC 2023 ). Since drought is one of the leading environmental stress factors, understanding how plants respond to water deficit conditions is crucial to predict future patterns of plant redistribution (Dyderski et al. 2018 ). Under water deficit conditions, plants reduce stomatal conductance (gs) and leaf transpiration (E) to limit the negative effects of soil dryness (Whitehead and Beadle 2004 ). Osmotic adjustment is one of the plant strategies that help sustain gas exchanges and maintain stomata openness as the soil becomes drier (Dichio et al. 2003 ). The modulation of leaf gas exchange under dry soil conditions reduces net photosynthesis (An), leading to excessive production of Reactive Oxygen Species (ROS), which cause oxidative damage to proteins, DNA and lipids (Cruz De Carvalho 2008; Tariq et al. 2017 ). The shift in metabolomic processes regulates the plant response to water deficit, including leaf gas exchange and cell turgor, which are mainly affected by primary metabolites, as well as secondary metabolism like accumulation. During drought, primary metabolites such as sugars (e.g., sucrose, glucose, fructose, raffinose, and mannitol) can osmoregulate cells and contribute to synthesizing antioxidants, such as ascorbic acid (Nishizawa-Yokoi et al. 2008 ; Rodríguez-Sánchez et al. 2010 ). When the risk of photodamage and the impairment of leaf photochemistry arises, increased levels of amino acids (e.g., proline or glutamine) can stabilize the cellular membranes and mitigate the negative effects of ROS accumulation (Ain-Lhout et al. 2001 ; Hu et al. 2015 ). Increasing metabolites with antioxidant and ROS-scavenging activities is a common plant strategy to limit oxidative damage and improve cell membrane stability (Sobrino-Plata et al. 2024 ). Many secondary metabolites act as osmoprotectants, reducing water loss and protecting the photosynthetic apparatus from damage (Escandón et al. 2021 ). To preserve green leaf tissues, plants accumulate phenolic compounds (e.g., flavonoids) and terpenoids, which act as antioxidants and ROS scavengers. Plants can also improve their tolerance to drought by increasing tannin levels as soil water content declines, especially under less favorable conditions (Top et al. 2017 ; Arab et al. 2020 ). Osmotic adjustment, secondary metabolite accumulation and antioxidant mechanisms in plants can be influenced by nutrient availability (Yang et al. 2018 ). According to Kleiner et al. ( 1992 ), fertilized chestnut oaks exhibit greater osmotic adjustment than those growing in nutrient-poor soils, while nitrogen (N) and phosphorus (P) deficiencies promote the accumulation of flavonoids (Stewart et al. 2001 ). Phosphorus, a key component of molecules such as nucleic acids, phospholipids and ATP, is a macronutrient in plant metabolism (Amtmann and Blatt 2009 ). Phosphorus deficiency inhibits photosynthesis, respiration, and the uptake and assimilation of nitrate in plants (Pilbeam et al. 1993 ). According to the literature, P stress promotes the accumulation of organic acids and phenolics (e.g., flavonoids), but it can reduce the levels of most sugars (Hermans et al. 2006 ; Liu et al. 2016 ; Zhang et al. 2021 ). Conversely, Tariq et al. ( 2018 ) reported that Alnus cremastogyne Burkill seedlings exposed to high P availability under drought conditions showed improved photosynthetic rates and enhanced antioxidant defense, with reduced lipid peroxidation. The activity of antioxidant enzymes such as superoxide dismutase, catalase and peroxidase, along with the accumulation of osmolytes and soluble proteins, can be improved by P input. However, drought events may cause nutrient deficiencies even in plants with high soil nutrient availability, as nutrient uptake declines with decreasing soil moisture. In the soil matrix, the phosphate ion primarily moves by diffusion. Thus, dry conditions limit its movement toward the root surface (da Silva et al. 2011 ). The availability of nutrients and water regulates plant growth, species distribution, and competition within ecosystems, ultimately influencing forest composition. Introducing alien species can further affect native biodiversity and biogeochemical processes (Angeloni et al. 2006 ). In Europe, the Northern American species Quercus rubra L. (Red Oak, RO) was introduced by humans at the end of the 17th century as an ornamental and valuable timber tree (Nicolescu et al. 2020 ). Due to its remarkable capacity for natural regeneration, RO is considered potentially invasive, or even fully invasive, in some European countries. In northern Italy, this alien species has colonized much of the native vegetation in the Po River plain ( Querco-carpinetum cenosis), a forest predominantly composed by Quercus robur L. (English Oak, EO) and Carpinus betulus L. (Terzuolo et al. 2006 ). The introduction of RO in the mixed broadleaved forests of Northwestern Italy can change the soil characteristics, affecting the bio-cycling of nutrients, especially the availability of P. In well-developed soils characterized by low P availability, introducing RO could further slowdown P turnover, potentially driving the ecosystem towards a no-return threshold for restoring the original forest composition (Bonifacio et al. 2015 ). Since RO is more effective in dominating nutrient-poor soils and dry conditions (Major et al. 2013 ; Lavnyy and Savchyn 2016) than EO, drought periods and nutrient deficiency may exacerbate competitive interactions between the two species. Rolando et al., ( 2025 ) observed that not only EO was less tolerant to drought, but it also exhibited a slower recovery from repeated severe drought events compared to RO, with its vulnerability being particularly pronounced in nutrient-limited environments. Their findings further indicate that P supplementation mitigates the adverse effects of drought on EO, improving plant recovery. In this context, the aim of this study is to investigate the metabolomic responses of Quercus robur (English Oak, EO) and Quercus rubra (Red Oak, RO) to the combined drought and P scarcity stress factors. Specifically, we hypothesize that i) the P supply enhances the gas exchange rates of EO, with limited effect on RO, due to interspecific differences in resource use efficiency, therefore ii) EO will exhibit better drought tolerance when soil P availability is increased, as higher soil P levels may enhance photosynthetic rates and antioxidant defense mechanisms, which help mitigate oxidative damage; iii) in contrast, the RO response to water scarcity will be not significantly influenced by nutrient availability, since the alien species has inherently low water and nutrient demands and is able to adopt adaptive strategies to cope with low soil fertility and water availability, iv) consequently, increasing P availability may improve the competitive strength of the native EO in relation to RO. To test these hypotheses, we conducted a mesocosm experiment in which seedlings of both EO and RO were transplanted into nutrient-poor soil collected from an area predominantly colonized by RO. To simulate different environmental conditions, we manipulated soil fertility by adding P and applied two distinct water management regimes to assess the effects of varying water availability and nutrient input on plant growth and metabolism. Materials and Methods Plant and soil material The experiment was conducted in a greenhouse at the Department of Agricultural, Forest and Food Science (DISAFA) of the University of Turin, Italy. In December 2021, two-year-old Quercus rubra L. (RO) and Quercus robur L. (EO) seedlings provided by a nursery were transplanted into 3.4 L cylindrical pots (28 cm height x 12.5 cm diameter). A total of 40 plants (20 RO and 20 EO) were employed in the study. The soil used in the experiment (0–20 cm layer) was collected from La Mandria Natural Park in Northwestern Italy (N 45.153213, E 7.581204), an area currently dominated by RO but formerly covered by EO and Carpinus betulus L.. The soil was classified as Oxyaquic Fragiudalfs (USDA, Soil Taxonomy) with the following initial characteristics: pH 4.4, organic carbon 29 g kg − 1 , total nitrogen (N) 2.2 g kg − 1 , available P 5.2 mg kg − 1 (P av ), 4.2 mg N-NO 3 − kg − 1 , and 36 mg N-NH 4 + kg − 1 . During the experiment, plants were maintained in a glass greenhouse under partially controlled climatic conditions, with an average air temperature of 25.4 ± 4.8°C and relative humidity of 67.7 ± 14.6%. Experimental design and treatments Nutrient inputs consisted of two conditions: one representing the original nutrient-poor soil (-P), and another where plants received 39.3 mg P kg − 1 provided as KH 2 PO 4 (+ P), on May 18th 2023 (Day of year - DOY- 138). Twenty-five days after nutrient inputs, 10 EO and 10 RO plants were regularly well-watered (denoted as WW), while the remaining 20 plants were subjected to water deficit conditions (denoted as D), followed by rewatering (RW). The experimental design followed a split-plot layout with five replications (R). The main plots consisted of the two plant species (SP, EO and RO), the subplots included two water management regimes (WM), and the sub-subplots involved two nutrient input levels (NI). The WW condition was maintained through daily irrigation, ensuring the field capacity. At the same time, 70% of the water lost from the previous day was restored in D plants until DOY 178 (15 days of drought). The D plants were then rewatered to reach and keep the field capacity of approximately 80% until DOY 193 (15 days of rewatering). All pots were weighed daily to adjust the WM accordingly. Soil analysis Soil samples were collected on DOYs 163 and 178 to assess variations in total carbon (TC), total nitrogen (TN), and available P (P av ) following the drought period. Only two soil samplings were carried out during the plant growth to minimize disturbance of the rhizosphere during the water stress phase. Samples were dried and sieved at 2.0 mm. A portion was ground to 0.5 mm for TC and TN analysis using high-temperature combustion in an elemental analyzer (Vario Isotope Select; Elementar Analysensysteme GmbH, Hanau, Germany). Available P was quantified according to Olsen et al. ( 1954 ). The extracted P was then measured colorimetrically using the malachite green method (Ohno and Zibilske 1991 ). Measurement of leaf gas exchange and stem water potential Leaf gas exchanges (gs, An and E) were measured on fully expanded leaves exposed to direct sunlight using a portable infrared gas analyzer (ADC-LCPro + system, The Analytical Development Company Ltd, Hoddesdon, UK). CO 2 levels were maintained at greenhouse conditions (400–450 ppm). Leaf gas exchange was monitored every two days in the morning (between 10:00 am and 12:30 pm) on three to five plants in each treatment (one leaf per plant) for the whole duration of the experimental trial. The Ψ stem was measured on leaves collected before imposing water stress conditions (DOY 163), after 4 (DOY 167) and 11 (DOY 174) days of drought, at the end of the water scarcity phase (DOY 178), and after 15 days of rewatering (DOY 193). Leaves were placed in humidified, aluminum foil-wrapped plastic bags for about 15 min prior to excision. After excision, the leaves were allowed to equilibrate for an additional 10 minutes before the water potential was measured using a Scholander-type pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, CA, USA). On DOY 178, the relative chlorophyll content (SPAD values) was measured with a SPAD meter (SPAD 502 Plus Chlorophyll Meter, Spectrum, Plainfield, IL, USA) to assess chlorophyll levels after drought. The Performance Index (PI) parameter was measured in each plant using a continuous excitation Pocket PEA fluorometer equipped with black leaf clips (Hansatech Instruments Ltd, King’s Lynn, UK). Leaves were dark-adapted for at least 30 minutes before measurement. Chemical characterization of leaves Leaves collected on DOYs 163 (watering initial phase), 167, 174, 178 (drought) and 193 (rewatering) were dried and grounded to 0.5 mm. Carbon (C) and Nitrogen (N) contents were determined by high-temperature combustion using an elemental analyzer (Vario Isotope Select; Elementar Analysensysteme GmbH, Hanau, Germany). For P analysis, samples were digested with 12.5 mL of H 2 SO 4 and 2.5 mL of HClO 4 , and quantified colorimetrically using the malachite green method (Ohno and Zibilske 1991 ). Metabolomics analysis of leaves The metabolomics analysis was conducted only on DOY 178 plants, where leaves were collected with liquid nitrogen and sampled at -20°C. Lyophilized plant tissue (100 mg) was dissolved in 1 mL of 80% methanol (v/v) acidified with 0.1% formic acid (v/v) and mechanically extracted using a Polytron PT 1200E homogenizer for 3 min at maximum power. The extracts were centrifuged at 5000 g for 15 min and filtered through a 0.22 µm cellulose membrane filter into vials for liquid chromatographic analysis. Untargeted profiling of samples was performed using the 6560-drift tube-ion mobility-quadrupole-time of flight-high resolution mass spectrometer (DTIM-UHPLC-QTOF-HRMS; Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was achieved under a water-acetonitrile (both LC-MS grade, from Sigma-Aldrich, Milan, Italy) gradient elution (6–94% acetonitrile in 32 min), flow rate of 0.2 mL/min and injection volume of 6 µL, using 0.1% (v/v) formic acid as phase modifier on an -Agilent Poroshell 120 PFP column (100 mm × 2.1 i.d., 1.9 µm particle size) -Agilent Zorbax Eclipse plus C18 analytical column (50 × 2.1 mm, 1.8 µm). The QTOF mass analyzer operated in positive mode (ESI+) for both MS and MS/MS acquisition with nitrogen as both sheath gas (12 L/min and 315 ◦C) and drying gas (14 L/min and 250 ◦C). The nebulizer pressure was 45 psi, nozzle voltage was 350 V, and the capillary voltage was 4.0 kV. For MS acquisition, the full scan mode was performed within range of the m/z 100–1200 (1 spectra/s), mass resolution of 30,000 full width at half maximum (FWHM), m/z = 200. The data-dependent mode was performed for precursor fragmentation (10, 20, and 40 eV) and acquisition of MS/MS data from QC samples, with a mass resolution of 30,000 (FWHM), selecting 8 precursors per cycle (1 Hz, m/z 80–1200, positive polarity, and active exclusion after 2 spectra). The Processing of the chromatograms was carried out using the MassHunter Qualitative Analysis software (version B.06.00, Agilent Technologies). The collected data (.d files) were processed using MS-DIAL software (version 4.70) for automatic peak finding, LOWESS normalization, and annotation via spectral matching, exploiting the comprehensive BMDMS-NP, Fiehn/Vaniya natural product library, and GNPS databases. The mass range 100–1200 m/z was searched for features with a minimum peak height of 10,000 cps, using an accurate mass tolerance for peak centroiding of 0.05 and 0.1 Da, for MS and MS/MS, respectively. Retention time information was excluded from the calculation of the total score. The identification step was based on mass accuracy, isotopic pattern, and spectral matching. These criteria were used to calculate a total identification score, using a minimum cut-off value of 70%, considering the most common HESI + adducts. The data annotation and MS/MS structural confirmations were conducted using MS-DIAL software - version 4.90 - (Tsugawa et al. 2015 ) for automated peak finding (against pooled QC) and putative annotation via spectral matching using the publicly available databases (e.g., BMDMS-NP, Fiehn/Vaniya natural product library, and GNPS). Compound annotation was carried out considering mass accuracy, isotopic profiles, and MS/MS spectral matching (where available), using a total identification score with a minimum cut-off of 70% to reach level 2 of confidence in identification (Blaženović et al. 2019 ). The mass range of 100–1200 m/z with a minimum peak height of 10,000 cps was considered, using accurate mass tolerances of 0.05 Da for MS and 0.1 Da for MS/MS. Data collection and statistical analysis Plant physiological data were recorded every two days from June 11 (163 DOY) to July 11 (DOY 193), 2023, to evaluate the status of WW, D and RW plants. The watering conditions before imposing drought (DOY 163), two representative time points during water stress (DOYs 167 and 174), the end of drought (DOY 178), and rewatering (DOY 193) were chosen to determine leaf gas exchanges, Ψ stem and chemical characterization of leaves (C, N, P). All the metabolomics (DOY 178) and soil (DOYs 163 and 178) data were statistically analyzed. For variables measured at multiple time points, the time (T) factor was incorporated into the analysis. All the statistical analyses were conducted using R 4.3.1 (R Code Team, 2022). The statistical analysis was organized into three distinct phases: baseline measurements (DOY 163), drought conditions (DOYs 167, 174, 178), and rewatering (DOY 193). First, a two–way variance (ANOVA) analysis with a linear model was performed for each physiological parameter and leaf chemical element recorded at DOY163, using the stats package (R Core Team 2023 ). The same statistical approach was applied to soil results, separating DOY 163 and DOY 178. During the drought phase, a four-way ANOVA with a mixed effect model was performed on each physiological parameter and leaf chemical element measured using the nlme package (Pinheiro et al. 2007 ). The fixed factors in this model were SP, WM, NI and T, while replicates (R) – represented by 3 out of 5 replicates – were treated as a random factor. Consequently, a three-ways ANOVA with a linear model was conducted on each parameter recorded on DOY 193 and for SPAD and performance index measurements recorded on DOY 178, again employing the stats package (R Core Team 2023 ). The distribution of measured variables was checked for normality and homogeneity of variance using the Shapiro–Wilk and Levene's tests, respectively. When ANOVA assumptions were violated, data were log-transformed, and the tests were repeated on the transformed data. The post–hoc test performed after each ANOVA was the Tukey's honestly significant difference (HSD) at a significance level of p-value < 0.05, with the multcomp packages (Hothorn et al. 2008 ). Afterwards, a multivariate analysis of variance (MANOVA) was conducted to assess the influence of SP, WM and NI on leaf gas exchange parameters and Ψ stem measured at the end of water stress (DOY 178) and rewatering (DOY 193). The same analysis was conducted on chemical data from leaves sampled on DOYs 178 and 193. Finally, the apparent P recovery (APR) of EO and RO leaves was calculated as follows: The metabolomics data were elaborated using the software Mass Profiler Professional 12.6 (Agilent Technologies). The raw data were log2 transformed, 75th percentile normalized, and baselined against the median of each compound. Sample patterns were investigated using unsupervised hierarchical cluster analysis (HCA) with Euclidean distance and Ward's linkage rule. Furthermore, a supervised ANOVA multi-block orthogonal partial least squares (AMOPLS) was carried out using the package rAMOPLS on R (v 4.2.1.) to select Variable Importance in Projection (VIP 2 ) markers associated with different treatments. The AMOPLS statistical significance was set at α = 0.01 and validated with 100 permutation processes. The results were expressed as the Relative Sum of Squares (RSS), representing the percentage of variability attributed to each factor; RSS p-value, statistical significance; and block contribution, in percentage, associated with each effect. Afterwards, the VIP markers associated with different factors were considered to build the pathways analysis, comparing [WW + P] vs. [WW -P], [D -P] vs. [WW -P], and [D + P] vs. [WW -P], separating for EO and RO species. The metabolite changes were defined using log Fold Change analysis comparing all the treatments with control well-watered and under phosphorus deficiency. Results Soil On DOY 163 (watering phase), the soil total C (TC) under EO -P (22.71 ± 2.71 g kg − 1 ) was similar to that of RO -P soil (23.39 ± 2.95 g kg − 1 ) (Fig. 1 a). However, with P input, TC differed between EO (25.03 ± 4.07 g kg − 1 ) and RO (22.74 ± 2.57 g kg − 1 ) with a remarkable SP*NI interaction effect on DOY 163. Conversely, drought nullified TC differences between EO + P and RO + P, though notable differences were still observed between soils under different species and low nutrient availability. For RO specifically, soil TC in WW -P decreased from DOY 163 (23.39 ± 2.95 g kg − 1 ) to DOY 178 (19.28 ± 1.96 g kg − 1 ), while the drought event increased soil TC under -P conditions (24.68 ± 3.08 g kg − 1 ) compared to WW at DOY 178. The SP and NI did not impact the soil TN during the watering phase, but nutrient content in the soil was affected by the interaction of SP*WM*NI during drought. The main difference was observed between EO D -P (1.72 ± 0.18 g kg − 1 ) and RO D -P (2.00 ± 0.19 g kg − 1 ), while P addition suppressed the differences observed between SP in D plants (Fig. 1 b). On DOY 178, as reported for soil TC, the TN content in RO D -P was higher than in RO WW -P soil (1.64 ± 0.11 g kg − 1 ). The available P pool was below 5 mg kg − 1 in -P soils, independently of WM and T. Nutrient availability increased with the addition of P, ranging between 28.58 ± 13.44 mg kg − 1 in RO WW to 43.10 ± 10.96 mg kg − 1 in EO D. In fact, ANOVA highlighted a significant effect of NI at both DOY 163 and 178 (Fig. 1 c). Physiological parameters During the initial watering phase, the Ψ stem values of EO -P and EO + P were − 0.40 ± 0.07 MPa and − 0.38 ± 0.10 MPa, respectively (Fig. 2 a). Similar values were measured for RO -P and RO + P without statistical differences on DOY 163. After 15 days of rewatering, Ψ stem of D plants was comparable to that of WW. However, ANOVA analysis highlighted a remarkable effect of SP and NI, revealing that the Ψ stem of EO was higher than that of RO. Regardless of species, RW + P plants had lower Ψ stem than RW -P. After 25 days from nutrient input, and prior to the imposition of drought conditions, adding P increased the gs by 112.5% compared to EO -P (Fig. 2 b). Conversely, gs in RO was not affected by NI. The gs in water-stressed EO and RO + P was 0.00 ± 0.00 mol H 2 O m − 2 s − 1 , while EO D -P and RO D -P exhibited minimal values of 0.02 ± 0.02 mol H 2 O m − 2 s − 1 and 0.01 ± 0.00 mol H 2 O m − 2 s − 1 , respectively. ANOVA analysis showed a significant effect of SP*WM*NI during D conditions, highlighting the role of P input in reducing gs in drought-stressed EO. Moreover, the SP*WM*NI interaction revealed interspecific differences between EO WW and RO WW from DOY 167 to 178. After 15 days of rewatering, both EO and RO fully recovered their gs (DOY 193). Similarly, at DOY 163, P input did not affect the An of RO, but it increased the An of the native species by 101.1% compared to EO -P (Fig. 2 c). The minimum An in both SP was measured on DOY 178. During water stress conditions, ANOVA denoted a significant effect of SP*WM*NI interaction, indicating interspecific differences in WW + P plants. Regardless of the time, P addition increased An in EO, while no effect was detected on RO. On DOY 178, the An of EO D + P plants (0.21 ± 0.07 µmol CO 2 m − 2 s − 1 ) was lower than that of EO D -P (2.25 ± 2.00 µmol CO 2 m − 2 s − 1 ). The rewatering restored An in D plants to levels comparable to WW plants in both species. ANOVA showed that SP was the only factor affecting An on DOY 193, with EO exhibiting higher values than RO. On DOY 163, P input affected all gas exchanges, including E (Figure S1 ). Results showed that P addition increased E in EO but did not affect RO. Drought conditions reduced E to 1.01 ± 0.44 mmol H 2 O m − 2 s − 1 in EO -P, and 0.33 ± 0.04 mmol H 2 O m − 2 s − 1 in RO -P. The lowest values were observed in + P plants, with EO D showing 0.20 ± 0.02 mmol H₂O m − 2 s − 1 and RO D at 0.17 ± 0.06 mmol H₂O m − 2 s − 1 . After 15 days of rewatering, E in RW plants remained lower than in WW, regardless of SP and NI. ANOVA identified remarkable differences between SP, showing that E in EO was higher than in RO, consistent with the other gas exchange parameters. On DOY 178, ANOVA on SPAD and PI revealed significant effects of SP and WM (Figure S2a and b). Both parameters were higher in EO (39.1 ± 3.4 SPAD; 4.5 ± 1.9 PI) compared to RO (36.1 ± 3.0 SPAD; 3.6 ± 1.7 PI). Regardless of SP, D plants exhibited reduced SPAD (36.2 ± 3.8) and PI (2.7 ± 1.1) compared WW plants (SPAD: 38.9 ± 2.5; PI: 5.5 ± 1.3). MANOVA on DOY 178 data showed that SP ( p-value = 4.54e-04), WM ( p-value = 3.76e-12), NI ( p-value = 8.84e-08) and all their interactions significantly affected leaf gas exchanges and Ψ stem (Table S1 ). Interspecific differences were observed between well-watered EO and RO, irrespective of NI. Profile plots separated EO WW -P and EO WW + P from RO WW -P and RO WW + P (Figure S3a). In contrast, the physiological status of EO D + P was similar to RO D + P, while EO and RO under combined water and nutrient stress were classified separately. On DOY 193, results displayed a remarkable effect of SP ( p-value = 7.95e-05), WM ( p-value = 2.80e-03) and NI ( p-value = 3.22e-03), as well as WM*NI ( p-value = 3.21e-02) and SP*WM*NI ( p-value = 1.82e-03; Table S1 ). In the profile plot, EO RW + P grouped with RO RW -P and RO RW + P, while during rewatering, RO WW + P also joined this group. EO RW -P exhibited a physiological status similar to EO WW, independent of NI (Figure S3b). Leaves chemical characterization At DOY 163 (watering initial phase), the C content in -P plants (EO -P: 451.1 ± 5.4 g kg − 1 ; RO -P: 456.1 ± 5.1 g kg − 1 ) was higher than in + P plants (EO: 447.8 ± 8.1 g kg − 1 ; RO: 451.0 ± 6.4 g kg − 1 ). Notably, NI was the only factor with a remarkable effect on C content at DOY 163 (Fig. 3 a). From DOY 167 to 178, EO increased its leaf C content regardless of WM and NI, reaching the highest value in WW + P (459.6 ± 4.7 g kg − 1 ) on DOY 178. No differences in C content were observed in RO, during drought. Although the SP*WM interaction did not significantly affect C content during drought, ANOVA indicated that WM alone had a significant effect, with D leaves exhibiting higher C content than WW plants. As reported for DOY 163, +P plants had higher C content than -P plants after rewatering. At DOY 163, the N content in EO -P leaves (25.8 ± 2.6 g kg − 1 ) was similar to that in RO -P (22.6 ± 2.3 g kg − 1 ) (Fig. 3 b). P addition did not affect N content in both SP. However, from DOY 163 to 178, ANOVA displayed a remarkable effect of SP*NI*T. In EO, the main differences were observed between 167 and 178, where -P plants showed an increase in N content from 24.8 ± 0.6 g kg − 1 to 26.8 ± 3.7 g kg − 1 in WW plants and from 26.8 ± 2.2 g kg − 1 to 28.2 ± 3.1 g kg − 1 in D plants. During the same period, RO + P increased N content to 26.0 ± 2.5 g kg − 1 in WW plants and 22.2 ± 1.3 g kg − 1 in D plants. After rewatering, the N content differed between species, with EO leaves having higher nutrient levels than RO. At DOY 163, the P content in the leaves of -P plants was 885.8 ± 99.5 mg kg − 1 for EO and 856.8 ± 62.8 mg kg − 1 for RO (Fig. 3 c). NI significantly affected the nutrient content in the plants, resulting in a higher P level in EO (1812.4 ± 169.2 mg kg − 1 ) compared to RO (1163.9 ± 154.9 mg kg − 1 ). ANOVA highlighted statistical differences between EO WW + P and RO WW -P. During drought conditions, the P content of EO + P remained higher than that of RO + P, regardless of WM. However, after rewatering, RO RW showed a lower P content than RO WW. At the same time, EO displayed no significant differences between RW and WW, maintaining higher values than RO regardless of NI. After P addition, EO absorbed more P than RO. Specifically, at DOY 163, the APR for EO was 8.5 ± 1.8%, while the apparent P recovery for RO was 3.6 ± 3.2% (Fig. 3 d). Drought did not affect the APR of EO, but it reduced the RO APR to -2.3 ± 3.4% on DOY 167. Consequently, from DOY 174 to 178, the RO APR values returned to the levels observed at DOY 163. Following rewatering, ANOVA revealed a significant effect of plant species on APR, consistent with the observations at DOY 163. At the beginning of the experiment, the C/N ratio of EO leaves (17.5 ± 1.8 in -P) was lower than that of RO (20.4 ± 2.2 in -P). ANOVA did not reveal a significant effect of NI on the C/N ratio at the initial watering phase. The interspecific difference persisted during drought and after rewatering (Fig. 4 a). The C/P ratio of EO -P leaves (516.8 ± 53.4) was comparable to that of RO -P leaves (536.1 ± 40.8) at DOY 163 (Fig. 4 b). However, P addition resulted in differences between species, with a decline of the ratio of EO + P down to 248.7.1 ± 26.5, while RO + P measured 390.1 ± 58.3. ANOVA displayed a significant effect of the interaction SP*NI at DOY 163, during the drought phase, and after 15 days of rewatering. From DOY 167 to 178, the C/N ratio increased regardless of SP, WM and NI. At DOY 163, the nutrient-stressed native and alien species exhibited similar N/P ratios. The P input decreased the N/P ratio to 13.1 ± 2.2 in EO and 18.9 ± 2.9 in RO (Fig. 4 c). ANOVA indicated a significant effect of SP*NI during drought and after rewatering, confirming the earlier observations based on DOY 163 results. Moreover, on DOY 193, the significant effect of SP*WM revealed that RO RW had a higher N/P ratio than both EO RW and WW plants. Metabolic response of EO and RO under nutrient and drought stress The metabolomics analysis was conducted on the DOY 178 plant stage using an untargeted approach, allowing us to annotate 1744 metabolites putatively. The list of identified features, including pathway classification, specific metabolite ontology, their abundances, and MS1 isotopic and MS/MS spectra (where they are confirmed), is listed in the supplementary Table S3 . Analyzing features obtained from the profiling of EO and RO leaves under nutrient and drought stress, the approach perfectly identified secondary metabolites as the most abundant class, followed by primary metabolites (e.g., fatty acids and lipids, carbohydrates, amino acids, and nucleotides, Table S3 ). To assess the overall similarities/dissimilarities among samples influenced by different factors (i.e., water management, nutrient supplementation, and species), a hierarchical clustering analysis (HCA) was performed. As reported in Figure S5 , the species factor resulted in the most differences, cauterizing into two branches. Concerning EO species, the HCA analysis clusters samples based on both nutrient supplementation and water management, suggesting specific metabolite profiles and abundances along treatments. Conversely, RO species reported less linearity between metabolic response and treatments, confirming our more resilient species hypothesis. Based on the unsupervised results, highlighting the species factor as the primary contributor to the overall variance, a supervised ANOVA multi-blocking orthogonal projection to latent structures discriminant analysis (AMOPLS-DA) was performed to focus on the detail about the nutrient and water management factors on the data variance, for both EO and RO species separately (Table 1 ). Table 1 Relative variability and block contributions of the ANOVA multi-blocking orthogonal projection to latent structures discriminant analysis (AMOPLS) of two Quercus spp metabolomic data. species (English Oak, EO; Red Oak, RO) affected by different water management (WM), phosphorus nutrient input (NI), and interaction between WM x NI. RSS: Relative sum of squares, Tp1–3: predictive components, To: orthogonal component. EO AMOPLS model Effect Name RSS RSS p-value R2Y p-value Tp1 Tp2 Tp3 To1 WM 20.9% 0.01 0.01 97.2% 0.2% 1.4% 18.4% NI 14.1% 1.00 0.01 0.8% 99.1% 1.7% 22.7% WM x NI 7.6% 1.00 0.01 1.0% 0.3% 94.5% 27.8% Residuals 57.5% - - 1.1% 0.4% 2.4% 31.0% RO AMOPLS model WM 12.8% 0.01 0.01 95.6% 1.7% 4.4% 23.1% NI 8.2% 1.00 0.01 1.4% 94.6% 4.7% 24.7% WM x NI 6.0% 1.00 0.01 1.4% 1.8% 85.8% 25.5% Residuals 73.0% - - 1.5% 1.9% 5.1% 26.7% Our results reported that the WM factor exerted the greatest influence on EO and RO species, showing 20.9% and 12.8% relative sum of squares (RSS) values, respectively. The discrimination between well-watered and drought-stress plants was perfectly made by component 1 (tp1). Nutrient supplementation and its interaction with WM were the secondary factors that influenced metabolic response in both species and discriminated using components 2 and 3 (tp2 and tp3), respectively. Interestingly, the supervised AMOPLS model highlighted a higher percentage of the observed metabolomic differences that could not be explained by our factors in RO than EO species, supporting the hypothesis of better correlation between treatments and metabolic response in EO as firstly suggested by HCA. Afterwards, the variable importance in the projection (VIP) analysis was carried out to identify biomarkers associated explicitly with differentiating samples based on factors. The 50 most discriminant metabolites per factor were selected and reported in the supplementary table, together with log fold change values for both EO ( Table S4 ) and RO ( Table S5 ). The specific metabolic pathways were then built using these VIP markers, considering biosynthetic pathways (Fig. 5 a) and further going into detail about primary (Fig. 5 b) and secondary metabolites biosynthesis (Fig. 5 c). Regarding the modulation of EO biosynthesis pathways, phosphorus supplementation under WW conditions down-modulated primary and secondary metabolite synthesis and nitrogen-, oxygen- and sulfur-containing metabolites (Fig. 5 a). The most affected metabolite classes belonging to primary metabolites were organic amino compounds and lipids (Fig. 5 b). Secondary metabolites were isoprenoids, nitrogen-, oxygen- and sulfur-containing metabolites (Fig. 5 c). In contrast, the drought stress up-modulated the biosynthesis of secondary metabolites, where the accumulation of phenylpropanoids, alkaloids, and isoprenoids was mainly represented (Fig. 5 c). Additionally, lipid and fatty acid synthesis were also affected, reporting a reduction in steroids and short- and medium-chain fatty acids, alongside an increase in long-chain fatty acid synthesis (Fig. 5 b). Interestingly, phosphorus supplementation under drought-stress conditions led to an increased abundance of specialized metabolites in plant tissues (Fig. 5 a), fully addressing the synthesis of phenylpropanoids. This evidence suggests a synergistic effect of phosphorus input in enhancing endogenous bioactive compounds to cope with abiotic stress. Concerning the modulation of RO biosynthesis pathways, nutrient and water management factors produced similar modulation patterns, differing mainly in the abundance of specific classes of metabolites. Indeed, the most relevant metabolite classes were secondary metabolites, followed by nitrogen-containing compounds and primary metabolites (Fig. 5 a and 5 b). Regarding secondary metabolites biosynthesis, drought stress selectively increased the accumulation of phenylpropanoids and alkaloids while reducing isoprenoids as a response strategy. At the same time, phosphorus input under the same condition maintained a high level of phenylpropanoids and was accompanied by the up-modulation of isoprenoids synthesis (Fig. 5 c). Profiling of phenylpropanoids composition of EO and RO under nutrient and drought stress Since the primary metabolic response of EO and RO under nutrient and water management stress factors was secondary metabolite accumulation, a deep clarification of specific subclasses of phenylpropanoid compounds was plotted based on log fold change values deriving from the pairwise comparison between treatments and control (Fig. 6 ). Regarding the EO species (Fig. 6 a), drought conditions, and at a higher extent combined with the phosphorus supplementation, increased flavonoid O -glycosides, O -methylated flavonoids, coumarins and derivatives, flavones, and hydrolyzable tannins. Specifically, the enhanced production of coumarins under drought + phosphorus was also confirmed by a clear down-accumulation of coumaric acid, their synthetic precursors. Focusing on RO species (Fig. 6 b), drought stress induced the accumulation of flavonoid O -glycosides, neoflavans, isoflavones, and high hydrolyzable tannins. As previously observed, the RO species resulted in less sensitivity to the phosphorus input under both well-watered and drought conditions. Discussion Interspecific plant differences due to nutrient status under watering conditions To understand the responses of EO and RO to water stress under varying levels of P availability, it is essential to assess the nutritional and physiological status of well-watered species under -P and + P soil. Twenty – five days after the P supply, before imposing a mild and long drought event, the stem water potential (Ψ stem ) and gas exchange value of EO and RO were consistent with the previous findings of Gori et al. ( 2023 ) and Cotrozzi et al. (2016), on well-watered Q. ilex and Quercus spp., respectively. Our results showed that Ψ stem was unaffected by P input, while increased gas exchange levels were observed for EO + P, in line with the findings from Rolando et al. ( 2025 ). The relationship between net photosynthesis and P has been studied in plants: P can modulate the assimilation of N for the synthesis of ribulose-1,5-bisphosphate carboxylase, the primary enzyme that catalyzes the first step of carbon dioxide fixation in photosynthesis (Spreitzer and Salvucci 2002 ; Warren et al. 2005 ). Moreover, P is a component of the adenosine triphosphate synthesis (ATP) and nicotinamide adenine dinucleotide (NADPH), which are, respectively, the primary sources of energy and reducing agents needed to drive the biochemical transformations of carbon dioxide into organic compounds in the Calvin cycle (Farooq et al. 2012 ). As a matter of fact, Kayoumu et al. ( 2023 ) observed that P shortage reduces the photosynthetic capacity and leaf transpiration. Therefore, increasing EO gas exchanges following P input could suggest that this soil element is the limiting factor for EO growth. Conversely, gas exchanges of RO were not modulated by soil P addition, probably because the alien species did not experience a P deficit, even in the depleted soil. This may be attributed to RO having a higher P use efficiency than EO, as previously observed (Rolando et al. 2025 ). This interspecific difference in resource utilization capacity could also be extended to photosynthesis. In fact, under adequate soil water content and regardless of P availability, the gas exchange rates of RO are lower than those of EO, suggesting a reduced CO₂ assimilation of RO. The native species absorbed and allocated more P in leaves than the alien species; thus, the apparent P recovery confirmed that EO's P demand was higher than RO's. Nevertheless, the soil P av pool did not show significant differences between + P soils under different species. This may be related to the dramatic soil low P levels: although a large amount of P (110 kg/ha) was added to mitigate nutrient deficiency in plants, a large part of the added P may have been retained by the mineral phase and released when needed, leading to equalize the P av pool in soil, independently of P species (Rolando et al., 2025 ). Evidence of this process is provided by the increase in total organic soil C, which suggested root exudation under our experimental conditions. Root exudates (e.g., organic acids, enzymes, phenolic compounds) can enhance P availability in soils, mobilizing P from stable pools (DeLuca et al. 2015 ; Menezes-Blackburn et al. 2016 ; Rafi et al. 2019 ). Preece et al. ( 2021 ) observed the same trend in + P soil under Q. Ilex . According to our first hypothesis, EO exhibited increased gas exchange following P input, likely due to its lower resource use efficiency than RO. This confirms that P is a limiting factor for EO growth and its competitive ability against RO in highly weathered soils. Effects of drought on plants as a function of soil P addition The combined application of physiological measurements and untargeted metabolomics shed light on plant response to drought and nutrient stresses, indicating that both EO and RO triggered contrasting effects at both physiological and metabolic levels, primarily depending on water availability. Water deficits disrupt crucial processes in plants, such as photosynthesis, transpiration, and nutrient uptake, with the effects varying depending on the severity of stress, plant genotype, and growth stage (Kirkham 2005 ). Under drought conditions, plants often produce ROS, leading to oxidative damage, carbohydrate and protein synthesis disruption, and lipid metabolism, which can ultimately cause cell death (Feng et al. 1994 ; Blum 2005 ). In response to these stresses, plants have evolved various adaptive mechanisms to mitigate damage, such as limiting transpiration through stomatal closure, accumulating osmoprotective solutes in the cytoplasm, and activating pathways that enhance water uptake, improving stomatal conductance, and increasing water-use efficiency (Mitchell et al. 2008 ; Peñuelas and Staudt 2010 ). Concerning EO, drought significantly reduced the stem water potential, but the decrease was less pronounced under P-limited conditions. This finding is consistent with previous studies, such as those by Burman et al., ( 2009 ), which highlighted that P fertilization could help mitigate the negative effects of water stress by promoting plant growth and leaf area expansion, increasing water demand and accelerating soil water consumption. However, the increased water consumption in P-fertilized plants could lead to a more significant reduction in water status during drought stress. Despite this, plants with higher P availability tend to show better recovery after rehydration, improved photosynthetic efficiency, and higher enzymatic activity, which is supported by our findings of improved gas exchange in EO with P supplementation during the initial watering phase (Burman et al. 2009 ). As drought progressed, EO plants showed a clear reduction in net photosynthesis, mainly when P was present, coinciding with stomata's closure, limiting CO₂ uptake. In contrast, EO -P plants maintained higher gas exchange rates during drought, indicating a potential trade-off between maintaining higher photosynthesis and tolerating stress. Although during watering conditions, P supply promoted photosynthesis by enhancing the biosynthesis of the primary source of energy (ATP), under drought, plants closed stomata, limiting the absorption of CO 2 from the atmosphere. This could be due to an imbalance between ATP and CO 2 concentrations, affecting the photosynthetic process. These physiological changes in EO were mirrored in the metabolomic profile, where we observed a shift towards synthesizing primary and secondary metabolites that could help mitigate oxidative damage and osmotic stress under the reduced water availability conditions (Street et al. 2006 ; Peñuelas and Staudt 2010 ). In this species, drought-induced stress led to an increase in the biosynthesis of phenylpropanoids, particularly hydroxylated phenylpropanoids (e.g., [6-[3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl and E)-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoate and dihydroxy B-ring-substituted flavonoids (e.g., luteolin 7-glucuronide and scutellarin). These metabolites are well-known for their antioxidant properties, as they scavenge ROS and help reduce oxidative damage (Gould et al. 2002 ; Hernandez et al. 2006 ; Agati et al. 2007 ; Hernández et al. 2009 ; Agati and Tattini 2010 ; Ferreres et al. 2011 ; Pollastri and Tattini 2011 ). Interestingly, adding P to drought-stressed EO led to the upregulation of primary and secondary metabolites, which act as antioxidants and osmoprotectants (Dos Santos et al. 2022 ; Ahmad et al. 2023 ). In particular, P addition under drought conditions overstimulated the production of phenolic compounds, including flavonoid O-glycosides, O-methylated flavonoids and coumarins. These compounds likely contribute to maintaining turgor pressure by decreasing osmotic potential in vacuoles, which helps mitigate the effects of water stress (Almeida et al. 2020 ). In addition to phenylpropanoids, an accumulation of saikosaponins, a class of triterpene saponins, was detected. Saikosaponins are known for their ability to scavenge ROS and protect cellular membranes from peroxidation, which is especially important under drought stress (Liu et al. 2005 ; Zhu et al. 2009 ). This finding supports the hypothesis that EO, when supplied with P, activates metabolic pathways that enhance its ability to cope with drought, possibly by increasing the production of stress-related metabolites. Among primary metabolites, increased levels of mannitol were found in the metabolic profile of EO under water stress and P supplementation. Mannitol helps stabilize proteins and cellular structures by interacting with the hydration shell around them and scavenging ROS produced in higher quantities under drought stress (Patel and Williamson 2016 ). Similarly, the metabolomic data also revealed an accumulation of 1,3,5-tricaffeoylquinic acid, a bioactive compound involved in antioxidant defense. Previous studies have shown that caffeic acids and their derivatives, such as 1,3,5-tricaffeoylquinic acid, are crucial in protecting plants from oxidative stress (Harrison et al. 2008 ; Kim et al. 2012 ). This metabolite may help protect EO from ROS damage, complementing the physiological data that suggest that EO’s photosynthetic system is less impaired under drought when P is available. Moreover, the input of P reprogrammed lipid and fatty acid metabolism in drought-stressed EO. The observed increase in long-chain fatty acids in EO + P under drought conditions has been previously interpreted as a plant strategy to maintain membrane integrity, potentially limiting cellular turgor loss (Ullah et al. 2022 ). Fatty acids are a crucial component of cellular membranes (Ruiz-Lopez et al. 2015 ), and their accumulation contributes to membrane rigidification (López-Pérez et al. 2009 ). In addition, EO's metabolomic profile revealed increased levels of gibberellic acid (GA). This plant hormone is known to be involved in response to various abiotic stresses, including water scarcity. GA helps regulate membrane permeability, osmolyte accumulation, and antioxidant systems, all enhancing stress tolerance (Shah et al. 2023 ). Our findings of increased GA accumulation under drought with P supplementation suggest that P might promote EO’s ability to manage stress through the modulation of plant growth and metabolism, which is reflected in the enhanced photosynthetic activity observed during the initial watering phase. On the other hand, RO data displayed a different response from EO for both physiological and metabolomic analyses under drought conditions. Regardless of P availability, water stress negatively impacted stem water potential and gas exchange values. Similarly, P supply did not lead to a higher production of defense secondary metabolites compared to P-limited conditions. In the absence of P, the metabolomic analysis displayed a remarkable increase in phenylpropanoids and alkaloids, suggesting that RO has a metabolic system capable of handling both water and nutrient limitations, possibly by activating specific stress-related pathways even under low P conditions. However, even without drought imposition, high levels of hydrolyzable tannins were detected in RO + P leaves. This points out the plant’s intrinsic capability to upregulate the synthesis of hydrolyzable tannins under adequate soil nutrient availability (Salminen et al. 2004 ). The lack of a strong effect of P on RO nutrient uptake during drought, particularly in terms of P accumulation, reinforces the idea that RO is less dependent on P compared to EO. This contrasts with the more pronounced effects of P on EO physiological and metabolic responses. In EO, P input acted as a stabilizer, enhancing both physiological and metabolomic responses to drought. Conversely, metabolic changes in RO were less influenced by P supplementation, suggesting that RO may operate at a baseline metabolic level that allows it to thrive in a wider range of environmental conditions. The metabolic reprogramming of both species under water-limited conditions was consistent with soil-plant nutrient bio-cycling. As previously shown by metabolomic data, EO was more responsive than RO to P addition, reflecting a greater P leave accumulation. On the other hand, P content in RO -P leaves was not linked to P input. Nevertheless, these results were observed regardless of water availability. In contrast to previous studies, the unaltered P concentration of RO leaves under water and nutrient-limited conditions and the increased content of soil total organic carbon suggested a higher root exudation responsible for P mobilization from the stable pool (DeLuca et al. 2015 ; Menezes-Blackburn et al. 2016 ; Rafi et al. 2019 ). This could be one of the competitive advantages of RO in dry and low fertility soils, as a strategy to gain an advantage over the native vegetation. Root exudates can help mobilize nutrients in the rhizosphere, offering a competitive advantage in nutrient-poor environments (Gargallo-Garriga et al. 2015 ). Moreover, the fact that RO leaves did not show a significant increase in P accumulation under drought conditions supports the idea that RO may have evolved to be less reliant on P input compared to EO, possibly due to its ability to regulate nutrient uptake and allocation more efficiently. This behavior aligns with the metabolic shifts observed in RO, where the accumulation of secondary metabolites likely supports its stress tolerance mechanisms under drought and nutrient limitation. Along with the increase of soil total C, the same trend was observed for total N in RO soil under both water and nutrient-limited conditions. However, these values were higher than those observed for EO under the same conditions. This could be due to EO's greater N uptake than RO during drought. In fact, in the absence of both water and P supply, N content in the leaves of RO tended to be lower than that of EO. Effect of rewatering on plants subjected to drought as a function of soil P addition After 15 days of rewatering, drought-stressed plants of both species increased the stem water potential and the gas exchanges to the level of plants under well-watered conditions, consistent with the literature (Pflug et al. 2018 ). Notably, gas parameter values, in agreement with the results of Jacobs et al. ( 2009 ) and Kebert et al. ( 2023 ), were higher in EO than RO, suggesting an interspecific difference irrespective of water supply. P input contributed to EO recovery, showing the highest values of net photosynthesis among all EO and RO plants regardless of nutrient and water availability. The modulation of net photosynthesis by P has been reported in the literature: drought-stressed plants supplied by P addition generally recover their photosynthetic system more quickly than P-deficient plants (Radin and Eidenbock 1984 ; Singh et al. 1997 ; Burman et al. 2009 ). Although the net photosynthesis of RO was lower than EO, higher levels of C in leaves were found in the alien species after rewatering. This suggests that RO may have exhibited a more rapid turnover of primary carbon metabolites compared to EO following rewetting (Zang et al. 2014 ). The chemical composition of leaves also pointed out an interspecific difference in plant nutrient demand. Even though P supply led to an increase in P leaves in both species, this allocation exhibited higher values in EO. During the water relief and throughout the entire experiment, higher P content was consistently observed in the leaves of P-supplied plants. This supports the hypothesis mentioned previously Rolando et al. ( 2025 ) that RO species have a lower nutrient demand/higher nutrient use efficiency than EO. Conclusions In this study, the response of two Quercus spp. (EO and RO) to the combined effects of drought and P addition, was investigated. The findings provide novel insights into the interplay between nutrient availability and water stress in modulating metabolomic pathways. Under drought stress, EO was dependent on P, with significant upregulation of phenylpropanoid pathways and increased biosynthesis of antioxidant metabolites in P-fertilized plants. Conversely, P had a minimal influence on the metabolomic profile of RO under drought, highlighting the species’ adaptability to drought conditions. The greater resilience of RO is likely related to its intrinsic ability to produce polyphenols and tannins, as well as to employ strategies for water uptake under dry conditions and optimize nutrient acquisition, maintaining a lower overall nutrient demand. This capacity allows RO to thrive in environments with limited water and nutrients, potentially giving it a competitive advantage over EO in drought-prone ecosystems. However, P addition may enhance EO competitiveness, which could limit the spread of the alien species in favor of the native one in ecosystems increasingly affected by drought due to climate change. Declarations ACKNOWLEDGEMENTS We thank “La Mandria” Natural Park for granting the permissions to conduct sampling within the park. Their support and cooperation were essential to this research. This study was carried out within the Agritech National Research Center and received funding from the NextGenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022). Funding European Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022), Spoke 6. Competing interests All the authors declare they have no financial interests. Author contributions All authors contributed to the study's conception and design. 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Ann For Sci 62:1–8. https://doi.org/10.1051/forest:2004088 Whitehead D, Beadle CL (2004) Physiological regulation of productivity and water use in Eucalyptus: a review. Forest Ecology and Management 193:113–140. https://doi.org/10.1016/j.foreco.2004.01.026 Yang L, Wen K-S, Ruan X, et al (2018) Response of Plant Secondary Metabolites to Environmental Factors. Molecules 23:762. https://doi.org/10.3390/molecules23040762 Zang U, Goisser M, Grams TEE, et al (2014) Fate of recently fixed carbon in European beech (Fagus sylvatica) saplings during drought and subsequent recovery. Tree Physiology 34:29–38. https://doi.org/10.1093/treephys/tpt110 Zhang X, Zhang M, Zhao T, Deng B (2021) Phosphate availability regulates flavonoid accumulation associated with photosynthetic carbon partitioning in Cyclocarya paliurus . Physiologia Plantarum 173:1956–1966. https://doi.org/10.1111/ppl.13539 Zhu Z, Liang Z, Han R, Wang X (2009) Impact of fertilization on drought response in the medicinal herb Bupleurum chinense DC.: Growth and saikosaponin production. Industrial Crops and Products 29:629–633. https://doi.org/10.1016/j.indcrop.2008.08.002 Supplementary Files Supplementarymaterialslegends.docx SupplementarymaterialsRolandoetal..xlsx Cite Share Download PDF Status: Published Journal Publication published 12 Jun, 2025 Read the published version in Plant and Soil → Version 1 posted Reviewers agreed at journal 17 Apr, 2025 Reviewers invited by journal 17 Apr, 2025 Editor invited by journal 11 Apr, 2025 Editor assigned by journal 10 Apr, 2025 First submitted to journal 10 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6420617","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444152699,"identity":"5e63d41d-d8b9-4ccc-9f57-15d0f3bf106b","order_by":0,"name":"Morena Rolando","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Morena","middleName":"","lastName":"Rolando","suffix":""},{"id":444152700,"identity":"e407ebfb-cba5-40d9-8b23-5d36d7389b4f","order_by":1,"name":"Paola Ganugi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA70lEQVRIiWNgGAWjYHACxgMw1oEPDAw8YAYhPXAFB2cAtYD0HCCkBy7PDFJO0Bpz9sMHDvxgqJU3l0i/eNi27Y6MPXsD4+EPeLRY9qQlHOxhOG64c0ZOweHctmc8PDwH8DvM4ECOwQEehmOMG27kJAC1HObhkUggoOX8G4ODfxiO2YO1WBKl5UaOwWEehprEDTfSDxxmJEaL5YxnCYdlDA4kbzjzhuFgzzmgljMHGw6cwaPFnD/54MM3FXW2G46nP/7wo+ywPXt78+EPFfgcBiEPAwkeA6gYYwMeDTAtDHVAzP4Ar8pRMApGwSgYuQAAkjtcNRgvP6sAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1930-7021","institution":"University of Turin Faculty of Agriculture: Universita degli Studi di Torino Scuola di Agraria e Medicina Veterinaria","correspondingAuthor":true,"prefix":"","firstName":"Paola","middleName":"","lastName":"Ganugi","suffix":""},{"id":444152701,"identity":"49c8af86-a1d0-4f8e-b010-8be19fc8f8fd","order_by":2,"name":"Leilei Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Leilei","middleName":"","lastName":"Zhang","suffix":""},{"id":444152702,"identity":"9fcf3d12-fb30-4e2f-b859-d30dc6a153d3","order_by":3,"name":"Francesca Secchi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Secchi","suffix":""},{"id":444152703,"identity":"ac041083-8180-44f6-abfd-5c2203412e84","order_by":4,"name":"Luigi Lucini","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Luigi","middleName":"","lastName":"Lucini","suffix":""},{"id":444152704,"identity":"1b8ddd46-c733-4ae2-a006-65f16ce937a0","order_by":5,"name":"Luisella Celi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Luisella","middleName":"","lastName":"Celi","suffix":""}],"badges":[],"createdAt":"2025-04-10 13:31:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6420617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6420617/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11104-025-07610-4","type":"published","date":"2025-06-12T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82135810,"identity":"b9917ef4-9149-41ee-bf36-b195dc6456d2","added_by":"auto","created_at":"2025-05-07 06:09:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178053,"visible":true,"origin":"","legend":"\u003cp\u003ea) total carbon (TC), b) total nitrogen (TN) and c) available phosphorous (P\u003csub\u003eav\u003c/sub\u003e) in soils affected by two different species (SP; English Oak, EO and Red Oak, RO) under different water management (WM; well-watered, WW and drought conditions, D) with different nutrient input (NI), at the initial watering phase (DOY 163) and the last day of drought imposition (DOY 178). According with ANOVA analysis and Tukey's post - hoc, the different letters show significant among factor (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/7dfd24188ac0bafee7aacb11.png"},{"id":82139166,"identity":"87da0b33-0b9c-4eae-a779-c7b766f47cfb","added_by":"auto","created_at":"2025-05-07 06:25:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1008241,"visible":true,"origin":"","legend":"\u003cp\u003ea) Stem water potential (Ψ\u003csub\u003estem\u003c/sub\u003e), b) stomatal conductance (gs) and net photosynthesis (An) of two different species (SP; English Oak, EO and Red Oak, RO) under different water management (WM; well-watered, WW and drought conditions, D; followed by rewatering, RW) with different nutrient input (NI) during time (T). According with ANOVA analysis and Tukey's post – hoc the different letters show significance between treatments (\u003cem\u003ep\u003c/em\u003e=0.05). The statistical analysis was performed separating the watering (DOY 163), drought (DOY 167, 174, 178) and rewatering phase (DOY 193).\u0026nbsp;\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/a012bed5aac897f5335f59e9.png"},{"id":82137385,"identity":"11036232-8620-428e-8452-26a0b01c0282","added_by":"auto","created_at":"2025-05-07 06:17:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":393139,"visible":true,"origin":"","legend":"\u003cp\u003ea) Carbon (C), b) nitrogen (N) and c) phosphorous (P) leaves content, d) apparent P recovery (APR) of two different species (SP; English Oak, EO and Red Oak, RO) under different water management (WM; well-watered, WW and drought conditions, D; followed by rewatering, RW) with different nutrient input (NI) during time (T). According with ANOVA analysis and Tukey's post – hoc the different letters show significance between treatments (\u003cem\u003ep\u003c/em\u003e=0.05). The statistical analysis was performed separating the watering (DOY 163), drought (DOY 167, 174, 178) and rewatering phase (DOY 193).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/fe0423d3ff3cf09fc8fdd771.png"},{"id":82137386,"identity":"607f232b-9e08-447a-b098-0e1f6b8dc6ff","added_by":"auto","created_at":"2025-05-07 06:17:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":975735,"visible":true,"origin":"","legend":"\u003cp\u003ea) C/N, b) C/P, c) N/P ratio leaves of two different species (SP; English Oak, EO and Red Oak, RO) under different water management (WM; well-watered, WW and drought conditions, D; followed by rewatering, RW) with different nutrient input (NI), during time (T). According with ANOVA analysis and Tukey's post – hoc the different letters show significance between treatments (\u003cem\u003ep\u003c/em\u003e=0.05). The statistical analysis was performed separating the watering (DOY 163), drought (DOY 167, 174, 178) and rewatering phase (DOY 193).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/0bd150e78a6fca5252291256.png"},{"id":82135812,"identity":"9b8af557-06a8-416d-aeea-d8457e86c7a4","added_by":"auto","created_at":"2025-05-07 06:09:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":773153,"visible":true,"origin":"","legend":"\u003cp\u003ePathway analysis of two \u003cem\u003eQuercus\u003c/em\u003e spp. species (English Oak, EO; Red Oak, RO) affected by different water management – i.e., well-watered (WW) and drought (D) and phosphorus nutrient input (P), in comparison to the initial experimental conditions (WW –P). (a) Biosynthesis of primary and secondary metabolites, as well as compounds potentially associated with both metabolic pathways, in EO and RO. (b) Superclass of primary metabolites and (c) superclass of secondary metabolites primarily affected in EO and RO under different water and nutrient availability conditions.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/e25d4a78e353d90560ce50c7.png"},{"id":82139168,"identity":"cb65a832-ddaa-45d3-9ab2-0aa47dd4016d","added_by":"auto","created_at":"2025-05-07 06:25:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":679125,"visible":true,"origin":"","legend":"\u003cp\u003ePhenylpropanoids profiling analysis of two \u003cem\u003eQuercus\u003c/em\u003e spp. species (English Oak, EO; Red Oak, RO) affected by different water management – i.e., well-watered (WW) and drought (D) and phosphorus nutrient input (P) for (a) EO and (b) RO species. syn = synthesis, deriv. = derivatives.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/d2b1b767f10022753bfa3924.png"},{"id":84726864,"identity":"f67f89f4-6167-4462-a457-0976b0d43610","added_by":"auto","created_at":"2025-06-16 16:08:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5120107,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/b3c78783-4440-42df-9630-8541df185f02.pdf"},{"id":82135808,"identity":"f48e6d23-9dcc-4a9d-9935-be4b18d48d08","added_by":"auto","created_at":"2025-05-07 06:09:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15059,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialslegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/00dd2169a3e8d5382d50dee8.docx"},{"id":82137389,"identity":"bcec3eeb-a19d-46bb-b5c4-b23464234464","added_by":"auto","created_at":"2025-05-07 06:17:54","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2580688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementarymaterialsRolandoetal..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6420617/v1/5401643367129547ff09bc71.xlsx"}],"financialInterests":"","formattedTitle":"The interplay between drought and P scarcity shapes resilience to stress in Quercus spp. by modulating metabolomic profiles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEcosystems increasingly face more frequent and prolonged extreme conditions due to climate change (IPCC \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Since drought is one of the leading environmental stress factors, understanding how plants respond to water deficit conditions is crucial to predict future patterns of plant redistribution (Dyderski et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Under water deficit conditions, plants reduce stomatal conductance (gs) and leaf transpiration (E) to limit the negative effects of soil dryness (Whitehead and Beadle \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Osmotic adjustment is one of the plant strategies that help sustain gas exchanges and maintain stomata openness as the soil becomes drier (Dichio et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The modulation of leaf gas exchange under dry soil conditions reduces net photosynthesis (An), leading to excessive production of Reactive Oxygen Species (ROS), which cause oxidative damage to proteins, DNA and lipids (Cruz De Carvalho 2008; Tariq et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe shift in metabolomic processes regulates the plant response to water deficit, including leaf gas exchange and cell turgor, which are mainly affected by primary metabolites, as well as secondary metabolism like accumulation. During drought, primary metabolites such as sugars (e.g., sucrose, glucose, fructose, raffinose, and mannitol) can osmoregulate cells and contribute to synthesizing antioxidants, such as ascorbic acid (Nishizawa-Yokoi et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rodr\u0026iacute;guez-S\u0026aacute;nchez et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). When the risk of photodamage and the impairment of leaf photochemistry arises, increased levels of amino acids (e.g., proline or glutamine) can stabilize the cellular membranes and mitigate the negative effects of ROS accumulation (Ain-Lhout et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Increasing metabolites with antioxidant and ROS-scavenging activities is a common plant strategy to limit oxidative damage and improve cell membrane stability (Sobrino-Plata et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Many secondary metabolites act as osmoprotectants, reducing water loss and protecting the photosynthetic apparatus from damage (Escand\u0026oacute;n et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To preserve green leaf tissues, plants accumulate phenolic compounds (e.g., flavonoids) and terpenoids, which act as antioxidants and ROS scavengers. Plants can also improve their tolerance to drought by increasing tannin levels as soil water content declines, especially under less favorable conditions (Top et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Arab et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOsmotic adjustment, secondary metabolite accumulation and antioxidant mechanisms in plants can be influenced by nutrient availability (Yang et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). According to Kleiner et al. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), fertilized chestnut oaks exhibit greater osmotic adjustment than those growing in nutrient-poor soils, while nitrogen (N) and phosphorus (P) deficiencies promote the accumulation of flavonoids (Stewart et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Phosphorus, a key component of molecules such as nucleic acids, phospholipids and ATP, is a macronutrient in plant metabolism (Amtmann and Blatt \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Phosphorus deficiency inhibits photosynthesis, respiration, and the uptake and assimilation of nitrate in plants (Pilbeam et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). According to the literature, P stress promotes the accumulation of organic acids and phenolics (e.g., flavonoids), but it can reduce the levels of most sugars (Hermans et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Conversely, Tariq et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that \u003cem\u003eAlnus cremastogyne\u003c/em\u003e Burkill seedlings exposed to high P availability under drought conditions showed improved photosynthetic rates and enhanced antioxidant defense, with reduced lipid peroxidation. The activity of antioxidant enzymes such as superoxide dismutase, catalase and peroxidase, along with the accumulation of osmolytes and soluble proteins, can be improved by P input. However, drought events may cause nutrient deficiencies even in plants with high soil nutrient availability, as nutrient uptake declines with decreasing soil moisture. In the soil matrix, the phosphate ion primarily moves by diffusion. Thus, dry conditions limit its movement toward the root surface (da Silva et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe availability of nutrients and water regulates plant growth, species distribution, and competition within ecosystems, ultimately influencing forest composition. Introducing alien species can further affect native biodiversity and biogeochemical processes (Angeloni et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In Europe, the Northern American species \u003cem\u003eQuercus rubra\u003c/em\u003e L. (Red Oak, RO) was introduced by humans at the end of the 17th century as an ornamental and valuable timber tree (Nicolescu et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to its remarkable capacity for natural regeneration, RO is considered potentially invasive, or even fully invasive, in some European countries. In northern Italy, this alien species has colonized much of the native vegetation in the Po River plain (\u003cem\u003eQuerco-carpinetum\u003c/em\u003e cenosis), a forest predominantly composed by \u003cem\u003eQuercus robur\u003c/em\u003e L. (English Oak, EO) and \u003cem\u003eCarpinus betulus\u003c/em\u003e L. (Terzuolo et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The introduction of RO in the mixed broadleaved forests of Northwestern Italy can change the soil characteristics, affecting the bio-cycling of nutrients, especially the availability of P. In well-developed soils characterized by low P availability, introducing RO could further slowdown P turnover, potentially driving the ecosystem towards a no-return threshold for restoring the original forest composition (Bonifacio et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Since RO is more effective in dominating nutrient-poor soils and dry conditions (Major et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lavnyy and Savchyn 2016) than EO, drought periods and nutrient deficiency may exacerbate competitive interactions between the two species.\u003c/p\u003e \u003cp\u003eRolando et al., (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) observed that not only EO was less tolerant to drought, but it also exhibited a slower recovery from repeated severe drought events compared to RO, with its vulnerability being particularly pronounced in nutrient-limited environments. Their findings further indicate that P supplementation mitigates the adverse effects of drought on EO, improving plant recovery.\u003c/p\u003e \u003cp\u003eIn this context, the aim of this study is to investigate the metabolomic responses of \u003cem\u003eQuercus robur\u003c/em\u003e (English Oak, EO) and \u003cem\u003eQuercus rubra\u003c/em\u003e (Red Oak, RO) to the combined drought and P scarcity stress factors. Specifically, we hypothesize that i) the P supply enhances the gas exchange rates of EO, with limited effect on RO, due to interspecific differences in resource use efficiency, therefore ii) EO will exhibit better drought tolerance when soil P availability is increased, as higher soil P levels may enhance photosynthetic rates and antioxidant defense mechanisms, which help mitigate oxidative damage; iii) in contrast, the RO response to water scarcity will be not significantly influenced by nutrient availability, since the alien species has inherently low water and nutrient demands and is able to adopt adaptive strategies to cope with low soil fertility and water availability, iv) consequently, increasing P availability may improve the competitive strength of the native EO in relation to RO. To test these hypotheses, we conducted a mesocosm experiment in which seedlings of both EO and RO were transplanted into nutrient-poor soil collected from an area predominantly colonized by RO. To simulate different environmental conditions, we manipulated soil fertility by adding P and applied two distinct water management regimes to assess the effects of varying water availability and nutrient input on plant growth and metabolism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003ePlant and soil material\u003c/h2\u003e\n \u003cp\u003eThe experiment was conducted in a greenhouse at the Department of Agricultural, Forest and Food Science (DISAFA) of the University of Turin, Italy. In December 2021, two-year-old \u003cem\u003eQuercus rubra\u003c/em\u003e L. (RO) and \u003cem\u003eQuercus robur\u003c/em\u003e L. (EO) seedlings provided by a nursery were transplanted into 3.4 L cylindrical pots (28 cm height x 12.5 cm diameter). A total of 40 plants (20 RO and 20 EO) were employed in the study.\u003c/p\u003e\n \u003cp\u003eThe soil used in the experiment (0\u0026ndash;20 cm layer) was collected from La Mandria Natural Park in Northwestern Italy (N 45.153213, E 7.581204), an area currently dominated by RO but formerly covered by EO and \u003cem\u003eCarpinus betulus\u003c/em\u003e L.. The soil was classified as Oxyaquic Fragiudalfs (USDA, Soil Taxonomy) with the following initial characteristics: pH 4.4, organic carbon 29 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, total nitrogen (N) 2.2 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, available P 5.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (P\u003csub\u003eav\u003c/sub\u003e), 4.2 mg N-NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 36 mg N-NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. During the experiment, plants were maintained in a glass greenhouse under partially controlled climatic conditions, with an average air temperature of 25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;4.8\u0026deg;C and relative humidity of 67.7\u0026thinsp;\u0026plusmn;\u0026thinsp;14.6%.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eExperimental design and treatments\u003c/h3\u003e\n\u003cp\u003eNutrient inputs consisted of two conditions: one representing the original nutrient-poor soil (-P), and another where plants received 39.3 mg P kg \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e provided as KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (+\u0026thinsp;P), on May 18th 2023 (Day of year - DOY- 138). Twenty-five days after nutrient inputs, 10 EO and 10 RO plants were regularly well-watered (denoted as WW), while the remaining 20 plants were subjected to water deficit conditions (denoted as D), followed by rewatering (RW). The experimental design followed a split-plot layout with five replications (R). The main plots consisted of the two plant species (SP, EO and RO), the subplots included two water management regimes (WM), and the sub-subplots involved two nutrient input levels (NI). The WW condition was maintained through daily irrigation, ensuring the field capacity. At the same time, 70% of the water lost from the previous day was restored in D plants until DOY 178 (15 days of drought). The D plants were then rewatered to reach and keep the field capacity of approximately 80% until DOY 193 (15 days of rewatering). All pots were weighed daily to adjust the WM accordingly.\u003c/p\u003e\n\u003ch3\u003eSoil analysis\u003c/h3\u003e\n\u003cp\u003eSoil samples were collected on DOYs 163 and 178 to assess variations in total carbon (TC), total nitrogen (TN), and available P (P\u003csub\u003eav\u003c/sub\u003e) following the drought period. Only two soil samplings were carried out during the plant growth to minimize disturbance of the rhizosphere during the water stress phase. Samples were dried and sieved at 2.0 mm. A portion was ground to 0.5 mm for TC and TN analysis using high-temperature combustion in an elemental analyzer (Vario Isotope Select; Elementar Analysensysteme GmbH, Hanau, Germany). Available P was quantified according to Olsen et al. (\u003cspan class=\"CitationRef\"\u003e1954\u003c/span\u003e). The extracted P was then measured colorimetrically using the malachite green method (Ohno and Zibilske \u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMeasurement of leaf gas exchange and stem water potential\u003c/h3\u003e\n\u003cp\u003eLeaf gas exchanges (gs, An and E) were measured on fully expanded leaves exposed to direct sunlight using a portable infrared gas analyzer (ADC-LCPro\u0026thinsp;+\u0026thinsp;system, The Analytical Development Company Ltd, Hoddesdon, UK). CO\u003csub\u003e2\u003c/sub\u003e levels were maintained at greenhouse conditions (400\u0026ndash;450 ppm). Leaf gas exchange was monitored every two days in the morning (between 10:00 am and 12:30 pm) on three to five plants in each treatment (one leaf per plant) for the whole duration of the experimental trial. The \u0026Psi; \u003csub\u003estem\u003c/sub\u003e was measured on leaves collected before imposing water stress conditions (DOY 163), after 4 (DOY 167) and 11 (DOY 174) days of drought, at the end of the water scarcity phase (DOY 178), and after 15 days of rewatering (DOY 193).\u003c/p\u003e\n\u003cp\u003eLeaves were placed in humidified, aluminum foil-wrapped plastic bags for about 15 min prior to excision. After excision, the leaves were allowed to equilibrate for an additional 10 minutes before the water potential was measured using a Scholander-type pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, CA, USA). On DOY 178, the relative chlorophyll content (SPAD values) was measured with a SPAD meter (SPAD 502 Plus Chlorophyll Meter, Spectrum, Plainfield, IL, USA) to assess chlorophyll levels after drought.\u003c/p\u003e\n\u003cp\u003eThe Performance Index (PI) parameter was measured in each plant using a continuous excitation Pocket PEA fluorometer equipped with black leaf clips (Hansatech Instruments Ltd, King\u0026rsquo;s Lynn, UK). Leaves were dark-adapted for at least 30 minutes before measurement.\u003c/p\u003e\n\u003ch3\u003eChemical characterization of leaves\u003c/h3\u003e\n\u003cp\u003eLeaves collected on DOYs 163 (watering initial phase), 167, 174, 178 (drought) and 193 (rewatering) were dried and grounded to 0.5 mm. Carbon (C) and Nitrogen (N) contents were determined by high-temperature combustion using an elemental analyzer (Vario Isotope Select; Elementar Analysensysteme GmbH, Hanau, Germany). For P analysis, samples were digested with 12.5 mL of H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and 2.5 mL of HClO\u003csub\u003e4 ,\u003c/sub\u003e and quantified colorimetrically using the malachite green method (Ohno and Zibilske \u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eMetabolomics analysis of leaves\u003c/h2\u003e\n \u003cp\u003eThe metabolomics analysis was conducted only on DOY 178 plants, where leaves were collected with liquid nitrogen and sampled at -20\u0026deg;C. Lyophilized plant tissue (100 mg) was dissolved in 1 mL of 80% methanol (v/v) acidified with 0.1% formic acid (v/v) and mechanically extracted using a Polytron PT 1200E homogenizer for 3 min at maximum power. The extracts were centrifuged at 5000 g for 15 min and filtered through a 0.22 \u0026micro;m cellulose membrane filter into vials for liquid chromatographic analysis. Untargeted profiling of samples was performed using the 6560-drift tube-ion mobility-quadrupole-time of flight-high resolution mass spectrometer (DTIM-UHPLC-QTOF-HRMS; Agilent Technologies, Santa Clara, CA, USA). The chromatographic separation was achieved under a water-acetonitrile (both LC-MS grade, from Sigma-Aldrich, Milan, Italy) gradient elution (6\u0026ndash;94% acetonitrile in 32 min), flow rate of 0.2 mL/min and injection volume of 6 \u0026micro;L, using 0.1% (v/v) formic acid as phase modifier on an -Agilent Poroshell 120 PFP column (100 mm \u0026times; 2.1 i.d., 1.9 \u0026micro;m particle size) -Agilent Zorbax Eclipse plus C18 analytical column (50 \u0026times; 2.1 mm, 1.8 \u0026micro;m). The QTOF mass analyzer operated in positive mode (ESI+) for both MS and MS/MS acquisition with nitrogen as both sheath gas (12 L/min and 315 ◦C) and drying gas (14 L/min and 250 ◦C). The nebulizer pressure was 45 psi, nozzle voltage was 350 V, and the capillary voltage was 4.0 kV. For MS acquisition, the full scan mode was performed within range of the m/z 100\u0026ndash;1200 (1 spectra/s), mass resolution of 30,000 full width at half maximum (FWHM), m/z\u0026thinsp;=\u0026thinsp;200. The data-dependent mode was performed for precursor fragmentation (10, 20, and 40 eV) and acquisition of MS/MS data from QC samples, with a mass resolution of 30,000 (FWHM), selecting 8 precursors per cycle (1 Hz, m/z 80\u0026ndash;1200, positive polarity, and active exclusion after 2 spectra). The Processing of the chromatograms was carried out using the MassHunter Qualitative Analysis software (version B.06.00, Agilent Technologies). The collected data (.d files) were processed using MS-DIAL software (version 4.70) for automatic peak finding, LOWESS normalization, and annotation via spectral matching, exploiting the comprehensive BMDMS-NP, Fiehn/Vaniya natural product library, and GNPS databases. The mass range 100\u0026ndash;1200 m/z was searched for features with a minimum peak height of 10,000 cps, using an accurate mass tolerance for peak centroiding of 0.05 and 0.1 Da, for MS and MS/MS, respectively. Retention time information was excluded from the calculation of the total score. The identification step was based on mass accuracy, isotopic pattern, and spectral matching. These criteria were used to calculate a total identification score, using a minimum cut-off value of 70%, considering the most common HESI\u0026thinsp;+\u0026thinsp;adducts.\u003c/p\u003e\n \u003cp\u003eThe data annotation and MS/MS structural confirmations were conducted using MS-DIAL software - version 4.90 - (Tsugawa et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e) for automated peak finding (against pooled QC) and putative annotation via spectral matching using the publicly available databases (e.g., BMDMS-NP, Fiehn/Vaniya natural product library, and GNPS). Compound annotation was carried out considering mass accuracy, isotopic profiles, and MS/MS spectral matching (where available), using a total identification score with a minimum cut-off of 70% to reach level 2 of confidence in identification (Blaženović et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The mass range of 100\u0026ndash;1200 m/z with a minimum peak height of 10,000 cps was considered, using accurate mass tolerances of 0.05 Da for MS and 0.1 Da for MS/MS.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eData collection and statistical analysis\u003c/h3\u003e\n\u003cp\u003ePlant physiological data were recorded every two days from June 11 (163 DOY) to July 11 (DOY 193), 2023, to evaluate the status of WW, D and RW plants. The watering conditions before imposing drought (DOY 163), two representative time points during water stress (DOYs 167 and 174), the end of drought (DOY 178), and rewatering (DOY 193) were chosen to determine leaf gas exchanges, \u0026Psi; \u003csub\u003estem\u003c/sub\u003e and chemical characterization of leaves (C, N, P).\u003c/p\u003e\n\u003cp\u003eAll the metabolomics (DOY 178) and soil (DOYs 163 and 178) data were statistically analyzed. For variables measured at multiple time points, the time (T) factor was incorporated into the analysis.\u003c/p\u003e\n\u003cp\u003eAll the statistical analyses were conducted using R 4.3.1 (R Code Team, 2022).\u003c/p\u003e\n\u003cp\u003eThe statistical analysis was organized into three distinct phases: baseline measurements (DOY 163), drought conditions (DOYs 167, 174, 178), and rewatering (DOY 193). First, a two\u0026ndash;way variance (ANOVA) analysis with a linear model was performed for each physiological parameter and leaf chemical element recorded at DOY163, using the \u003cem\u003estats\u003c/em\u003e package (R Core Team \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). The same statistical approach was applied to soil results, separating DOY 163 and DOY 178. During the drought phase, a four-way ANOVA with a mixed effect model was performed on each physiological parameter and leaf chemical element measured using the \u003cem\u003enlme\u003c/em\u003e package (Pinheiro et al. \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). The fixed factors in this model were SP, WM, NI and T, while replicates (R) \u0026ndash; represented by 3 out of 5 replicates \u0026ndash; were treated as a random factor.\u003c/p\u003e\n\u003cp\u003eConsequently, a three-ways ANOVA with a linear model was conducted on each parameter recorded on DOY 193 and for SPAD and performance index measurements recorded on DOY 178, again employing the \u003cem\u003estats\u003c/em\u003e package (R Core Team \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe distribution of measured variables was checked for normality and homogeneity of variance using the Shapiro\u0026ndash;Wilk and Levene\u0026apos;s tests, respectively. When ANOVA assumptions were violated, data were log-transformed, and the tests were repeated on the transformed data. The post\u0026ndash;hoc test performed after each ANOVA was the Tukey\u0026apos;s honestly significant difference (HSD) at a significance level of \u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, with the \u003cem\u003emultcomp\u003c/em\u003e packages (Hothorn et al. \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAfterwards, a multivariate analysis of variance (MANOVA) was conducted to assess the influence of SP, WM and NI on leaf gas exchange parameters and \u0026Psi; \u003csub\u003estem\u003c/sub\u003e measured at the end of water stress (DOY 178) and rewatering (DOY 193). The same analysis was conducted on chemical data from leaves sampled on DOYs 178 and 193.\u003c/p\u003e\n\u003cp\u003eFinally, the apparent P recovery (APR) of EO and RO leaves was calculated as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eThe metabolomics data were elaborated using the software Mass Profiler Professional 12.6 (Agilent Technologies). The raw data were log2 transformed, 75th percentile normalized, and baselined against the median of each compound. Sample patterns were investigated using unsupervised hierarchical cluster analysis (HCA) with Euclidean distance and Ward\u0026apos;s linkage rule. Furthermore, a supervised ANOVA multi-block orthogonal partial least squares (AMOPLS) was carried out using the package rAMOPLS on R (v 4.2.1.) to select Variable Importance in Projection (VIP\u003csup\u003e2\u003c/sup\u003e) markers associated with different treatments. The AMOPLS statistical significance was set at \u0026alpha;\u0026thinsp;=\u0026thinsp;0.01 and validated with 100 permutation processes. The results were expressed as the Relative Sum of Squares (RSS), representing the percentage of variability attributed to each factor; RSS p-value, statistical significance; and block contribution, in percentage, associated with each effect. Afterwards, the VIP markers associated with different factors were considered to build the pathways analysis, comparing [WW\u0026thinsp;+\u0026thinsp;P] \u003cem\u003evs.\u003c/em\u003e [WW -P], [D -P] \u003cem\u003evs.\u003c/em\u003e [WW -P], and [D\u0026thinsp;+\u0026thinsp;P] \u003cem\u003evs.\u003c/em\u003e [WW -P], separating for EO and RO species. The metabolite changes were defined using log Fold Change analysis comparing all the treatments with control well-watered and under phosphorus deficiency.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSoil\u003c/h2\u003e \u003cp\u003eOn DOY 163 (watering phase), the soil total C (TC) under EO -P (22.71\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was similar to that of RO -P soil (23.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). However, with P input, TC differed between EO (25.03\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and RO (22.74\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) with a remarkable SP*NI interaction effect on DOY 163. Conversely, drought nullified TC differences between EO\u0026thinsp;+\u0026thinsp;P and RO\u0026thinsp;+\u0026thinsp;P, though notable differences were still observed between soils under different species and low nutrient availability. For RO specifically, soil TC in WW -P decreased from DOY 163 (23.39\u0026thinsp;\u0026plusmn;\u0026thinsp;2.95 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) to DOY 178 (19.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while the drought event increased soil TC under -P conditions (24.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to WW at DOY 178.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe SP and NI did not impact the soil TN during the watering phase, but nutrient content in the soil was affected by the interaction of SP*WM*NI during drought. The main difference was observed between EO D -P (1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and RO D -P (2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while P addition suppressed the differences observed between SP in D plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). On DOY 178, as reported for soil TC, the TN content in RO D -P was higher than in RO WW -P soil (1.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eThe available P pool was below 5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in -P soils, independently of WM and T. Nutrient availability increased with the addition of P, ranging between 28.58\u0026thinsp;\u0026plusmn;\u0026thinsp;13.44 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in RO WW to 43.10\u0026thinsp;\u0026plusmn;\u0026thinsp;10.96 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in EO D. In fact, ANOVA highlighted a significant effect of NI at both DOY 163 and 178 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological parameters\u003c/h2\u003e \u003cp\u003eDuring the initial watering phase, the Ψ \u003csub\u003estem\u003c/sub\u003e values of EO -P and EO\u0026thinsp;+\u0026thinsp;P were \u0026minus;\u0026thinsp;0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 MPa and \u0026minus;\u0026thinsp;0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 MPa, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Similar values were measured for RO -P and RO\u0026thinsp;+\u0026thinsp;P without statistical differences on DOY 163. After 15 days of rewatering, Ψ \u003csub\u003estem\u003c/sub\u003e of D plants was comparable to that of WW. However, ANOVA analysis highlighted a remarkable effect of SP and NI, revealing that the Ψ \u003csub\u003estem\u003c/sub\u003e of EO was higher than that of RO. Regardless of species, RW\u0026thinsp;+\u0026thinsp;P plants had lower Ψ \u003csub\u003estem\u003c/sub\u003e than RW -P.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 25 days from nutrient input, and prior to the imposition of drought conditions, adding P increased the gs by 112.5% compared to EO -P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Conversely, gs in RO was not affected by NI. The gs in water-stressed EO and RO\u0026thinsp;+\u0026thinsp;P was 0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 mol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, while EO D -P and RO D -P exhibited minimal values of 0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 0.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 mol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. ANOVA analysis showed a significant effect of SP*WM*NI during D conditions, highlighting the role of P input in reducing gs in drought-stressed EO. Moreover, the SP*WM*NI interaction revealed interspecific differences between EO WW and RO WW from DOY 167 to 178. After 15 days of rewatering, both EO and RO fully recovered their gs (DOY 193).\u003c/p\u003e \u003cp\u003eSimilarly, at DOY 163, P input did not affect the An of RO, but it increased the An of the native species by 101.1% compared to EO -P (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The minimum An in both SP was measured on DOY 178. During water stress conditions, ANOVA denoted a significant effect of SP*WM*NI interaction, indicating interspecific differences in WW\u0026thinsp;+\u0026thinsp;P plants. Regardless of the time, P addition increased An in EO, while no effect was detected on RO. On DOY 178, the An of EO D\u0026thinsp;+\u0026thinsp;P plants (0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u0026micro;mol CO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was lower than that of EO D -P (2.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00 \u0026micro;mol CO\u003csub\u003e2\u003c/sub\u003e m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The rewatering restored An in D plants to levels comparable to WW plants in both species. ANOVA showed that SP was the only factor affecting An on DOY 193, with EO exhibiting higher values than RO.\u003c/p\u003e \u003cp\u003eOn DOY 163, P input affected all gas exchanges, including E (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Results showed that P addition increased E in EO but did not affect RO. Drought conditions reduced E to 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44 mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in EO -P, and 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 mmol H\u003csub\u003e2\u003c/sub\u003eO m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in RO -P. The lowest values were observed in +\u0026thinsp;P plants, with EO D showing 0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mmol H₂O m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and RO D at 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mmol H₂O m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. After 15 days of rewatering, E in RW plants remained lower than in WW, regardless of SP and NI. ANOVA identified remarkable differences between SP, showing that E in EO was higher than in RO, consistent with the other gas exchange parameters.\u003c/p\u003e \u003cp\u003eOn DOY 178, ANOVA on SPAD and PI revealed significant effects of SP and WM (Figure S2a and b). Both parameters were higher in EO (39.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 SPAD; 4.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 PI) compared to RO (36.1\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 SPAD; 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.7 PI). Regardless of SP, D plants exhibited reduced SPAD (36.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8) and PI (2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1) compared WW plants (SPAD: 38.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5; PI: 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3).\u003c/p\u003e \u003cp\u003eMANOVA on DOY 178 data showed that SP (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.54e-04), WM (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.76e-12), NI (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.84e-08) and all their interactions significantly affected leaf gas exchanges and Ψ \u003csub\u003estem\u003c/sub\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Interspecific differences were observed between well-watered EO and RO, irrespective of NI. Profile plots separated EO WW -P and EO WW\u0026thinsp;+\u0026thinsp;P from RO WW -P and RO WW\u0026thinsp;+\u0026thinsp;P (Figure S3a). In contrast, the physiological status of EO D\u0026thinsp;+\u0026thinsp;P was similar to RO D\u0026thinsp;+\u0026thinsp;P, while EO and RO under combined water and nutrient stress were classified separately. On DOY 193, results displayed a remarkable effect of SP (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.95e-05), WM (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.80e-03) and NI (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.22e-03), as well as WM*NI (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.21e-02) and SP*WM*NI (\u003cem\u003ep-value\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.82e-03; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In the profile plot, EO RW\u0026thinsp;+\u0026thinsp;P grouped with RO RW -P and RO RW\u0026thinsp;+\u0026thinsp;P, while during rewatering, RO WW\u0026thinsp;+\u0026thinsp;P also joined this group. EO RW -P exhibited a physiological status similar to EO WW, independent of NI (Figure S3b).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLeaves chemical characterization\u003c/h2\u003e \u003cp\u003eAt DOY 163 (watering initial phase), the C content in -P plants (EO -P: 451.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.4 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; RO -P: 456.1\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was higher than in +\u0026thinsp;P plants (EO: 447.8\u0026thinsp;\u0026plusmn;\u0026thinsp;8.1 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; RO: 451.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Notably, NI was the only factor with a remarkable effect on C content at DOY 163 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom DOY 167 to 178, EO increased its leaf C content regardless of WM and NI, reaching the highest value in WW\u0026thinsp;+\u0026thinsp;P (459.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.7 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on DOY 178. No differences in C content were observed in RO, during drought. Although the SP*WM interaction did not significantly affect C content during drought, ANOVA indicated that WM alone had a significant effect, with D leaves exhibiting higher C content than WW plants. As reported for DOY 163, +P plants had higher C content than -P plants after rewatering.\u003c/p\u003e \u003cp\u003eAt DOY 163, the N content in EO -P leaves (25.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was similar to that in RO -P (22.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). P addition did not affect N content in both SP. However, from DOY 163 to 178, ANOVA displayed a remarkable effect of SP*NI*T. In EO, the main differences were observed between 167 and 178, where -P plants showed an increase in N content from 24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.7 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in WW plants and from 26.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 28.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in D plants. During the same period, RO\u0026thinsp;+\u0026thinsp;P increased N content to 26.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ein WW plants and 22.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003ein D plants. After rewatering, the N content differed between species, with EO leaves having higher nutrient levels than RO.\u003c/p\u003e \u003cp\u003eAt DOY 163, the P content in the leaves of -P plants was 885.8\u0026thinsp;\u0026plusmn;\u0026thinsp;99.5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for EO and 856.8\u0026thinsp;\u0026plusmn;\u0026thinsp;62.8 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for RO (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). NI significantly affected the nutrient content in the plants, resulting in a higher P level in EO (1812.4\u0026thinsp;\u0026plusmn;\u0026thinsp;169.2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) compared to RO (1163.9\u0026thinsp;\u0026plusmn;\u0026thinsp;154.9 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). ANOVA highlighted statistical differences between EO WW\u0026thinsp;+\u0026thinsp;P and RO WW -P.\u003c/p\u003e \u003cp\u003eDuring drought conditions, the P content of EO\u0026thinsp;+\u0026thinsp;P remained higher than that of RO\u0026thinsp;+\u0026thinsp;P, regardless of WM. However, after rewatering, RO RW showed a lower P content than RO WW. At the same time, EO displayed no significant differences between RW and WW, maintaining higher values than RO regardless of NI.\u003c/p\u003e \u003cp\u003eAfter P addition, EO absorbed more P than RO. Specifically, at DOY 163, the APR for EO was 8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8%, while the apparent P recovery for RO was 3.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Drought did not affect the APR of EO, but it reduced the RO APR to -2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4% on DOY 167. Consequently, from DOY 174 to 178, the RO APR values returned to the levels observed at DOY 163. Following rewatering, ANOVA revealed a significant effect of plant species on APR, consistent with the observations at DOY 163.\u003c/p\u003e \u003cp\u003eAt the beginning of the experiment, the C/N ratio of EO leaves (17.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8 in -P) was lower than that of RO (20.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 in -P). ANOVA did not reveal a significant effect of NI on the C/N ratio at the initial watering phase. The interspecific difference persisted during drought and after rewatering (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe C/P ratio of EO -P leaves (516.8\u0026thinsp;\u0026plusmn;\u0026thinsp;53.4) was comparable to that of RO -P leaves (536.1\u0026thinsp;\u0026plusmn;\u0026thinsp;40.8) at DOY 163 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). However, P addition resulted in differences between species, with a decline of the ratio of EO\u0026thinsp;+\u0026thinsp;P down to 248.7.1\u0026thinsp;\u0026plusmn;\u0026thinsp;26.5, while RO\u0026thinsp;+\u0026thinsp;P measured 390.1\u0026thinsp;\u0026plusmn;\u0026thinsp;58.3. ANOVA displayed a significant effect of the interaction SP*NI at DOY 163, during the drought phase, and after 15 days of rewatering. From DOY 167 to 178, the C/N ratio increased regardless of SP, WM and NI.\u003c/p\u003e \u003cp\u003eAt DOY 163, the nutrient-stressed native and alien species exhibited similar N/P ratios. The P input decreased the N/P ratio to 13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.2 in EO and 18.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 in RO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). ANOVA indicated a significant effect of SP*NI during drought and after rewatering, confirming the earlier observations based on DOY 163 results. Moreover, on DOY 193, the significant effect of SP*WM revealed that RO RW had a higher N/P ratio than both EO RW and WW plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMetabolic response of EO and RO under nutrient and drought stress\u003c/h2\u003e \u003cp\u003eThe metabolomics analysis was conducted on the DOY 178 plant stage using an untargeted approach, allowing us to annotate 1744 metabolites putatively. The list of identified features, including pathway classification, specific metabolite ontology, their abundances, and MS1 isotopic and MS/MS spectra (where they are confirmed), is listed in the \u003cb\u003esupplementary Table S3\u003c/b\u003e. Analyzing features obtained from the profiling of EO and RO leaves under nutrient and drought stress, the approach perfectly identified secondary metabolites as the most abundant class, followed by primary metabolites (e.g., fatty acids and lipids, carbohydrates, amino acids, and nucleotides, \u003cb\u003eTable S3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo assess the overall similarities/dissimilarities among samples influenced by different factors (i.e., water management, nutrient supplementation, and species), a hierarchical clustering analysis (HCA) was performed. As reported in \u003cb\u003eFigure S5\u003c/b\u003e, the species factor resulted in the most differences, cauterizing into two branches. Concerning EO species, the HCA analysis clusters samples based on both nutrient supplementation and water management, suggesting specific metabolite profiles and abundances along treatments. Conversely, RO species reported less linearity between metabolic response and treatments, confirming our more resilient species hypothesis. Based on the unsupervised results, highlighting the species factor as the primary contributor to the overall variance, a supervised ANOVA multi-blocking orthogonal projection to latent structures discriminant analysis (AMOPLS-DA) was performed to focus on the detail about the nutrient and water management factors on the data variance, for both EO and RO species separately (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelative variability and block contributions of the ANOVA multi-blocking orthogonal projection to latent structures discriminant analysis (AMOPLS) of two Quercus spp metabolomic data. species (English Oak, EO; Red Oak, RO) affected by different water management (WM), phosphorus nutrient input (NI), and interaction between WM x NI. RSS: Relative sum of squares, Tp1\u0026ndash;3: predictive components, To: orthogonal component.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003eEO AMOPLS model\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEffect Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRSS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRSS p-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eR2Y p-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTp1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTp2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTp3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTo1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e22.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWM x NI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e94.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e27.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eResiduals\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRO AMOPLS model\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWM\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e23.1%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e94.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWM x NI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.4%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e85.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eResiduals\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.9%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.1%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e26.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOur results reported that the WM factor exerted the greatest influence on EO and RO species, showing 20.9% and 12.8% relative sum of squares (RSS) values, respectively. The discrimination between well-watered and drought-stress plants was perfectly made by component 1 (tp1). Nutrient supplementation and its interaction with WM were the secondary factors that influenced metabolic response in both species and discriminated using components 2 and 3 (tp2 and tp3), respectively. Interestingly, the supervised AMOPLS model highlighted a higher percentage of the observed metabolomic differences that could not be explained by our factors in RO than EO species, supporting the hypothesis of better correlation between treatments and metabolic response in EO as firstly suggested by HCA. Afterwards, the variable importance in the projection (VIP) analysis was carried out to identify biomarkers associated explicitly with differentiating samples based on factors. The 50 most discriminant metabolites per factor were selected and reported in the supplementary table, together with log fold change values for both EO (\u003cb\u003eTable S4\u003c/b\u003e) and RO (\u003cb\u003eTable S5\u003c/b\u003e). The specific metabolic pathways were then built using these VIP markers, considering biosynthetic pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) and further going into detail about primary (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and secondary metabolites biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the modulation of EO biosynthesis pathways, phosphorus supplementation under WW conditions down-modulated primary and secondary metabolite synthesis and nitrogen-, oxygen- and sulfur-containing metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The most affected metabolite classes belonging to primary metabolites were organic amino compounds and lipids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Secondary metabolites were isoprenoids, nitrogen-, oxygen- and sulfur-containing metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In contrast, the drought stress up-modulated the biosynthesis of secondary metabolites, where the accumulation of phenylpropanoids, alkaloids, and isoprenoids was mainly represented (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Additionally, lipid and fatty acid synthesis were also affected, reporting a reduction in steroids and short- and medium-chain fatty acids, alongside an increase in long-chain fatty acid synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Interestingly, phosphorus supplementation under drought-stress conditions led to an increased abundance of specialized metabolites in plant tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), fully addressing the synthesis of phenylpropanoids. This evidence suggests a synergistic effect of phosphorus input in enhancing endogenous bioactive compounds to cope with abiotic stress.\u003c/p\u003e \u003cp\u003eConcerning the modulation of RO biosynthesis pathways, nutrient and water management factors produced similar modulation patterns, differing mainly in the abundance of specific classes of metabolites. Indeed, the most relevant metabolite classes were secondary metabolites, followed by nitrogen-containing compounds and primary metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Regarding secondary metabolites biosynthesis, drought stress selectively increased the accumulation of phenylpropanoids and alkaloids while reducing isoprenoids as a response strategy. At the same time, phosphorus input under the same condition maintained a high level of phenylpropanoids and was accompanied by the up-modulation of isoprenoids synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProfiling of phenylpropanoids composition of EO and RO under nutrient and drought stress\u003c/h2\u003e \u003cp\u003eSince the primary metabolic response of EO and RO under nutrient and water management stress factors was secondary metabolite accumulation, a deep clarification of specific subclasses of phenylpropanoid compounds was plotted based on log fold change values deriving from the pairwise comparison between treatments and control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Regarding the EO species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), drought conditions, and at a higher extent combined with the phosphorus supplementation, increased flavonoid \u003cem\u003eO\u003c/em\u003e-glycosides, \u003cem\u003eO\u003c/em\u003e-methylated flavonoids, coumarins and derivatives, flavones, and hydrolyzable tannins. Specifically, the enhanced production of coumarins under drought\u0026thinsp;+\u0026thinsp;phosphorus was also confirmed by a clear down-accumulation of coumaric acid, their synthetic precursors. Focusing on RO species (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), drought stress induced the accumulation of flavonoid \u003cem\u003eO\u003c/em\u003e-glycosides, neoflavans, isoflavones, and high hydrolyzable tannins. As previously observed, the RO species resulted in less sensitivity to the phosphorus input under both well-watered and drought conditions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInterspecific plant differences due to nutrient status under watering conditions\u003c/h2\u003e \u003cp\u003eTo understand the responses of EO and RO to water stress under varying levels of P availability, it is essential to assess the nutritional and physiological status of well-watered species under -P and +\u0026thinsp;P soil. Twenty \u0026ndash; five days after the P supply, before imposing a mild and long drought event, the stem water potential (Ψ\u003csub\u003estem\u003c/sub\u003e) and gas exchange value of EO and RO were consistent with the previous findings of Gori et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and Cotrozzi et al. (2016), on well-watered \u003cem\u003eQ. ilex\u003c/em\u003e and \u003cem\u003eQuercus\u003c/em\u003e spp., respectively.\u003c/p\u003e \u003cp\u003eOur results showed that Ψ\u003csub\u003estem\u003c/sub\u003e was unaffected by P input, while increased gas exchange levels were observed for EO\u0026thinsp;+\u0026thinsp;P, in line with the findings from Rolando et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The relationship between net photosynthesis and P has been studied in plants: P can modulate the assimilation of N for the synthesis of ribulose-1,5-bisphosphate carboxylase, the primary enzyme that catalyzes the first step of carbon dioxide fixation in photosynthesis (Spreitzer and Salvucci \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Warren et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Moreover, P is a component of the adenosine triphosphate synthesis (ATP) and nicotinamide adenine dinucleotide (NADPH), which are, respectively, the primary sources of energy and reducing agents needed to drive the biochemical transformations of carbon dioxide into organic compounds in the Calvin cycle (Farooq et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As a matter of fact, Kayoumu et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) observed that P shortage reduces the photosynthetic capacity and leaf transpiration. Therefore, increasing EO gas exchanges following P input could suggest that this soil element is the limiting factor for EO growth. Conversely, gas exchanges of RO were not modulated by soil P addition, probably because the alien species did not experience a P deficit, even in the depleted soil. This may be attributed to RO having a higher P use efficiency than EO, as previously observed (Rolando et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This interspecific difference in resource utilization capacity could also be extended to photosynthesis. In fact, under adequate soil water content and regardless of P availability, the gas exchange rates of RO are lower than those of EO, suggesting a reduced CO₂ assimilation of RO.\u003c/p\u003e \u003cp\u003eThe native species absorbed and allocated more P in leaves than the alien species; thus, the apparent P recovery confirmed that EO's P demand was higher than RO's. Nevertheless, the soil P\u003csub\u003eav\u003c/sub\u003e pool did not show significant differences between +\u0026thinsp;P soils under different species. This may be related to the dramatic soil low P levels: although a large amount of P (110 kg/ha) was added to mitigate nutrient deficiency in plants, a large part of the added P may have been retained by the mineral phase and released when needed, leading to equalize the P\u003csub\u003eav\u003c/sub\u003e pool in soil, independently of P species (Rolando et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Evidence of this process is provided by the increase in total organic soil C, which suggested root exudation under our experimental conditions. Root exudates (e.g., organic acids, enzymes, phenolic compounds) can enhance P availability in soils, mobilizing P from stable pools (DeLuca et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Menezes-Blackburn et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rafi et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Preece et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) observed the same trend in +\u0026thinsp;P soil under \u003cem\u003eQ. Ilex\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAccording to our first hypothesis, EO exhibited increased gas exchange following P input, likely due to its lower resource use efficiency than RO. This confirms that P is a limiting factor for EO growth and its competitive ability against RO in highly weathered soils.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of drought on plants as a function of soil P addition\u003c/h2\u003e \u003cp\u003eThe combined application of physiological measurements and untargeted metabolomics shed light on plant response to drought and nutrient stresses, indicating that both EO and RO triggered contrasting effects at both physiological and metabolic levels, primarily depending on water availability.\u003c/p\u003e \u003cp\u003eWater deficits disrupt crucial processes in plants, such as photosynthesis, transpiration, and nutrient uptake, with the effects varying depending on the severity of stress, plant genotype, and growth stage (Kirkham \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Under drought conditions, plants often produce ROS, leading to oxidative damage, carbohydrate and protein synthesis disruption, and lipid metabolism, which can ultimately cause cell death (Feng et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Blum \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In response to these stresses, plants have evolved various adaptive mechanisms to mitigate damage, such as limiting transpiration through stomatal closure, accumulating osmoprotective solutes in the cytoplasm, and activating pathways that enhance water uptake, improving stomatal conductance, and increasing water-use efficiency (Mitchell et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Pe\u0026ntilde;uelas and Staudt \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConcerning EO, drought significantly reduced the stem water potential, but the decrease was less pronounced under P-limited conditions. This finding is consistent with previous studies, such as those by Burman et al., (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), which highlighted that P fertilization could help mitigate the negative effects of water stress by promoting plant growth and leaf area expansion, increasing water demand and accelerating soil water consumption. However, the increased water consumption in P-fertilized plants could lead to a more significant reduction in water status during drought stress. Despite this, plants with higher P availability tend to show better recovery after rehydration, improved photosynthetic efficiency, and higher enzymatic activity, which is supported by our findings of improved gas exchange in EO with P supplementation during the initial watering phase (Burman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs drought progressed, EO plants showed a clear reduction in net photosynthesis, mainly when P was present, coinciding with stomata's closure, limiting CO₂ uptake. In contrast, EO -P plants maintained higher gas exchange rates during drought, indicating a potential trade-off between maintaining higher photosynthesis and tolerating stress. Although during watering conditions, P supply promoted photosynthesis by enhancing the biosynthesis of the primary source of energy (ATP), under drought, plants closed stomata, limiting the absorption of CO\u003csub\u003e2\u003c/sub\u003e from the atmosphere. This could be due to an imbalance between ATP and CO\u003csub\u003e2\u003c/sub\u003e concentrations, affecting the photosynthetic process.\u003c/p\u003e \u003cp\u003eThese physiological changes in EO were mirrored in the metabolomic profile, where we observed a shift towards synthesizing primary and secondary metabolites that could help mitigate oxidative damage and osmotic stress under the reduced water availability conditions (Street et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Pe\u0026ntilde;uelas and Staudt \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this species, drought-induced stress led to an increase in the biosynthesis of phenylpropanoids, particularly hydroxylated phenylpropanoids (e.g., [6-[3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-3,4,5-trihydroxyoxan-2-yl]methyl and E)-3-(4-hydroxy-3-methoxyphenyl) prop-2-enoate and dihydroxy B-ring-substituted flavonoids (e.g., luteolin 7-glucuronide and scutellarin). These metabolites are well-known for their antioxidant properties, as they scavenge ROS and help reduce oxidative damage (Gould et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Hernandez et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Agati et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hern\u0026aacute;ndez et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Agati and Tattini \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ferreres et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pollastri and Tattini \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, adding P to drought-stressed EO led to the upregulation of primary and secondary metabolites, which act as antioxidants and osmoprotectants (Dos Santos et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ahmad et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In particular, P addition under drought conditions overstimulated the production of phenolic compounds, including flavonoid O-glycosides, O-methylated flavonoids and coumarins. These compounds likely contribute to maintaining turgor pressure by decreasing osmotic potential in vacuoles, which helps mitigate the effects of water stress (Almeida et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition to phenylpropanoids, an accumulation of saikosaponins, a class of triterpene saponins, was detected. Saikosaponins are known for their ability to scavenge ROS and protect cellular membranes from peroxidation, which is especially important under drought stress (Liu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis finding supports the hypothesis that EO, when supplied with P, activates metabolic pathways that enhance its ability to cope with drought, possibly by increasing the production of stress-related metabolites.\u003c/p\u003e \u003cp\u003eAmong primary metabolites, increased levels of mannitol were found in the metabolic profile of EO under water stress and P supplementation. Mannitol helps stabilize proteins and cellular structures by interacting with the hydration shell around them and scavenging ROS produced in higher quantities under drought stress (Patel and Williamson \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, the metabolomic data also revealed an accumulation of 1,3,5-tricaffeoylquinic acid, a bioactive compound involved in antioxidant defense. Previous studies have shown that caffeic acids and their derivatives, such as 1,3,5-tricaffeoylquinic acid, are crucial in protecting plants from oxidative stress (Harrison et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). This metabolite may help protect EO from ROS damage, complementing the physiological data that suggest that EO\u0026rsquo;s photosynthetic system is less impaired under drought when P is available.\u003c/p\u003e \u003cp\u003eMoreover, the input of P reprogrammed lipid and fatty acid metabolism in drought-stressed EO. The observed increase in long-chain fatty acids in EO\u0026thinsp;+\u0026thinsp;P under drought conditions has been previously interpreted as a plant strategy to maintain membrane integrity, potentially limiting cellular turgor loss (Ullah et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Fatty acids are a crucial component of cellular membranes (Ruiz-Lopez et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and their accumulation contributes to membrane rigidification (L\u0026oacute;pez-P\u0026eacute;rez et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, EO's metabolomic profile revealed increased levels of gibberellic acid (GA). This plant hormone is known to be involved in response to various abiotic stresses, including water scarcity. GA helps regulate membrane permeability, osmolyte accumulation, and antioxidant systems, all enhancing stress tolerance (Shah et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our findings of increased GA accumulation under drought with P supplementation suggest that P might promote EO\u0026rsquo;s ability to manage stress through the modulation of plant growth and metabolism, which is reflected in the enhanced photosynthetic activity observed during the initial watering phase.\u003c/p\u003e \u003cp\u003eOn the other hand, RO data displayed a different response from EO for both physiological and metabolomic analyses under drought conditions. Regardless of P availability, water stress negatively impacted stem water potential and gas exchange values. Similarly, P supply did not lead to a higher production of defense secondary metabolites compared to P-limited conditions. In the absence of P, the metabolomic analysis displayed a remarkable increase in phenylpropanoids and alkaloids, suggesting that RO has a metabolic system capable of handling both water and nutrient limitations, possibly by activating specific stress-related pathways even under low P conditions.\u003c/p\u003e \u003cp\u003eHowever, even without drought imposition, high levels of hydrolyzable tannins were detected in RO\u0026thinsp;+\u0026thinsp;P leaves. This points out the plant\u0026rsquo;s intrinsic capability to upregulate the synthesis of hydrolyzable tannins under adequate soil nutrient availability (Salminen et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe lack of a strong effect of P on RO nutrient uptake during drought, particularly in terms of P accumulation, reinforces the idea that RO is less dependent on P compared to EO. This contrasts with the more pronounced effects of P on EO physiological and metabolic responses. In EO, P input acted as a stabilizer, enhancing both physiological and metabolomic responses to drought. Conversely, metabolic changes in RO were less influenced by P supplementation, suggesting that RO may operate at a baseline metabolic level that allows it to thrive in a wider range of environmental conditions.\u003c/p\u003e \u003cp\u003eThe metabolic reprogramming of both species under water-limited conditions was consistent with soil-plant nutrient bio-cycling. As previously shown by metabolomic data, EO was more responsive than RO to P addition, reflecting a greater P leave accumulation. On the other hand, P content in RO -P leaves was not linked to P input. Nevertheless, these results were observed regardless of water availability.\u003c/p\u003e \u003cp\u003eIn contrast to previous studies, the unaltered P concentration of RO leaves under water and nutrient-limited conditions and the increased content of soil total organic carbon suggested a higher root exudation responsible for P mobilization from the stable pool (DeLuca et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Menezes-Blackburn et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Rafi et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This could be one of the competitive advantages of RO in dry and low fertility soils, as a strategy to gain an advantage over the native vegetation. Root exudates can help mobilize nutrients in the rhizosphere, offering a competitive advantage in nutrient-poor environments (Gargallo-Garriga et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, the fact that RO leaves did not show a significant increase in P accumulation under drought conditions supports the idea that RO may have evolved to be less reliant on P input compared to EO, possibly due to its ability to regulate nutrient uptake and allocation more efficiently. This behavior aligns with the metabolic shifts observed in RO, where the accumulation of secondary metabolites likely supports its stress tolerance mechanisms under drought and nutrient limitation.\u003c/p\u003e \u003cp\u003eAlong with the increase of soil total C, the same trend was observed for total N in RO soil under both water and nutrient-limited conditions. However, these values were higher than those observed for EO under the same conditions. This could be due to EO's greater N uptake than RO during drought. In fact, in the absence of both water and P supply, N content in the leaves of RO tended to be lower than that of EO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eEffect of rewatering on plants subjected to drought as a function of soil P addition\u003c/h2\u003e \u003cp\u003eAfter 15 days of rewatering, drought-stressed plants of both species increased the stem water potential and the gas exchanges to the level of plants under well-watered conditions, consistent with the literature (Pflug et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, gas parameter values, in agreement with the results of Jacobs et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Kebert et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), were higher in EO than RO, suggesting an interspecific difference irrespective of water supply.\u003c/p\u003e \u003cp\u003eP input contributed to EO recovery, showing the highest values of net photosynthesis among all EO and RO plants regardless of nutrient and water availability. The modulation of net photosynthesis by P has been reported in the literature: drought-stressed plants supplied by P addition generally recover their photosynthetic system more quickly than P-deficient plants (Radin and Eidenbock \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Burman et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Although the net photosynthesis of RO was lower than EO, higher levels of C in leaves were found in the alien species after rewatering. This suggests that RO may have exhibited a more rapid turnover of primary carbon metabolites compared to EO following rewetting (Zang et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe chemical composition of leaves also pointed out an interspecific difference in plant nutrient demand. Even though P supply led to an increase in P leaves in both species, this allocation exhibited higher values in EO.\u003c/p\u003e \u003cp\u003eDuring the water relief and throughout the entire experiment, higher P content was consistently observed in the leaves of P-supplied plants. This supports the hypothesis mentioned previously Rolando et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) that RO species have a lower nutrient demand/higher nutrient use efficiency than EO.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, the response of two \u003cem\u003eQuercus\u003c/em\u003e spp. (EO and RO) to the combined effects of drought and P addition, was investigated. The findings provide novel insights into the interplay between nutrient availability and water stress in modulating metabolomic pathways. Under drought stress, EO was dependent on P, with significant upregulation of phenylpropanoid pathways and increased biosynthesis of antioxidant metabolites in P-fertilized plants. Conversely, P had a minimal influence on the metabolomic profile of RO under drought, highlighting the species\u0026rsquo; adaptability to drought conditions. The greater resilience of RO is likely related to its intrinsic ability to produce polyphenols and tannins, as well as to employ strategies for water uptake under dry conditions and optimize nutrient acquisition, maintaining a lower overall nutrient demand. This capacity allows RO to thrive in environments with limited water and nutrients, potentially giving it a competitive advantage over EO in drought-prone ecosystems.\u003c/p\u003e \u003cp\u003eHowever, P addition may enhance EO competitiveness, which could limit the spread of the alien species in favor of the native one in ecosystems increasingly affected by drought due to climate change.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank \u0026ldquo;La Mandria\u0026rdquo; Natural Park for granting the permissions to conduct sampling within the park. Their support and cooperation were essential to this research. This study was carried out within the Agritech National Research Center and received funding from the NextGenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) \u0026ndash; MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 \u0026ndash; D.D. 1032 17/06/2022, CN00000022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEuropean Union Next-Generation EU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) \u0026ndash; MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 \u0026ndash; D.D. 1032 17/06/2022, CN00000022), Spoke 6.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll the authors declare they have no financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All authors contributed to the study\u0026apos;s conception and design. Conceptualization: Morena Rolando, Francesca Secchi, Luisella Celi. Data curation and visualization; Morena Rolando, Paola Ganugi, Luisella Celi, Francesca Secchi, Leilei Zhang. Formal analysis and investigation: Morena Rolando, Paola Ganugi, Francesca Secchi, Luisella Celi, Leilei Zhang, Luigi Lucini. Methodology: Morena Rolando, Luisella Celi, Francesca Secchi, Paola Ganugi, Luigi Lucini, Leilei Zhang. Supervision: Luisella Celi, Francesca Secchi. Project administration: Luisella Celi. Writing - original draft: Morena Rolando, Paola Ganugi, Leilei Zhang. Writing - review \u0026amp; editing: all authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgati G, Matteini P, Goti A, Tattini M (2007) Chloroplast‐located flavonoids can scavenge singlet oxygen. 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Industrial Crops and Products 29:629\u0026ndash;633. https://doi.org/10.1016/j.indcrop.2008.08.002\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"drought, abiotic stress, phosphorous, Quercus spp., metabolism, resilience","lastPublishedDoi":"10.21203/rs.3.rs-6420617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6420617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Aims\u003c/h2\u003e \u003cp\u003ePlant resilience to drought conditions is modulated by physiological and metabolic responses, including the accumulation of antioxidant metabolites. These mechanisms can be in influenced by nutrient availability, especially phosphorus (P). In European mixed forests, native species with high resource demands often compete with more efficient alien species, whose advantages under abiotic stress can enhance their invasiveness. This study investigates how drought and P availability affect the resilience of two \u003cem\u003eQuercus\u003c/em\u003e species differing in stress tolerance and resource use efficiency.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe conducted a mesocosm experiment in which the native \u003cem\u003eQ. robur\u003c/em\u003e (EO) and alien \u003cem\u003eQ. rubra\u003c/em\u003e (RO) were grown under well-watered or drought conditions, with or without P supplementation. Leaf gas exchange and stem water potential were measured, leaves and soil were chemically characterized, and metabolite biosynthesis was investigated using an omics approach.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDuring drought, the metabolism of P-supplied EO shifted toward the accumulation of phenylpropanoids, flavonoids, saikosaponins, mannitol, and long-chain fatty acids, which are compounds known for their antioxidant, osmoprotectant, and membrane-stabilizing functions. Conversely, RO plants displayed a more conservative metabolic profile, with limited changes in response to P supply. Under P deficiency, RO accumulated secondary metabolites such as phenylpropanoids and alkaloids, highlighting its ability to withstand with combined nutrient and water stress.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur results reveal contrasting adaptive strategies: EO is more susceptible to drought stress, but its resilience can be modulated by P availability, whereas RO maintains more stable physiological and metabolic functions, consistent with higher nutrient-use efficiency.\u003c/p\u003e","manuscriptTitle":"The interplay between drought and P scarcity shapes resilience to stress in Quercus spp. by modulating metabolomic profiles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 06:09:49","doi":"10.21203/rs.3.rs-6420617/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-17T08:16:06+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T04:56:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2025-04-11T09:44:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-11T02:06:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2025-04-10T09:29:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5678e64a-4a44-47be-b7fb-ed43ce0288dc","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:07:21+00:00","versionOfRecord":{"articleIdentity":"rs-6420617","link":"https://doi.org/10.1007/s11104-025-07610-4","journal":{"identity":"plant-and-soil","isVorOnly":false,"title":"Plant and Soil"},"publishedOn":"2025-06-12 15:57:26","publishedOnDateReadable":"June 12th, 2025"},"versionCreatedAt":"2025-05-07 06:09:49","video":"","vorDoi":"10.1007/s11104-025-07610-4","vorDoiUrl":"https://doi.org/10.1007/s11104-025-07610-4","workflowStages":[]},"version":"v1","identity":"rs-6420617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6420617","identity":"rs-6420617","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}