Phosphorus and nitrogen physiology of two contrasting poplar genotypes when exposed to phosphorus and/or nitrogen starvation

Tree Physiology, Jan 2016

Phosphorus (P) and nitrogen (N) are the two essential macronutrients for tree growth and development. To elucidate the P and N physiology of woody plants during acclimation to P and/or N starvation, we exposed saplings of the slow-growing Populus simonii Carr (Ps) and the fast-growing Populus × euramericana Dode (Pe) to complete nutrients or starvation of P, N or both elements (NP). P. × euramericana had lower P and N concentrations and greater P and N amounts due to higher biomass production, thereby resulting in greater phosphorus use efficiency/N use efficiency (PUE/NUE) compared with Ps. Compared with the roots of Ps, the roots of Pe exhibited higher enzymatic activities in terms of acid phosphatases (APs) and malate dehydrogenase (MDH), which are involved in P mobilization, and nitrate reductase (NR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), which participate in N assimilation. The responsiveness of the transcriptional regulation of key genes encoding transporters for phosphate, ammonium and nitrate was stronger in Pe than in Ps. These results suggest that Pe possesses a higher capacity for P/N uptake and assimilation, which promote faster growth compared with Ps. In both poplars, P or NP starvation caused significant decreases in the P concentrations and increases in PUE. Phosphorus deprivation induced the activity levels of APs, phosphoenolpyruvate carboxylase and MDH in both genotypes. Nitrogen or NP deficiency resulted in lower N concentrations, amino acid levels, NR and GOGAT activities, and higher NUE in both poplars. Thus, in Ps and Pe, the mRNA levels of PHT1;5, PHT1;9, PHT2;1, AMT2;1 and NR increased in the roots, while PHT1;9, PHO1;H1, PHO2, AMT1;1 and NRT2;1 increased in the leaves during acclimation to P, N or NP deprivation. These results suggest that both poplars suppress P/N uptake, mobilization and assimilation during acclimation to P, N or NP starvation.

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Phosphorus and nitrogen physiology of two contrasting poplar genotypes when exposed to phosphorus and/or nitrogen starvation

Tree Physiology Volume Phosphorus and nitrogen physiology of two contrasting poplar genotypes when exposed to phosphorus and/or nitrogen starvation Honghao Gan 0 Yu Jiao 1 Jingbo Jia 0 Xinli Wang 1 Hong Li 2 Wenguang Shi 0 Changhui Peng 1 Andrea Polle 3 Zhi-Bin Luo 0 1 0 College of Life Sciences and State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University , Yangling, Shaanxi 712100 , PR China 1 Key Laboratory of Environment and Ecology in Western China of Ministry of Education, College of Forestry, Northwest A&F University , Yangling, Shaanxi 712100 , PR China 2 College of Plant Protection, Northwest A&F University , Yangling, Shaanxi 712100 , PR China 3 Bu?sgen-Institute, Department of Forest Botany and Tree Physiology , Georg- Phosphorus (P) and nitrogen (N) are the two essential macronutrients for tree growth and development. To elucidate the P and N physiology of woody plants during acclimation to P and/or N starvation, we exposed saplings of the slow-growing Populus simonii Carr (Ps) and the fast-growing Populus ? euramericana Dode (Pe) to complete nutrients or starvation of P, N or both elements (NP). P. ? euramericana had lower P and N concentrations and greater P and N amounts due to higher biomass production, thereby resulting in greater phosphorus use efficiency/N use efficiency (PUE/NUE) compared with Ps. Compared with the roots of Ps, the roots of Pe exhibited higher enzymatic activities in terms of acid phosphatases (APs) and malate dehydrogenase (MDH), which are involved in P mobilization, and nitrate reductase (NR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH), which participate in N assimilation. The responsiveness of the transcriptional regulation of key genes encoding transporters for phosphate, ammonium and nitrate was stronger in Pe than in Ps. These results suggest that Pe possesses a higher capacity for P/N uptake and assimilation, which promote faster growth compared with Ps. In both poplars, P or NP starvation caused significant decreases in the P concentrations and increases in PUE. Phosphorus deprivation induced the activity levels of APs, phosphoenolpyruvate carboxylase and MDH in both genotypes. Nitrogen or NP deficiency resulted in lower N concentrations, amino acid levels, NR and GOGAT activities, and higher NUE in both poplars. Thus, in Ps and Pe, the mRNA levels of PHT1;5, PHT1;9, PHT2;1, AMT2;1 and NR increased in the roots, while PHT1;9, PHO1;H1, PHO2, AMT1;1 and NRT2;1 increased in the leaves during acclimation to P, N or NP deprivation. These results suggest that both poplars suppress P/N uptake, mobilization and assimilation during acclimation to P, N or NP starvation. nutrient deficiency; nutrient use efficiency; phosphate transporter; phosphorus metabolism; transcriptionalregulation; tree Introduction Phosphorus (P) and nitrogen (N) are the two essential macronutrient elements required for plant growth and development. Phosphorus is a component of numerous metabolites in plants, such ?These authors contributed equally to this work. as DNA, RNA, phospholipids, adenosine triphosphate (ATP), adenosine diphosphate and nicotinamide adenine dinucleotide phosphate. Similarly, N is also an essential component in proteins, nucleic acids, chlorophylls and many secondary metabolites in plants. However, although plants require these macronutrients, plant roots (Rennenberg and Herschbach 2013) . However, the supply of these essential nutrients is often limited in the soil. organic P compounds in the soil can release Pi via the actions of Inorganic phosphate (Pi) is the main P resource for plants in acid phosphatases (APs) and excreted organic acids such as the soil solution (Figure 1) (Plassard and Dell 2010). However, malic acid and citric acid, which are derived from the glycolysis most of the Pi in soils can be precipitated or complexed by catpathway and the citric acid cycle, where the key enzymes ions (e.g., Ca2+ and Mg2+), thereby making it unavailable to involved are phosphoenolpyruvate carboxylase (PEPC) and plants. The other P pool in the soil comprises organic P commalate dehydrogenase (MDH) (Figure 1) (Li et al. 2002, Shane pounds derived from the degradation of plant litter, microbial et al. 2013) . Previously, it was shown that organic P accounted detritus and soil organic matter (Shen et al. 2011) . It is currently for 70?90% of the mobile P in a rendzic forest soil (Kaiser et al. considered that organic P compounds cannot be taken up by 2003). Therefore, both Pi and organic P compounds are key players in total P homeostasis in natural and agricultural ecosystems. Due to the low availability of Pi in the soil solution, plants often suffer from Pi deficiencies. Plants subjected to Pi deficiency can exhibit inhibited growth of the primary roots, decreased P concentrations in different tissues and lower photosynthetic rates, but increased photosynthetic P use efficiency (PPUEi, i.e., the ratio of the instantaneous photosynthetic carbon assimilation rate per unit foliar P) (L?pez-Arredondo et al. 2014) . Currently, our knowledge of the responses of plants to Pi deficiency is based mainly on experimental evidence obtained using the herbaceous model plant, Arabidopsis thaliana, which has P nutrition strategies that are distinct from those of woody plants (Rennenberg and Herschbach 2013) . In contrast, limited information is available about the physiological and molecular mechanisms of woody plants during acclimation to Pi deficiency (Rennenberg and Herschbach 2013) . Inorganic phosphate entry into root cells, its further intracellular distribution and intercellular mobilization are controlled mainly by Pi transporters (Figure 1) (L?pez-Arredondo et al. 2014) . In Arabidopsis, PHT1s, PHT2s, PHO1s and PHO2s are important gene families that encode Pi transporters. AtPHT1;5 and AtPHT1;9 are high-affinity Pi transporters, which function mainly at low Pi concentrations (L?pez-Arredondo et al. 2014) . AtPHT1;5 is involved in the mobilization of Pi from source to sink organs, according to developmental cues and the P status (Nagarajan et al. 2011) , whereas AtPHT1;9 is responsible for Pi acquisition at the root?soil interface and the translocation of Pi from the roots to the shoot (Remy et al. 2012, Lapis-Gaza et al. 2014) . AtPHT2;1 is located in the chloroplast inner envelope membrane where it functions as a low-affinity Pi transporter that mediates Pi allocation in the whole plant (Daram et al. 1999, Versaw and Harrison 2002) . AtPHO1;H1 contributes to the transfer of Pi to root xylem vessels (Stefanovic et al. 2007) . AtPHO2 encodes a ubiquitin conjugase E2 that prevents the overaccumulation of Pi in the shoots and it is a target of the microRNA, miR399, which is involved in Pi signaling (Chiou et al. 2006, Kant et al. 2011) . These genes exhibit differential transcriptional expression in Arabidopsis according to the internal and external P status (L?pez-Arredondo et al. 2014) . Homologs of these Arabidopsis genes have been identified in the sequenced genome of the woody plant, Populus trichocarpa (Tuskan et al. 2006) . The structure and expression profiles of the phosphate PHT1 transporter gene family have been characterized in mycorrhizal P. trichocarpa (Loth-Pereda et al. 2011) ; however, little information is available about the transcriptional regulation of these genes in Populus species in response to Pi deficiency. Compared with Pi, the uptake and assimilation of ammonium (NH4+ ) and nitrate (NO3? ), the two major inorganic N resources for plants in the soil solution, have been characterized much more comprehensively in woody plants (Figure 1). NH4+ and NO3? enter plant cells via specific transporters, i.e., ammonium (AMTs) and nitrate (NRTs) transporters, respectively (Rennenberg et al. 2010) . In the sequenced genome of P. trichocarpa, 14 AMTs and 79 NRTs have been putatively identified (Couturier et al. 2007, Plett et al. 2010, Bai et al. 2013) . Among these putative inorganic N transporters, AMT1;1, AMT2;1, NRT1;1 and NRT2;1 might play crucial roles in the uptake of NH4+ and NO3? by Populus species because the transcript levels of these genes change significantly in response to altered N availability (Dluzniewska et al. 2007, Ehlting et al. 2007, Li et al. 2012b, Luo et al. 2013a) . After uptake, NO3? is reduced to NH4+ by nitrate reductase (NR) and nitrite reductase (Xu et al. 2012) , and NH4+ is further converted into glutamine and glutamate catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT), respectively. In addition, glutamate can be produced via glutamate dehydrogenase (GDH). Previous studies have shown that the enzymatic activities of NR, GOGAT and GDH were decreased under low N supply conditions, whereas they increased with high N availability in poplar roots and/or leaves (Li et al. 2012b, Luo et al. 2013a) . However, limited information is available regarding the transcriptional regulation of these transporters and the activities of these enzymes in woody plants exposed to P or N starvation. Populus species are fast-growing woody plants, which are used to generate biomass in the production of biofuels and pulp as well as for mitigating CO2 emissions (Luo and Polle 2009, Studer et al. 2011) . Poplar plantations are often cultivated on marginal land where the soil is P- and/or N-limited. However, limited information is available about the physiological and transcriptional regulation mechanisms of P and N assimilation in poplars in response to P, N or both (NP) deficiency. In a preliminary experiment, we identified a slow-growing poplar genotype (Populus simonii Carr (Ps)) and a fast-growing poplar genotype (Populus ? euramericana Dode (Pe)) under sufficient nutrient conditions. To identify the differences in P/N physiology that underlie the contrasting growth characteristics of both poplar genotypes with a sufficient nutrient supply, as well as to elucidate the physiological acclimation mechanisms of both poplars in response to P and/or N starvation, we exposed Ps and Pe saplings to complete nutrients and starvation of P, N or both elements (NP). We hypothesized that (i) the fastgrowing poplar genotype would possess a greater capacity for the uptake and assimilation of P/N and (ii) both poplar genotypes would suppress P/N absorption and metabolism processes in response to P, N or NP starvation. In particular, we addressed the following questions. (i) What are the physiological differences in P/N uptake and assimilation that underlie the contrasting growth characteristics of both poplar genotypes? (ii) What are the physiological and transcriptional regulation mechanisms of both poplar genotypes that are activated in response to P, N or NP starvation? Materials and methods Plant materials and nutritional treatments Cuttings (?20 cm in length, 2 cm in diameter, 1-year-old stems) of the slow-growing Ps and the fast-growing Pe were rooted and subsequently planted in pots (10 l) filled with fine sand, with three holes of 1 cm diameter at the bottom of the pot for drainage. Plantlets were cultivated in a greenhouse (natural light; day/ night temperate: 25/20 ?C; relative humidity: 75%). Plantlets were irrigated with 50 ml of one-eighth strength Hoagland solution (8 mM NH4NO3, 2 mM KH2PO4, 1 mM MgSO4 ? 7H2O, 2 mM CaCl2 ? 2H2O, 25 ?M KCl, 12.5 ?M H3BO3, 1 ?M MnSO4 ? H2O, 1 ?M ZnSO4 ? 7H2O, 0.25 ?M CuSO4 ? 5H2O, 0.25 ?M Na2MoO4 ? 2H2O and 53.7 ?M EDTA-FeNa ? 3H2O; pH 5.5) every day. After cultivation for 5 weeks (the amount of supplied P and N was 14 and 49 mg, respectively, during this period), plants that exhibited similar height (?40 cm) and growth performance in each genotype were selected for nutritional treatments. For each genotype, 24 plants were divided into four groups with six plants in each. The plants in each group for each genotype were treated with modified Hoagland solution (1 mM MgSO4 ? 7H2O, 2 mM CaCl2 ? 2H2O, 25 ?M KCl, 12.5 ?M H3BO3, 1 ?M MnSO4 ? H2O, 1 ?M ZnSO4 ? 7H2O, 0.25 ?M CuSO4 ? 5H2O, 0.25 ?M Na2MoO4 ? 2H2O and 53.7 ?M EDTA-FeNa ? 3H2O; pH 5.5) containing one of the following P and N concentrations: complete nutrients (8 mM NH4NO3 and 2 mM KH2PO4, Con), and starvation of P (8 mM NH4NO3 and 0 mM KH2PO4, P?), N (0 mM NH4NO3 and 2 mM KH2PO4, N?) or both elements (0 mM NH4NO3 and 0 mM KH2PO4, NP?). In the P? and NP? treatments, KCl was used to replace KH2PO4 to avoid K deficiency. Each plant was slowly irrigated to avoid runoff with 100 ml of distilled water and 50 ml of modified Hoagland solution every other day. The nutritional treatments were continued for 8 weeks in order to obtain sufficient plant material for physiological analyses. Hence, during the period of 8 weeks, each plant from the complete nutrient treatment received 87 mg P and 314 mg N. Although nutrient leaching might have occurred in this pot experimental system, this was unable to affect the physiological processes of plants exposed to starvation of P, N or NP. Thus, nutrient leaching was not considered further in this study. At the beginning of the nutritional treatment, the apex of each plant was marked using a laboratory marker to distinguish the shoots formed during the nutritional treatment. Gas exchange, harvesting and ectomycorrhizal examination Before harvest, the gas exchange in three mature leaves (leaf plastochron index = 9?11) formed during nutritional treatments was determined for each plant. The net photosynthetic rates (A), stomatal conductance (gs) and transpiration rates (E) were determined with a portable photosynthesis system (LiCor-6400, LI-COR Inc., Lincoln, NE, USA) and an attached LED light source (6400-02) as described by He et al. (2011) . The instantaneous PPUEi was calculated using the following formula: PPUEi = A ? [PS]LfoAliar , where SLA and [P]foliar denote the specific leaf area and foliar P concentration, respectively. The instantaneous photosynthetic N use efficiency (PNUEi) was calculated as described by Li et al. (2012b). Subsequently, each plant was harvested by separating the roots and shoots formed before and during the nutritional treatment into wood, bark and leaf tissues. The root system of each plant was washed well with the corresponding nutritional solution. The root system was then wrapped in laboratory tissue paper to remove any water from the root surfaces. The fresh weights of the roots were recorded. Parts of the roots (?1 g) from each plant were excised from the root system, scanned and analyzed using a WinRHIZO Root Analyzer System (WinRHIZO 2012b, Regent Instruments Inc., Montreal, Canada), as described by Luo et al. (2013b). Leaf discs for determining the SLA were also collected from leaves formed during the nutritional treatments, and the SLA was calculated according to the method of Cao et al. (2012) . Subsequently, the samples were wrapped with tinfoil and frozen immediately in liquid nitrogen. The frozen samples were ground into fine powder in liquid nitrogen using a mortar and pestle, and stored at ?80 ?C until further analysis. Frozen powder (?60 mg) from each tissue was dried at 65 ?C for 72 h to determine the fresh : dry mass ratio. The biomass of each plant was estimated based on the fresh weight of each tissue and the fresh : dry mass ratio. Poplar trees are often colonized by ectomycorrhizal fungi under natural conditions and ectomycorrhizal roots may significantly enhance the uptake and assimilation of P and N (Plassard and Dell 2010) , so root subsamples from poplars cultivated in sand culture were also collected and examined to determine the presence of ectomycorrhizae under a light microscope (Eclipse E200, Nikon, Tokyo, Japan), as described previously (Luo et al. 2009) . Determination of chlorophylls and anthocyanin The concentrations of chlorophylls and carotenoids in the leaves were determined spectrophotometrically, as described by Li et al. (2012b). The anthocyanin concentrations in the leaves formed during the nutritional treatments were analyzed spectrophotometrically, according to the method of Lei et al. (2011) . Determination of P, N and phosphorus/nitrogen use?efficiency To determine the concentrations of P and N in poplar samples, fine powder (?100 mg DW) was digested in a mixture of 5 ml 98% H2SO4 and 1 ml H2O2 (300 g l?1), as described by Wang et al. (2009) . Subsequently, the P concentration in the digested solution was determined spectrophotometrically at 700 nm based on the molybdenum blue method (Sun et al. 2012) . The N concentrations in the digested solution were analyzed using a Continuous-Flow Analyzer (AA3, Bran-Luebbe, Hamburg, Germany) at 660 nm, according to the method of Schortemeyer et al. (1993) . The P/N amounts in the tissues were calculated as the concentrations of P or N multiplied by the biomass of the relevant tissue. Phosphorus use efficiency/N use efficiency (PUE/NUE) was defined according to the formula: PUE (NUE) = biomass/P (N) uptake, as proposed by Veneklaas et al. (2012) and Li et al. (2012b). The biomass was the total biomass of each plant. The P (N) uptake for each plant was estimated using the P (N) concentration multiplied by the total dry mass of each plant. Determination of ATP and free amino acids The ATP concentrations were determined in poplar roots and leaves according to the method described by Lundin and Thore (1975) . Briefly, each frozen powdered poplar sample (?50 mg) was extracted with 1 ml of cold 5% trichloroacetic acid for 5 min. Next, the pH of the extract solution was adjusted to 7.5 and the extract solution was diluted with 1 ml distilled water, before subjecting the extract solution to centrifugation (13,000g, 10 min, 4 ?C). The ATP concentration in the supernatant was measured using an ATP-Lite assay kit (Vigorous, Beijing, China). A standard curve was established using a series of diluted solutions of the ATP standard (Vigorous). Free amino acids in the roots and leaves were analyzed by liquid chromatography?mass spectrometry (LTQ-XL, Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the method proposed by Cao et al. (2014) . Determination of enzymatic activities The activities of APs (EC 3.1.3.2) were assayed in poplar roots and leaves based on the method of Lei et al. (2011) . In brief, ?50 mg of fine powdered poplar roots or leaves was extracted in 1.8 ml of cold extraction buffer (0.04 M sodium acetate, pH 6.5) on ice. After centrifugation (19,000g, 10 min, 4 ?C), the supernatant was used to assay the APs. To determine the activities of APs, 100 ?l of the extract was mixed with 600 ?l of 15 mM disodium 4-nitrophenyl phosphate and 1.3 ml of 40 mM sodium acetate, before incubating in the dark at 37 ?C for 30 min. The absorbance of the mixture was then recorded spectrophotometrically at 412 nm. The activities of PEPC (EC 4.1.1.31) and MDH (EC 1.1.1.37) were determined in poplar roots and leaves according to the methods of Gajewska et al. (2013) and L? et al. (2012) , respectively. Briefly, each frozen powdered sample (?100 mg) was extracted in extraction buffer (0.1 M Tris?HCl (pH 8.0), 5 mM ?-mercaptoethanol, 10% (v/v) glycerol, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM MgCl2 ? 6H2O and 1% (w/v) poly(vinylpolypyrrolidone)), and after centrifugation (20,000g, 20 min, 4 ?C), the supernatant was used to assay PEPC and MDH. To determinate the activity of PEPC, 100 ?l of the extract was mixed with 900 ?l of reaction solution (25 mM Tris?HCl (pH 8.0), 5 mM MgCl2 ? 6H2O, 1 mM D-dithiothreitol, 2 mM KHCO3, 0.1 mM NADH, 3 U MDH and 2.5 mM phosphoenolpyruvate), and the mixture was incubated at 37 ?C for 5 min. Subsequently, the decrease in the absorbance of the mixture was monitored spectrophotometrically at 340 nm for 5 min. To determine the MDH activity, 900 ?l of the reaction mixture (25 mM Tris?HCl (pH 8.0), 0.1 mM NADH and 0.15 mM oxaloacetate (OAA)) was preincubated at 37 ?C for 5 min and the reaction was started by the addition of 100 ?l plant extract. The decrease in the absorbance of the mixture was monitored spectrophotometrically at 340 nm for 5 min. The activities of NR (EC 1.7.99.4), GOGAT (EC 1.4.7.1) and GDH (EC 1.4.1.2) were analyzed in poplar roots and leaves according to the methods described by Luo et al. (2013a). Analysis of the transcript levels of key genes involved in P and N uptake and assimilation The transcriptional changes in the key genes involved in P and N uptake and assimilation were analyzed by quantitative reverse transcription polymerase chain reaction based on the method described by Li et al. (2012b). Briefly, total RNA was extracted from fine powdered fresh samples (?100 mg) and purified with a plant RNA extraction kit (R6827, Omega Bio-Tek, Norcross, GA, USA). To remove DNA contamination, the purified total RNA was digested with deoxyribonuclease from a TURBO DNA-free kit (AM1907, Ambion, Thermo-Fisher, Beijing, China), according to the manufacturer?s instructions. The success of the DNAfree treatment was evaluated by a control real-time PCR procedure. After DNA-free treatment, the RNA (?1 ?g) was used to synthesize cDNA with a PrimeScript RT reagent kit (RR047A, Takara, Dalian, China) where the reaction system comprised 20 ?l based on the instructions provided by the manufacturer. Quantitative PCR was performed in a 20-?l reaction volume, which contained 10 ?l of 2? SYBR Green Premix EX Taq II (DRR820A, Takara), 2 ?l of cDNA and 0.2 ?l of 20 mM primers, which were designed specifically for each gene (see Table S1 available as Supplementary Data at Tree Physiology Online). Quantitative PCR was performed using an IQ5 realtime system (Bio-Rad, Hercules, CA, USA). Actin2/7 was used as a reference gene (see Table S1 available as Supplementary Data at Tree Physiology Online) (Brunner et al. 2004) . To ensure the primer specificity, PCR products were sequenced and aligned with homologs from P. trichocarpa and other model plants (see Figure S1 available as Supplementary Data at Tree Physiology Online). PCR was performed in triplicate together with a dilution series of the reference gene. The efficiencies of all PCRs ranged between 101 and 108% (see Table S1 available as Supplementary Data at Tree Physiology Online). Statistical analysis Statistical tests were performed using Statgraphics (STN, St Louis, MO, USA), where the data were tested for normality prior to the statistical analysis. The effects of genotypes and nutritional treatments on variables were analyzed by two-way analysis of variance (ANOVA). Data were considered to be significantly different when P < 0.05 in the ANOVA F-test. The data obtained from quantitative PCR were normalized using the program REST (Pfaffl et al. 2002) . The gene expression heatmap was generated using the heatmap.2 () command in the package ?gplots? in R (http://www.r-project.org/), as described previously (Luo et al. 2013a) . Results Root characteristics and growth performance The root characteristics were analyzed in both poplar genotypes (see Table S2 and Figure S2 available as Supplementary Data at Tree Physiology Online). The fast-growing Pe exhibited greater length, surface area and volume of the total fine roots compared with the slow-growing Ps (see Table S2 available as Supplementary Data at Tree Physiology Online). Long-term P, N or NP starvation resulted in greater decreases in length, surface area and volume of the total fine roots in Pe compared with Ps (see Table S2 available as Supplementary Data at Tree Physiology Online). Ectomycorrhizae were not observed in the root systems of both poplars (see Figure S2 available as Supplementary Data at Tree Physiology Online). The growth performance was also assessed in both poplars (see Tables S3 and S4 and Figure S3 available as Supplementary Data at Tree Physiology Online). The developmental stage of the two poplar genotypes did not differ before the harvest and they still grew slowly under P, N or NP deprivation (see Figure S3 available as Supplementary Data at Tree Physiology Online). Pe had higher E and lower anthocyanin levels than Ps with a sufficient supply of nutrients (see Table S3 available as Supplementary Data at Tree Physiology Online). Deprivation of P, N or NP caused greater reductions in A, gs and E, but higher increases in the anthocyanin levels of Pe than those of Ps (see Table S3 available as Supplementary Data at Tree Physiology Online). Similarly, Pe had a greater root and leaf biomass, and SLA compared with Ps under the control nutrient conditions (see Table S4 available as Supplementary Data at Tree Physiology Online). Nitrogen starvation led to a greater increase in the root biomass of Pe than that of Ps (see Table S4 available as Supplementary Data at Tree Physiology Online). Phosphorus, N or NP deficiency led to greater decreases in the leaf mass, leaf area and SLA in Pe compared with those in Ps (see Table S4 available as Supplementary Data at Tree Physiology Online). Phosphorus status, PPUEi, PUE and ATP We analyzed the P status, PPUEi and PUE in both poplar genotypes (Figure 2). Pe had lower P concentrations but greater amounts of P in the roots and leaves than Ps (Figure 2a?d), which were attributable to the greater biomass of Pe compared with that of Ps. Furthermore, Pe had higher PPUEi and PUE values than Ps (Figure 2e and f). Phosphorus or NP starvation caused greater reductions in the P concentrations in the roots and leaves of Ps than those of Pe (Figure 2a and b). Nitrogen deprivation led to greater increases in the P concentrations in the roots of Ps compared with those of Pe (Figure 2a), but it caused no changes in the foliar P levels of Ps and decreased the P concentration in the leaves of Pe (Figure 2b). Deprivation of P or NP led to greater reductions in the amounts of P in the roots and leaves of Pe compared with those in Ps (Figure 2c and d). Nitrogen starvation resulted in similar increases in the amounts of P in the roots of Ps and Pe and a greater decrease in the amount of P in the leaves of Pe compared with Ps (Figure 2c and d). Phosphorus, N or NP starvation resulted in greater decreases in PPUEi for Pe than Ps (Figure 2e). Deprivation of P or NP led to significant increases in PUE, but N starvation caused reductions in PUE in both poplar genotypes (Figure 2f). ATP is a P-rich compound in plants that is sensitive to nutritional changes in the soil. Thus, the ATP concentrations were determined in the roots and leaves of both poplar genotypes (Figure 3). The ATP concentrations were lower in the roots of Pe than those of Ps (Figure 3a), but this pattern was not observed in the leaves of both genotypes (Figure 3b). Phosphorus or NP starvation significantly reduced the ATP concentrations in the roots and leaves of both poplars, although P deprivation had no impact on the ATP level in Pe roots (Figure 3). Interestingly, N deficiency led to higher ATP concentrations in the roots of both poplars and in Ps leaves, but lower ATP level in Pe leaves, compared with the corresponding ATP levels under the complete nutrient supply (Figure 3). Activities of enzymes involved in P assimilation The activities of enzymes involved in P assimilation were assayed in the roots and leaves of both poplar genotypes (Figure 4). The activities of APs were higher in the roots and leaves of Pe than those of Ps (Figure 4a and b). Phosphorus starvation induced increases in the activities of APs in the roots and leaves of both poplar genotypes (Figure 4a and b). Nitrogen deprivation resulted in higher activities of APs in the roots of Ps, but lower activities of APs in the roots and leaves of Pe compared with those when provided complete nutrients (Figure 4a and b). Starvation of both elements (NP?) caused marked decreases in the activities of APs in the roots and leaves of both poplars compared with the complete nutrient control (Figure 4a and b). The PEPC activities were similar in the roots or leaves of both poplar genotypes (Figure 4c and d). The PEPC activity was induced under P deprivation, but inhibited under N or NP starvation in the roots and leaves of both poplars compared with those under complete nutrients (Figure 4c and d). The MDH activities were higher in the roots of Pe than those of Ps, whereas the opposite pattern was observed in the leaves of both genotypes (Figure 4e and f). The MDH activities were induced in the roots and leaves of both poplars when exposed to P starvation compared with those under complete nutrients (Figure 4e and f). In contrast, the MDH activities were inhibited in the roots and leaves of both genotypes under N or NP deprivation compared with those in the complete nutrients control (Figure 4e and f). Nitrogen status, PNUEi, NUE and free amino acids The N status, PNUEi, NUE and free amino acids were assessed in the roots and leaves of poplars (Figures 5 and 6). The N concentrations were lower in the roots and leaves of Pe than those of Ps (Figure 5a and b). In contrast, the amounts of N were greater in the roots and leaves of Pe than those of Ps (Figure 5c and d). Phosphorus starvation increased the N concentrations in the roots of both poplars, but decreased the N levels in the leaves of Ps and Pe compared with the complete nutrients conditions (Figure 5a and b). Nitrogen or NP starvation caused marked reductions in the N concentrations in the roots and leaves of both poplars compared with the supply of complete nutrients (Figure 5a and b). Phosphorus deprivation caused increases in the amounts of N in the roots but decreases in the amounts of N in the leaves of both poplars (Figure 5c and d). Nitrogen or NP starvation caused significant reductions in the amounts of N in the roots and leaves of both poplars (Figure 5c and d). The PNUEi value was similar in both poplar genotypes (Figure 5e), but NUE was significantly higher in Pe than Ps (Figure 5f). Phosphorus, N or NP starvation resulted in marked decreases in PNUEi for both poplar genotypes compared with the complete nutrients treatment (Figure 5e). However, N or NP deprivation significantly increased the NUE value for both poplars compared with the supply of complete nutrients (Figure 5f). Seventeen free amino acids were identified in the roots and leaves of both poplars (see Table S5 available as Supplementary Data at Tree Physiology Online), where Lys and Arg were the most abundant (see Table S5 available as Supplementary Data at Tree Physiology Online). Under the complete nutrients treatment, the concentration of total free amino acids was lower in the roots of Pe than those of Ps, but the total free amino acid levels were similar in the roots of Pe and Ps when exposed to P, N or NP starvation (Figure 6a). Similarly, the concentration of total free amino acids was lower (?22%) in the leaves of Pe than those of Ps when supplied sufficient nutrients (Figure 6b). Phosphorus deprivation increased the levels of total free amino acids in the roots and leaves of Pe and Ps, whereas N or NP starvation led to marked decreases in concentrations of total free amino acids in the roots and leaves of both genotypes compared with the complete nutrients treatment (Figure 6). Activities of enzymes involved in N assimilation After the uptake of NH4+ and NO3? , these N ions are further converted into intermediates and precursors of N-containing compounds, where these processes are catalyzed by several enzymes, including NR, GOGAT and GDH. Therefore, we analyzed the activities of these enzymes in the roots and leaves of Ps and Pe (Figure 7). The NR activities were higher in the roots and leaves of Pe than those of Ps (Figure 7a and b). Phosphorus or N starvation caused marked reductions in the NR activities in the roots and leaves of both poplars, and NP deprivation resulted in further decreases in the NR activities in the roots and leaves of Ps and Pe compared with the complete nutrients treatment (Figure 7a and b). The GOGAT activities were higher in the roots of Pe than those of Ps, whereas the opposite was found in the leaves of both genotypes (Figure 7c and d). Phosphorus, N or NP deprivation caused significant inhibition of the GOGAT activities in the roots and leaves of Ps and Pe compared with the complete nutrients treatment (Figure 7c and d). The GDH activities were higher in the roots of Pe than those of Ps, whereas the opposite pattern was found in the leaves of both genotypes (Figure 7e and f). The GDH activities were inhibited significantly in the roots of Ps and Pe when exposed to P, N or NP starvation compared with those receiving the complete nutrients treatment (Figure 7e). In contrast, the foliar GDH activities were increased in Ps when exposed to N or NP deprivation compared with the complete nutrients treatment (Figure 7f). Changes in the transcript levels of key genes involved in P/N assimilation The transcriptional regulation of key genes played a key role in the physiological differences between both poplars during their acclimation to P, N or NP deprivation. Based on our preliminary experiments, we assessed the mRNA levels of genes encoding phosphate transpor ters (i.e., PHT1;5, PHT1;9, PHT2;1, PHO1;H1 and PHO2), purple AP 1 (PAP1), ammonium and nitrate transporters (i.e., AMT1;1, AMT2;1, NRT1;1 and NRT2;1) and NR (Figure 8). In the roots, the genes analyzed in Pe exhibited much higher transcriptional responsiveness to P, N or NP starvation than those in Ps (Figure 8a). The mRNA levels of PHT1;5 were increased significantly by 3- to 23-fold in the roots of Ps when exposed to P or NP starvation and in those of Pe when subjected to P, N or NP deprivation (Figure 8a). Greater increases in the transcript levels of PHT1;9 were found in the roots of Pe than Ps under P, N or NP starvation (Figure 8a). The transcript levels of PHT2;1 and PHO1;H1 increased slightly in the roots of Ps and Pe when exposed to nutritional starvation (Figure 8a). The transcript levels of PAP1 increased the most (?10- to 18-fold) in the roots of Ps when exposed to P, N or NP deprivation and in those of Pe under P or NP starvation (Figure 8a). The mRNA level of AMT1;1 was upregulated by fivefold in the N-starved roots of Ps, but downregulated (ca half to one-tenth) in the roots of Ps under P or NP deprivation and in those of Pe when subjected to P, N or NP starvation (Figure 8a). NRT1;1 and NRT2;1 exhibited a similar transcriptional regulation pattern to AMT1;1 in response to nutritional stress (Figure 8a). The transcript levels of NR increased slightly in the roots of Ps and Pe when exposed to N or NP starvation (Figure 8a). Similar to the roots, the changes in the mRNA level of genes in the leaves of Pe were greater than those of Ps in response to P, N or NP deprivation (Figure 8b). In the leaves, the mRNA levels of PHT1;9 increased slightly in Ps when subjected to P or N starvation and in Pe when exposed to P or NP deficiency (Figure 8b). The transcript levels of PHT2;1 were induced in the leaves of Pe under N or NP starvation (Figure 8b). The mRNA levels of PHO1;H1 were upregulated in the leaves of Ps when subjected to P or NP deprivation and those of Pe when exposed to P, N or NP starvation (Figure 8b). Increased transcript levels of PHO2 were detected in the leaves of Ps and Pe when exposed to P, N or NP deprivation (Figure 8b). PAP1 had a similar regulation pattern to PHO2 in response to nutritional changes (Figure 8b). The mRNA levels of AMT1;1 were also induced in the leaves of Ps and Pe under N or NP starvation (Figure 8b). The transcript levels of NRT1;1 and NR were downregulated in the leaves of Pe with N or NP starvation (Figure 8b). Discussion Differences in P/N acquisition and assimilation between Ps and Pe Under treatment with complete nutrients and P, N or NP starvation, the length, surface area, volume of the total fine roots and root biomass were higher in Pe compared with Ps, which suggest that Pe has a greater capacity for exploiting nutrients in the soil, and thus, the root characteristics of Pe were more sensitive to P, N or NP deprivation than those of Ps. For Pe, this greater capacity for nutrient acquisition may be critical for its fast growth. The faster growth of plants may also result in the consumption of more nutrients during the synthesis of metabolites (Lawlor 2002) . The greater biomass of Pe clearly required more P and N. Thus, the amounts of P and N (39?108%) were consistently higher in the roots and leaves of Pe than those of Ps. Plant productivity is mainly reliant on photosynthesis. During photosynthetic CO2 assimilation, P-containing compounds are required for physiological processes, such as the biosynthesis of ATP from ADP, the production of triose-P and the regeneration of ribulose-1,5-bisphosphate (RuBP) (Hidaka and Kitayama 2013) . Thus, the efficient utilization of P during photosynthesis is a potentially important determinant of plant PUE. In the current study, the PPUEi value for Ps under the treatment with complete nutrients was close to the global average value of 103 ?mol CO2 g?1 P s?1 (Wright et al. 2004), whereas the PPUEi value for Pe was much higher (?70%), thereby indicating that P utilization is more efficient during photosynthesis by Pe. Woody plants can maintain higher PPUEi values by allocating more P to the metabolites that participate in photosynthesis and less P to compounds such as nucleic acids and phospholipids, which are not involved directly in carbon fixation (Lambers et al. 2012, Hidaka and Kitayama 2013) . Therefore, the higher PPUEi value of Pe compared with Ps indicates that Pe may allocate more P to the photosynthetic pathway than Ps. This was supported by the higher foliar ATP level and enzymatic activity of the APs in Pe compared with those in Ps. In agreement with higher PPUEi of Pe, the PUE value of Pe was higher than that of Ps. Phosphorus use efficiency is defined as the ratio of the biomass relative to the P absorbed by plants, so more P investment in growth can lead to a higher PUE, while greater P storage in plant cells can lead to a lower PUE. Thus, the higher PUE value of Pe compared with that of Ps suggests that Pe probably invests more P in growth, whereas Ps channels more P to vacuoles for storage. This was supported by the lower P concentrations in the roots and leaves of Pe compared with those of Ps. Similar to P, N is also critical for photosynthetic processes because N is an essential constituent of the photosynthetic machinery (Xu et al. 2012) . Thus, the NUE in photosynthesis may have an important effect on the whole-plant NUE. In this study, PNUEi was similar in Pe and Ps. The lower foliar N concentration, higher foliar N amount and higher NUE in Pe compared with those in Ps under treatment with sufficient nutrients suggest that Pe can utilize N more efficiently to promote biomass production. This was supported by the lower levels (?22%) of total free foliar amino acids and greater activity (85%) of foliar NR in Pe compared with those in Ps under treatment with sufficient nutrients. The higher NR activities in Pe may accelerate N assimilation to yield more precursors such as amino acids and proteins for biomass production. Nitrogen use efficiency is an essential characteristic of agricultural cops, which is evaluated extensively during cultivar selection (Good et al. 2004, Ju et al. 2009, Xu et al. 2012) . In contrast, little information is available about the NUE for woody plants such as poplars (Rennenberg et al. 2010). However, previous studies indicate that slowgrowing poplars will accumulate N because increased N cannot be used to stimulate growth, thereby decreasing the NUE (Euring et al. 2012, Li et al. 2012b) . In the current study, our results indicated that Pe was superior to Ps in terms of utilizing N to produce biomass under the conditions with complete nutrient, which was probably attributable to lower N storage by Pe compared with Ps. The accelerated P and N physiological processes in Pe agreed well with the transcriptional regulation of the key genes involved in the assimilation of P and N. PHT1;5 and PHT1;9 play important roles in the uptake and translocation of Pi in plants (Nagarajan et al. 2011, Remy et al. 2012, Lapis-Gaza et al. 2014) . The increased transcriptional levels of PHT1;5 and PHT1;9 in the roots, as well as PHT1;9 in the leaves, of Pe under treatment with complete nutrients compared with Ps agreed with the greater amounts of P in Pe than those in Ps. Previous studies have demonstrated that AMT1;1, AMT2;1, NRT1;1, NRT2;1 and NR are involved in N uptake, translocation and assimilation in poplars (Couturier et al. 2007, Li et al. 2012b, Luo et al. 2013a) . The induced mRNA levels of AMT2;1 in the roots, and ATM1;1, NRT1;1, NRT2;1 and NR in the leaves, of Pe when supplied with complete nutrients compared with those in Ps agreed with the greater amounts of N, the higher levels of free amino acids and the higher NR activity in Pe. The differential expression of transcripts of these genes demonstrated that Pe possesses a more efficient system for taking up, translocating and assimilating P and N compared with Ps. Overall, these results suggest that Pe possesses a greater capacity for P/N uptake and assimilation, thereby promoting faster growth compared with Ps, which is attributable to the greater fine root surface area, higher activities of APs and NR and increased mRNA levels of genes involved in P/N absorption and metabolism in Pe. Physiological and transcriptional regulation mechanisms of P/N assimilation in poplars during acclimation to P, N or NP starvation Woody plants are often colonized by ectomycorrhizal fungi and the presence of ectomycorrhizae can significantly improve P/N nutrition in the hosts under natural conditions, particularly in low P/N soil (Plassard and Dell 2010) . However, ectomycorrhizae were not formed when both poplar genotypes were cultivated in sand, which simplified our experimental system. Therefore, in the present study, we avoided the possible interference by ectomycorrhizae during P/N nutrition in host trees. The findings of this study improve our understanding of the response of nonmycorrhizal woody plants to nutritional deprivation. The plant root architecture is highly responsive to changes in P and N availability (Linkohr et al. 2002) . In Arabidopsis, the growth of primary roots was inhibited by P starvation, but lateral root growth was stimulated in a short-term (8?14 days) experiment (Cerutti and Delatorre 2013) . Similarly, Arabidopsis roots exhibited increased outgrowth of lateral roots under limiting N conditions by adopting an ?active-foraging strategy? (Ruffel et al. 2011) . Many other plants stimulate their fine root surface area under temporary N deficiency, whereas they inhibit root growth under conditions of long-term limiting N availability due to the lack of internal N reserves (Kraiser et al. 2011) . Our results demonstrated that the total fine root surface area of both poplar genotypes decreased after exposure to nutrient deprivation compared with those provided sufficient nutrients, which was probably related to the relatively long-term (8 weeks) P, N or NP starvation leading to a lack of internal nutrients. However, P, N or NP starvation did not suppress the root biomass in Ps and Pe, which suggests that roots still play an important role in the acquisition of other nutrients and water under long-term P, N or NP deprivation. Phosphorus and N are key components of the metabolites involved in photosynthetic processes, so the supply of these macronutrients is essential for the maintenance of high CO2 assimilation rates and the rapid growth by plants (Rennenberg et al. 2010, L?pez-Arredondo et al. 2014) . Phosphorus and N starvation in plants can lead to decreases in the energy supply (such as ATP and ADP) and intermediates (e.g., amino acids and RuBP), as well as altering the enzymatic activities related to biochemical reactions during photosynthetic processes. As a consequence, reductions in A and changes in PPUEi and PNUEi may occur in plants. The significant decreases in A, PPUEi and PNUEi in Ps and Pe exposed to P, N or NP deprivation are consistent with the results obtained from other plant species (Brooks et al. 1988, Fredeen et al. 1990) . The decreases in A and PPUEi for both poplars when exposed to P or NP starvation were probably related to reductions in the ATP supply for these plants under these conditions. Similarly, the decreases in A and PNUEi under N or NP deprivation can also be ascribed to lower levels of N-containing precursors such as free amino acids and the reduced activities of enzymes involved in photosynthetic processes. Phosphorus use efficiency and NUE reflect the biomass production per unit mass of P and N, respectively. At the plant level, in both poplars, the decreased PUE under N starvation and NUE under P deprivation were due to the accumulation of P and N in these conditions, respectively. In contrast, for both poplars at the plant level, the increases in PUE under P or NP starvation and NUE under N or NP deprivation were probably associated with the complete utilization of stored P or N for biomass production in these conditions. In agreement, under P or N deprivation, the P or N stored in the vacuoles of plant cells can be released into the cytosol for biomass synthesis (Sharkey et al. 1986, Mimura 1995, Lambers et al. 2012) . Several enzymes play pivotal roles in P/N acquisition and assimilation in plants, including APs, PEPC, MDH, NR, GOGAT and GDH (Rennenberg et al. 2010, Rennenberg and Herschbach 2013, L?pez-Arredondo et al. 2014) . The extracellular and intracellular APs in plants are involved in the acquisition of external Pi from the soil and the recycling of internal Pi (Duff et al. 1994). The extracellular APs are located in the cell walls or secreted by roots into the rhizosphere, thereby releasing Pi from organic phosphorylated compounds, whereas the intracellular APs often occur in the vacuoles, which can remove Pi from endogenous P-containing metabolites (Duff et al. 1994) . Increases in the enzymatic activities of extra- and intracellular APs often occur when plants are exposed to P deficiency (Tian et al. 2012, Zhang et al. 2015) . Similarly, the increased enzymatic activities of APs in P-starved poplars suggest that Ps and Pe optimize Pi uptake and internal recycling. Phosphoenolpyruvate carboxylase is a ubiquitous and tightly regulated cytosolic enzyme, which catalyzes phosphoenolpyruvate to form OAA and Pi (Shane et al. 2013). The enhanced activity of PEPC can accelerate the synthesis of OAA, thereby leading to further overproduction of malate via MDH. Malate is a precursor involved in the tricarboxylic acid (TCA) cycle, which can produce citrate. Malate and citrate can also be excreted into the extracellular space to chelate metal ions such as Ca2+ and Al3+, which immobilize Pi in the rhizosphere to enhance the availability of soluble Pi for the roots (Vance et al. 2003, Shane et al. 2013) . Increases in the activities of PEPC and MDH often occur in plants exposed to P deficiency, including poplar species (Aono et al. 2001, Pe?aloza et al. 2005, Desai et al. 2014) . Therefore, the elevated activities of PEPC and MDH in the roots and leaves of Ps and Pe under P deprivation suggest that both poplars can induce the activities of enzymes involved in Pi cycling and mobilization in response to P starvation. In contrast, the activities of NR, GOGAT and GDH, which are involved in N assimilation, were inhibited in the roots and leaves of Ps and Pe under N deprivation conditions. This was probably related to the shortage of substrates for these enzymes in both poplars when exposed to N starvation because the activities of NR, GOGAT and GDH are induced by the substrate (Li et al. 2012b, Luo et al. 2013a) . The transcriptional regulation of genes involved in the uptake and assimilation of P and N plays a crucial role in herbaceous plants in response to nutritional deficiency (Krouk et al. 2010, Krapp et al. 2011, Ruffel et al. 2011, Schluter et al. 2012, Bonneau et al. 2013, Secco et al. 2013) , but limited information is available about the transcriptional modulation that underlies the physiological responses of woody plants to P and/or N starvation (Rennenberg et al. 2010, Loth-Pereda et al. 2011, Rennenberg and Herschbach 2013) . In this study, the transcriptional induction of several Pi transporters encoded by PHT1;5, PHT1;9 and PHT2;1 in the roots, as well as PHT1;9, PHO1;H1 and PHO2 in the leaves, of Ps and Pe in P or NP starvation conditions indicates that both poplars enhance the mRNA levels of these Pi transporters, which probably increases the capacity for Pi uptake in the roots and mobilization in the leaves. In agreement with our results, P starvation resulted in marked increases in the transcript levels of PHT1;5 and PHT1;9 in the roots of three poplar genotypes in a previous study, i.e., P. trichocarpa, Populus deltoides and P. deltoides ? P. trichocarpa (Loth-Pereda et al. 2011) . The PAPs comprise a subfamily of APs in plants (Li et al. 2002). Several PAP genes are transcriptionally induced when herbaceous plants are exposed to P starvation (del Pozo et al. 1999, Hurley et al. 2010, Li et al. 2012a) . We found that the increased mRNA levels of PAP1 in the roots and leaves of Ps and Pe when treated with P or NP deprivation agreed with the enhanced enzymatic activities of APs, which may contribute to the activities of PAPs under nutritional deficiency. Similarly, the induced mRNA levels of AMT2;1 and NR in the roots, as well as AMT1;1 and NRT2;1 in the leaves, of Ps and Pe when exposed to N or NP deprivation indicate that both poplars increase the mobilization and assimilation of NH4+ and NO3? during acclimation to N or NP deprivation. Indeed, the transcriptional induction of genes encoding AMTs and NRTs has been reported in the roots and leaves of other poplar genotypes when exposed to low N levels or N starvation (Selle et al. 2005, Couturier et al. 2007, Ehlting et al. 2007, Luo et al. 2013a) . Overall, these results demonstrate that Ps and Pe overexpress transcripts of PHT1;5, PHT1;9, PHT2;1, AMT2;1 and NR in the roots, and of PHT1;9, PHO1;H1, PHO2, AMT1;1 and NRT2;1 in the leaves, during acclimation to P, N or NP deprivation. Obviously, these genes will be good candidates for manipulation during the breeding of transgenic trees with tolerance to P/N deficiency in future studies. Our results also suggest that both poplar genotypes suppressed P and N uptake and assimilation during acclimation to P, N or NP deprivation, which were associated with the inhibition of fine root growth, A, PPUEi, PNUEi, and the activities of NR, GOGAT and GDH, as well as enhanced PUE, NUE, and the activities of APs, PEPC and MDH, and induced transcript levels of genes involved in P/N metabolism. In conclusion, the fast-growing Pe had lower P/N concentrations and greater amounts of P/N due to higher biomass production, thereby resulting in a higher PUE/NUE value compared with the slow-growing Ps. The roots of Pe had higher enzymatic activities of APs and MDH, which are involved in P mobilization, and NR, GOGAT and GDH, which participate in N assimilation, compared with the roots of Ps. Thus, the transcriptional regulation responsiveness of key genes encoding transporters for phosphate (PHTs and PHOs), ammonium (AMTs) and nitrate (NRTs) was stronger in Pe than Ps. These results suggest that Pe possesses a greater capacity for P/N uptake and assimilation, which promotes faster growth compared with Ps, and this is associated with a greater fine root surface area, higher activities of APs and NR and increased mRNA levels of genes involved in P/N absorption and metabolism in Pe. Phosphorus or NP starvation caused significant decreases in the P concentrations and increases in PUE for both poplars. Phosphorus deprivation increased the activities of APs, PEPC and MDH in both genotypes. Nitrogen or NP deficiency reduced the N concentrations, amino acid levels and activities of NR and GOGAT, and increased NUE in both poplars. In agreement, Ps and Pe had increased mRNA levels of PHT1;5, PHT1;9, PHT2;1, AMT2;1 and NR in the roots, as well as PHT1;9, PHO1;H1, PHO2, AMT1;1 and NRT2;1 in the leaves, during acclimation to P, N or NP deprivation. Thus, our results suggest that both poplars suppress P/N uptake, mobilization and assimilation during acclimation to P, N or NP starvation. Supplementary data Supplementary data for this article are available at Tree Physiology Online. Confilct of interest None declared. Funding This research was jointly supported by the State Key Basic Research Development Program (grant no. 2012CB416902), the National Natural Science Foundation of China (grant no. 31100481, 31270647 and 31470618), the Specialized Research Fund for the Doctoral Program of Higher Education of China (grant no. 20130204110012) and the Fundamental Research Funds for the Central Universities of China (grant no. YQ2013005, QN2013013). Research conducted in A.P.?s laboratory was supported by DFG funding Po362/22-1 in the framework of SPP 1685?Ecosystem nutrition. Aono T , Kanada N , Ijima A , Oyaizu H ( 2001 ) The response of the phosphate uptake system and the organic acid exudation system to phosphate starvation in Sesbania rostrata . Plant Cell Physiol 42 : 1253 - 1264 . Bai H , Euring D , Volmer K , Janz D , Polle A ( 2013 ) The nitrate transporter (NRT) gene family in poplar . PLoS One 8 : e72126 . 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Gan, Honghao, Jiao, Yu, Jia, Jingbo, Wang, Xinli, Li, Hong, Shi, Wenguang, Peng, Changhui, Polle, Andrea, Luo, Zhi-Bin. Phosphorus and nitrogen physiology of two contrasting poplar genotypes when exposed to phosphorus and/or nitrogen starvation, Tree Physiology, 2016, 22-38, DOI: 10.1093/treephys/tpv093