Physiological responses of Robinia pseudoacacia L. of different origins to soil nitrogen supply

Abstract

• Robinia pseudoacacia L. is a widely distributed tree species capable of adapting to different nitrogen (N) supplies in the soil. However, provenance‐specific responses of Robinia to different soil N supplies are largely unexplored.

• To investigate the importance of soil N availability alone for the performance of Robinia, this study examined the physiological responses of five Robinia provenances to different N levels under conditions without exogenous inoculation. The provenances selected for this study originated from four provinces in China with different climates, i.e., Liaoning (LN), Ningxia (NX), Jiangxi (JX), and Shandong (L155 and L5001), and were cultivated under normal (1.4 mM), high (14.3 mM), and low (0 mM) N supplies. After 8 months (N treatments were conducted over the following three and a half months) of cultivation, CO₂ and H₂O gas exchange, the activities of enzymes involved in N metabolism, N contents and partitioning, and the accumulation and scavenging of reactive oxygen species were analyzed.

• The results revealed significant differences in biomass, photosynthesis, and N partitioning across provenances. JX and L155 exhibited improved N metabolism at high N supply, whereas L5001 and LN showed enhanced N metabolism under both high and low N supplies. Additionally, at low N, L155, L5001, and NX showed greater oxidative stress resistance and membrane stability compared to JX and LN.

• These physiological differences between provenances were found to be driven by local climate parameters, such as mean annual temperature and precipitation. Long‐term adaptation to the climate at the site of origin plays a crucial role in shaping the performance of Robinia at different N supplies.

The present study demonstrates that variation in mineral N supply triggered coordinated and provenance‐specific adjustments in nitrogen assimilation, carbon gain, oxidative stress and biomass allocation among the five Robinia provenances analysed. Our results are therefore particularly relevant for nursery production and early plantation establishment, where mineral N availability critically influences seedling survival and initial biomass accumulation.

INTRODUCTION

Nitrogen (N) is a crucial element in many biological molecules, including amino acids, proteins, nucleotides and chlorophyll, making it indispensable for key biological processes such as photosynthesis and protein synthesis (Mu & Chen ²⁰²¹). As a consequence, N availability affects growth, nutrient partitioning and abiotic stress resistance of plants (Pandey 2018; Wang et al. ²⁰²⁵). In their environment, plants are exposed to varying N availability depending on soil type, anthropogenic fertilizer application and climate (Ahmedi et al. ²⁰¹⁰; Gavit et al. ²⁰¹⁹). In croplands, the spatial and temporal dynamics of soil N are complex due to human activities and biogeochemical processes (Deng et al. ²⁰²⁰). For instance, in East China, total soil N levels in agricultural systems range from 0.25–3.64 g kg⁻¹, thus showing considerable variation (Deng et al. ²⁰²⁰). The N level in grassland was found to be higher than in cropland, as moisture accumulation promotes the build‐up of soil organic matter (SOM) (Wiesmeier et al. ²⁰¹³). For forests, the majority of N is derived from atmospheric deposition and N₂ fixation (Johnson & Turner ²⁰¹⁴). While external organic N input stimulates N₂O emission from soil nitrification, soil denitrification can limit this process by the conversion of N₂O to N₂ (Gao et al. ²⁰¹⁵; Rennenberg & Dannenmann ²⁰¹⁵), further contributing to the wide variability of N levels in forest soils.

Clearly, the soil nutrient status is closely linked to plant growth and development (Rennenberg & Dannenmann ²⁰¹⁵; Meller et al. ²⁰²⁰). This is largely due to long‐term plant adaptation during evolution in original habitats, which result in differing nutrient limitations across plant provenances (Sun et al. ²⁰²³). To cope with external N, plants have developed a huge N sensory system accompanied by physiological responses (Gent & Forde ²⁰¹⁷). Tree species differ strongly in their physiological responses to soil N availability depending on their functional type, particularly between N₂‐fixing and non‐N₂‐fixing species (Marron et al. ²⁰¹⁸; Marron & Epron ²⁰¹⁹). In non‐N₂‐fixing trees, increased soil N supply generally stimulates photosynthesis, leaf N content and growth, but may also reduce N use efficiency and induce oxidative stress under excessive N availability (Wang et al. ^{2010, 2018a, 2018b}). In contrast, N₂‐fixing species often downregulate symbiotic N₂ fixation when soil N is abundant, while relying more strongly on biological N₂ fixation under low N availability (Liu et al. ²⁰²⁴), leading to distinct patterns of N partitioning, enzyme activities and C–N balance (Mantovani et al. ²⁰¹⁵). Such physiological responses vary not only between different plant species (Chen et al. ²⁰⁰⁵), but also among different provenances of the same species, as observed, for example in Pinus tabulaeformis (Song & Hou ²⁰²⁰) and Machilus pauhoi (Zou et al. ^{2022a, 2022b}). Provenance based differences in root and leaf responses to varying N levels are attributed to adaptation strategies, which affect N allocation and utilization (Song & Hou ²⁰²⁰; Zou et al. ^{2022a, 2022b}). N deficiency or excess can disturb cellular redox homeostasis, leading to excessive production of reactive oxygen species (ROS) that damage lipids, proteins, and membranes (Anjum et al. ²⁰¹⁵; Zakari et al. ²⁰²⁰). N limitation may restrict protein synthesis and antioxidant enzyme production, whereas excessive N can enhance nitrate reduction and photorespiratory activity, both of which may increase ROS generation (Zhong et al. ²⁰¹⁷; Mu & Chen ²⁰²¹). Consequently, alteration in N availability are closely linked to oxidative stress responses in plants. Such oxidative damage is commonly reflected by increased H₂O₂ and malondialdehyde (MDA) levels (Xia et al. ²⁰²⁵) as indicators of oxidative stress and membrane lipid peroxidation, reflecting cellular damage rather than direct evidence of stress resistance. Provenances with more efficient antioxidant systems or N use may therefore maintain lower oxidative damage, thereby sustaining photosynthesis and growth (Goncharuk et al. ²⁰²²; Xia et al. ²⁰²⁵). Where growth performance is maintained despite elevated oxidative stress markers, this indicates a capacity to tolerate or compensate oxidative stress, rather than providing direct proof of resistance.

Black locust (Robinia pseudoacacia L.), a widely distributed N‐fixing tree species, is known for its economic importance and strong abiotic stress resistance (Liu et al. ²⁰²⁰). As a consequence, its cultivation is an important factor of forest economy in many countries (Liu et al. ²⁰²⁰) for timber (Rédei et al. ²⁰⁰⁸), leaf fodder (Papanastasis et al. ²⁰⁰⁸) and honey production (Farkas & Zajácz ²⁰⁰⁷). It thrives in environments with varying nitrogen (N) levels, ranging from N‐deficient saline soils (Mao et al. ²⁰¹⁶; Gavit et al. ²⁰¹⁹) to loam with high N contents (Nicodemus et al. ²⁰⁰⁸). Additionally, due to its symbiotic nitrogen fixation (SNF) with rhizobia, Robinia indirectly serves as a N source for non‐N₂‐fixing plants in mixed plantings by improving soil N contents (Hu et al. ²⁰¹⁷). Hence, Robinia has a remarkable adaptability to different N availability in the soil. For this adaptation of N nutrition, long‐term adaptation to original habitats is thought to be closely linked to genetic variation (Ramírez‐Valiente et al. ²⁰¹⁴; Song & Hou ²⁰²⁰), suggesting that even the same species in different locations may exhibit distinct characteristics. However, previous studies on Robinia were more related to SNF and abiotic stress than provenance‐specific responses to N nutrition (Wang et al. ^{2021a, 2021b}; Xue et al. ²⁰²⁴). Consequently, there is limited understanding of how different Robinia provenances respond to varying nitrogen supplies, particularly regarding to intrinsic differences in N uptake, metabolism and allocation that are independent of symbiotic N₂ fixation.

In this study, we hypothesized that (i) N partitioning of Robinia provenances is related to the capacity of N assimilation and metabolism, which depend on the sensitivity of different provenances to N supply; (ii) reduced oxidative damage and improve membrane stability facilitate Robinia provenances to cope with stress by low and high N availability; (iii) the capacity of carbon (C) and N metabolism of different Robinia provenances is influenced by mean annual temperature and mean annual precipitation at the site of origin of these provenances.

MATERIALS AND METHODS

Plant material and soil composition

The present experiments were conducted in the College of Resources and Environment, Southwest University, Chongqing (29°49′ N, 106°25′ E) at the Center of Molecular Ecophysiology (CMEP) with five different Robinia pseudoacacia L. provenances from four provinces in China. LN originated from Liaoning province (41°48′N, 123°25′ E) with a temperate continental monsoonal climate and brown soil; NX originated from Ningxia province (38°17′N, 105°50′ E) with a continental climate and loessial soil; JX originated from Jiangxi province (27°18′N, 116°00′ E) with a humid subtropical monsoon climate and red soil; L155 and L5001 originated from Shandong province (36°24′N, 118°24′ E) with a warm temperate monsoon climate and brown soil (Hui et al. ²⁰²⁵). Mean annual temperature and precipitation during the last 6 years at these provinces are shown in Table S1. The seeds of LN, NX and JX were purchased from nurseries that collect and propagate seeds for the regional tree population for reforestation and afforestation purposes. L155 (No. ROPSE3701810015) seeds were obtained from the Forest Tree Germplasm Resource Center (Shandong, China); L5001 (No. ROPEU3707720003) seeds were obtained from the Forestry Research Institute of the China Academy of Forestry Sciences (Beijing, China). All seeds were collected from adult, reproductive Robinia trees (ca. 10 years old) from nurseries specific to each provenance.

Seeds were visually screened prior to germination to ensure uniformity. Only fully developed, undamaged seeds of similar size and shape were selected, while visibly shrivelled or irregular seeds were excluded. This procedure was applied consistently across all provenances. Selected seeds were then immersed in sterile water for 24 h and subsequently boiled in water for 5 min. The seeds were then moved to culture trays with moist sterilized paper covering them, and they were put in a climate chamber (RLD‐1000E‐4, Ledian Ltd., Ningbo, China) with a temperature of 25 °C and a photosynthetic active radiation illumination of 400 μmol m⁻² s⁻¹ for 12 h. Uniform seedlings were moved into plastic pots that were 20 cm in diameter and 20 cm in height once they had grown to a length of 5 to 6 cm. They were then raised in a greenhouse. All pots contained 2.5 kg mixed substrate composed of 95% commercial garden soil (total nitrogen 1.61 g kg⁻¹, total phosphorus 0.73 g kg⁻¹, total potassium 22.69 g kg⁻¹, organic matter 82.24 g kg⁻¹ and pH = 7.34) and 5% perlite that were sterilized by carbendazim (Liu et al. ²⁰²⁴; Chen et al. ²⁰²⁵).

Seedling cultivation and experimental design

Seedlings were cultivated for a total of 240 d (8 months) in the greenhouse (temperature range 25 ~ 35 °C, about 500 μmol m⁻² s⁻¹ photosynthetic active radiation for 12 h, humidity was determined by daily differences of the local climate). During the first 135 d, 300 mL nutrient solution (0.05 mM K₂SO₄, 0.25 mM MgSO₄·7H₂O, 1 mM CaCl₂, 6 mM KNO₃, 1 mM NH₄H₂PO₄, 2 μM H₃BO₃, 1 μM MnSO₄·H₂O, 0.2 μM CuSO₄·5H₂O, 0.1 μM CoSO₄·7H₂O, 0.5 μM ZnSO₄·7H₂O, 0.1 μM Na₂MoO₄·2H₂O) was applied every 14 d for each seedling. According to the greenhouse condition, each seedling was watered with 300 mL of deionized water every 3 ~ 5 d to ensure normal growth (Broughton & Dilworth ¹⁹⁷¹; Fotelli et al. ²⁰¹¹).

Before different N treatments, seedlings of uniform size and vigour were selected to minimize variation in initial growth status. The selection was applied consistently across all provenances and aimed to reduce the influence of early development differences on treatment responses. Such pre‐selection was a common practice in controlled experiments and is not expected to introduce systematic bias among provenances, but rather to improve the comparability of physiological responses to N supply. The selected seedlings were then assigned to different N treatments in a completely randomized block design with low N (LN: 0 mM) representing the degraded soil at the N‐poor forest sites of the Loess Plateau, northwest China, normal N (NN: 1.4 mM), and high N (HN: 14.3 mM) for 105 d with 4 plants for each treatment. KNO₃ and NH₄H₂PO₄ were used as N sources as previously described (Hu et al. ²⁰¹⁷). Other elements were continued to be supplied as indicated above. This experimental design resulted in a total of 60 seedlings, and four replicates were used for all determinations in each treatment. To exclude the influence of symbiotic N₂ fixation, the growth substrate was sterilized and seedlings were grown without rhizobial inoculation. Under these conditions, plant N acquisition depended solely on the N treatments.

Determination of CO₂ and H₂O gas exchange

An open gas exchange system (Li‐6800, Li‐Cor, Lincoln, Nebraska, USA) was used to measure the net photosynthetic rate (P _n ), stomatal conductance (G _s ), intercellular CO₂ concentration (C _i ) and transpiration rate (T _r ) of Robinia leaves the day before plant harvest. The parameters were as follows: leaf chamber temperature of 25 °C, relative humidity of 60%, photosynthetic active radiation of 1500 μmol m⁻² s⁻¹ and CO₂ concentration of 400 μmol mol⁻¹. Water use efficiency (WUE) was calculated as the ratio of P _n to T _r (Liu et al. ²⁰¹³). Three mature leaves in the middle and top sections of the four Robinia plants that were grown were measured for each seed source and all N supply. Six data points were noted for every leaf.

Harvest and determination of water content

After harvest, the seedlings were carefully washed with running water to remove adhering soil particles and subsequently rinsed 3 ~ 4 times with distilled water (Sun et al. ²⁰²³). Each plant was separated into roots, stems and leaves and weighed to determine fresh weight (FW). For dry weight determination, plant tissues were frozen in liquid N, ground into a fine powder using a cryomill (JXFSTPRP‐CL, Shanghai Jing Xin) and thoroughly homogenized. Subsamples (1 g) of the homogenized tissue were then dried at 65 °C for 48 h until constant weight was achieved. Drying at 65 °C was widely used for plant tissue, including woody species, as it ensures complete removal of water while minimizing thermal degradation of organic compounds (Sun et al. ²⁰²³; Liu et al. ²⁰²⁴). The use of homogenized material ensures that the subsamples are representative of the whole tissue. The following formula was used to determine the water content:

$$\text{Water content}\left(\mathrm{mg}\mathrm{H}2\mathrm{O}/\mathrm{mg}\mathrm{FW}\right)=\frac{\mathit{FW}-\mathit{DW}}{\mathit{FW}}\times 100\%$$

Determination of total C and total N contents, δ¹³C and δ¹⁵N abundance

Tin capsules were filled with 100 ~ 300 μg of dry leaf and root powder (less than 2% water content) that had been weighed (IVA Analysentechnik, Meerbusch, Germany). In order to quantify the δ¹³C and δ¹⁵N signatures as well as the total C and N contents, element and stable isotope analyses were performed using a C/N elemental analyser connected to a Stable Isotope Ratio Mass Spectrometer (EA‐IRMS) [Sercon HS‐2022, Sercon Ltd, Cheshire, UK], as previously detailed by Simon et al. (2011). The Hangzhou Research Interest Information Technology Co. (Hangzhou, China) conducted the measurements.

Quantification of nitrate and ammonium in plant tissues

Nitrate and ammonium were extracted and determined by plant nitrate and ammonium kits (Comin, Biotechnology Co. Ltd., Suzhou, China) following the manufacturer's instructions. For both nitrate and ammonium determination, fresh plant samples were mixed at a mass (g): distilled water (mL) ratio of 1: 5 at room temperature. For nitrate determination, the mixtures were extracted in a 90 °C water bath for 30 min with continuous shaking. After cooling to room temperature, samples were centrifuged at 12,000 g for 15 min and the supernatants were used for nitrate analysis. For ammonium determination, the mixtures were centrifuged at 12,000 g and 4 °C for 10 min and the supernatants were used for ammonium analysis. Using a spectrophotometer (AOE instrument, Shanghai, China), nitrate and ammonium contents were determined at 410 nm and 625 nm absorption, respectively, and calculated by comparison with standards. Standard curves: y = 0.0156x + 0.0073, R² = 0.9997 (nitrate); y = 0.0374x – 0.0623, R² = 0.9989 (ammonium). Plant samples for each treatment and provenance were analysed with four biological replicates (n = 4), and each extract was technically measured in triplicate to ensure analytical precision. Calibration curves were prepared using standard solutions provided in the kits and were run for each measurement batch. The linearity of the standard curves was confirmed prior to sample analysis.

Quantification of total protein in plant tissues

About 0.15 g of polyethylene polypyrrolidone (PVPP) was combined with 0.05 g of leaf or root powder to eliminate phenolic compounds. The mixture was incubated at 4 °C for 30 min and centrifuged at 4 °C at 14,000 g for 10 min after 1.5 mL of buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA, 15% glycerol (v/v), 900 μL phenylmethylsulfonyl fluoride (PMSF, 0.1 M), 450 μL dithiothreitol (DTT, 1 M) and 90 μL 1‰ Triton X‐100) was added. After adding 500 μL of supernatant to 500 μL of room temperature 10% trichloroacetic acid and incubating for 10 min, the mixture was centrifuged for 10 min at 4 °C and 14,000 g. At room temperature, 500 μL of supernatant was added to 500 μL of 10% trichloroacetic acid, incubated for 10 min, and then centrifuged for 10 min at 4 °C and 14,000 g. The precipitate was then dissolved in 1 mL of 1 M KOH at 4 °C for 30 min while being shaken. Using a UV‐1800 spectrophotometer (AOE instrument, Shanghai, China), 200 μL extract aliquots were combined with 4 mL Bradford reagent (0.1 g Coomassie brilliant blue G‐250) in 50 mL 90% ethanol and 100 mL 85% (w/v) phosphoric acid. The mixture was then examined at 595 nm. The standard utilized was bovine serum albumin. Protein N was calculated as previously described (Hu et al. ²⁰¹³; Xia et al. ²⁰²⁵).

Quantification of total amino acids in plant tissues

About 0.2 mL of Hepes buffer (containing 20 mM Hepes, 5 mM EGTA, 10 mM NaF, pH 7.0) and 1 mL of a methanol/chloroform combination (3.5:1.5, v:v) were combined with 0.05 g of plant powder. Following a 30 min incubation period in an ice bath, 600 μL of distilled water was added, and the mixture was centrifuged for 5 min at 4 °C and 14,000 g. Aliquots of 240 μL supernatant were incubated for 30 min at 100 °C with 240 μL of ninhydrin reagent (solutions A and B: 1:1; solution A: comprising 2.1 g monohydrate citric acid and 0.067 g anhydrous stannous chloride in 20 mL 1 M NaOH in distilled water at a constant volume of 50 mL, pH 5.0; solution B: 2 g ninhydrin in 50 mL ethylene glycol monomethyl ether). About 3 mL of 50% isopropyl alcohol was added once the mixture had cooled to room temperature, and it was then left to incubate for 15 min in the dark. The combination was then examined at 570 nm using a UV‐1800 spectrophotometer. The standard was L‐glutamine. Total amino acid N was calculated as previously reported (Hu et al. ²⁰¹³, Xia et al. ²⁰²⁵).

Calculation of structural N

Nitrate N, ammonium N, protein N and total amino acid N were subtracted from total N to determine the structural N content of plant tissues (Hu et al. ²⁰¹³). The dry weight of the plant samples served as the basis for all computations.

Determination of malondialdehyde and hydrogen peroxide content

The modified glucosinolate/barbituric acid test, previously published (Li et al. ²⁰⁰⁸; Liu et al. ²⁰²⁴), was used to measure the amount of malondialdehyde (MDA). Five millilitres of 5% trichloroacetic acid (TCA) were combined with 0.5 g of dry powder in aliquots, and the resulting homogenate was centrifuged for 5 min at 3,000 rpm. After adding aliquots of 2 mL supernatant to 2 mL of 0.67% thiobarbituric acid (TBA), the mixture was heated for 30 min at 100 °C in a water bath, allowed to cool to room temperature and then centrifuged once more. A UV‐1800 spectrophotometer was used to measure the supernatant's absorbance at 450, 532 and 600 nm. The MDA concentration was then computed as follows:

$$\mathrm{MDA}\text{content}\left(\text{μmol}\mathrm{L}-1\right)=\frac{6.45\left(A_{532}-A_{600}\right)-0.56A_{450}}{\mathit{FW}*1000}\times V$$

A: absorption at the wavelength indicated; V: volume of the extract.

The technique outlined by Nxele et al. (2017) was used to determine the hydrogen peroxide (H₂O₂) contents. For this, 1.5 mL of 0.1% TCA (w:v) was combined with 0.1 g of plant tissue powder. The mixture was centrifuged for 15 min at 12,000 g and 4 °C. To 0.5 mL of potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI, an aliquot of 0.5 mL supernatant was added. A UV‐1800 spectrophotometer was used to detect the absorption at 390 nm after the combination was left in the dark for an hour. The amount of H₂O₂ was determined by comparing it to a standard calibration curve.

Determination of enzyme activities

The activities of nitrate reductase (NR), glutamine synthetase (GS), oxo‐glutarate aminotransferase (NADH‐GOGAT) and oxo‐glutarate aminotransferase (Fd‐GOGAT) were all measured by assay kits (Comin, Biotechnology Co. Ltd., Suzhou, China).

NR catalyses the reduction of nitrate to nitrite by consumption of NADH. Since NADH exhibits maximum absorbance at 340 nm, NR activity was expressed as the rate of NADH oxidation, measured by the decrease in absorbance at 340 nm. For NR extraction, approximately 0.1 g of plant sample and 1 mL extraction solution were homogenized in an ice bath. The homogenate was centrifuged at 8,000 g and 4 °C for 10 min and the supernatant was mixed with the reagents for the determination of NR activity. The absorbance of the mixture was measured at 340 nm after 1 and 6 min using a UV‐1800 spectrophotometer. Based on the difference in absorbance between the two time points, nitrate reductase activity was computed.

GS catalyses the synthesis of glutamine from ammonium and glutamate in the presence of ATP and Mg²⁺. The glutamine produced in this reaction can be converted to γ‐glutamylhydroxamate, which forms a coloured complex with iron under acidic conditions. This complex displays maximum absorbance at 540 nm, allowing GS activity to be determined spectrophotometrically. For GS extraction, approximately 0.1 g of fresh plant sample was added to 1 mL extraction solution and homogenized in an ice bath. The homogenate was centrifuged at 8,000 g and 4 °C for 10 min. Subsequently, the supernatant. was mixed with the reagents provided by the assay kit according to the instruction manual, incubated at room temperature for 10 min and centrifuged at 8,000 g for 10 min. The absorbance of the supernatant was measured in a UV‐1800 spectrophotometer at 540 nm. GS activity was calculated as the difference between the complete reaction mixture and its respective control reaction.

GOGAT catalyses the transfer of the amino group from glutamine to α‐ketoglutarate, yielding two molecules of glutamate. For leaf samples, the glutamate generated is oxidized by glutamate dehydrogenase in a coupled reaction, leading to the simultaneous production of NADH. The resulting NADH reduces WST‐8 to an orange‐coloured formazan dye. The formation of this dye is monitored by the increase in absorbance at 450 nm. With this approach, interference with leaf pigments is avoided. For root samples, GOGAT activity was measured by the reverse reaction, with the oxidation of NADH to NAD⁺ coupled to glutamate synthesis. Consequently, GOGAT activity in roots is reflected by the rate of decrease in NADH absorbance at 340 nm. For NADH‐GOGAT extraction, approximately 0.1 g of fresh root sample was added to 1 mL extraction solution, homogenized in an ice bath and centrifuged at 8,000 g and 4 °C for 10 min. The supernatant was mixed with the reagents of the assay kit as indicated by the manufacturer. The absorbance of the mixture was measured by using a UV‐1800 spectrophotometer at 340 nm after 20 s and 5 min 20 s. The difference in absorbance between the two points was used to calculate the root enzyme activity. For Fd‐GOGAT extraction from leaf material, approximately 0.1 g of fresh plant sample was added to 1 mL of extraction solution, homogenized in an ice bath and centrifuged at 10,000 g and 4 °C for 10 min. The supernatant was collected and mixed with the reagents provided in the assay kit according to the instructions of the manufacturer. The mixture was incubated at 30 °C for 30 min, followed by incubation in a 95 °C water bath for 5 min. After cooling, samples were centrifuged at 10,000 g and 4 °C for 5 min. The supernatant was mixed with the reaction solution of the assay kit and incubated at 25 °C for 30 min. The absorbances of the samples and controls were then measured at 450 nm using a UV‐1800 spectrophotometer. One control was set up for each sample, Fd‐GOGAT activity was calculated based on the differences between the two absorbances.

Statistical analysis

Microsoft Excel was used to process all of the data. Using two‐way ANOVA and SPSS 26.0 software, differences between N supply and provenances were examined (SPSS Inc., Chicago, USA). Initially, the Shapiro–Wilk test was used to check if the raw data had a normal distribution. Logarithmic or square root transformations were used to non‐normally distributed data. Tukey's test was used to examine the statistical significance of differences at a significance level of P < 0.05. GraphPad Prism 9.0 (San Diego, CA, USA) was used to visualize the data.

RESULTS

Biomass and water contents in roots and leaves

Compared to normal N supply, high N significantly increased root biomass in the JX and LN provenances, but reduced it in NX to the lowest level recorded (Table 1). At low N supply, root biomass decreased notably in L155 and NX, but increased in L5001 to the highest level (Table 1). High N substantially boosted leaf biomass of L5001 and LN, whereas low N caused a sharp decline in L155 leaf biomass, but increased leaf biomass in L5001 and LN (Table 1). When considering overall biomass accumulation (root and leaf biomass combined), L155 showed a strong dependence on soil N supply, while L5001 maintained or even increased total biomass under low N supply. N supply had no significant effect on root water content across most Robinia provenances, except for a decrease in L5001 at low N supply (Table 1). In contrast, leaf water content significantly increased in JX at high N supply and remained unaffected in the other provenances. Overall, changes in N supply influenced biomass accumulation more strongly than tissue water contents.

Table 1.

Biomass and water contents in roots and leaves of different Robinia provenances in response to N supply.

origin	treatment	biomass (g)		water content (%)
		roots	leaves	roots	leaves
JX	LN	9.52 ± 1.05 Bc	12.63 ± 0.40 Ab	0.67 ± 0.057 Aab	0.71 ± 0.018 ABa
	NN	11.93 ± 0.35 Bbc	14.32 ± 2.13 Ab	0.65 ± 0.019 Aa	0.67 ± 0.085 Ba
	HN	15.47 ± 1.93 Aa	14.12 ± 2.32 Abc	0.64 ± 0.037 Ac	0.74 ± 0.022 Aa
L155	LN	8.97 ± 0.47 Bc	9.97 ± 1.37 Bc	0.63 ± 0.055 Bb	0.67 ± 0.074 Ba
	NN	13.92 ± 1.86 Ab	14.39 ± 1.48 Ab	0.71 ± 0.062 ABb	0.71 ± 0.026 ABa
	HN	15.73 ± 0.73 Aa	13.61 ± 0.51 Ac	0.75 ± 0.027 Aa	0.74 ± 0.017 Aa
L5001	LN	17.89 ± 1.63 Aa	13.55 ± 0.34 Bab	0.66 ± 0.069 Aab	0.71 ± 0.043 Aa
	NN	11.74 ± 1.72 Bbc	11.06 ± 0.66 Cc	0.71 ± 0.049 Aa	0.71 ± 0.071 Aa
	HN	12.48 ± 1.47 Bb	17.81 ± 1.18 Aa	0.68 ± 0.062 Aabc	0.74 ± 0.028 Aa
LN	LN	8.03 ± 1.38 Bc	10.15 ± 0.24 Bc	0.73 ± 0.056 Aa	0.71 ± 0.018 Aa
	NN	9.48 ± 1.64 Bc	7.74 ± 0.67 Cd	0.67 ± 0.076 Aa	0.73 ± 0.028 Aa
	HN	12.33 ± 1.04 Ab	12.53 ± 0.31 Ac	0.65 ± 0.054 Abc	0.69 ± 0.059 Aa
NX	LN	11.85 ± 0.68 Bb	14.85 ± 1.64 Aa	0.70 ± 0.055 Aab	0.73 ± 0.021 Aa
	NN	18.89 ± 1.88 Aa	18.84 ± 0.99 Aa	0.73 ± 0.042 Aa	0.71 ± 0.026 Aa
	HN	9.38 ± 0.77 Cc	17.34 ± 2.96 Aab	0.73 ± 0.036 Aab	0.72 ± 0.040 Aa

JX indicates Robinia originating from the Jiangxi province, L155 and L5001 indicate two provenances of Robinia originating from the Shandong province, LN (in the first column) indicates Robinia originating from the Liaoning province, and NX indicates Robinia originating from Ningxia. NN, normal nitrogen supply, as the control group; HN, high nitrogen supply; LN, low nitrogen supply (second column). All data are means (± SE) (n = 4). Different lowercase letters indicate significant differences between Robinia from different origins at the same N supply (P < 0.05). Different capital letters indicate significant differences between different N supplies of the same Robinia provenance (P < 0.05).

Foliar CO₂ and H₂O gas exchange

Compared to normal N supply, foliar photosynthesis (P _n ), transpiration (T _r ) and stomatal conductance (G _s ) significantly decreased in JX, L5001, LN and NX under high N supply, but increased in L155, where these parameters were highest (Table 2). Under low N supply, P _n , T _r and G _s decreased significantly in all Robinia provenances except for P _n of L5001 and T _r of L155 (Table 2). Across all treatments, absolute values of G _s and P _n were comparatively low, suggesting a general limitation of gas exchange, potentially related to stomatal regulation under the experimental conditions. For intracellular carbon dioxide concentration (C _i ), JX showed a significant decrease, while NX experienced a notable increase at high N supply (Table 2). Low N supply increased C _i in L155 and NX, while reducing C _i in JX and L5001 (Table 2). Among all provenances, L155 had the highest and LN the lowest C _i at low N supply, whereas LN had the lowest C _i at high N supply (Table 2). These results indicated that high N supply inhibited photosynthetic CO₂ fixation and transpiration in the Robinia provenances except for the L155 provenance. In this provenance, photosynthetic CO₂ fixation increased with increasing N supply.

Table 2.

CO₂ and H₂O gas exchange of different Robinia provenances in response to N supply.

origin	treatment	P n (μmol m−2 s−1)	T r (mmol m−2 s−1)	G s (mol m−2 s−1)	C i (μmol mol−1)	WUE (μmol CO2 mmol−1 H2O)
JX	LN	2.87 ± 0.39 Bc	0.25 ± 0.02 Bb	0.0025 ± 0.0003 Cb	179.32 ± 10.92 Cc	11.50 ± 1.29 Aa
	NN	4.31 ± 0.64 Abc	0.39 ± 0.09 Ab	0.0340 ± 0.0160 Aa	298.55 ± 27.54 Aa	11.17 ± 0.91 Aa
	HN	0.91 ± 0.25 Cd	0.12 ± 0.03 Cd	0.0110 ± 0.0030 Bb	223.43 ± 36.70 Bb	7.69 ± 1.28 Aab
L155	LN	1.32 ± 0.19 Cd	0.39 ± 0.04 Ba	0.0044 ± 0.0004 Cab	350.54 ± 20.35 Aa	3.40 ± 0.12 Ac
	NN	1.78 ± 0.28 Bd	0.46 ± 0.08 Bb	0.0140 ± 0.0050 Bb	277.38 ± 15.01 Ba	3.95 ± 0.36 Ac
	HN	2.51 ± 0.51 Aa	0.60 ± 0.06 Aa	0.0200 ± 0.0018 Aa	258.18 ± 31.06 Ba	4.21 ± 0.44 Ac
L5001	LN	4.88 ± 0.58 Aa	0.48 ± 0.07 Ba	0.0051 ± 0.0006 Ca	183.42 ± 10.67 Bc	10.25 ± 0.84 Aab
	NN	4.45 ± 0.28 Ab	0.69 ± 0.11 Aa	0.0350 ± 0.0120 Aa	297.59 ± 21.54 Aa	6.59 ± 0.59 Bb
	HN	1.62 ± 0.27 Cc	0.25 ± 0.02 Cc	0.0097 ± 0.0018 Bb	274.92 ± 13.92 Aa	6.45 ± 0.23 Babc
LN	LN	2.89 ± 0.27 Bc	0.25 ± 0.07 Bb	0.0025 ± 0.0003 Cb	151.10 ± 8.86 Ad	12.24 ± 1.43 Aa
	NN	5.19 ± 0.66 Aa	0.47 ± 0.05 Ab	0.0170 ± 0.0048 Ab	147.17 ± 17.38 Ab	11.01 ± 0.58 Aa
	HN	2.03 ± 0.47 Cb	0.23 ± 0.03 Bc	0.0070 ± 0.0047 Bb	148.99 ± 14.60 Ac	9.18 ± 1.16 Aa
NX	LN	3.22 ± 0.37 Bb	0.43 ± 0.03 Ba	0.0045 ± 0.0005 Cab	210.15 ± 8.12 Bb	7.34 ± 0.44 Ab
	NN	4.14 ± 0.46 Ac	0.62 ± 0.05 Aa	0.0200 ± 0.0018 Ab	159.69 ± 16.84 Cb	6.67 ± 0.16 Ab
	HN	2.06 ± 0.26 Cb	0.41 ± 0.06 Bb	0.0100 ± 0.0018 Bb	233.02 ± 31.17 Ab	5.10 ± 0.57 Bbc

JX indicates Robinia originating from the Jiangxi province, L155 and L5001 indicate two provenances of Robinia originating from the Shandong province, LN (in the first column) indicates Robinia originating from the Liaoning province, and NX indicates Robinia originating from Ningxia. NN, normal nitrogen supply, as the control group; HN, high nitrogen supply; LN, low nitrogen supply (second column). Net photosynthetic rate (P _n ), transpiration (T _r ), stomatal conductance (G _s ), intracellular carbon dioxide concentration (C _i ) and water use efficiency (WUE) are presented. All data are means (± SE) (n = 3). Different lowercase letters indicate significant differences between Robinia from different origins under the same N supply (P < 0.05). Different capital letters indicate significant differences between different N supplies of the same Robinia provenance (P < 0.05).

Instantaneous water use efficiency (WUE), calculated as the ratio of P _n to T _r , differed significantly between provenances and N treatments (Table 2). Under normal N supply, JX and LN exhibited comparatively high WUE, whereas L155 showed lower WUE. High N supply reduced WUE in JX and NX, while L155 maintained a relatively stable WUE across treatments. At low N supply, WUE decreased markedly in JX but remained high in L5001 and LN (Fig. 1).

Fig. 1.

Distribution map of different Robinia provenances.

Total N and C contents and their isotope composition of roots and leaves

In L5001 roots, total N content significantly increased at low N supply, but was lowest at high N supply (Fig. 2A). In leaves of LN, total N content significantly decreased with high N supply (Fig. 2B). Both high and low N significantly reduced ¹⁵N abundances in JX and NX roots, but increased this parameter in the leaves (Fig. 2C,D). In contrast, ¹⁵N abundances in L5001 and LN roots increased at high N supply, but decreased at low N supply (Fig. 2C). Low N also elevated ¹⁵N abundances in L5001 leaves, while both high and low N reduced ¹⁵N abundances in L155 and LN leaves (Fig. 2D). The NX provenance consistently had the highest ¹⁵N abundances in roots and leaves across all N supplies (Fig. 2C,D). For total C contents, different N supply had no effect except for an increase in L5001 leaves at low N supply (Fig. 2E,F). ¹³C abundances were not affected by different N supply and provenances (Fig. 2G,H). Since the C contents in Robinia leaves and roots were constant, changes in the C/N ratio were mainly due to N alterations. The root C/N ratio in L5001 roots was significantly lower at low N and highest at high N supply, whereas NX roots had the highest ratio at low N supply (Fig. 2I). High N also increased the C/N ratio in JX leaves. These findings show an efficient N utilization particularly by the NX provenance, with increased total N content at increasing N supply, while maintaining total C contents.

Fig. 2.

Total carbon (C) and nitrogen (N) contents, stable isotope signatures and C/N ratios in roots and leaves of different Robinia provenances in responses to N supply. (A, B) Total N contents in roots and leaves. (C, D) ¹⁵N isotopic abundances in roots and leaves. (E, F) Total C contents in roots and leaves. (G, H) ¹³C isotopic abundances in roots and leaves. (I, J) C/N ratio in roots and leaves. JX indicates Robinia originating from the Jiangxi province, L155 and L5001 indicate two provenances of Robinia originating from the Shandong province, LN (in the horizontal coordinates) indicates Robinia originating from the Liaoning province, and NX indicates Robinia originating from Ningxia. NN, normal nitrogen supply, as the control group; HN, high nitrogen supply; LN, low nitrogen supply (top in coordinates). All data are means (± SE) (n = 4). Different lowercase letters indicate significant differences between Robinia from different origins at the same N supply (P < 0.05). Different capital letters indicate significant differences between different N supplies of the same Robinia provenance (P < 0.05).

N partitioning in roots and leaves

In the L155 provenance, low N significantly increased protein N contents in roots, while high N raised it in the leaves (Fig. 3A,B). Also, NX leaves showed an increase in protein N at low N supply (Fig. 3B). The total amino acid N increased in NX roots at low N, while high N elevated this N fraction in L155 and L5001 leaves (Fig. 3C,D). At low N supply, amino acid N also increased in the leaves of L155 and LN (Fig. 3D). Among all Robinia provenances, the total amino acid N content of L155 was highest in leaves at high N supply (Fig. 3D).

Fig. 3.

N partitioning in roots and leaves of different Robinia provenances in response to N supply. (A, B) Protein N contents in roots and leaves. (C, D) Total amino acid N contents in roots and leaves. (E, F) Ammonium N contents in roots and leaves. (G, H) Nitrate N contents in roots and leaves. (I, J) Structural N contents in roots and leaves. JX indicates Robinia originating from the Jiangxi province, L155 and L5001 indicate two provenances of Robinia originating from the Shandong province, LN (in the horizontal coordinates) indicates Robinia originating from the Liaoning province, and NX indicates Robinia originating from Ningxia. NN, normal nitrogen supply, as the control group; HN, high nitrogen supply; LN, low nitrogen supply (top in coordinates). All data are means (± SE) (n = 4). Different lowercase letters indicate significant differences between Robinia from different origins at the same N supply (P < 0.05). Different capital letters indicate significant differences between different N supplies of the same Robinia provenance (P < 0.05).

For inorganic N, the ammonium N content decreased in L5001 and NX roots at low N, and both, low and high N levels reduced it in L155 leaves (Fig. 3E,F). Nitrate N content increased in LN and NX roots at high N, and in NX roots at low N (Fig. 3G). Low N boosted nitrate N in L5001 leaves (Fig. 3H). The structural N content increased at low N in L5001 roots and leaves, while high N elevated it in L5001 leaves, but decreased it in LN leaves (Fig. 3I,J). These results demonstrated that organic N contents were more sensitive to N supply, with distinct responses in roots and leaves.

Activities of enzymes involved in N metabolism

In roots of JX, L155 and LN, NR activity increased significantly at both high and low N supply (Fig. 4A), while in L5001 the activity of this enzyme decreased at high N and peaked at low N supply (Fig. 4A). NX roots also showed a marked increase in NR activity at both low and high N, while in L155 roots NR activity was highest at high and lowest at low N supply (Fig. 4A). In leaves, NR activity decreased with increasing N supply in JX and NX, but increased in L155, where highest levels were recorded at high N (Fig. 4B). JX had the highest leaf NR activity at low N supply (Fig. 4B).

Fig. 4.

Activities of nitrate reductase (NR), NADH‐glutamate: oxo‐glutarate aminotransferase (NADH‐GOGAT), FD‐glutamate: oxo‐glutarate aminotransferase (Fd‐GOGAT) and glutathione reductase (GR) in roots and leaves of different Robinia provenances in response to N supply. (A, B) NR activity in roots and leaves. (C, D) NADH‐GOGAT activity in roots and Fd‐GOGAT activity in leaves. (E, F) GR activity in roots and leaves. JX indicates Robinia originating from the Jiangxi province, L155 and L5001 indicate two provenances of Robinia originating from the Shandong province, LN (in the horizontal coordinates) indicates Robinia originating from the Liaoning province, and NX indicates Robinia originating from Ningxia. NN, normal nitrogen supply, as the control group; HN, high nitrogen supply; LN, low nitrogen supply (top in coordinates). All data are means (± SE) (n = 4). Different lowercase letters indicate significant differences between Robinia from different origins at the same N supply (P < 0.05). Different capital letters indicate significant differences between different N supplies of the same Robinia provenance (P < 0.05).

NADH‐GOGAT activity increased at high N supply significantly in JX and L155 roots, while it decreased in L5001, LN and NX roots, with highest activity in L155 and lowest activity in NX roots at high N supply (Fig. 4C). At low N supply, NADH‐GOGAT activity increased in JX but decreased in L5001 roots, with JX showing the highest activity (Fig. 4C). In leaves, low N significantly boosted Fd‐GOGAT activity in JX, while the NX provenance exhibited the highest foliar Fd‐GOGAT activity at low N supply (Fig. 4D).

GS activity decreased in L155 and L5001 roots at high N, and in L155 and LN roots at low N supply (Fig. 4E). In leaves, GS activity dropped notably in LN at high N and in L5001 at low N supply, where it reached its lowest level (Fig. 4F). These results indicate that JX and L155 had enhanced N assimilation capacity at high N supply, while L5001 and LN had reduced N assimilation capacity at both high and low N supply.

MDA and H₂O₂ contents in roots and leaves

At high N supply, LN roots had the highest H₂O₂ contents, while low N increased H₂O₂ in the roots of L155, LN, and NX (Fig. 5A). In leaves, H₂O₂ levels decreased in L155 at high N, but increased in L5001 and NX at low N supply (Fig. 5B). MDA contents increased in LN roots at high N supply, while at low N supply, MDA levels decreased in L155 roots but increased in LN and NX roots (Fig. 5C). In leaves, high N reduced MDA levels in L5001 and NX, and low N further decreased this parameter in L155, L5001 and NX, with L5001 showing the lowest foliar MDA level (Fig. 5D). These results show that L155, L5001 and NX exhibited lower MDA or H₂O₂ accumulation at low N supply, indicating reduced oxidative damage and improved membrane stability compared to the other provenances.

Fig. 5.

Contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) in roots and leaves of different Robinia provenances in response to N supply. (A, B) H₂O₂ contents in roots and leaves. (C, D) MDA contents in roots and leaves. JX indicates Robinia originating from the Jiangxi province, L155 and L5001 indicate two provenances of Robinia originating from the Shandong province, LN (in the horizontal coordinates) indicates Robinia originating from the Liaoning province, and NX indicates Robinia originating from Ningxia. NN, normal nitrogen supply, as the control group; HN, high nitrogen supply; LN, low nitrogen supply (top in coordinates). All data are means (± SE) (n = 4). Different lowercase letters indicate significant differences between Robinia from different origins at the same N supply (P < 0.05). Different capital letters indicate significant differences between different N supplies of the same Robinia provenance (P < 0.05).

Correlations of root and leaf traits with local climate

Correlations were found between traits of the Robinia provenances and local climate (mean annual temperatures and annual precipitations). Positive correlations with mean annual temperature were identified for the C/N ratio of L5001 roots and ammonium N contents as well as P _n and G _s in JX leaves (Fig. 6). Negative correlations with mean annual temperature were observed for total N content and structural N content of LN roots and water content of NX roots (Fig. 6).

Fig. 6.

Correlation heatmaps between Robinia traits and local climate (i.e. root traits and mean annual temperature, root traits and mean annual precipitation, leaf traits and mean annual temperature, leaf traits and mean annual precipitation). The shades of red and blue indicate the correlations between Robinia traits and local climate, values in boxes indicate Pearson correlation coefficients, positive values indicate positive correlations and negative values indicate negative correlations. Asterisks indicate significant correlations (*P < 0.05; **P < 0.01). Both L155 and L5001 correspond to the climate of Shandong province, JX corresponds to the climate of Jiangxi province, LN corresponds to the climate of Liaoning province, and NX corresponds to the climate of Ningxia province. Mean annual temperature and precipitation data are used for the last 6 years (2019–2024), and the original data were from NCEI (https://www.ncei.noaa.gov/).

Precipitation was positively correlated in L155 with root water content, in L5001 roots with MDA and H₂O₂ contents, in L5001 leaves with ¹⁵N abundance, and in LN leaves with Fd‐GOGAT activity (Fig. 6). Negative correlations were noted between precipitation and GS activity in L155 roots, between precipitation and ¹³C abundance in LN leaves, and between precipitation and NR activity as well as C _i in NX leaves (Fig. 6). These results indicated that Robinia provenances adjust N metabolism and water content based on local mean annual temperature and precipitation, reflecting climate‐driven physiological adaptations.

DISCUSSION

In this experiment, physiological changes in N metabolism and ROS production and scavenging were comparatively analysed in five Robinia provenances under three N supplies. The results showed the physiological responses of different Robinia provenances to high and low N supplies differed significantly, which could be particularly linked to the variability of climatic factors at the sites of origin.

Adaptation of N metabolism to N supply differs between Robinia provenances

During plant growth, inorganic N is converted to organic N by reduction and assimilation processes (Foyer & Zhang ²⁰¹¹). As the first enzyme of N assimilation, NR reduces NO₃ ⁻ to NO₂ ⁻ that is converted to NH₄ ⁺ by nitrite reductase (NiR) (Ramírez‐Valiente et al. ²⁰¹⁴). Subsequently, NH₄ ⁺ is assimilated into amino acids by the GS/GOGAT cycle (Zhang et al. ²⁰¹³; Dong et al. ²⁰²¹). Generally, in most herbaceous plants, NR activity is low in roots while high in leaves, indicating preferential N reduction in leaves (Hikosaka et al. ¹⁹⁹⁸; Darnell & Stutte ²⁰⁰¹; Dovrat et al. ²⁰²⁰). However, N reduction of woody plants occurs preferentially in roots (Herschbach et al. ²⁰¹²). Hence, high amounts of amino acids are transported from roots to leaves in the xylem of many woody plants. However, also the use of different inorganic N sources in the soil (nitrate vs. ammonium) differs between plant species and provenances (He et al. ²⁰¹⁷; Dawood et al. ²⁰²³; Sun et al. ²⁰²³) and further interacts in N₂ fixing legumes and actinorhizal plants with the symbiotic assimilation of atmospheric N₂ (Troelstra et al. ¹⁹⁹²; Guinet et al. ²⁰¹⁸; Sun et al. ²⁰²³). Therefore, it is essential in legumes and actinorhizal plants to disentangle the consequences of N availability in the soil from the interaction of these consequences with symbiotic N₂ fixation to identify the processes involved. The present study aimed to identify the consequences of N availability in the soil in different provenances of the legume tree species Robinia. For this purpose, the present study was performed in the absence of symbiotic N₂ fixation with non‐nodulated plants.

NR activity has been reported to vary between different plant species and provenances (Hänsch et al. ²⁰⁰¹; Alt et al. ²⁰¹⁷; Sun et al. ²⁰²¹). In the present study, low N availability in the soil increased NR activity of roots in all Robinia provenances (Fig. 4A), a common regulatory pattern achieved by promoting the expression of NR‐related genes (Kavoosi et al. ²⁰¹⁴; Wang et al. ^{2018a, 2018b}). In this context, NIA1 (encoding NR) was reported to show an initial decrease in expression but gradually recovered after prolonged N deficiency (Park et al. ²⁰¹¹; Berger et al. ²⁰²⁰). Differences in NR activity are typically accompanied by changes in N content; however, the effects of NR activity on N content and partitioning are complex (Kaiser et al. ²⁰⁰²; Fu et al. ²⁰¹⁸). In the present study, enhanced NR activity at low N availability stimulated N uptake by roots (Fig. 4A), but only L5001 roots had significantly higher total N contents (Table 1, Fig. 2A) indicating more balanced N acquisition in relation to growth in the other provenances as in line with our hypothesis (i).

High N contents have been reported to promote root biomass by enhancing total root length, root surface area and volume (Chen et al. ²⁰²⁰). The stable total N at increased organic N contents of L155 and NX roots at low N supply and the stable total N content and N partitioning in JX roots observed in the present study at high N supply (Figs. 2A and 3) indicate that inorganic N uptake by the roots was balanced in these provenances to root growth (Walker et al. ²⁰⁰¹; Seginer ²⁰⁰⁴). However, the responses of NR activity in different Robinia provenances to high N supply were not consistent (Fig. 4A) indicating the significance of other enzymes of N assimilation in determining organic N production (Nicodemus et al. ²⁰⁰⁸; Zhang & Du ²⁰¹⁶; Xiong et al. ²⁰²¹; Machado et al. ²⁰²³). In addition, the high N level in Shandong soil may indirectly explain the high NR activity of this Robinia provenance, indicating an environmental adaptation to N availability at the origin of this provenance (Ge et al. ²⁰¹³; Li et al. ²⁰¹⁶).

In addition to NR activity, the activities of the GS/GOGAT cycle enzymes play an important role in organic N production (Zhang et al. ²⁰¹³, Dong et al. ²⁰²¹). In the present study, GOGAT changed more actively in roots than in leaves in response to N supply (Fig. 4C,D) supporting the view of preferential N assimilation in the roots of Robinia. This conclusion was also indicated by much higher transcript abundances of NADH‐GOGATs in roots compared to the leaves (Cao et al. ²⁰²³). As observed for NR, NADH‐GOGAT was particularly active in L155 roots (Fig. 4A,C) consistent with the high biomass accumulation of this provenance (Table 1). In previous studies, high nitrate availability decreased the GS activity in Salvia splendens, but did not affect the activity of the enzyme in Petunia hybrida, while high ammonium availability reduced the GS activity in Ageratum houstonianum (Song et al. ²⁰²²). Similarly, in both roots and leaves, GS activity either decreased or was not affected in the present study at high and low N supplies (Fig. 4E,F). Apparently, N assimilation in the GS/GOGAT cycle is determined by GOGAT activity and GS activity is present in access and can even be down‐regulated at enhanced N assimilation.

Overall, the provenances differed in their capacity to modulate N assimilation pathways under contrasting N supplies. JX and L155 showed a stronger stimulation of N‐assimilation‐related enzyme activities under high N availability, whereas L5001 and LN maintained comparatively stable N metabolic performance across both low and high N treatments. These coordinated adjustments in enzyme activities and N partitioning were reflected by differences in biomass accumulation, indicating provenance‐specific N acquisition and utilization strategies.

ROS production and scavenging of Robinia provenances are modified by N supply

Balanced ROS production and scavenging are essential traits for growth and development of plants as well as plant responses to biotic and abiotic stresses (Bailey‐Serres & Mittler ²⁰⁰⁶). As the most stable ROS, excess H₂O₂ would attack unsaturated fatty acids in cell membranes, leading to peroxidation of membrane lipids, which produces MDA in a series of catabolic reactions (Choudhury et al. ²⁰¹³; Saxena et al. ²⁰¹⁶). Thus, high H₂O₂ and MDA levels indicate enhanced oxidative stress and lipid peroxidation, reflecting membrane damage (Sahoo et al. ²⁰²⁰; Hasanuzzaman et al. ²⁰²¹; Du et al. ²⁰²⁴). However, ROS can be scavenged by the antioxidative system via chemical and enzymatic reactions (Du et al. ²⁰²⁴). Hence, balanced ROS production and scavenging determine plant health in a permanently changing environment.

Previous studies reported that the capacity of the antioxidant system is strongly linked to nutrient availability (Shin et al. ²⁰⁰⁵; Tewari et al. ²⁰⁰⁷). Extremely high N supply was regarded as toxic, as indicated by the accumulation of phenolic compounds and H₂O₂ (Sánchez et al. ²⁰⁰⁰). In the present study, the LN provenance was far less resistant to high and low N availability than other Robinia provenances indicated by enhanced H₂O₂ and MDA contents (Fig. 5A,C). A similar situation was observed for the NX provenance at low N supply (Fig. 4). These responses may be due to the toxicity of excessive accumulation of nitrite and ammonium by increased NR with low NADH‐GOGAT and GS activities in the roots (Fig. 4, Zhang et al. ²⁰²², Marc et al. ²⁰²⁴). In addition, they reflect the long‐term adaptation of these Robinia provenances to local soil properties (Dorey et al. ²⁰²⁴) that are between the high and normal N supply used in the present study (Wang et al. ^{2017a, 2017b}). Apparently, LN and NX provenances can be characterized by poor regulation of N acquisition at extreme N supplies due to long‐term adaptation to moderate soil N contents (Heinrichs et al. ²⁰²¹; Marc et al. ²⁰²⁴; Salord et al. ²⁰²⁴).

Surprisingly, L155 roots showed low MDA at increased H₂O₂ contents at low N supply (Fig. 5A,C). As a signalling molecule, H₂O₂ promotes root growth, while lower MDA levels reflect reduced oxidative damage and improved cellular health (Sahoo et al. ²⁰²⁰; Hasanuzzaman et al. ²⁰²¹), consistent with the enhanced biomass accumulation of L155 roots. The simultaneous reduction in MDA and H₂O₂ contents in the leaves of the L155, L5001, and NX at high and low N (Fig. 5B,D) indicates effective ROS detoxification to varying degrees at high and low N in aboveground tissues of these provenances. As in agreement with our hypothesis (ii), these results show that different Robinia provenances possess different ROS production and scavenging capacities at different N supplies (Ying et al. ²⁰¹⁵). Together, N limitation or excess altered oxidative stress in a provenance‐dependent manner, as reflected by H₂O₂ and MDA levels. Importantly, lower oxidative damage in certain provenances coincided with better maintenance of growth‐related traits, suggesting a greater capacity to buffer N‐induced redox imbalance and sustain physiological performance.

N supply modifies leaf C traits of Robinia provenances

Plant C contents are closely linked to photosynthesis (Zou et al. ^{2022a, 2022b}). With increasing P _n plants can absorb more CO₂ and convert it to organic matter (e.g. sugars, cellulose), thereby increasing C content and biomass (Zhang et al. ²⁰²⁰; Zou et al. ^{2022a, 2022b}). In this study, unlike other Robinia provenances, the total leaf C content of L5001 was notably elevated at low N supply, a result attributed to enhanced C assimilation driven by high P _n (Table 2, Fig. 2). This increase in total C content contributed to greater leaf biomass of L5001, further highlighting the role of photosynthetic performance in biomass allocation (Table 1, Table 2). It is noteworthy that both G _s and P _n were relatively low across treatments. Such low values may indicate a general stomatal limitation of gas exchange, as G _s is a key regulator of CO₂ diffusion into leaves and thus directly influences photosynthesis (Harrison et al. ²⁰²⁰). Importantly, reduced G _s can occur even in the absence of severe drought stress, as stomatal behaviour is also regulated by environmental conditions and intrinsic physiological traits (Pirasteh‐Anosheh et al. ²⁰¹⁶). Factors such as light intensity, temperature and plant developmental stage are known to influence gas exchange rates and may contribute to the relatively low absolute values observed in this study (Pereira 1995; Driesen et al. ²⁰²⁰). In this context, the parallel variation of G _s and P _n suggests that photosynthetic performance was at least partly constrained by stomatal regulation rather than solely by biochemical limitations. Therefore, differences among provenances should be interpreted primarily in a relative sense, rather than based on absolute gas exchange rates. The distinct responses of C content and biomass accumulation of Robinia provenances to varying N supply underscore the genetic variability in their adaptation strategies. Previous studies have shown that N limitation accelerates leaf senescence, which is closely tied to the balance of C and N (Chen et al. ²⁰¹⁵). Senescent leaves often exhibit higher C/N ratios (Chen et al. ²⁰¹⁵) due to elevated total C contents. This was also observed in L5001 leaves without symptoms of senescence at low N supply in the present study (Fig. 2). Generally, N supply significantly affects gas exchange and biomass allocation, with higher N availability causing greater leaf area and biomass due to improved photosynthetic capacity and enhanced nutrient uptake (Hauer et al. ²⁰²¹; Munjonji & Ayisi ²⁰²¹). However, changes in biomass cannot be solely attributed to P _n . A comprehensive evaluation that considers CO₂ and H₂O exchange parameters and genotype specific traits is necessary to evaluate differences in biomass accumulation (Munjonji & Ayisi ²⁰²¹). In this context, the largely stable root water content observed across N treatments shows that root water status was not strongly affected by changes in N availability. In contrast, variation in leaf water content together with changes in T _r and G _s indicates adjustments in water transport and gas exchange regulation among provenances. These differences in water content between roots and leaves in response to N supply suggest that modified water allocation and transport processes contributed to the observed differences in biomass accumulation under contrasting N supplies. The observed differences in WUE further reflect provenance‐specific strategies in balancing C assimilation and water loss under varying N supplies. Provenances L5001 and LN, which maintained relatively high WUE under low N supply appear to coordinate photosynthetic C gain and transpiration more effectively than the other provenances studied. In contrast, the reduction of WUE in JX and NX under high or low N supply suggests a less favourable adjustment between C acquisition and water loss under N stress. In this study, the five Robinia provenances studied displayed varying responses in leaf biomass to N supply, likely due to genetic differences and sensitivities to N availability developed through adaptation to their respective environments (Cuervo‐Alarcon et al. ²⁰¹⁸; Sun et al. ²⁰²³). Factors such as precipitation and latitude have been reported to affect leaf traits of legume trees (Diatta et al. ²⁰²²). Thus, while increasing N availability generally promotes physiological properties of leaves, the extent of this effect varies between leaf traits and among Robinia provenances. This variability reflects the unique N requirements and adaptation of each provenance, shaped by its environmental and genetic background. Across provenances, differences in biomass accumulation in the present study were best explained by coordinated shifts in C gain and water loss, including P _n , T _r , G _s and WUE, rather than by a single gas exchange parameter alone. These patterns indicate distinct physiological strategies in balancing productivity and WUE under contrasting N supplies.

Correlation of local climate with root and leaf traits of Robinia provenances

Plant roots and leaves can interact directly with environmental changes, and their timely responses reflect the adaptability of plants to local environmental conditions (Chang et al. ²⁰²⁴). These adaptive traits involve intricate relationships between local climate and plant physiology, driven by the combined effects of multiple climate factors rather than a single variable (Meng et al. ²⁰¹⁷). Previous studies showed that differences in climate can explain the spatial variability of plant N and phosphorus (P) contents more effectively than nutrient availability in the soil, since the patterns of nutrients in plant leaves across different life forms are shaped by the interplay of climate and soil (Meier & Leuschner ²⁰⁰⁸; Wang et al. ²⁰²²). In this context, environmental drivers significantly influence root traits (Joswig et al. ²⁰²¹). Notably, precipitation and temperature have been shown to alter the expression of genes of N metabolism and microbial community composition, underscoring the impact of climate on nutrient dynamics (Alkorta et al. ²⁰¹⁷; Ku et al. ²⁰²²). In the present study, root traits exhibited more distinct responses compared to leaf traits, with stronger positive correlations between indicators of N metabolism and mean annual temperature and precipitation. Still the specific number and strength of these correlations varied among provenances (Fig. 6). For example, L5001 demonstrated robust positive correlations between nearly all N metabolism indicators and mean annual temperature and precipitation (Fig. 6). This effect may originate from adaptation to high temperature and precipitation, thereby enhancing the abundance and activity of nitrifying and denitrifying bacteria and facilitating the conversion of N into plant available forms. Additionally, local precipitation patterns may influence soil N leaching, leading to provenance specific N utilization strategies (He et al. ²⁰²¹). Compared to other climate factors, temperature and precipitation often showed stronger correlations with leaf traits (Meng et al. ²⁰¹⁷; Caldararu et al. ²⁰¹⁹; Solé‐Medina et al. ²⁰²²). Also in this study, leaf traits correlated with mean annual temperature and precipitation across all Robinia provenances, particularly traits associated with C and N metabolism as in line with our hypothesis (iii). Furthermore, global vegetation models indicate that C metabolism strongly correlates with temperature, thereby exerting a significant influence on N metabolism (Wujeska et al. ²⁰¹⁹). Compared to the other provenances analysed in this study, L155 exhibited weaker correlations of leaf and root traits with mean annual temperature and precipitation (Fig. 6), suggesting that L155 may possess superior adaptability to climate variability. When considering all measured traits collectively, a coherent provenance‐specific response pattern emerges. Variation in mineral N supply was accompanied by coordinated changes in N assimilation and partitioning, leaf gas exchange (including WUE), oxidative stress and biomass allocation. These interrelated physiological adjustments indicate that differences between provenance are expressed not at the level of single traits, but as integrated response. Importantly, because Robinia is an N₂‐fixing species, the present experiment, conducted without nodulation, isolates intrinsic mineral‐N‐driven responses that may otherwise partially be buffered by symbiotic N₂ fixation under field conditions.

Thus, the observed relationships between Robinia provenances and climatic parameters suggest that long‐term exposure to differing temperature and precipitation regimes may have contributed to the divergence in physiological traits. However, these correlations do not imply causality, and genetic differentiation independent of climate may also play an important role. In this context, it is noteworthy that the two provenances originating from Shandong province (L155 and L5001) also displayed distinct physiological responses despite their similar geographic origin. This observation indicates that regional climate alone cannot fully explain the observed variation. Differences in local soil conditions, microhabitat characteristics and genetic differentiation may contribute to the divergence between these provenances. Therefore, climate should be considered as one driver among multiple interacting factors shaping provenance‐specific responses. From an applied perspective, these provenance‐specific physiological responses guide the deployment and management of black locust. Provenances such as L5001 and LN, which maintained efficient N metabolism and low oxidative damage across contrasting N supplies, appear suitable for sites with variable or uncertain soil N availability. In contrast, provenances showing optimal performance under high N supply (e.g. JX and L155) may be preferentially deployed on fertile or fertilized sites to maximize productivity, whereas NX may be better suited for low‐N or nutrient‐poor soils. Overall, matching Robinia provenances to site‐specific soil N and climatic conditions could improve plantation performance and resource‐use efficiency. These findings are particularly relevant for nursery production and early plantation establishment, where mineral N availability strongly influences seedling survival and initial biomass accumulation. Selecting provenance‐site combinations based on N response patterns may help reduce fertilizer input and improve N use efficiency in managed plantations. In afforestation programmes on degraded or heterogeneous soils, the use of N‐resilient provenances such as L5001 or LN may enhance establishment success, stabilize early growth and potentially reduce long‐term management costs.

This study was conducted under controlled pot conditions and without rhizobial inoculation, enabling the isolation of mineral N‐driven physiological differences among Robinia provenances. Nevertheless, pot experiments inherently limit root development, soil heterogeneity, and plant–soil–microbe interactions compared to natural forest ecosystems. Under field conditions, trees explore larger soil volumes and interact with complex microbial communities, which may substantially modify nutrient acquisition strategies and growth performance. In particular, symbiotic N₂ fixation may buffer soil N limitation, regulate internal N metabolism, and potentially alter the relative performance ranking of provenances observed under mineral‐N‐only conditions. Effective nodulation under field conditions may partially compensate for low soil N availability and reduce the magnitude of provenance differences observed under controlled mineral‐N treatments. The interaction between genetic background and rhizobial symbiosis therefore represents a key mechanism requiring validation under field conditions. Furthermore, as the present study focused on seedlings, the identified physiological response patterns are most directly relevant to the establishment phase of Robina plantations. During early development, mineral N availability critically influences survival and initial biomass accumulation. Whether these provenance‐specific N response strategies persist or shift during later ontogenetic stages in mature trees remains to be determined through long‐term field studies.

CONCLUSION

The present study demonstrates that variation in mineral N supply triggered coordinated and provenance‐specific adjustments in nitrogen assimilation, carbon gain, oxidative stress, and biomass allocation among the five Robinia provenances analysed. Differences in growth performance were not attributable to a single physiological parameter, but rather to integrated shifts in N partitioning, enzyme activities, gas exchange (including water use efficiency) and oxidative stress. L5001 and LN maintained comparatively stable N metabolic performance across contrasting N supplies, whereas JX and L155 exhibited enhanced N assimilation capacity primarily under high N availability. In contrast, NX displayed traits indicative of efficient N utilization but showed increased oxidative stress under certain N conditions. These findings indicate that differences between provenances are expressed as distinct physiological response strategies to soil N variability. Because the experiment was conducted on non‐nodulated seedlings, the observed patterns reflect intrinsic mineral‐N‐driven differences in N uptake, metabolism and stress responses, independent of symbiotic N₂ fixation. In natural or plantation systems, however, rhizobial symbiosis may partially buffer soil N limitation and potentially modify provenance‐specific response rankings. The present results are therefore particularly relevant for nursery production and early plantation establishment, where mineral N availability critically influences seedling survival and initial biomass accumulation. Whether these provenance‐specific response strategies persist in mature trees with larger N storage pools as well as more extended and deeper root systems remains to be determined through long‐term field studies integrating rhizobial interactions.

AUTHOR CONTRIBUTIONS

Data analysis, graphic production and original manuscript writing were performed by TP. Material preparation, data collection, and analysis were performed by HW, RL, ZX, JJ and CX. BH and HR contributed to the conception and design of this study and the finalization and revision of the manuscript.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

Table S1. Mean annual temperature and precipitation during 2018–2023 at Liaoning, Ningxia, Jiangxi, and Shandong provinces.

DATA AVAILABILITY

The data that support the findings of this study are available from the corresponding author upon reasonable request.