Rice varieties with contrasting nitrogen use efficiency present different expression of amino acid transporters and ammonium transporters

ABSTRACT We evaluated the effects of isoforms of ammonium transporters (AMT) and amino acid transporters (AAT) on three rice varieties cultivated with different levels of ammonium (NH4 +) and its biochemical responses. The rice varieties used were IAC-47 (improved), Manteiga and Piauí (landraces), cultivated under 0.2 mM NH4 + (low N, LN), 1 mM NH4 + (sufficient N, SN) or resupply with 1 mM of NH4 + after three days without nitrogen (N). When cultivated with LN, the Piauí variety presented a higher expression of AAT (Lysine-Histidine Transporter 4 (OsLHT4) and GABA Transporters (OsGAT1)) in roots, while Manteiga and IAC-47 presented higher expression of the Amino Acid Permease 1 (OsAAP1) and Ammonium Transporter 1.1 (OsAMT1.1), respectively. Additionally, Piauí had the higher expression of glutamine synthetase (OsGS1.1 and OsGS1.3) in shoots cultivated with LN or resupply. The Piauí variety displayed higher glutamine synthetase activity and amino acid content under resupply. Manteiga and Piauí presented a lower reduction in fresh weight of leaves and sheaths under LN compared to IAC-47. The results suggest that the improved variety IAC-47 is more efficient when cultivated with SN, while, under LN or resupply, the landrace varieties were superior. Overall, the molecular responses were different among the varieties, which may affected their nitrogen metabolism.


Introduction
The global population is increasing with projections of an additional 1 billion people in 12 years (Prinsi et al. 2020). Thus, the biggest agricultural challenge will be to provide enough food for the growing population and increase production (Coulter 2020). In the last few decades, agricultural production was associated with the increased cultivated area, fertilizer consumption and environmental damage (Alexandratos and Bruinsma 2012). High rates of fertilizers containing nitrogen (N-fertilizer) can promote pollution in superficial and underground water (Wang et al. 2011). The application of N fertilizers increased after the 'green revolution', demanding around 1% of the current global energy production to meet the demand for N fertilizers. Nitrogen (N) is a highly required essential nutrient and is a constituent of important cellular molecules, such as amino acids, proteins, nucleic acids, ATP, NAD(P)H, chlorophyll, hormones and secondary metabolites (Souza and Fernandes 2018). Although N is highly used as fertilizers (Fageria and Baligar 2005), only about 35% of it is absorbed and used by plants (Omara et al. 2019). The nonabsorbed fraction of N fertilizer can cause environmental damage and lower profits.
Tropical regions in Brazil are remarked by low soil organic matter (SOM) content and fast SOM mineralization, demanding higher application of N fertilizer. Ammonium (NH 4 + ) is a cationic form of N and can be adsorbed by the soil negative charges (cation exchange capacity), reducing leaching, compared to the anionic nitrate (NO 3 − ) (Amberger 1993). Otherwise, rice (Oryza sativa) is an NH 4 +tolerant plant (Kronzucker et al. 1999).
Root NH 4 + uptake is mediated by ammonium transporters family (AMTs) (Sonoda et al. 2003) and assimilated into amino acids by glutamine synthetase (GS) and glutamine-2-oxoglutaric amino transferase (GOGAT) (Xu et al. 2012). Glutamate dehydrogenase (GDH) may catalyze NH 4 + assimilation; however, GDH is important in the reverse reaction (deamination of glutamate), important in leaf senescence (Boggio et al. 2000). Plant amino acids distribution and remobilization are mediated by amino acid transporters (AATs) (Zhao et al. 2012). The AAT is a multigene family transporting several amino acids with distinct affinity, specificity and expression (Lu et al. 2012). Zhao et al. (2012) demonstrated in rice that OsAPP15 is highly expressed in leaves; meanwhile, OsLHT4, OsANT4, OsGAT1, OsGAT4 and OsAAP1 are mostly expressed in roots. The tissue-specific expression highlights the importance of different AAT isoforms to rice growth and production. Rice overexpression of OsAAP1 increased growth and grain yield (Ji et al. 2020).
Rice is an important staple food worldwide, and Brazil is the largest grain producer outside Asia (Gadal et al. 2019). Rice production in Brazil is divided into flooded soils (88.2%) and upland soils (11.2%) (Embrapa Arroz e Feijão 2017). Rice production in upland areas is performed mainly by small farmers, and NO 3 − is the predominant N form in aerated soils (Girsang et al. 2020). However, landrace varieties cultivated in upland soil in the Maranhão state (Brazil) absorb NH 4 + present in the soils after intense raining and SOM decomposition (Fernandes 2014). The seasonal flush of NH 4 + requires efficient uptake and affects plant growth and production (Fernandes 2014). The landrace varieties were selected by small farmers and possess higher nitrogen use efficiency (NUE) (Junior et al. 1997;Dos Santos et al. 2003;de Sampaio et al. 2004;Santos et al. 2009;Coelho et al. 2016). In addition, landrace varieties present a high grain protein content (10% for Manteiga and 11% for Piauí) (Ferraz et al. 2001).
Since expression levels of AMT and ATT may affect nitrogen metabolism and plant growth at distinct NH 4 + levels, the objective of this work is to evaluate if the landrace varieties cultivated with low N input in the Maranhão state (Brazil), Manteiga and Piauí, present distinct phenotypic, physiological and molecular responses compared to the IAC-47 variety (improved) when cultivated with low and high NH 4 + levels.

Plant growth conditions and treatments
Plant growth was performed using a growth chamber with the following settings: light cycle 14 h/ 10 h (light/dark), photosynthetic photon flux of 400 µmoles m −2 s −1 , relative humidity of 70% and temperature of 28/24°C (day/night). Three rice varieties (Oryza sativa) were used in the experiment: Manteiga and Piauí (landraces from Maranhão state-Brazil) and IAC-47 (improved variety to high N levels). The experimental set-up was a completely randomized design with three varieties, three treatments and four repetitions. The rice seeds were disinfected using 2% hypochlorite solution and germinated in distilled water using gauze to prevent seed immersion. Ten days after germination (DAG), the seedlings were transferred to pots (0.7 L) containing four plants per pot and modified ½ total ionic strength Yoshida solution (Yoshida et al. 1976) with 0.2 mM NH 4 + and pH 6.0. Teen DAG the pots received total ionic strength Yoshida solution (10 mg L −1 phosphor, 40 mg L −1 potassium, 40 mg L −1 calcium, 40 mg L −1 magnesium, 2 mg L −1 iron, 0.5 mg L −1 manganese, 0.01 mg L −1 zinc, 0.01 mg L −1 copper, 0.2 mg L −1 boron and 0.05 mg L −1 molybdenum) containing three NH 4 + treatments: T1 -constant supply of 0.2 mM NH 4 + , T2constant supply of 1 mM NH 4 + and T3 -resupply of 1 mM NH 4 + at 30 DAG after three days without N (constant supply of 1 mM NH 4 + for 17 days). The solution was refreshed every three days. At 30 DAG, five hours after the last solution change, plants were harvested and divided into root, sheaths and leaf. After weighting the fresh biomass, samples were collected for total RNA extraction, protein extraction and soluble metabolic (NH 4 + , free-amino N and soluble sugars). A detailed scheme depicting the experiment is shown in Figure 1.

Total RNA extraction and gene expression analysis
The total RNA was extracted according to Gao et al. (2001) with modifications (Sperandio et al. 2011) using NTES buffer (0.2 mM Tris-HCl pH 8.0; 25 mM EDTA pH 8.0; 0.3 mM NaCl; 2% SDS) and quantified using Nanodrop (Thermo Scientific). The quality was verified by A260/A230 and A260/A280 ratio and gel electrophoresis (agarose 1%). Total RNA was treated with DNase I (Life Technologies) according to the manufacturer's instructions. The cDNA synthesis was performed with 'TaqMan Reverse Transcription Reagents' (Applied Biosystems, Inc.) and oligo dT primer according to the manufacturer's instructions.
Real-time PCR reactions were performed in duplicates using a 'SYBR Green PCR Master Mix' kit (Applied Biosystems, Inc.) according to the manufacturer's instructions. The genes OsAct1 (LOC_Os03 g50885.1) and OsEF-1α (LOC_Os03 g08020.1) (Jain et al. 2006) were used as endogenous control. The primers were design using NCBI tool Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/ primer-blast/) (Supplement Table S1). Primer specificity was determined experimentally at the end of the Real Time PCR (melting curve). All primers presented high specificity to the target gene. Relative gene expression was calculated according to Livak and Schmittgen (2001).

Protein extraction and quantification
After the experiment harvest, 0.5 g of root and leaf was grounded with liquid nitrogen for 3 min using mortar and pestle followed by the addition of extraction buffer (1 M Tris-HCl pH 8.0; 0.5 M EDTA pH 8.0; 1.5 g of polyvinylpolypyrrolidone, 0.154 g of dithiothreitol, and 0.2 M phenylmethyl sulphonyl fluoride). The samples were centrifuged at 14.000 × g for 30 min (4°C), and the supernatants were collected. The protein content was determined according to Bradford (1976) and stored at −80°C.

Glutamine synthetase and glutamate dehydrogenase activity
The glutamine synthetase (GS) was determined according to Farnden and Robertson (1980). γ-Glutamyl hydroxamate (GHD) was determined measuring the absorbance in 540 nm.
The glutamate dehydrogenase (GDH) activity was determined according to Turano et al. (1996). The GDH amination activity was performed using 500 µL of solution with 100 mM Tris-HCl (pH 8.0), 50 mM (NH 4 ) 2 SO 4 , 13 mM alpha-ketoglutarate, 0.25 mM NADH, 1 mM CaCl 2 and 50 µg of protein. The reaction was started by adding protein, and the activity was determined by the rate of decrease in absorbance measured at 340 nm. The GDH deamination activity was performed using 500 µL of solution with 100 mM Tris-HCl (pH 9.3), 35 mM L-glutamate, 0.25 mM NAD + , 1 mM CaCl 2 , and 50 µg of protein. The reaction was started by adding protein, and the activity was determined by the rate of increase in absorbance measured at 340 nm.

Analysis of NH 4 + , free amino-N and soluble sugar content in plant tissues
Samples of root, sheath and leaves (0.5 g) were grounded using ethanol 80%, and chloroform was used for fractionation (Fernandes 1984). The soluble fraction was used for NH 4 + (Felker 1977), free amino-N (Yemm et al. 1955) and soluble sugar content determination (Yemm and Willis 1957).

Statistical analysis
The experiment was in a completely randomized design with four (4) repetitions. The data were submitted to normality and homogeneity and then to the F test (p < 0.05). The averages were compared by the Tukey test (p < 0.05). The data were ordered by Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA). The data analysis was performed using R software (R Core Team 2019).

Real-time PCR analysis of genes related to uptake and assimilation of NH 4 + and amino acid flux
To evaluate the differences among the rice varieties in gene expression related to N metabolism, the following genes were analyzed: OsAMT1.1 and OsAMT1.3 (NH 4 + uptake and signaling, Tabuchi  The IAC-47 variety presented higher expression of OsAMT1.1 in roots in both treatments analyzed (Figure 2(a)), as well as higher expression of OsAMT1.3 with resupply ( Figure 2(b)). IAC-47 roots also presented a higher expression of OsAAP1, OsGAT1 and OsGAT4 under resupply (Figure 2(c,e,f)). On the other hand, Piauí roots cultivated with constant levels of NH 4 + (0.2 mM) presented a higher expression of amino acid transporters (Figure 2(d-f)), except for OsAAP1 with higher expression in the Manteiga variety (Figure 2(c)). The Hierarchical Cluster Analysis (HCA) indicates the Piauí gene expression cluster separate from IAC-47 and Manteiga when cultivated with constant NH 4 + supply (Figure 2(g)). Meanwhile, plants cultivated with NH 4 + resupply presented the IAC-47 cluster separately from Piauí and Manteiga (Figure 2(h)).
The Piauí variety cultivated with constant NH 4 + supply presented a higher expression of genes related to N assimilation and amino acid transporters (Figure 3(a,b,f)), highlighting OsANT4 (Figure 3  (b)). Under NH 4 + resupply, Piauí also presented a higher expression of all analyzed genes (Figure 3(a, b,c,e,f)), except OsGS1.3 with a higher expression in IAC-47 ( Figure 3(d)). The higher expression of genes related to N assimilation and amino acid transport was further evidenced by HCA analysis (Figure 3(g,h)). Altogether, the gene expression analysis suggests that IAC-47 (improved variety) presented a higher expression of genes related to NH 4 + uptake. The landrace varieties presented a higher expression of genes related to NH 4 + assimilation and amino acid transport.

Glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activity in rice root and leaves
Roots presented higher GS activity (Figure 4(a-c)) and GDH (amination and deamination) compared to leaves ( Figure 5). The GS activity in plants cultivated with 0.2 mM NH 4 + did not differ statistically (Figure 4(a,d)). On the other hand, Piauí presented higher GS activity in roots and leaves when cultivated with 1 mM NH 4 + and NH 4 + resupply (Figure 4). Roots of the Piauí variety presented higher GDH amination activity when cultivated with 0.2 mM NH 4 + and NH 4 + resupply ( Figure 5). Roots of Piauí also presented higher GDH deamination when cultivated with NH 4 + resupply.

Relative expression
Relative expression

Plants' fresh weight and root/shoot ratio
Plants cultivated with 0.2 mM NH 4 + presented a lower fresh weight, and plants cultivated with 1 mM NH 4 + a higher fresh weight (Table 1). IAC-47 presented a higher fresh weight compared to Manteiga and Piauí. NH 4 + resupply leads to a higher R/S ratio in IAC-47, Piauí and Manteiga. Manteiga presented a higher R/S ratio compared to IAC-47 and Piauí when cultivated with constant NH 4 + supply and NH 4 + starvation. Overall, the results highlight the higher biomass production in IAC-47 and higher R/S in Manteiga.

NH 4 + , amino-N and soluble sugar content in plant tissues
The IAC-47 variety showed a higher NH 4 + content in roots compared to Manteiga and Piauí (Figure 6  (a,b)). However, Manteiga and Piauí had higher NH 4 + levels in leaves compared to IAC-47 under NH 4 + resupply ( Figure 6(c)). Under constant 1 mM NH 4 + supply and NH 4 + resupply, the Piauí variety showed a higher NH 4 + content (Figure 6(b,c)). NH 4 + supply affected the soluble sugar content in roots of IAC-47 cultivated with constant NH 4 + supply and leaves of Manteiga cultivated with NH 4 + resupply (Figure 6(d-f)).

Principal component analysis
The principal component analysis (PCA) highlighted the distinct response among the varieties at different NH 4 + levels (Figure 7). IAC-47 cultivated with 0.2 mM NH 4 + was related to root parameters (fresh weight, soluble sugar, amino-N and NH 4 + levels) (Figure 7(a) and (b)). On the other hand, Manteiga was related to leaves parameters (amino-N, soluble sugar and NH 4 + levels) and Piauí was related to sheaths parameter (amino-N).
IAC-47 plants cultivated to 1 mM NH 4 + had parameters related to the plant fresh weight and NH 4 + (roots) (Figure 7(c) and (d)). Manteiga was related to leaves parameters (sugar, NH 4 + , amino-N), and Piauí was related to sheaths parameters (NH 4 + and amino-N). The resupply with 1 mM NH 4 + grouped the IAC-47 parameters into the plant fresh weight and soluble sugars (Figure 7(e) and (f)). The levels of NH 4 + (roots and leaves) and soluble sugars (sheaths and leaves) were related to Manteiga. Piauí was related to amino-N (root and sheaths) and NH 4 + (sheaths). Overall, the PCA showed the IAC-47 fresh weight in response to differential NH 4 + supply, while the Landrace varieties demonstrated responses in amino-N levels (Piauí) and NH 4 + and soluble sugars (Manteiga).

Discussion
Plant molecular responses to NH 4 + are essential to enhance uptake and amino acids translocation in plant-growing areas (Zhang et al. 2015). Additionally, NH 4 + is an important N source for rice growth and production since this crop is grown in flooded soils where NH 4 + is the N main form (Hao et al. 2016). Rice is also cultivated in upland soils, and NH 4 + may be present in considerable concentration (Fernandes 2014). The molecular responses to NH 4 + are variable among rice varieties, and the investigation of gene expression to different NH 4 + levels may provide essential information to rice breeding programs. In this study, we showed that improved and landrace rice varieties presented differential molecular responses to NH 4 + levels, as well as biochemical and morphological responses. Root NH 4 + uptake is mediated by the AMT family (ammonium transporters) located in the plasma membrane (Li et al. 2017). The high affinity system operates when NH 4 + levels are low (< 1 mM), and the low affinity system when NH 4 + levels are high (> 1 mM) (Souza and Fernandes 2018). Rice OsAMT1.1 is constitutively expressed in roots and shoot, and it is important in soil with high NH 4 + levels (Souza and Fernandes 2018). Rice OsAMT1.3 is specific to roots and induced by low NH 4 + levels (Sperandio et al. 2011;Ferreira et al. 2015). In the present study, the IAC-47 variety showed a higher expression of OsAMT1.1 and OsAMT1.3 in roots regardless of NH 4 + levels compared to Piauí and Manteiga varieties (Figure 2(a,b)) and this may be an important trait to NH 4 + uptake in soils with a low or variable concentration of NH 4 + . Rice overexpressing OsAMT1.1 presented improved growth and grain yield, suggesting that OsAMT1.1 is important for NH 4 + uptake in low levels (Ranathunge et al. 2014). The OsAMT1.3 expression in Xenopus oocytes demonstrated that OsAMT1.3 has high NH 4 + affinity (five times more than OsAMT1.1) and may be important to adapt to variable NH 4 + levels (Hao et al. 2016). The IAC-47 variety also presented increased plant growth (Table 1), indicating that IAC-47 was efficient to use NH 4 + for biomass production. The Manteiga variety presented a higher root/shoot (R/S) ratio compared to IAC-47 and Piauí (Table 1), which may be an adaptive response to N levels. Higher plasticity of root growth increases the ability to adapt to abiotic stresses, such as nutritional stress (Des Marais et al. 2013;Pereira et al. 2021).
The main pathway to NH 4 + assimilation to form amino acids involves GS/GOGAT enzymes (Lewis et al. 1983;Taiz and Zeiger 2017). The GS1 isoform located in cytosol is important for NH 4 + assimilation in roots and protein turnover in leaves (Thomsen et al. 2014;Guan et al. 2016). Alternatively, NH 4 + can be assimilated by GDH and forming glutamate, and the reverse reaction (deamination) liberates NH 4 + (Souza and Fernandes 2018). In our experiment, the Piauí variety presented a higher expression of OsGS1.1 and OsGS1.3 compared to Manteiga and IAC-47 ( Figure  3(c,d)). Despite the lower expression of OsGS1.1 and OsGS1.3, Manteiga presents GS activity in root and shoot under constant 1 mM NH 4 + and NH 4 + resupply (Figure 4(a,c,f)). Gene expression and GS activity were not simultaneous. This may be due to post-translational regulations (Coelho et al. 2016). Landrace varieties under NH 4 + resupply had higher GS activity and GDH amination (p < 0.05) ( Figure  5(e)). GS activity is essential for rice growth as it supplies amino acids and reduces the toxic effects of NH 4 + . High levels NH 4 + can dissipate proton motive force across the plasma membrane and affect respiration and photosynthesis (Holzschuh et al. 2009;Taiz and Zeiger 2017). The overexpression of OsGS1.1 and OsGS1.2 in rice increases GS activity, amino acids content, soluble protein and total-N (Cai et al. 2009). Thus, the higher GS activity in landrace varieties cultivated with 1 mM NH 4 + and NH 4 + resupply suggests an interesting trait towards N assimilation.
Higher GS activity in landrace varieties did not increase biomass; however, it increased the free amino acid content in leaves and sheaths (Figure 6(h,i)). The NH 4 + assimilation is important for rice growth, but NH 4 + accumulation is toxic for plants (Liu and Von Wirén 2017). Rice landrace varieties cultivated with high levels of NH 4 + (150 mg L −1 ) presented a high free amino acid content and reduced growth due to the use of α-ketoglutarate generated by sugar catabolism (Fernandes 1984).   In our study, the Piauí variety presented lower levels of soluble sugar in roots cultivated with constant 1 mM NH 4 + (Figure 6(f)), suggesting higher sugar consumption in NH 4 + assimilation, which leads to lower plant growth, since the sugar content is essential for plant growth (Rocha et al. 2014).
The amino acids formed by NH 4 + assimilation in roots are transported to shoot by xylem (Zhang et al. 2015); meanwhile, the transport of amino acids from leaves to drain tissues occurs by phloem (Tegeder and Masclaux-Daubresse 2018). The efficiency of amino acid transport among tissues is dependent on specific transporters (AAT) (Fan et al. 2017). In our study, the Piauí variety presented a higher expression of AAT genes (Figure 2(g), Figure 3(g) and (h)), indicating a strategy of partition of the amino acids from NH 4 + assimilation, especially under NH 4 + resupply ( Figure  6(i)).
On the other hand, the IAC-47 variety presented a higher expression of OsAAP1, OsGAT1 and OsGAT4 in roots (Figure 2(h)) and lower levels of free amino acid in root and shoot under NH 4 + resupply ( Figure 6(i)). These results indicate the efficiency of the IAC-47 variety in using NH 4 + for vegetative growth. Lu et al. (2012) suggests that expression of OsAAP1, OsGAT1 and OsGAT4 in roots is important for plant growth. The overexpression of OsAAP1 in pea increased biomass (Zhang et al. 2015), suggesting that the higher expression of OsAAP1 in the IAC-47 variety is related to increased biomass in our study.
Principal component analysis (PCA) was performed to gain insight on how the three rice varieties responded to differential NH 4 + supply (Ferreira et al. 2020;Pereira et al. 2022). The PCA highlights the efficiency of IAC-47 to use NH 4 + for vegetative growth, maintaining low NH 4 + levels (Table 1 and Figure 7). The NH 4 + levels, free amino acid and sugar levels in Manteiga and Piauí varieties highlight the biochemical differences regarding NH 4 + use by rice varieties. The breeding program may favor biomass accumulation in the vegetative phase to improve grain production, as observed in the IAC-47 variety; however, Landrace varieties may not use N to increase vegetative growth but instead accumulate N to maintain growth under seasonal N flush in soil (Coelho et al. 2016). Our results demonstrate that the differential pattern of gene expression of AAT and AMT genes in IAC-47 (improved) and landrace varieties (Manteiga and Piauí) may influence the N responses to growth and amino acid content.