Water Uptake and Hormone Modulation Responses to Nitrogen Supply in Populus simonii under PEG-Induced Drought Stress

Abstract

In the present study, the effects of nitrogen (N) supply on water uptake, drought resistance, and hormone regulation were investigated in Populus simonii seedlings grown in hydroponic solution with 5% polyethylene glycol (PEG)-induced drought stress. While acclimating to drought, the P. simonii seedlings exhibited a reduction in growth; differential expression levels of aquaporins (AQPs); activation of auxin (IAA) and abscisic acid (ABA) signaling pathways; a decrease in the net photosynthetic rate and transpiration rate; and an increase in stable nitrogen isotope composition (δ¹⁵N), total soluble substances, and intrinsic water use efficiency (WUEi), with a shift in the homeostasis of reactive oxygen species (ROS) production and scavenging. A low N supply (0.01 mM NH₄NO₃) or sufficient N supply (1 mM NH₄NO₃) exhibited distinct morphological, physiological, and transcriptional responses during acclimation to drought, primarily due to strong responses in the transcriptional regulation of genes encoding AQPs; higher soluble phenolics, total N concentrations, and ROS scavenging; and lower transpiration rates, IAA content, ABA content, and ROS accumulation with a sufficient N supply. P. simonii can differentially manage water uptake and hormone modulation in response to drought stress under deficient and sufficient N conditions. These results suggested that increased N may contribute to drought tolerance by decreasing the transpiration rate and O₂⁻ production while increasing water uptake and antioxidant enzyme activity.

1. Introduction

Drought is a major limiting factor for plant growth on the Loess Plateau [1]. Forests in such areas often suffer from drought stress due to continued deficit of rain [2]. Increased forest mortality may be due to persistent drought stresses around the world [3]. In the past few decades, drought stress has caused widespread die-off of Populus tremuloides [4]. To ensure the sustainable development of forest plantations, it is essential to better understand the responses of woody species growing on poor soils.

P. simonii is an important poplar species on the Loess Plateau of China and is widely used for afforestation. However, the soil is often poor and water deficiency is also frequent in these regions [4]. Recently, we found that drought stress stunts nitrogen assimilation and N deficiency stimulated root development in P. simonii. However, how water uptake responds to N supply and drought stress remains unclear.

Drought stress often leads to physiological and molecular responses in plants [5,6]. A schematic model of responses to drought in plants has been well documented [7,8,9]. Poplar species (Populus spp.) are very sensitive to water availability and drought stress often decreases photosynthesis, alters root morphology, and inhibits enzyme activities. For instance, drought-induced stomatal closure often leads to an inhibited CO₂ assimilation rate [10,11]. Drought stress also can trigger the overproduction of reactive oxygen species (ROS) and eventually lead to oxidative damage. Against the harmful effects of ROS, plants’ defence systems can rely on the oxygen scavenging systems (SOD, APX, POD, and CAT) and osmotic adjustment compounds (soluble sugars, soluble phenolics, and amino acid compounds) [12,13]. In addition, water stress triggers signal transduction pathways, including the abscisic acid (ABA) and auxin (IAA) signaling pathway [14,15]. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) and the protein phosphatase 2C (PP2C) play key roles in ABA biosynthesis and ABA-mediated signaling pathways [8,16]. In auxin biosynthesis, flavin-containing monooxygenase (YUCC) and tryptophan aminotransferase (TAA) are essential components. Transcriptional regulation of the key genes involved in the ABA and/or IAA signal transduction chains are important for detecting changes in soil water availability [7,17]. Furthermore, plants transport water by apoplastic and/or cell-to-cell (symplastic and transmembrane) pathways, and aquaporins (AQPs) play crucial roles in transmembrane water uptake [18]. The expression levels of AQPs, including the plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), and small basic intrinsic proteins (SIPs) can be modulated to regulate the water uptake in various soil water availability [19,20]. However, the role of AQPs in water transport under drought condition and N supply is unclear.

Previous studies have found that carbon and nitrogen metabolism can be altered by drought stress. Indeed, soluble sugars, soluble phenolics, total C, and total N can be altered and play important roles in osmoregulation in response to drought stress [8,21]. In addition, N uptake is often coupled with water uptake because NH₄⁺ and/or NO₃⁻ must be dissolved in water, and then absorbed by roots [8]. The N supply (via irrigation) affects plant growth under drought conditions by increasing soluble sugars and the activities of antioxidant enzymes [22].

P. simonii is widely distributed on the Loess Plateau, and the soil in this area suffers from drought and N deficiencies. In a previous study, it was found that an increase in N contributed to drought tolerance by increasing NH₄⁺ uptake and decreasing nitrogen metabolism in P. simonii [2]. We investigated several morphological (root characteristics), physiological (photosynthesis, C metabolism, N metabolism, ROS accumulation, and scavenging), and molecular (transcript levels of representative genes involved in water uptake and hormone metabolism) changes in response to N supply under drought condition. We hypothesized that the negative effects of drought stress on water uptake and hormone metabolism could be eased by a high N supply.

2. Materials and Methods

2.1. Plant Materials and Treatments

P. simonii cuttings (approximately 15 cm in length and 20 mm in diameter) were rooted in fine sandy soil and vermiculite (1:1). The plants were cultivated in a greenhouse as described previously [23]. One sprout was left for each plant. After 4 weeks, the substrate were removed and similar saplings (approximately 20 cm) were cultivated under hydroponic conditions in Hoagland solution (10 μM EDTA·FeNa, 0.5 μM H₂MoO₄, 30 μM H₃BO₃, 1 μM CuSO₄·5H₂O, 1 μM ZnSO₄·7H₂O, 5 μM MnSO₄·H₂O, 1 mM MgSO₄·7H₂O, 1 mM KH₂PO₄, 1 mM Na₂SO₄, and 1 mM CaCl₂) containing 0.01 mM NH₄NO₃ (low N supply) or 1 mM NH₄NO₃ (sufficient N supply), and the pH was adjusted to 5.5 for each solution. Subsequently, a total of 36 plants were subjected to drought stress via the addition of 5% PEG-6000 (osmotic stress, −0.362 MPa) to the modified Hoagland’s nutrient solution; 36 plants that had been grown in the nutrient solution without PEG-6000 served as the control. During the experiment, the aerated nutrient solutions were renewed every two days. The treatments were maintained under hydroponic cultivation for 4 weeks prior to harvest.

2.2. Measurement of Gas Exchange, Photosynthetic Pigments, and Root Growth

Gas exchange was determined on three mature leaves (leaf plastochron index = 8–10) with a portable photosynthesis system (Li-Cor-6400, Li-Cor, Inc., Lincoln, NE, USA). The measurements of net photosynthetic rates (A), stomatal conductance (g_s), and transpiration rates were carried out from 9:00 to 11:00. The instantaneous water use efficiency (WUE_i) was calculated as the ratio between A and g_s. The chlorophyll content was measured with a portable chlorophyll meter (SPAD 502 Meter, Minolta Corporation, Tokyo, Japan).

Parts of roots (approximately 2 g) were excised from the root system, scanned with a scanner (HP Scanjet G4050), and analyzed with a WinRHIZO root analyzer system (WinRHIZO version 2007b, Regent Instruments, Quebec, QC, Canada). Six leaves from each plant were submerged in water for 24 h to measure the leaf saturated weight (SW). The remaining roots and leaves were wrapped with tinfoil, ground into fine powder in liquid N with a mortar and pestle, and stored at −80 °C. Frozen powder (c. 100 mg) from each plant was dried at 60 °C for 72 h to determine dry biomass and the fresh mass to dry mass ratio.

2.3. RWC, MDA Content, and Rate of Superoxide Anion Radical (O₂⁻) Production

The RWC was defined according to Guo et al. [23]. RWC (%) = (FW − DW)/(SW − DW) × 100%, where FW, DW, and SW are the dry weight, fresh weight, and saturated weight, respectively.

The rate of O₂⁻ production was quantified according to Meng et al. [10]. Fine powder (100 mg) was homogenized in 50 mM potassium phosphate buffer (pH 7.8). After centrifugation (10,500× g, 20 °C,10 min), 1 mL of 1 mM hydroxylamine hydrochloride and 0.5 mL of 50 mM phosphate buffer (pH 7.8) were added to 1 mL supernatant and the mixture was incubated at 25 °C for 60 min. Then, 1 mL of 17 mM sulfanilamide and 1 mL of 7 mM α-naphthylamine were added to the mixture. After 20 min, the O₂⁻ concentration was quantified spectrophotometrically at 530 nm.

The MDA content was determined according to the methods of Dhinsa and Matowe [24]. The fine powder (100 mg) was extracted in 10% (w/v) trichloroacetic acid (TCA) and centrifuged (10,000× g, 20 °C, 10 min). Then, 2 mL of TCA containing 0.6% (w/v) thiobarbituric acid (TBA) was added and mixed with 1 mL of the supernatant. After incubating at 95 °C for 30 min, the mixture was centrifugated (10,000× g, 4 °C, 10 min). The absorbance of the supernatant was measured at 532 and 600 nm.

2.4. Determination of Soluble Sugars, Soluble Phenolics, and Amino Acid Compounds

The concentration of total soluble sugars was analyzed using the anthrone method [12]. Briefly, approximately 100 mg fine powder was homogenized in 4 mL of 80% ethanol. The mixture was incubated at 80 °C for 30 min. After centrifugation (6000× g, 25 °C, 10 min), 10 mg of activated charcoal was added for 30 min at 80 °C to decolorize the supernatant. Then, 5 mL of anthrone reagent was added. The mixture was incubated in boiling water for 10 min. After the mixture was cooled to room temperature, the absorbance of the supernatant was determined spectrophotometrically at 620 nm (Shimadzu, UV-3600, Kyoto, Japan).

The soluble phenolics in the plant materials were determined as described by Pritchard [25]. Approximately 100 mg fine powder was extracted in 1.5 mL of 80% methanol and shaken (150 rpm, 25 °C, 12 h) in the dark. After centrifugation (12,000× g, 25 °C, 10 min), the supernatant was collected. The soluble phenolics were determined spectrophotometrically at 765 nm.

The soluble amino acid compounds were determined as described previously [26]. A total of 100 mg of the powder was extracted in an extraction solution (methanol/ chloroform (7:3, v/v), 20 mM HEPES, 5 mM ethylene glycol tetraacetic acid, 10 mM NaF, pH 7.0) and incubated on ice for 30 min. Then, the mixture was extracted with distilled water, freeze-dried (Alpha 2–4, Christ, Osterode, Germany), and finally dissolved in lithium citrate buffer (pH 2.2). Free amino acids in the samples were analyzed using an automated amino acid analyzer (L-8900, Hitachi High-Technologies Corporation, Kyoto, Japan).

2.5. Analysis of Total C, Total N, ¹³C, and ¹⁵N

The samples were dried in an oven at 80 °C. The isotopic ratio was analyzed using an elemental analyzer (NA 1110, CE Instruments, Rodano, Italy) coupled to a GVI IsoPrime isotope ratio mass spectrometer (IRMS).

The stable C isotope composition was calculated as:

δ¹³C = (Rsac − Rsdc)/Rsdc × 1000 [‰]

where Rsac and Rsdc are the ratios of ¹³C to ¹²C in the sample and the standard, respectively.

The stable N isotope composition was calculated as:

δ¹⁵N = (Rsan − Rsdn) /Rsdn × 1000 [‰]

where Rsan and Rsdn are the ratios of ¹⁵N to ¹⁴N in the sample and the standard, respectively. The reference standard was N₂ in air.

2.6. Antioxidant Enzymes Activities

Due to space limitations, the detailed methods for determining antioxidant enzyme activities (SOD, POD, CAT, and APX) are provided in Supplementary Materials and Methods.

2.7. IAA and ABA Contents

IAA and ABA were extracted, purified, and quantified according to the method described by Diego [27]. The fine powder (approximately 0.5 g) was extracted in 4.0 mL of isopropanol/hydrochloric acid and shaken for 30 min at 4 °C before 10 mL of dichloromethane was added. The mixture was shaken again for 30 min at 4 °C. After centrifugation (13,000 rpm, 5 min, 4 °C), the lower organic phase was dried under N₂, dissolved in 150 μL of methanol (0.1% methane acid), and passed through a 0.22 μm membrane. Then, the filtrate was subjected to high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) using a ZORBAX SB-C18 (Agilent Technologies, Santa Clara, CA, USA) column. The MS conditions were as follows: the spray voltage was 4500 V; the pressure of the air curtain, nebulizer, and aux gas were 15, 65, and 70 psi, respectively; and the atomizing temperature was 400 °C.

To quantify ABA, endogenous ABA was extracted from 50 mg of each sample using 0.5 mL of homogenization buffer (70% methanol and 0.1% formic acid); as an internal standard, 2 ng of ABA-d6 (Olchemim, Olomouc, Czech Republic) was added to the extracts. The mixture was diluted two times with deionized water, and the ABA content was analyzed with the ultrahigh performance liquid chromatography-triple TOF 5600+ system (Sciex, Concord, ON, Canada).

2.8. Analysis of Transcript Levels

Total RNA was extracted and purified with a plant RNA extraction kit (R6827, Omega Bio-Tek, Norcross, GA, USA); trace genomic DNA was digested by DNase I (E1091, Omega Bio-Tek); and 1 μg of total RNA was used to synthesize cDNA with the PrimeScript RT reagent kit (DRR037S, Takara, Dalian, China). Quantitative PCR was performed in a 20 μL solution reaction volume using 10 μL 2× SYBR Green Premix Ex Taq II, 2 μL of cDNA, and 0.2 μM primers (Supplementary Table S1) in a real-time platform (LightCycler 96 System, Roche, Basel, Swiss). The conservative actin2/7 was used as a reference gene [28]. Key genes involved in water uptake and hormone modulation were selected for the gene transcription analysis [8,17]. Primers were designed using the Primer 3 online tool. The qRT-PCR experiments were performed in triplicate for each sample. The efficiencies of the PCR reactions ranged between 95 and 105% (Supplementary Table S1).

2.9. Data Processing and Statistical Analysis

Statistical tests were performed with the SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). The effects of N supply and drought treatments on variables were analyzed by two-way analysis of variance (ANOVA). The means were compared on the basis of least significant differences (p = 0.05). The gene expression heatmap was generated by the command heatmap.2() using the ‘gplots’ package in R, as described by Luo et al. [13].

3. Results

3.1. Growth Parameters

Drought stress led to serious growth inhibition (decreased height and biomass) under both 0.01 mM NH₄NO₃ and 1 mM NH₄NO₃ (Supplementary Table S2 and Figure S1). Drought stress also markedly reduced the total length, surface area, and volume of the roots. N availability had a different effect on plant growth under drought stress and control conditions (Supplementary Table S2 and Figure S1). Under drought stress, the plants supplied with sufficient N had a greater biomass, chlorophyll content, and root development as compared with the plants supplied with less N (Supplementary Table S2 and Figure S1). Unexpectedly, plants under 0.01 mM NH₄NO₃ grew better under control conditions (without drought stress), exhibiting greater height and increased root development when they were supplied with 0.01 mM NH₄NO₃ than when they were supplied with 1 mM NH₄NO₃.

3.2. RWC, Photosynthesis, MDA, and the Rate of O₂⁻ Production

Drought stress significantly decreased the RWC, net photosynthetic rates, stomatal conductance, and transpiration rate under both 0.01 mM and 1 mM NH₄NO₃ (Table 1). The WUEi, MDA, and O₂⁻ in leaves increased significantly in response to drought stress in the plants under 0.01 mM NH₄NO₃, whereas these variables were not significantly affected in the plants under 1 mM NH₄NO₃ (Table 1). Drought stress increased the root MDA content under both 0.01 mM and 1 mM NH₄NO₃ (Table 1).

Under control conditions, N supply had minor effect on RWC, photosynthesis, electrolyte leakage, MDA, and ROS (Table 1). However, under PEG-induced drought stress, 0.01 mM NH₄NO₃ resulted in significant increases in transpiration rate, WUEi, MDA, and O₂⁻ in leaves as compared with 1 mM NH₄NO₃ (Table 1).

3.3. Soluble Sugars, Soluble Phenolics, and Amino Acid Compounds

Soluble sugars and phenolics were significantly increased in response to drought stress in both the roots and leaves (Figure 1). N level had no significant effect on soluble sugars in roots, whereas 0.01 mM NH₄NO₃ decreased foliar soluble sugars (Figure 1A,B). Soluble phenolics (particularly in the leaves) were increased in response to 0.01 mM NH₄NO₃, regardless of the drought treatment (Figure 1C,D).

The total amino N concentrations and the composition of amino compounds changed in response to osmotic stress and N concentration (Supplementary Table S3). The 17 common amino acids were sorted into five groups based on biosynthetic origin [26]. In the 0.01 mM NH₄NO₃ treatment, the increase in the total amino acid content in response to osmotic stress in the roots was primarily due to a significant increase in the aspartate group (Figure 2A). This proportional increase in the aspartate group (from 28% to 37%) was compensated by a proportional decrease in the glutamate group (from 31% to 25%). Asparagine and glutamine were the main components of the aspartate group and the glutamate group, respectively (Figure 2A). Similarly, the total amount of amino compounds was higher in response to osmotic stress in the roots of plants grown with 1 mM NH₄NO₃, and the increase was primarily due to the changes in the aspartate, alanine, and glutamate groups. The proportion of aspartate compounds increased (from 28% to 39%), whereas the proportions of alanine compounds and glutamate compounds decreased (from 15% to 11% and from 34% to 29%, respectively) under drought conditions (Figure 2A); under both drought and control conditions, 0.01 mM NH₄NO₃ decreased the total amino acid content but had a little effect on the relative proportion of groups (Figure 2A).

In the leaves, the serine group showed a higher absolute (1.5-fold) and proportional (from 6% to 14%) content in response to drought stress under 0.01 mM NH₄NO₃ (Figure 2B). Osmotic stress also increased the total amino acid content of most biosynthetic groups (except the tryptophan group) under 1 mM NH₄NO₃ (Figure 2B). Under 1 mM NH₄NO₃, the proportions of the serine group and the glutamate group increased (from 7% to 16% and from 11% to 24%, respectively), whereas the proportion of the tryptophan group decreased (from 22% to 8%) in response to drought stress (Figure 2B). Under the control conditions, N supply had no significant effect on the total amino acid concentration and relative content (Figure 2B). However, under the drought treatment, the total amino concentrations were higher for all biosynthetic groups under the 1 mM NH₄NO₃ treatment than under the 0.01 mM NH₄NO₃ treatment (Figure 2B).

3.4. Total C, Total N, ¹³C, and ¹⁵N

Drought stress significantly decreased the total C in roots, whereas it had no effect on foliar total C and δ¹³C under 0.01 mM and 1 mM NH₄NO₃ (Figure 3). The total C and δ¹³C in roots and leaves were not affected by N levels (Figure 3).

Drought stress had little effect on the total N concentration of roots, while the foliar total N concentration under 1 mM NH₄NO₃ decreased in response to the drought treatment (Figure 4A,B). There was a significant increase in δ¹⁵N under the drought treatment as compared with the control in both roots and leaves (Figure 4C,D).

The N levels also affected the total N concentration of both roots and leaves (Figure 4A,B). Generally, the total N concentration of both roots and leaves was lower under 0.01 mM NH₄NO₃ as compared with that under 1 mM NH₄NO₃ under both drought stress and control conditions (Figure 4A,B). In contrast, 0.01 mM NH₄NO₃ led to higher δ¹⁵N in roots (Figure 4C), 0.01 mM NH₄NO₃ also led to higher δ¹⁵N in the leaves under control conditions, whereas it had no significant effect on δ¹⁵N under drought stress (Figure 4D).

3.5. Antioxidant Enzymes Activities

The SOD activity in roots was not affected by drought stress, whereas drought stress significantly increased SOD activity under both 0.01 and 1 mM NH₄NO₃ treatments (Figure 5A,B). Drought stress also led to increased APX activity in both roots and leaves, except, root APX activity under 0.01 mM NH₄NO₃ remained unaltered in response to drought stress (Figure 5C,D).

Root SOD activity was not affected by N availability under either drought or control conditions (Figure 5A). N levels also had no impact on SOD activity in the control treatment; however, the 1 mM NH₄NO₃ treatment led to a higher SOD activity under drought stress as compared with the 0.01 mM NH₄NO₃ treatment (Figure 5B). Similar results were observed for root APX activity (Figure 5C). The APX activity in leaves was not affected by N levels under either drought or control conditions (Figure 5D).

Drought stress increased CAT activity in roots and leaves under the 1 mM NH₄NO₃ treatment but had no effect under 0.01 mM NH₄NO₃ (Figure 5E,F). The POD activity in roots and leaves was also stimulated by drought stress, except, the root POD activity remained unchanged under 0.01 mM NH₄NO₃ (Figure 5G,H). In the PEG-treated plants, CAT and POD activities increased significantly when the N supply was increased. However, CAT and POD activities were not affected by N availability in the control plants (Figure 5E,H).

3.6. IAA and ABA Concentrations

Drought stress had no effect on root IAA concentration under 0.01 mM NH₄NO₃ (Figure 6A). However, the root IAA concentration under 1 mM NH₄NO₃ was increased under the drought stress (Figure 6A). IAA concentration was decreased by drought stress under 0.01 mM NH₄NO₃ but remained unaltered under 1 mM NH₄NO₃ (Figure 6B). ABA concentrations in both roots and leaves were higher under drought stress as compared with the control conditions (Figure 6C,D).

The 0.01 mM NH₄NO₃ treatment induced a higher IAA concentration in both PEG-treated and control plants (Figure 6A,B). Additionally, ABA concentrations in both roots and leaves were higher in response to 0.01 mM NH₄NO₃ under the control conditions (Figure 6C,D). Under drought stress, root ABA concentration was not influenced by N supply; however, 0.01 mM NH₄NO₃ significantly increased foliar ABA concentration (Figure 6C,D).

3.7. PCA of Morphological and Physiological Responses

PC1 and PC2 accounted for 49 and 18% of the variation, respectively (Figure 7 and Supplementary Table S4). PC1 uncoupled the effect of drought, and PC2 clearly separated the variation due to N supply. Root ABA content and the soluble sugar content of roots and leaves were key contributors to PC1, whereas both total N concentration and the IAA content of roots and leaves were important factors influencing PC2.

3.8. Transcript Levels of AQPs and Representative Genes Involved in IAA and ABA Signaling Pathways

The cluster analysis separated the effects of drought stress and N availability based on the responsiveness of transcript levels (Figure 8). Under 1 mM NH₄NO₃, the mRNA levels of most genes (except TIP1;6) in roots were upregulated in response to drought stress (Figure 8A). The transcript abundance of NCED3, PIP2;1, TIP2;1, PP2C, and PIP1;2 in roots was similarly increased by drought conditions under 0.01 mM NH₄NO₃ (Figure 8A). However, under the 0.01 mM NH₄NO₃ treatment, YUCCA1, YUCCA3, and TIP1;6 were suppressed, whereas TAA1 and SIP1;2 remained unchanged in response to drought stress in roots (Figure 8A). Additionally, the mRNA levels of genes related to auxin biosynthesis (TAA1, YUCCA1, and YUCCA3) increased under 0.01 mM NH₄NO₃ as compared with under 1 mM NH₄NO₃ under control conditions, whereas this effect was not significant under drought conditions (Figure 8A).

In the leaves, the transcript abundances of almost all of the investigated genes (except NCED3 and TAA1) was also increased by drought stress under the 1 mM NH₄NO₃ treatment (Figure 8B). Under the 0.01 mM NH₄NO₃ treatment, drought stress also upregulated the expression of TIP2;1, PIP2;1, TIP1;6, and PP2C but had little effect on the mRNA levels of other genes in the leaves (Figure 8B). Under control conditions, 0.01 mM NH₄NO₃ resulted in a significant decrease in the PP2C and TIP1;6 transcript levels in the leaves. However, the mRNA levels of YUCCA1 were increased by 0.01 mM NH₄NO₃ under control conditions (Figure 8B). Under drought conditions, the transcript abundances of almost all of the investigated genes (YUCCA1, NCED3, and TAA1) was higher under 1 mM NH₄NO₃ than under 0.01 mM NH₄NO₃ (Figure 8B).

4. Discussion

Plants have developed strategies to adapted to drought stress. For instance, the inhibition of physiological activity may change the redistribution of limited resources and improve plant survival [29,30]. Consistent with many previous studies, drought stress has detrimental effects on the root morphology and photosynthesis of P. simonii [31,32,33]. Quite unexpectedly, under control conditions (no drought stress), nitrogen deficiency stimulated the root growth of P. simonii (Supplementary Table S2 and Figure S1). Generally, high nitrogen supply significantly increases root growth parameters in plants [2,30]. In this study, however, P. simonii seedlings showed better root development under 0.01 mM NH₄NO₃ under control conditions, exhibiting a greater root biomass and root surface area. These results may be explained by adaptation to the environment that P. simonii often grows on the Loess Plateau under conditions of nitrogen deficiency in Northwest China. P. simonii may have developed a mechanism (such as root development) to take up nitrogen efficiently, even from nutrient-deficient soil [2]. However, the PEG-treated plants showed improved growth when the N level was increased, suggesting that an increased N supply may help P. simonii survive under drought stress.

Drought stress often induces changes in cell membranes (increased permeability and decreased stability) [34]. Thus, RWC was used to indicate the degree of dehydration and membrane stability. In this study, decreased RWC was observed in the leaves of P. simonii under drought stress, which could reduce turgor pressure and enhance membrane permeability. The increase in MDA and O₂⁻ in leaves in response to drought stress reflects oxidative damage to membrane lipids and other vital substances. These results indicated that drought stress damaged sensitive biological macromolecules, impaired their functions, and harmed membrane [35]. To maintain turgor pressure in the cytoplasm, the accumulation of soluble substances plays an important role in osmoregulation. In our study, the notable increase in soluble sugars, phenolics, and amino acids suggested that more soluble substances were synthesized. Significant increases were observed in the aspartate, serine, and glutamate biosynthesis groups in response to drought stress. Aspartate and glutamine play important roles in the storage and/or transport of N from source to sink tissues [26,36], an intensive N reallocation under stress. Cysteine (the precursor of which is serine), glutamate, and glycine are used for the synthesis of glutathione (GSH) and the elimination of free radicals under stress [37]. Additionally, proline (belonging to the glutamate group) is a non-toxic osmoprotectant which is 300 times more soluble in water than other amino acids and may play an important role in cell pressure adjustment [38].

In this study, 1 mM NH₄NO₃ compensated for the negative effects of drought on osmolytes, O₂⁻ production, and the MDA content of leaves. Many studies have also found that nitrogen supply can decrease the MDA concentration, adjust osmotic pressure, and increase membrane stability to prevent damage caused by drought stress [39,40]. For example, N supply (100 and 200 kg/ha) prevented cell membrane damage and enhanced osmotic regulation in Agrostis palustris Huds. under water stress [41]. Nitrogen fertilizer could decrease MDA content of maize (Zea mays L.) under drought conditions [42]. These results indicated that the detrimental effects of drought stress on P. simonii were somehow eased by 1 mM NH₄NO₃.

After detecting reduced soil water availability, altered homeostasis of ROS production and scavenging can occur in plants during acclimation to drought [43]. In the enzymatic systems, SOD and APX can convert O₂⁻ to H₂O₂ [44], and POD and CAT can decompose H₂O₂ to H₂O at different cellular locations. The ROS production and the activities of oxygen scavenging systems determine whether oxidative signaling and/or damage will occur [45]. In this study, high activities of SOD, POD, CAT, and APX were observed (particularly under the 1 mM NH₄NO₃ treatment) in response to drought stress. Under drought conditions, the activities of most antioxidant enzymes were significantly increased in response to 1 mM NH₄NO₃ as compared with 0.01 mM NH₄NO₃, indicating that N supply improved the drought resistance of P. simonii.

Metabolites of C and N in plants can also act as osmoprotectants or signal molecules under drought stress [12,46]. Water deprivation can lead to a reduction in total C concentration as a result of decreased CO₂ assimilation in plants [47,48]. In the present study, long-term drought treatment also led to decreased concentrations of total C and N in P. simonii. Plants face a dilemma under drought stress: On the one hand, plants must prevent water loss via stomatal closure, and thus, the photosynthesis is limited. on the other hand, they need photosynthates to support key processes necessary for survival [49,50]. Thus, plants must increase WUE during acclimation to drought conditions. The δ¹⁵N at natural abundance levels acts as a tracer, and significant discrimination is positive in most biological systems [8]. Plants retard their N uptake and assimilation processes and are forced to utilize the ¹⁵N to meet N demands and are enriched in the tissues under 0.01 mM NH₄NO₃. The δ¹³C and δ¹⁵N values show positive linear correlations with WUEi in plants and δ¹³C and δ¹⁵N enrichment are closely associated with water consumption and can be considered to be indicators of WUE [51,52]. The δ¹³C of roots and leaves was not affected by drought stress. However, δ¹⁵N increased in the roots and leaves of P. simonii under drought conditions as compared with the control. These results reflect the acclimation of P. simonii to drought conditions via increased WUE.

The transcriptional regulation of PIPs is crucial for water transport across the plasma membrane and acclimation to drought in poplar plants [53,54,55]. Most of the AQP genes in roots and/or leaves have been reported to be upregulated in response to drought stress, suggesting that these AQPs can play key roles in facilitating water movement and redistribution under dynamic environmental water conditions. However, AQPs have been reported to be upregulated, downregulated, or unchanged in various poplar genotypes in response to drought stress [8,53,56]. In this study, the mRNA levels of AQPs (especially PIP1;2 and TIP2;1) in P. simonii were significantly upregulated in response to drought stress and similar results were found in Populus cathayana [8]. These results indicate that P. simonii has developed the AQPs expression modulation strategy to acclimate to drought stress on the Loess Plateau and PIP1;2 and TIP2;1 may play decisive roles in the strategy.

Poplar plants can perceive changes in soil water availability via the IAA and ABA signaling pathways [9]. At physiological concentrations, auxin induces the opening of stomata [57,58]. To minimize water loss, root auxin concentrations are often increased and leaf auxin concentrations are often decreased during acclimation to drought conditions [59,60]. Indeed, in the present study, the root IAA concentrations under 1 mM NH₄NO₃ were increased, whereas the IAA concentrations were decreased by drought stress. In addition, 0.01 mM NH₄NO₃ induced higher IAA concentrations in the roots and leaves of P. simonii under both drought and control conditions, which was consistent with the root growth stimulated by N deficiency. In auxin biosynthesis, TAAs and YUCs play essential roles: The TAA of amino transferases converts tryptophan to indole-3-pyruvate (IPA), and then the IPA is converted to IAA by the YUCCA (YUC) family of flavin monooxygenases [61]. Indeed, the representative genes (TAA1, YUCCA, 1 and YUCCA3) in P. simonii were induced by drought stress in the roots of P. simonii under 1 mM NH₄NO₃. However, the mRNA levels of these genes in leaves (except YUCCA1) under 0.01 mM NH₄NO₃ were significantly decreased by drought stress and were consistent with the IAA concentration. In particular, the high levels of TAA1, YUCCA1, and YUCCA3 observed under 0.01 mM NH₄NO₃ suggest that P. simonii might be sensitive to N deficiency and thus, may accelerate IAA synthesis to stimulate root development, which is consistent with the observed root characteristics.

Additionally, the ABA concentrations in both roots and leaves of P. simonii were higher under drought stress as compared with control conditions. The ABA concentrations in poplars are often increased by drought stress and can recover to previous levels after re-watering [62,63,64]. The significant increase in ABA concentration in response to drought stress may be an important mechanism for transmitting a chemical signal that indicates the water status of the soil and leads to a cascade of reactions via regulation of leaf stomatal conductance [65]. Accordingly, NCED and PP2C, which are key genes involved in the ABA signaling pathway, were downregulated in response to drought treatment. A similar result was reported in Populus euramericana and Populus cathayana, which suggests that NCED3 and PP2C may play decisive roles in the ABA signaling pathway during acclimation to limited water availability [65].

In summary, P. simonii demonstrated decreased height; differential expression of aquaporins (AQPs); activation of the IAA and ABA signaling pathways; decreased net photosynthetic rate and transpiration rate; increased δ¹⁵N, total soluble substances and WUEi; and altered homeostasis of reactive oxygen species (ROS) production and scavenging (Figure 9). These data suggest that P. simonii slows the process of water acquisition during acclimation to drought stress. Moreover, plants with a sufficient N supply exhibited more soluble compounds and greater antioxidant enzyme activities, lower transpiration rates (E), IAA content, ABA content and ROS production, and greater responsiveness of transcriptional regulation of 10 genes involved in water uptake and hormone modulation than plants with a low N supply under drought stress (Figure 9). These results suggest that P. simonii can differentially manage water uptake and hormone modulation under different N supply conditions. This is important when selecting poplar species for different soil conditions.

Author Contributions

Writing—original draft preparation, Z.L., X.W., Y.L., Z.Q. and Z.B.; writing—review and editing, Z.L., X.W. and Y.Z.; visualization, Z.L., X.W., Z.L. and N.W. Resources, Z.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data relevant to the study are included in the article.

Acknowledgments

We thank numerous students and lab staff from the Chinese Academy of Forestry for their assistance in laboratory.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

AQP, aquaporin; IAA, auxin; ABA, abscisic acid; δ¹³C, stable carbon isotope composition; δ¹⁵N, stable nitrogen isotope composition; WUEi, intrinsic water use efficiency; ROS, reactive oxygen species; O₂⁻, superoxide anion radical; SOD, superoxide dismutase; APX, ascorbate peroxidase; CAT, catalase; POD, peroxidase; MDA, malonaldehyde; NCED3, 9-cis-epoxycarotenoid dioxygenase 3; PP2C, protein phosphatase 2C; YUCC, flavin-containing monooxygenase; TAA, tryptophan aminotransferases; PCA, principal component analysis.

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Figures and Table

Figure 1. Soluble sugars and soluble phenolics in roots (A,C) and leaves (B,D) of Populus simonii as affected by nitrogen supply and drought stress. Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant differences. N, N supply; D, drought treatment; N × D, interaction of N supply and drought treatment. **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 2. Amino compounds in roots (A) and leaves (B) of Populus simonii as affected by nitrogen supply and drought stress. Amino acids deriving from the same pathway are grouped together. The percent distributions of these biosynthetic groups are presented, as well as the mean values of sums of amino compounds of each group of three plants and SE. (Striped) aspartate, threonine, isoleucine, methionine, lysine; (black) serine, glycine, cysteine; (white) glutamate, histidine, arginine, proline; (light grey) phenylalanine, tyrosine; (dark grey) alanine, leucine, valine.

Figure 3. Total C concentration and δ13C in roots (A,C) and leaves (B,D) of Populus simonii as affected by nitrogen supply and drought stress. Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant differences. N, N supply; D, drought treatment; N × D, interaction of N supply and drought treatment. ***, p < 0.001; ns, not significant.

Figure 4. Total N concentration and δ15N in roots (A,C) and leaves (B,D) of Populus simonii as affected by nitrogen supply and drought stress. Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant differences. N, N supply; D, drought treatment; N × D, interaction of N supply and drought treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 5. Activities of SOD, APX, CAT, and POD enzymes in roots (A,C,E,G) and leaves (B,D,F,H) of Populus simonii as affected by nitrogen supply and drought stress. Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant differences. N, N supply; D, drought treatment; N × D, interaction of N supply and drought treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 6. IAA and ABA contents in roots (A,C) and leaves (B,D) of Populus simonii as affected by nitrogen supply and drought stress. Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant differences. N, N supply; D, drought treatment; N × D, interaction of N supply and drought treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 7. PCA plots of the first two principal components in roots and leaves of Populus simonii as affected by nitrogen supply and drought stress. PCA was conducted based on data (both values were averaged in the same treatment) of parameters presented in Supplementary Table S4.

Figure 8. Fold changes of transcript levels of AQPs and representative genes involved in IAA and ABA signaling pathways in roots (A) and leaves (B) of Populus simonii as affected by nitrogen supply and drought stress. Signal intensities were calibrated according to a constitutively expressed poplar actin gene [10].

Figure 9. A schematic model of responses to nitrogen supply in Populus simonii under drought stress. While acclimating to drought, the P. simonii exhibited a reduction in growth; a decrease in the photosynthetic rate and transpiration rate; and an increase in δ15N, soluble substances, ROS scavenging; and water use efficiency. Strong response in the transcriptional regulation of AQPs; higher soluble phenolics; lower transpiration rates, IAA and ABA content, and ROS accumulation and scavenging were observed with a sufficient N supply.

Table 1

The RWC, photosynthesis, MDA, and the rate of O₂⁻ production of P. simonii as affected by drought and nitrogen supply.

N Supply(mM)	DroughtTreatment	RWC (%)	A (μmol CO2 m−2 s−1)	gs (mmol m−2 s−1)	E (mmol H2O m−2 s−1)	WUEi (μmol CO2 mol−1 H2O)	MDA in Roots(μmol g−1 FW)	MDA in Leaves(μmol g−1 FW)	O2− in Roots(nmol g−1 FW min−1)	O2− in Leaves(nmol g−1 FW min−1)
0.01	Control	88.65 ± 0.95a	11.9 ± 0.4a	221.67 ± 9.10a	4.85 ± 0.15a	53.87 ± 2.51b	12.18 ± 1.90b	11.22 ± 1.47c	0.11 ± 0.02a	0.04 ± 0.01b
	Drought	77.57 ± 1.34b	2.7 ± 0.2c	33.33 ± 4.22b	1.28 ± 0.16b	83.54 ± 6.44a	35.75 ± 3.65a	28.62 ± 2.12a	0.12 ± 0.01a	0.14 ± 0.02a
1	Control	90.41 ± 1.29a	9.8 ± 0.2b	255.00 ± 9.92a	4.97 ± 0.19a	38.48 ± 1.63bc	15.23 ± 3.09b	15.57 ± 1.07bc	0.14 ± 0.02a	0.04 ± 0.01b
	Drought	80.01 ± 1.10b	2.4 ± 0.4c	71.67 ± 7.49b	0.26 ± 0.02c	33.60 ± 4.77c	30.32 ± 2.08a	20.63 ± 1.65b	0.12 ± 0.01a	0.06 ± 0.00b
p-values	N	ns	*	***	**	***	ns	ns	ns	**
	D	***	***	***	***	**	***	***	ns	***
	N × D	ns	ns	ns	**	***	ns	**	ns	**

Data indicate mean ± SE (n = 6). Different letters in the same column indicate significant differences. p-values of the ANOVAs of drought, nitrogen supply, and their interaction are indicated. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/f13060907/s1, Supplementary materials and methods, Supplementary Table S1: Primers used for qRT-PCR, Supplementary Table S2: Growth parameters of P. simonii, Supplementary Figure S1: The typical root state of P. simonii, Supplementary Table S3: The concentrations of 17 free amino acids in fine roots and leaves, Supplementary Table S4: PCA of morphological and physiological responses. References [66,67,68,69] are cited in Supplementary Materials.

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