The importance of the urea cycle and its relationships to polyamine metabolism during ammonium stress in Medicago truncatula.

The ornithine-urea cycle (urea cycle) makes a significant contribution to the metabolic responses of lower photosynthetic eukaryotes to episodes of high nitrogen availability. In this study, we compared the role of the plant urea cycle and its relationships to polyamine metabolism in ammonium-fed and nitrate-fed Medicago truncatula plants. High ammonium resulted in the accumulation of ammonium and pathway intermediates, particularly glutamine, arginine, ornithine, and putrescine. Arginine decarboxylase activity was decreased in roots, suggesting that the ornithine decarboxylase-dependent production of putrescine was important in situations of ammonium stress. The activity of copper amine oxidase, which releases ammonium from putrescine, was significantly decreased in both shoots and roots. In addition, physiological concentrations of ammonium inhibited copper amine oxidase activity in in vitro assays, supporting the conclusion that high ammonium accumulation favors putrescine synthesis. Moreover, early supplementation of plants with putrescine avoided ammonium toxicity. The levels of transcripts encoding urea-cycle-related proteins were increased and transcripts involved in polyamine catabolism were decreased under high ammonium concentrations. We conclude that the urea cycle and associated polyamine metabolism function as important protective mechanisms limiting ammonium toxicity in M. truncatula. These findings demonstrate the relevance of the urea cycle to polyamine metabolism in higher plants.

Introduction

Ammonium (NH4+) as the sole source of nitrogen (N) is toxic for many plant species, leading to physiological and morphological disorders that affect plant growth and development (Britto and Kronzucker, 2002; Bittsánszky ; Esteban ). Indeed, plants subjected to periods of high NH4+ availability develop imbalances of essential cations (Ariz ) and changes in the content of N-rich compounds, such as amino acids of low carbon/nitrogen (C/N) ratio (Ariz ; Vega-Mas ) and polyamines (PAs) (Houdusse , 2008; Ariz ; Zhu ).

It has been proposed that the ornithine–urea cycle (urea cycle) might constitute an important control point of N metabolism in plants subjected to high NH4+ conditions (Moschou ; Esteban ), since in lower photosynthetic eukaryotic phyla, the urea cycle significantly contributes to the metabolic responses under high N availability (Allen ). Nevertheless, the urea cycle is not considered complete in plants because the carbamoyl phosphate synthase-type I, the enzyme that synthesizes urea directly from NH4+, is absent. In contrast, plants possess a functional carbamoyl phosphate synthase-type II (CPSII, EC 6.3.5.5) that synthesizes urea from glutamine (Gln) instead of NH4+ (Zhou ; Brady ). In the urea cycle, the synthesis of arginine (Arg) from Gln is catalyzed by three sequential enzymes, ornithine transcarbamylase (OTC, EC 2.1.3.3), argininosuccinate synthase (AS, EC 6.3.4.5), and argininosuccinate lyase (AL, EC 4.3.2.1) (Micallef and Shelp, 1989; Slocum, 2005). Then, Arg is converted to ornithine (Orn) through the action of arginase (ARG, EC 3.5.3.1), with the concomitant production of urea (Kang and Cho, 1990; Winter ), and urea is further degraded by plant urease (URE, 3.5.1.5) to form NH4+ (Polacco and Winkler, 1984; Witte, 2011).

Furthermore, once Arg and Orn are produced in the urea cycle, the translocation of N towards the biosynthesis of the diamine putrescine (Put) occurs (see Esteban ) through the consecutive action of the enzymes arginine decarboxylase (ADC, EC 4.1.1.19), agmatine iminohydrolase (AIH, EC 3.5.3.12), and N-carbamoyl-putrescine amidohydrolase (NCPAH, EC 3.5.1.53), and/or the enzyme ornithine decarboxylase (ODC, EC 4.1.1.17), respectively (Fuell ). The ADC pathway is present in plants but absent in metazoans (Slocum ), while the ODC pathway is considered to be universal (Michael ) except in some members of the Brassicaceae family, including Arabidopsis thaliana (Hanfrey ). Put is successively aminopropylated to form the triamine spermidine (Spd) in a reaction catalyzed by spermidine synthase (SPDS), and Spd is further converted to spermine (Spm) by the action of spermine synthase (SPMS) (Slocum ).

Plants finely regulate PA homeostasis at the level of synthesis, conjugation, turnover, transport, and catabolism (Moschou ; Tiburcio ). PA catabolism is mediated by numerous amine oxidases (AOs), including copper-containing amine oxidases (CuAOs, EC 1.4.3.6) and flavin-containing polyamine oxidases (PAOs, EC 1.5.3.3) (Cona ; Alcázar ; Wang ), with specific functions in plant tissue differentiation and development (Tavladoraki ). Additionally, PA catabolism contributes to several physiological processes as a source of stress-related molecules such as γ-aminobutyric acid (GABA) and hydrogen peroxide (H2O2). Particularly, PA-derived H2O2 contributes to generate oxidative stress within plant tissues or to activate antioxidative defense responses and cell-wall lignification (Su ; Angelini ; Gupta ).

Currently, there is little information on the overall effect of NH4+ on the urea cycle and PA metabolism, regardless of the remarkable accumulation of Put that NH4+ nutrition induces (Houdusse , 2008; Belastegui-Macadam ; Ariz ; Zhu ). Indeed, there are four enzymatic steps releasing NH4+ and, therefore, NH4+ may act as a feedback regulator of these pathways. Filling in this knowledge gap could be essential to understand the onset of NH4+ stress, since the urea cycle–PA metabolism may represent a regulatory pathway in the management of N and, specifically, under NH4+ nutrition.

Here, we have analyzed the metabolite contents, transcript levels, and enzyme activities of the urea cycle and PA ­metabolism in Medicago truncatula plants growing in the presence of NH4+ as the only N source, in comparison to nitrate (NO3–)-fed plants. We found an accumulation of the intermediates Gln, Arg, Orn, and Put, an induction of the transcript levels of the urea-cycle-related genes and Put biosynthetic genes, and a decrease of CuAO activity during NH4+ stress, which highlighted the metabolic implications of these two interconnected routes. We have also addressed the question of whether Put led to the NH4+ stress or whether it is part of the mechanism of stress alleviation.

Materials and methods

Plant material and growth conditions

Seeds of Medicago truncatula Gaertn. ecotype Jemalong A17 were scarified with 95% sulfuric acid for 8 min, washed with sterile water, sterilized with 50% (v/v) sodium hypochlorite solution for 5 min, and consecutively washed with sterile water until the pH reached 7. After being kept overnight at 4 °C in darkness, seeds were germinated on Petri dishes containing 0.4% (w/v) plant agar for 72 h at 14 °C in darkness. Then, five sprouts were transferred to glass jars under axenic conditions, which contained 100 ml of modified Fahraeus media with 5 g l–1 of phytagel (Esteban ; Buezo ). The growth medium contained 0.9 mM CaCl2, 0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM Na2HPO4, 20 μM ferric citrate, 0.8 μM MnCl2, 0.6 μM CuSO4, 0.7 μM ZnCl2, 1.6 μM H3BO3, 0.5 μM Na2MoO4, and 1 mM or 25 mM of N applied as either Ca(NO3)2 or (NH4)2SO4. Both 1 mM and 25 mM NH4+-fed plants were supplemented, respectively, with 0.5 mM and 12.5 mM CaSO4 to compensate for the Ca2+ supplied together with the NO3– treatments.

To assess the effect of Put in the response to NH4+ nutrition, we supplemented plants with Put at 0.5 mM and we evaluated plant biomass as a marker of NH4+ stress. We used the following controls: a control with 2 mM NH4+ to discard the possibility that the Put effect relied partially on the concentration of N added to the culture medium, since adding Put implied the addition of an extra 1 mM of N; and a control with both 1 mM NH4+ and 1 mM NO3– to assess whether the positive effect of exogenous Put was different from that of NO3–. No control was used at the high N concentration, as the difference between 25 mM and 26 mM of N was not considered relevant.

After a growth period of 14 d, shoots and roots were separately harvested, and both fresh weight and dry weight were measured. They were then frozen in liquid nitrogen, and stored at –80 °C for further analyses.

Determination of inorganic soluble cation content

Ionic content was detected using a DIONEX-DX500 ion chromatograph equipped with an AS40 autosampler and ED40 electrochemical detector (Dionex Corporation, Sunnyvale, CA, USA) as described by Ariz . Frozen plant tissue (200 mg) was incubated in 1 ml of milli-Q water for 5 min at 80 °C in a water bath. The soluble ionic fraction was obtained by centrifugation at 16 000 g for 30 min. The supernatants, stored at –20 °C, were diluted 1:10 for injection. Ion Pac CG12A and Ion Pac CG12A were used as the stationary phase and 30% 100 mM NaOH and 70% milli-Q water were used as the mobile phase at 1.5 ml min–1 flow rate for 15 min. Soluble cations (Na+, K+, Mg2+, Ca2+, and NH4+) were determined using 20 mM methanosulfonic acid as the mobile phase for 13 min.

Determination of amino acid and protein content

Determination of amino acids was performed using high-performance capillary electrophoresis in a Beckman Coulter PA-800 (Coulter Inc., Brea, CA, USA) equipped with a laser-induced fluorescence detector (argon ion: 488 nm) as described in Ariz . Frozen plant tissue (100 mg) was ground with a mortar and pestle using liquid nitrogen, homogenized with 1.5 ml of 1 M HCl, incubated in ice for 10 min, and centrifuged at 13 000 g for 10 min at 4 °C. The recovered supernatants were neutralized with NaOH and stored at –80 °C. Amino acids were derivatized with fluorescein isothiocyanate dissolved in 20 mM ­acetone/borate, pH 10, at room temperature for 12–16 h. Samples were injected in a migration buffer composed of 80 mM borax and 45 mM α-cyclodextrin, pH 9.2, using a pressurized method (5 s, 3.45 kPa). Single amino acids were eluted in a 50 µm internal diameter × 43/53.2 cm fused silica capillary at a voltage of 30 kV and 20 °C. Norvaline and homoglutamic acid were used as internal standards. Protein content was measured using a dye-binding Bradford microassay (Bio-Rad, Watford, UK) with bovine serum albumin as a standard.

Determination of polyamine content

The content of free PAs was measured using a Waters 575 High-Performance Liquid Chromatography Pump controlled by a Waters Pump Control Module and equipped with a Waters 474 fluorescence detector (Waters, Milford, MA, USA) as described in Ariz with some modifications. Frozen plant tissue (200 mg) was ground with liquid nitrogen and homogenized with 10:1 (v/w) of extraction buffer (5% aqueous HClO4, w/w):plant tissue, containing 0.1:1 (v/w) of 2 mM 1,6-hexanediamine:plant tissue as an internal standard solution. Samples were shaken at 24 000 rpm for 15 s, incubated for 1 h at 4 °C, and centrifuged at 15 000 g for 15 min at 4 °C. Volumes of 200 µl of the recovered supernatant were mixed with 400 µl of 3 M aqueous Na2CO3 and 400 µl of 0.12 M dansyl chloride in acetone, and incubated in darkness for 1 h at 60 °C. The reaction was quenched by the addition of 100 µl of 0.87 M proline and further incubation for 30 min. Then, PAs were extracted using 5 ml of ethyl acetate. Samples were centrifuged at 3000 g for 5 min at room temperature, organic layers were recovered, and the solvent was removed under reduced pressure at 40 °C. PAs were dissolved in 0.5 ml of methanol and filtered with a 0.45 µm pore nylon filter. The content of PAs was determined from 25 µl of extract. A Tracer Excel 120-ODSA column (3 µm 4.6 × 150 mm, Teknokroma, Barcelona, Spain) at 30 °C was used as the stationary phase. The mobile phase consisted of solvent A (water) and B (methanol), with a constant flux of 0.5 ml min–1. An increasing concentration gradient was used for solvent B, from 58% to 100% over 44 min, and then remained constant for 4 min. The concentration of solvent B was then gradually decreased to 58% for 3 min and allowed to rest for an additional 3 min. The fluorescence detector was set at λex=350 nm, λem=515 nm. Retention times were 29.88 min for Put, 45.06 min for Spd, and 48.85 min for Spm.

Determination of ADC and OCD enzymatic activities

The activities of ADC and ODC enzymes were determined according to Wu . Frozen plant tissue (200 mg) was ground with liquid nitrogen and homogenized with 1.5 ml of 0.1 M cold phosphate buffer, pH 6.3, which contained 5 mM EDTA, 1 mM pyridoxal phosphate, 0.01 mM polyvinyl pyrrolidone, 10 mM dithiothreitol, and 0.43 mM sodium thiosulfate. The homogenates were centrifuged at 12 000 g for 40 min at 4 °C. A volume of 0.8 ml of the recovered supernatant was mixed with 1 ml of reaction mix, composed of 0.1 M Tris–HCl buffer, pH 7.5, 5 mM EDTA, 40 μM pyridoxal phosphate, and 5 mM dithiothreitol, and 0.2 ml of either 25 mM l-Arg (for ADC activity) or 25 mM l-Orn (for ODC activity). To calculate the specific activity, control samples in which l-Arg or l-Orn were replaced by perchloric acid were used. A volume of 0.5 ml of the sample was mixed with 1 ml of 2 M NaOH and 10 μl benzoyl chloride, stirred for 20 s, and incubated for 30 min at 37 °C. A volume of 2 ml of saturated NaCl and 2 ml of 100% ether was added and the mixture was centrifuged at 1500 g for 5 min at 4 °C. Subsequently, 1 ml of the ether phase extraction was evaporated at 50 °C in a water bath, and the remainder was dissolved in 3 ml of 100% methanol. The reaction was measured at 254 nm in a GeneQuant 1300 spectrophotometer (Harvard Bioscience Inc., Holliston, MA, USA). An increase in absorbance of 1.0 at 254 nm for 1 min was considered one activity unit of enzyme.

Determination of CuAO and PAO enzymatic activities

Amine oxidase activity was determined as described by Su with some modifications. Plant frozen tissue (200 mg) was ground with liquid nitrogen and homogenized with 2:1 (v/w) 100 mM sodium phosphate buffer, pH 6.5. The homogenates were centrifuged at 12 000 g for 20 min at 4 °C. A volume of 10 µl of the recovered supernatant was mixed with the reaction mix, composed of 10 µl of 15 mM 4-aminoantipyrine/0.2% (v/v) N,N-dimethylaniline, 250 U ml–1 horseradish peroxidase, and 20 mM PAs. The AO activity was assayed using Put, Spd, or Spm as substrate. To calculate the specific activity, control samples without PAs were used. The reaction was measured at 555 nm in a SpectraMax 340pc microplate reader (Molecular Devices, San Jose, CA, USA), after being incubated at room temperature for 30 min. A change of 0.01 in absorbance was considered one activity unit of AO enzyme (Su ).

To calculate the inhibition parameters of CuAO activity by NH4+, increasing concentrations of (NH4)2SO4 from 1 mM to 100 mM were added to the reaction mixture. The maximum rate (Vmax) and Michaelis constant (Km) values were determined from Michaelis–Menten plots, and the inhibitor constant (Ki) values were obtained from a non-linear regression fitting of data for a mixed inhibition model performed with GraphPad Prism (version 8.4.0).

Determination of H2O2 content

The content of H2O2 was quantified using an Amplex Red H2O2 assay according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Frozen plant tissue (70 mg) was ground with liquid nitrogen and homogenized with 4:1 (v/w) 50 mM sodium phosphate buffer, pH 7.4. Samples were centrifuged at 12 000 g for 20 min at 4 °C. Volumes of 15 µl of plant extract were mixed with 85 µl of reaction mix, composed of 25 µM Amplex Red reagent and 0.2 U ml–1 horseradish peroxidase. The mixture was shaken at 450 rpm for 1 min and then incubated for 50 min at 30 °C. Samples were read every 10 min to follow the reaction kinetics at 570 nm in a SpectraMax 340pc microplate reader (Molecular Devices, San Jose, CA, USA). An H2O2 standard curve was prepared to quantify the H2O2 content of every sample.

Gene identification, RNA isolation, and gene expression analyses

The selection of urea cycle- and PA-related genes was done in the following databases linked to the M. truncatula genome (Tang ): GenBank, Phytozome, Uniprot, and QuickGO. In total, 25 genes encoding for CPSII (Medtr4g103830 and Medtr2g093280), OTC (Medtr1g022420 and Medtr3g112050), AS (Medtr3g088970 and Medtr5g042880), AL (Medtr3g100220), ARG (Medtr4g024960), URE (Medtr3g085640), ODC (Medtr3g114870), ADC (Medtr3g113910 and Medtr4g072020), AIH (Medtr4g112810), NCPAH (Medtr2g086600), CuAO (Medtr1g104590, Medtr4g117610, Medtr3g080500, Medtr3g077080, Medtr5g033170, Medtr8g069505, and Medtr1g104550), and PAO (Medtr3g033000, Medtr5g090300, Medtr3g064370, and Medtr2g039160) were studied.

RNA was extracted from 100 mg of frozen plant tissue powder using TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and RQ1 RNase-free DNase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The concentration and integrity of RNA were verified by the 260 nm/280 nm absorption ratio in an ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA (1 µg) was retrotranscribed into cDNA with the PrimeScriptTM RT kit (Takara Bio Inc., Kusatsu, Shiga, Japan).

The specific primers (Supplementary Table S1) were designed using the Primer-BLAST bioinformatics tool. Gene expression was determined from 2 µl of cDNA diluted 1:5 in a 20 µl reaction volume using SYBR® Premix Ex TaqTM (Takara Bio Inc., Kusatsu, Shiga, Japan) in a Step One Plus Real Time PCR System (Applied Biosystems, Foster City, CA, USA) as described in Buezo . The PCR program was 95 °C for 5 min, 40 cycles of 15 s at 94 °C followed by 1 min at 60 °C, and a final melting curve was programmed to confirm the absence of contamination with genomic DNA. Ubiquitin carrier protein 4 (Medtr3g062450) and 26S proteasome regulatory subunit S5A_2 (Medtr5g022440) were used as M. truncatula reference genes (Larrainzar ).

Phylogenetic analysis

The amino acid sequences of CuAOs and PAOs were retrieved by a BLASTp analysis using A. thaliana orthologous proteins as query sequences to retrieve those of M. truncatula by sequence similarity. Multiple amino acid sequence alignments were performed using the MAFFT 7 E-INS-I algorithm, which included the characterized orthologous proteins from A. thaliana (Møller and McPherson, 1998; Tavladoraki ; Takahashi ; Fincato ; Wimalasekera , 2011; Planas-Portell ; Ghuge ; Qu ; Fraudentali ; Liu ), Lens culinaris (Rossi ), Malus domestica (Zarei ), Oryza sativa (Ono ; Liu ), and Pisum sativum (Tipping and McPherson, 1995; Laurenzi ; Petřivalský ; Sagor ) (Supplementary Table S2). The resulting phylogenetic tree was obtained through the neighbor-joining method and the statistical bootstrap test with 1000 replications (Katoh ) and plotted by using iTOL (Letunic and Bork, 2021).

Statistical analysis

All statistical analyses were conducted using R studio (version 3.6.2) (R Core Team, 2020). Independent replicates (n=3–8) of each treatment were analyzed in the experiments. All data were tested for normality with the Shapiro–Wilk test and for homogeneity of variances with the Bartlett test. Differences among treatments were evaluated with one-way ANOVA and post hoc Student–Newman–Keuls test. For non-parametric data, Welch’s ANOVA and Games–Howell tests were performed. Differences were considered statistically significant at P<0.05.

Results

High NH4+ nutrition led to NH4+ over-accumulation but did not affect K+ concentration in M. truncatula plants

Plants supplied with high doses of either NO3– or NH4+ exhibited the highest content of soluble cations in shoots, whereas solely 25 mM NO3– nutrition increased the total soluble cation content in roots (Fig. 1A). At the shoot level, 25 mM NH4+ nutrition led to a large increase in NH4+ internal content in comparison with the 1 mM and 25 mM NO3– treatments (8.9- and 8.5-fold, respectively). A similar increase in NH4+ internal content was found in roots (7.7- and 3.6-fold, respectively). Besides, the NH4+ content in roots was greater in plants fed 1 mM NH4+ than 1 mM NO3– plants (2.8-fold) (Fig. 1B).

Fig. 1.

Effect of different sources and concentrations of N nutrition on the content of total soluble cations (A), NH4+ (B), K+ (C), Na+ (D), Ca2+ (E), and Mg2+ (F) in shoots and roots of 14-day-old M. truncatula seedlings. Data represent means ±SE values (n=4). Different letters denote statistically significant differences at P<0.05.

Regarding essential cations, the K+ content was higher in roots of 25 mM NO3–-fed plants than in roots of 1 mM NO3–-fed plants, while it remained unchanged in 1 mM or 25 mM NH4+-fed plants relative to 1 mM NO3–-fed plants (Fig. 1C). The Na+ content decreased in both shoots and roots as the N dose increased, but no significant differences in Na+ content were detected among plants grown with different N sources (Fig. 1D). Moreover, Ca2+ and Mg2+ contents were lower in roots of NH4+-fed plants than in roots of NO3–-fed plants (Fig. 1E, F).

NH4+ nutrition highly induced the accumulation of the urea cycle metabolites Gln, Arg, and Orn and the diamine Put

The highest content of total amino acids was found in both shoots and roots of 25 mM NH4+-fed plants. By contrast, the soluble protein content increased only in shoots of 25 mM NH4+-fed plants in comparison with NO3–-fed plants (Supplementary Fig. S1).

Gln content was highly increased in shoots and roots of 25 mM NH4+-fed plants (7.6- and 30.6-fold, respectively) compared with NO3–-fed plants (Fig. 2A; Supplementary Fig. S2). Furthermore, the contents of Arg and Orn, pivotal N compounds within the urea cycle and precursors of PAs, increased remarkably in shoots (8.2- and 3.9-fold, respectively) and roots (28.4- and 3.6-fold, respectively) of plants subjected to high NH4+ nutrition relative to plants grown in the presence of 1 mM NO3– (Fig. 2B, C). By contrast, NH4+ treatment at both concentrations caused a 30% decrease in the content of the non-protein amino acid GABA in shoots, but not in roots (Fig. 2D).

Fig. 2.

Effect of different sources and concentrations of N nutrition on the urea-cycle-related amino acids Gln (A), Arg (B), Orn (C), and GABA (D) in shoots and roots of 14-day-old M. truncatula seedlings. Data represent means ±SE values (n=4). Different letters denote statistically significant differences at P<0.05.

Plants grown under 25 mM NH4+ showed 9.2- and 2.1-fold larger contents of Put (Fig. 3A) and Spd (Fig. 3B) in shoots, respectively, compared with 25 mM NO3-fed plants. Similarly, roots of 1 mM and 25 mM NH4+-fed plants showed significantly higher Put content than NO3–-fed plants (7.6- and 87.2-fold, respectively) (Fig. 3A). No significant differences in Spm content were observed between NO3–- and NH4+-fed plants (Fig. 3C).

Fig. 3.

Effect of different sources and concentrations of N nutrition on the content of the polyamines Put (A), Spd (B), and Spm (C) in shoots and roots of 14-day-old M. truncatula seedlings. Data represent means ±SE values (n=3). Different letters denote statistically significant differences at P<0.05.

NH4+ nutrition decreased ADC and CuAO activities, but induced ODC activity

In roots, NH4+-fed plants showed a decrease in ADC activity at both NH4+ concentrations, relative to NO3–-fed plants (Fig. 4A). By contrast, ODC activity increased in both 1 mM and 25 mM NH4+-fed plants, respectively (Fig. 4B). No significant differences in the activity of either enzyme were observed in shoots (Fig. 4A, B). The Put-dependent AO activity was significantly lower by 57% in shoots and 53% in roots of 25 mM NH4+-fed plants compared with 25 mM NO3–-fed plants (Fig. 5A). Spd-dependent PAO activity was also lower in shoots of 25 mM NH4+-fed plants in comparison to 25 mM NO3–-fed plants, whereas no significant difference between the two treatments was observed in roots (Fig. 5B). Spm-dependent PAO activity did not vary significantly between tissues or treatments (Fig. 5C).

Fig. 4.

Effect of different sources and concentrations of N nutrition on ADC (A) and ODC (B) enzymatic activity in shoots and roots of 14-day-old M. truncatula seedlings. The enzymatic activity is expressed in enzyme units min–1 mg–1 protein. Arg was used as substrate in the ADC assay, while Orn was used as the substrate for ODC activity. Data represent means ±SE values (n=3–4). Different letters denote statistically significant differences at P<0.05.

Fig. 5.

Effect of different sources and concentrations of N nutrition on Put-dependent CuAO (A), Spd-dependent PAO (B), and Spm-dependent PAO (C) enzymatic activity in shoots and roots of 14-day-old M. truncatula seedlings. The enzymatic activity is expressed in enzyme units min–1 mg–1 protein. Put was used as the substrate in the CuAO assay (A), while Spd (B) or Spm (C) were used as substrates for PAO activity. Data represent means ±SE values (n=3). Different letters denote statistically significant differences at P<0.05.

To test whether NH4+ may act as a feedback regulator of Put oxidation, we analyzed in vitro the AO activity in the presence of NH4+. The inhibition studies with NH4+ evidenced that CuAO activity was competitively affected when the NH4+ concentration was <50 mM, since Vmax remained unchanged while the Km increased. The measured Ki for NH4+ was 44.6 mM in shoots (Fig. 6A, B) and 25.7 mM in roots (Fig. 6C, D). From the fresh and dry weight data we could estimate a water content of 90% for shoots and 94% for roots. The internal concentration of NH4+ in the root ranged around 25 mM (Fig. 1), which was also in the range of the Ki measured for NH4+ in roots. These results evidenced that CuAO ­activities were inhibited by the increase of internal NH4+ content, especially in 25 mM NH4+-fed plants.

Fig. 6.

Inhibitor constant (Ki) of NH4+ on CuAO activity in extracts of M. truncatula shoots (A, B) and roots (C, D) grown under 1 mM NO3– for 14 days. Michaelis–Menten plots were obtained using Put as the substrate and NH4+ as an inhibitor (A, C). Maximum rate (Vmax) and Michaelis constant (Km) are plotted against the concentration of inhibitor from 0 to 100 mM NH4+ (B, D). Data represent means ±SE values (n=3).

The H2O2 content was measured since H2O2 is one of the AO reaction products. Both shoots and roots of M. truncatula plants exhibited a significant increase in H2O2 content under conditions of high N, with the greatest increase being observed in plants grown in the presence of 25 mM NH4+ (Fig. 7). These results contrasted the reduction of AO activity under conditions of high NH4+ (Supplementary Fig. S3). Thus, it is possible that other H2O2-producing enzymes, different from CuAOs and PAOs, contribute to the increase of H2O2 content under high NH4+ conditions.

Fig. 7.

Effect of different sources and concentrations of N nutrition on H2O2 content in shoots and roots of 14-day-old M. truncatula seedlings. Data represent means ±SE values (n=3). Different letters denote statistically significant differences at P<0.05.

Taken together, these results demonstrated that NH4+ nutrition at a high dose had important effects on the content of many of the urea cycle–PA metabolism intermediates (i.e. Gln, Arg, Orn, and Put) as well as the activities of the enzymes (i.e. ADC, ODC, and CuAO), in comparison with plants grown under a high dose of NO3– (Fig. 8).

Fig. 8.

The urea cycle and PA metabolism in plants. Enzymes are represented in bold text, and metabolites in regular text (after Esteban ). Changes, when comparing NH4+ versus NO3– nutrition, are depicted in green when either metabolite content or enzymatic activity decreased, and in red when they increased. Shoot and root tissues are represented by the upper and lower squares, respectively. Illustration created with BioRender 2022.

NH4+ nutrition modulated the transcript levels of genes involved in the urea cycle and polyamine metabolism in M. truncatula plants

In both shoots and roots, 1 mM and 25 mM NH4+ nutrition increased the transcript levels of the urea cycle genes MtOTC2, MtAS2, and MtAL, in comparison to NO3– nutrition at the respective doses. Moreover, transcript levels of MtARG were diminished by NH4+ treatment at a low dose but increased by 25 mM NH4+ nutrition compared with NO3–. No changes were observed in the expression level of MtURE. Transcript levels of the genes MtADC1, MtADC2, and MtODC, involved in Put biosynthesis, were strongly increased in plants grown under NH4+ conditions relative to NO3–-fed plants in both shoot and root tissues (Fig. 9A).

Fig. 9.

Effect of different concentrations of NH4+ nutrition on the transcript levels of the genes involved in the urea cycle and Put biosynthesis (A) and PA catabolism (B) assayed from RNA samples of 14-day-old M. truncatula seedlings (n=3–4). Data were calculated relative to transcript levels in seedlings exposed to 1 mM and 25 mM NO3– treatments, respectively.

Regarding genes involved in PA catabolism, in the M. truncatula genome seven CuAO genes have been identified in in silico analyses. An interspecific phylogenetic tree generated from predicted amino acid sequences indicated that MtCuAO1, MtCuAO2, and MtCuAO7 belonged to Clade I (Tavladoraki ) together with AtCuAOα1, AtCuAOα3, AtCuAOβ, LcAO, and PsAO. MtCuAO3 was grouped in Clade II together with AtCuAOγ1 and MdAO2. MtCuAO4, MtCuAO5, and MtCuAO6 belonged to Clade III together with AtCuAOζ and MdAO1 (Fig. 10A, B).

Fig. 10.

Interspecific phylogenetic tree of M. truncatula copper amine oxidases and polyamine oxidases. Amino acid sequences were aligned with the MAFFT 7 E-INS-I algorithm. The phylogenetic tree was performed by using the neighbor-joining method and the statistical bootstrap test with 1000 replications. The obtained bootstrap values are indicated at the nodes (A). The predicted substrate preference, involvement in back-conversion (BC) or terminal catabolism (TC), subcellular localization, and function of each enzyme based on the already characterized orthologous genes, and the prediction made by the Gene Ontology Annotation database (B).

Four PAO genes were present. In particular, MtPAO1 belonged to Clade I of the PAO phylogenetic tree (Salvi and Tavladoraki, 2020) along with AtPAO1, OsPAO6, and OsPAO7. MtPAO4 was grouped in Clade II together with AtPAO5 and OsPAO1. MtPAO2 and MtPAO3 belonged to Clade III along with AtPAO2, AtPAO3, AtPAO4, OsPAO3, OsPAO4 and OsPAO5 (Fig. 10A, 10B).

Transcript levels of MtCuAO2 and MtCuAO7 were lower in shoots of 25 mM NH4+-fed plants compared with 25 mM NO3–-fed plants. In contrast, MtCuAO1 and MtPAO3 transcript levels were higher in these plants. In roots, transcript ­levels of CuAOs were slightly lower in 25 mM NH4+-fed plants, except for MtCuAO3 and MtCuAO5, which were higher in comparison to plants grown under 25 mM NO3–; the same pattern was also observed for MtPAO4 (Fig. 9B).

Exogenous supplementation with Put improved biomass accumulation under NH4+ nutrition

The growth of M. truncatula plants in the presence of NH4+ appeared strongly reduced compared with the growth in the presence of NO3–. Indeed, biomass production in NH4+-fed plants was lower than that in NO3–-fed plants. At low dose, NH4+-fed plants had 34% and 29% lower shoot (Fig. 11A) and root (Fig. 11B) dry biomass, respectively, than NO3–-fed plants. At high dose, the decrease in the dry biomass of NH4+-fed plants relative to NO3–-fed plants was 40% in shoots and 51% in roots. The addition of Put to the NH4+-containing medium alleviated the reduction in plant growth (Fig. 11C), whereas it did not affect plant growth when added to the NO3–-containing nutrient solution. The effect of Put in plants grown in NH4+-containing medium was observed at both shoot and root levels, as shown by measuring shoot and root dry weight (Fig. 11). Adding Put to the growth medium induced a similar effect on plant biomass than that of 1 mM NO3– supplementation in the nutrient solution. As Put at 0.5 mM represented an additional 1 mM of N supplemented to the plant, we included a 2 mM NH4+ control. The Put treatment differed from both the 1 mM and 2 mM NH4+ controls, so the positive effect of Put was not due to an increase in N fertilization (Fig. 11; Supplementary Fig. S4).

Fig. 11.

Effect of different sources and concentrations of N nutrition on the dry biomass of 14-day-old M. truncatula seedlings. (A, B) Distribution of plant biomass of shoots (A) and roots (B) expressed as dry weight (DW) per plant subjected to different N treatments and supplemented with Put. Data represent means ±SE values (n=8). Different letters denote statistically significant differences at P<0.05. Representative image of NH4+-fed plants (C). Scale bar=1 cm.

Discussion

The accumulation of urea cycle intermediates and Put in M. truncatula plants under NH4+ nutrition relates to cation homeostasis and C/N regulation

The accumulation of NH4+ is considered to be the main biomarker of NH4+ toxicity (González-Moro ), observed in pea (Ariz ) lettuce, spinach, lupine (Cruz ), tomato (Vega-Mas ), wheat (Vega-Mas ), A. thaliana (Sarasketa ), and also for M. truncatula plants subjected to high NH4+ conditions compared with NO3–-fed plants (Fig. 1). Even though NH4+-fed M. truncatula plants showed the ionic imbalance previously recognized as the main cause of NH4+ toxicity (Britto and Kronzucker, 2002; Esteban ), they were able to counterbalance K+ homeostasis under NH4+ stress conditions in both shoots and roots. Hence, the maintenance of K+ homeostasis may be linked to a greater tolerance towards NH4+ stress (Zhang ), as previously observed in pea plants, which reverse the NH4+ toxicity when transferred to high-irradiance conditions (Ariz ). Pea plants grown under high NH4+ and high irradiance received a provision of extra C, triggering an ameliorated response to NH4+ stress by decreasing the Arg pool and increasing Put content, compared with low-irradiance-treated plants (Ariz ). Thus, the accumulation of Arg in high-NH4+-fed M. truncatula plants (Fig. 2) may be a response to limited C availability, as observed in plants grown under low irradiance (Ariz ). However, M. truncatula plants showed the increase in Put content (Fig. 3) that was previously associated with the tolerance response to NH4+ in high-irradiance-treated plants (Ariz ).

The channeling of surplus N into amino acids (Supplementary Fig. S1), particularly the urea-cycle metabolites with low C/N ratio such as Gln, Arg, Orn, and Put, in shoots and roots of M. truncatula seedlings grown in high NH4+ (Fig. 2; Supplementary Fig. S2) reveals a mechanism of N redistribution to cope with high-NH4+ conditions. The sequestration of NH4+ excess into low C/N molecules such as Arg, which is the amino acid with the lowest C/N ratio and a major Put precursor, helps plant cells to maintain the endogenous NH4+ content below toxic levels (Ueda ). In addition, the availability of C provided by several anaplerotic reactions is also considered critical in legumes (Ariz ) and cereal plants (Vega-Mas ). Therefore, there is evidence that the urea cycle is a likely C/N regulatory control point under NH4+ stress as it represents an NH4+ sink, as well as a connection between C and N metabolism (Esteban ).

Enhanced tolerance to abiotic stress is usually accompanied by increased contents of PAs in conditions such as salinity, drought, and low/high temperature (Alcázar ; Wang ), and also under NH4+ stress (Belastegui-Macadam ). Unlike other stress conditions, where Spd and/or Spm have been reported to primarily accumulate ­(Alcázar ; Wang ), our data indicate that high NH4+ content induced an increase in Put and, to a lesser extent, Spd, but it did not affect the Spm content in M. truncatula roots (Fig. 3). This observation is consistent with the recently proposed adaptive role that Put may play under K+ deficiency conditions (Cui ), by controlling ion channels and H+-ATPases (Pottosin ), and it may well explain previous results of studies of plants subjected to NH4+ stress. Thus, it was described that high irradiance induced the synthesis of Put in high-NH4+-fed pea plants (Ariz ), which maintained or even improved the internal K+ content in both shoots and roots (Ariz ).

The role of the urea cycle in connection to PA metabolism

Put has been revealed as an essential metabolite in plant response to stress conditions (Cui ). Indeed, the activities of enzymes involved in either Put biosynthesis (Fig. 4) or Put catabolism (Figs 5, 6) were affected by NH4+. In other types of abiotic stress, Put accumulation resulted from enhanced ADC expression (Alcázar ). Regarding the synthesis of Put in NH4+-fed plants, our data showed a switch in the functioning of the urea cycle in roots of NH4+-fed from the ADC to the ODC pathway (Fig. 4). It seems that this switch to ODC activity is a characteristic of NH4+ stress in legumes, as the increase in Put in soybean seedlings under NH4+ has been also reported using radiolabeled Orn (Le Rudulier and Goas, 1977). Indeed, González-Hernández recently suggested a role for the ODC pathway in the NH4+ tolerance response of tomato plants, since plants overexpressing ODC showed improved growth parameters. Interestingly, the NH4+-sensitive A. thaliana does not possess its own ODC gene (Hanfrey ), which suggests that the ODC enzyme may provide greater plasticity to plants to cope with NH4+ stress.

Regarding the gene expression levels, except for MtCPSII1 in roots, all the urea-cycle genes were induced during high-NH4+ conditions in both shoots and roots (Fig. 9). The remarkable increase in transcript levels of MtADC1, MtADC2, and MtODC in shoots, and MtADC2 and MtODC in roots, highlighted the relevance of the interconnection between the urea cycle and PA metabolism. The strong increase in ADC transcript levels in M. truncatula plants grown under NH4+ nutrition contrasted with the decrease in ADC activity. This suggests the existence of post-transcriptional regulatory mechanisms as the described regulation of the ADC enzymes by upstream open reading frames (Jiménez-Bremont ). However, we cannot exclude that the reaction catalyzed by ADC was affected by the following AIH and NCPAH activities, as both release NH4+. In any case, the importance of the pathway has also been reflected in ODC-silencing tomato plants under NH4+ nutrition, which required compensation by ADC induction. Furthermore, both ADC-silencing and ODC-overexpressing mutants showed amelioration of NH4+ toxicity syndrome (González-Hernández ).

Regarding PA catabolism, the lower total activities of the CuAOs confirmed the deceleration of the Put catabolic reactions at a high NH4+ dose (Fig. 5), and evidenced the existence of a feedback inhibitory mechanism of CuAO by NH4+ (Fig. 6), as proposed in Esteban . Accordingly, exogenous GABA has been shown to alleviate hypoxia (Wang ) and drought damage (Yong ) by inducing PA accumulation as well as preventing PA degradation, which supports a feedback regulation of CuAO activity. Therefore, the increase of Put in high-NH4+-fed plants may be attributed to an increase in ODC activity and a reduction in CuAO activity as the cellular NH4+ content increases (Fig. 8).

In contrast to the urea-cycle genes, the transcript levels of the PA-catabolism genes showed a general decrease, except for MtCuAO1 and MtPAO3 in shoots, and MtCuAO3 and MtPAO4 in roots (Fig. 9). In accordance with the reduced CuAO activity observed in 25 mM NH4+-fed plants, the transcriptional analysis showed that high NH4+ conditions decreased the predicted apoplastic MtCuAO2 and MtCuAO7 in shoots, while the predicted apoplastic MtCuAO3 was up-regulated in roots of NH4+-fed plants in comparison to plants grown under NO3– nutrition. It is possible that MtCuAO3 and MtPAO4 are involved in processes unrelated to the urea cycle–PA metabolism connection (Fig. 10). Indeed, in A. thaliana, knockout of the MtCuAO3 orthologous gene AtCuAOγ1 contributes to both PA-induced H2O2 production and nitric oxide biosynthesis involved in the abscisic acid signal transduction pathway (Wimalasekera ). Furthermore, the MtPAO4 orthologous gene in A. thaliana AtPAO5 participates in plant development and xylem differentiation, interfering with the auxin/cytokinin interplay (Alabdallah ).

The activities of AOs could contribute to the reported increase in reactive oxygen species in plants grown under high NH4+ (Yang ) through the production of H2O2 (Gupta ). However, M. truncatula plants treated with higher doses of N showed increases in H2O2 content (Fig. 7), which correlated inversely with the activities of CuAO in both shoots and roots, and with PAO in shoots (Supplementary Fig. S3). Thus, there must exist mechanisms of H2O2 production other than AOs under NH4+ toxicity conditions.

The alleviating effect of Put on M. truncatula biomass under NH4+ nutrition

The internal content of Put has been correlated with the reduction in growth of wheat and pepper plants grown under NH4+ nutrition (Houdusse ), which we also observed in M. truncatula plants grown in axenic conditions (Fig. 11; Supplementary Fig. S4). Conversely, in this work we show a remarkable alleviation of NH4+ toxicity symptoms in M. truncatula seedlings when we applied Put early. This effect was similar to the addition of NO3– in terms of dry biomass (Houdusse ), which was considered a signaling effect (Hachiya ). This observation is in agreement with the results of studies of other stresses, where exogenous Put mitigated cadmium (Zhu ) and aluminum (Zhu ) toxicity in rice, iron deficiency in A. thaliana (Zhu ), and salinity stress in cucumber (Shu ; Yuan , 2019), and also improved drought tolerance in wheat (Doneva ). The alleviation by Put of the growth reduction was especially evident during high-stress conditions at 25 mM NH4+. These results open up the question of how Put mitigates NH4+ stress, but they also reveal that the common role of Put in the alleviation of stresses works also for NH4+ toxicity. Furthermore, this function involves a metabolic connection to the urea cycle, highlighting the importance of this route in M. truncatula.

Conclusions

Few studies have dealt with the urea cycle as it has been considered to be incomplete in plants. Our results evidenced that high-NH4+ conditions increased the transcript levels of genes involved in the urea cycle and the content of intermediates of this cycle. The low C/N ratio intermediates Gln, Arg, Orn, and Put accumulated, evidencing a C limitation under high-NH4+ conditions. Furthermore, high NH4+ content altered PA metabolism, leading to a high accumulation of Put, which helps the plant to tolerate the stress. The increase of Put content may be attributed to an increase in ODC activity and a reduction in CuAO activity, probably through an inhibitory effect of NH4+. Finally, this study shows that exogenously supplied Put leads to alleviation of the growth reduction provoked by NH4+. The remarkable changes in both the urea cycle and the PA pathway during NH4+ toxicity provide a clue to its importance as a protection mechanism and as a regulator of C/N metabolism.

Supplementary data

The following supplementary data are available at JXB online.

Table S1. Primers designed for amplification of urea cycle and PA metabolism genes of M. truncatula by RT–qPCR.

Table S2. Plant CuAOs and PAOs used for the phylogenetic analysis.

Fig. S1. Effect of different N nutrition on the total amino acid and soluble protein contents.

Fig. S2. Effect of different N nutrition on the amino acid content in shoots and roots of M. truncatula seedlings.

Fig. S3. Pearson correlation of the analyzed urea cycle and PA metabolism components.

Fig. S4. Representative image of plants subjected to different N nutrition and supplemented with 0.5 mM Put.

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