Ectopic Expression of Sugarcane ScAMT1.1 Has the Potential to Improve Ammonium Assimilation and Grain Yield in Transgenic Rice under Low Nitrogen Stress

In China, nitrogen (N) fertilizer is excessively used in sugarcane planting areas, while the nitrogen use efficiency (NUE) of sugarcane is relatively low. Mining and identifying the key genes in response to low N stress in sugarcane can provide useful gene elements and a theoretical basis for developing sugarcane varieties with high NUE. In our study, RNA-Seq combined with qRT-PCR analysis revealed that the ScAMT1.1 gene responded positively to low N stress, resulting in the stronger low N tolerance and high NUE ability of sugarcane cultivar ROC22. Then, ScAMT1.1 was cloned from sugarcane. The full-length cDNA of the ScAMT1.1 gene is 1868 bp, containing a 1491 bp open reading frame (ORF), and encoding 496 amino acids. ScAMT1.1 belongs to the AMT superfamily and shares 91.57% homologies with AMT1.1 from Oryza sativa. Furthermore, it was stably overexpressed in rice (O. sativa). Under low N treatment, the plant height and the fresh weight of the ScAMT1.1-overexpressed transgenic rice were 36.48% and 51.55% higher than that of the wild-type, respectively. Both the activity of ammonium assimilation key enzymes GS and GDH, and the expression level of ammonium assimilation key genes, including GS1.1, GS1.2, GDH, Fd-GOGAT, and NADH-GOGAT2 in the transgenic plants, were significantly higher than that of the wild-type. The grain number and grain yield per plant in the transgenic rice were 6.44% and 9.52% higher than that of the wild-type in the pot experiments, respectively. Taken together, the sugarcane ScAMT1.1 gene has the potential to improve ammonium assimilation ability and the yield of transgenic rice under low N fertilizer conditions. This study provided an important functional gene for improving sugarcane varieties with high NUE.


Introduction
Nitrogen (N) is one of the essential elements for crop growth and development [1]. N supply is a critical limiting factor of high yield in crop production, resulting in much chemical N fertilizer used in crop production worldwide. Sugarcane (Saccharum spp. hybrids) is the most important sugar crop. As a tall perennial grass, sugarcane requires a large quantity of N for normal growth, and excessive chemical N fertilizer has been applied to increase sugarcane yield, leading to the low nitrogen use efficiency (NUE), with only 20-40% absorbed of the N application amount in sugarcane production [2][3][4], especially in China and India, two of the top three sugarcane producing countries [5]. This not only causes soil acidification and compaction but also reduces the sugar content of the sugarcane. The breeding of high NUE varieties is an effective way to reduce N fertilizer consumption [6]. Identification of the key genes and studying the physiological

The ScAMT1.1 Gene Responded Positively to Low N Stress in Sugarcane
From samples of leaf and root in two sugarcane varieties ROC22 and Badila treated with low N stress, five differentially expressed genes (DEGs) of the AMT family were identified based on the previous mRNA transcriptomic datasets generated by RNA-Seq [23]. Their expression patterns in sugarcane under low N stress were evaluated by both RNA-Seq and quantitative real-time PCR (qRT-PCR) using the samples of roots and leaves, indicating the consistency of their expression patterns in both detections ( Figure 1). In detail, ScAMT1.2 or ScAMT2.1 was upregulated in the roots of both ROC22 and Badila, as well as in the leaves ( Figure 1B,C). Contrary to ScAMT1.2 and ScAMT2.1, ScAMT3.2 presented a downregulated pattern in two tissues of both high-and low-NUE sugarcane cultivars ( Figure 1D). In addition, ScAMT3.3 was specifically downregulated in the roots ( Figure 1E). Moreover, the expression levels of ScAMT1.2, ScAMT2.1, ScAMT3.2, and ScAMT3. 3 were not significantly different between the two cultivars under low N conditions. Interestingly, ScAMT1.1 expression profiles were different from the other four ScAMTs. Within those AMT family members, only ScAMT1.1 was kept at higher levels in ROC22 than that in Badila under low N concentration ( Figure 1A). The higher expression level of ScAMT1.1 might contribute to improving the low N tolerance and high NUE ability of ROC22.

The ScAMT1.1 Gene Responded Positively to Low N Stress in Sugarcane
From samples of leaf and root in two sugarcane varieties ROC22 and Badila treated with low N stress, five differentially expressed genes (DEGs) of the AMT family were identified based on the previous mRNA transcriptomic datasets generated by RNA-Seq [23]. Their expression patterns in sugarcane under low N stress were evaluated by both RNA-Seq and quantitative real-time PCR (qRT-PCR) using the samples of roots and leaves, indicating the consistency of their expression patterns in both detections ( Figure  1). In detail, ScAMT1.2 or ScAMT2.1 was upregulated in the roots of both ROC22 and Badila, as well as in the leaves ( Figure 1B,C). Contrary to ScAMT1. 2 and ScAMT2.1, ScAMT3.2 presented a downregulated pattern in two tissues of both high-and low-NUE sugarcane cultivars ( Figure 1D). In addition, ScAMT3.3 was specifically downregulated in the roots ( Figure 1E). Moreover, the expression levels of ScAMT1.2, ScAMT2.1, ScAMT3.2, and ScAMT3. 3 were not significantly different between the two cultivars under low N conditions. Interestingly, ScAMT1.1′ expression profiles were different from the other four ScAMTs. Within those AMT family members, only ScAMT1.1 was kept at higher levels in ROC22 than that in Badila under low N concentration ( Figure 1A). The higher expression level of ScAMT1.1 might contribute to improving the low N tolerance and high NUE ability of ROC22.  Error bars represent ± standard error from the mean of three biological replicates. Different letters above columns indicate significant differences (p < 0.05).

Gene Cloning and Bioinformatics Analysis of ScAMT1.1 Gene
In the present study, a ScAMT1.1 gene was cloned from the sugarcane cultivar ROC22 (Supplementary Figure S1). Its full-length cDNA was 1868 bp, containing a 1491 bp open reading frame (ORF) and encoding 496 amino acids (Supplementary Figure S2). A conservative AMT1.1 domain, belonging to the AMT superfamily, was located at the middle region (from 36th to 459th amino acid residues) of the ScAMT1.1 protein sequence ( Figure 2). The theoretical molecular weight of the ScAMT1.1 protein was 124.47 kDa, which belongs to the acidic hydrophobic protein and contains transmembrane domains with the greatest probability of localization in the plasma membrane. quence ( Figure 2). The theoretical molecular weight of the ScAMT1.1 protein was 124.47 kDa, which belongs to the acidic hydrophobic protein and contains transmembrane domains with the greatest probability of localization in the plasma membrane.
The ScAMT1.1 from sugarcane and the AMT family from the other four species, i.e., A. thaliana, O. sativa, S. bicolor, and S. spontaneum, were used to generate a phylogenetic tree. The results showed that the genetic relationship coincided with the plant AMT class ( Figure 3). ScAMT1.1 was classed into the AMT1.1 group and had the closest genetic relationship with SbAMT1.1 ( Figure 3). The multiple sequence alignment result was similar to the phylogenetic analysis. The amino acid sequence of ScAMT1. 1 Figure 4). Sugarcane is one of the monocotyledonous C4 crops, alongside S. bicolor and Z. mays. Therefore, the phylogenetic analysis and sequence alignment results were consistent with their biological classification features (Supplementary Figure S3).

Identification of the ScAMT1.1 Overexpressed Rice
The seeds from 20 transgenic rice lines were screened for response to herbicide treatment at germination, as well as the three-leaf stage. Among them, 19 lines were determined as the positive transgenic plants by seed germination and plant sprayed using Basta solution (Figure 5 A,B) and detected using PCR ( Figure 5C).    H  H  H  Q   I  L  I  V  V  V  I   I  I  I  I  I  V  I   Q  Q  Q  Q  Q  L  Q  i   I  I  I  I  I  I  I

Identification of the ScAMT1.1 Overexpressed Rice
The seeds from 20 transgenic rice lines were screened for response to herbicide treatment at germination, as well as the three-leaf stage. Among them, 19 lines were determined as the positive transgenic plants by seed germination and plant sprayed using Basta solution ( Figure 5A,B) and detected using PCR ( Figure 5C).  Figure 6A), and thus they were used to investi traits in the following assay. The transgenic lines showed a better pe wild-type plants under low N stress. After culturing for 16 d under lo tion, transgenic rice seedlings of T-5, T-6, and T-12 grew significan wild-type ( Figure 6B), and their plant height was 46.83%, 26.58%, and that of the wild-type ( Figure 6C), respectively. In addition, their fresh 43.02%, and 52.33% higher than that of the wild-type, respectively (Fi

Phenotypic Index Analysis Shows Overexpressing ScAMT1.1 Enhances Plants' Height and Fresh Weight in the Transgenic Rice under Low N Stress
Four homozygous T3 generation transgenic lines (T-2, T-5, T-6, and T-12) were obtained according to the methods described in 4.4. Compared to the other transgenic lines, higher ScAMT1.1 expression levels in the transgenic lines of T-5, T-6, and T-12 were found by qRT-PCR ( Figure 6A), and thus they were used to investigate the agronomic traits in the following assay. The transgenic lines showed a better performance than the wild-type plants under low N stress. After culturing for 16 d under low N nutrient solution, transgenic rice seedlings of T-5, T-6, and T-12 grew significantly better than the wild-type ( Figure 6B), and their plant height was 46.83%, 26.58%, and 36.03% higher than that of the wild-type ( Figure 6C), respectively. In addition, their fresh weight was 59.30%, 43.02%, and 52.33% higher than that of the wild-type, respectively ( Figure 6D).

The Four Ammonium Assimilation Related Enzymes Were More Active in th Lines Than That in the Wild-Type Plants
Co-expression analysis results showed that the expression levels of nium assimilation-related genes, except for the NADH-dependent glutam gene (NADH-GOGAT1), were significantly upregulated in the transgenic li N condition ( Figure 7A). Similar results were obtained from the experimen activities ( Figure 7B). No significant differences were found in the activity mate synthase (GOGAT) enzyme between the transgenic lines and the wi ( Figure 7B). However, the activity of the other enzymes (glutamine synth glutamate dehydrogenase, GDH) in the transgenic lines was significantl those in the wild-type ( Figure 7B).

The Four Ammonium Assimilation Related Enzymes Were More Active in the Transgenic Lines Than That in the Wild-Type Plants
Co-expression analysis results showed that the expression levels of all the ammonium assimilation-related genes, except for the NADH-dependent glutamate synthase gene (NADH-GOGAT1), were significantly upregulated in the transgenic lines under low N condition ( Figure 7A). Similar results were obtained from the experiments on enzyme activities ( Figure 7B). No significant differences were found in the activity of the glutamate synthase (GOGAT) enzyme between the transgenic lines and the wild-type plants ( Figure 7B). However, the activity of the other enzymes (glutamine synthetase, GS, and glutamate dehydrogenase, GDH) in the transgenic lines was significantly higher than those in the wild-type ( Figure 7B).

Overexpressed ScAMT1.1 Enhanced Grain Number and Grain Yield of the Transgenic Plants in the Pot Experiment under Low N Condition
Under low N conditions, transgenic lines of the ScAMT1.1 overexpressed rice and the wild-type were planted in the planting pots. The ripening time of transgenic lines was extended by about 15 d compared to the wild-type. However, the transgenic lines grew better than the wild-type during the whole growth duration of the rice. Figure 7A showed the different phenotypes of the wild-type and the ScAMT1.1 transgenic rice at the grain filling stage. At harvesting time, the grain number per plant of the transgenic lines T-5, T-6, and T-12 was 6.87%, 4.72%, and 7.72% higher than that of the wild-type ( Figure 8B), and the grain yield per plant was 11.80%, 7.64%, and 9.12% higher than that of the wild-type ( Figure 8C), respectively. In the pot experiment, the ScAMT1.1 overexpressed transgenic rice is more productive than the wild-type under low N conditions because its grain number and grain yield were 4.72-7.72% and 7.64-11.80% higher than that of the wild-type, respectively ( Figure 8B,C). because its grain number and grain yield were 4.72-7.72% and 7.64-11.80% higher than that of the wild-type, respectively ( Figure 8B,C).
Within the AMT family, AMT1.1 was an important member of the high-affinity transport system [1,9,16]. As a type of low ammonia concentration stress-sensitive gene, AMT1.1 was found to express preponderantly in the roots in both A. thaliana [18] and O. sativa [20] under low N stress. Sugarcane is one of the few ammonium-preferring crops. In this study, one AMT1.1 gene, termed ScAMT1.1, was cloned from the low N-tolerant sugarcane cultivar ROC22. Similar to the AMT1.1 from A. thaliana or O. sativa, ScAMT1.1 was induced under low N stress and highly expressed in the roots of sugarcane ( Figure  1).
Within the AMT family, AMT1.1 was an important member of the high-affinity transport system [1,9,16]. As a type of low ammonia concentration stress-sensitive gene, AMT1.1 was found to express preponderantly in the roots in both A. thaliana [18] and O. sativa [20] under low N stress. Sugarcane is one of the few ammonium-preferring crops. In this study, one AMT1.1 gene, termed ScAMT1.1, was cloned from the low N-tolerant sugarcane cultivar ROC22. Similar to the AMT1.1 from A. thaliana or O. sativa, ScAMT1.1 was induced under low N stress and highly expressed in the roots of sugarcane (Figure 1).
The other four members of the AMT family studied here exhibited a differential expression pattern in sugarcane under low N stress. ScAMT2.1 was significantly upregulated in the leaves of sugarcane under low N treatment (0.6 mM N) for six hours (Figure 1). However, ScAMT3.2 was significantly downregulated in the leaves at the same condition ( Figure 1). The opposite expression trends were also found between SsAMT2.1 and SsAMT3.2 in S. spontaneum under low N stress (0.1 Mm N) [7]. Moreover, we found that the expression trends of ScAMT2.1 and ScAMT3.2 were similar under low N stress between two contrasting NUE sugarcane cultivars (Figure 1). However, it is worth noting that the expression level of ScAMT1.1 was significantly higher in the low N-tolerant cultivar ROC22 than that in the low N-sensitive cultivar Badila (Figure 1). It could be speculated that ScAMT1.1 played a positive role in the N absorption process in sugarcane. N is typically supplied by a mixture of NH 4 + and NO 3 − in natural environments [2]. Most crops strongly preferred NO 3 − [7]. While some crops, e.g., rice and sugarcane, showed a stronger preference towards NH 4 + [7][8][9]. However, the reasons for the preference for NH 4 + or NO 3 − remain unclear [27]. AMTs are most likely involved in ammonium transport in a crop, which might be partially associated with the answer [28]. Therefore, the functions and molecular regulatory mechanisms of AMTs in NH 4 + uptake cause particular attention. AMT1.1 is an important ammonium transporter gene under low N stress. Whether overexpression of AMT1.1 in rice can lead to an increase in the NH 4 + absorption and yield production remained controversial.
Kumar et al. [21] and Huang et al. [22] overexpressed OsAMT1.1 in rice. The NH 4 + concentration was too high for transgenic plants to assimilate in time, thus resulting in toxicity and damage to plant growth. However, the results in another report were different [9]. The transgenic rice overexpressing OsAMT1.1 showed significant enrichment of OsAMT1.1 transcript under the nutrient solution of 30 µM (NH 4 ) 2 SO 4 , which was 20 times higher than the wild-type [9]. At the same time, the expression levels of ammonium assimilation key genes (GS1.1, GS1.2, GS2.1, Fd-GOGAT, NADH-GOGAT, and GDH) in the transgenic plants were significantly increased [9]. After ammonium is absorbed into the roots, it is converted into glutamine (Gln) through GS. Gln and α-Ketoglutaric acid generate glutamic acid by the GS/GOGAT cycle, and inorganic N is converted into organic N. GS1.2 and NADH-GOGAT were the key genes in the NH 4 + assimilation of plant roots, and GS1.1 and Fd-GOGAT were the key genes for N utilization in plants [9,29]. Therefore, the yield of the transgenic rice was about 30% higher than that of the wild-type [9].
To identify the function of ScAMT1.1 under N-deficient conditions, homozygous transgenic rice lines overexpressed ScAMT1.1 were developed in the present study. Our results were similar to Ranathunge et al. [9]. Heterologously expressed ScAMT1.1 in rice enhanced the ammonium assimilation ability of the transgenic plants. The evidence was as follows. Firstly, the activity of ammonium assimilation key enzymes GS, and GDH, as well as the expression levels of ammonium assimilation-related genes (GS1.1, GS1.2, GDH, Fd-GOGAT, and NADH-GOGAT2), were all significantly higher in the transgenic plants than those in the wild-type (Figure 7). Secondly, the plant height and fresh weight of the transgenic rice seedlings were 36.48% and 51.55% higher than those of the wild-type under N-deficient conditions ( Figure 6). Thirdly, grain number and grain yield per plant of the transgenic rice were 6.44% and 9.52% higher than those of the wild-type, respectively ( Figure 8). These demonstrated that the ammonium assimilation ability of the transgenic rice was higher than that of the wild-type. It could be speculated that enhancement of NH 4 + uptake in ScAMT1.1-overexpressed rice plants promotes the activity of enzymes involved in ammonium assimilation under low ammonium conditions. The NH 4 + content in the wild-type and the transgenic rice, together with its absorption and transport mechanism in the transgenic rice, need to be determined in the future. Additionally, the potential for improved grain yield in the ScAMT1.1 transgenic rice under low N stress also needs to be further detected in the field.

Materials and Treatments
Sugarcane leaf and root samples obtained from low N treatment and used in qRT-PCR were the same as those used in the previous study of RNA-Seq [23]. The leaves (low N treatment for 0 h, 6 h) and roots (low N treatment for 0 h, 3 h) of sugarcane cultivar ROC22 (low N-tolerant) and Badila (low N-sensitive) plantlets under low N condition (0.6 mM N) were selected. The samples of the leaves and roots from the cultivars ROC22 and Badila under low N treatment for 0 h were selected as the control, respectively.
The culture experiment of the transgenic rice under low N stress: seeds of the transgenic and wild-type rice were germinated and cultivated in the seedling trays. The plantlets at the three-leaf stage with uniform size were selected as experimental materials and divided into two groups as follows: Group one, plantlets were cultured for 16 5.8), and the whole plant samples were collected. Ten randomly selected plantlets were pooled into a biological duplicate. Among them, six seedlings were used for physiological index determination, and the other four seedlings were frozen rapidly in liquid N for qRT-PCR of ammonium assimilation key genes. The experiments were done in three biological replicates. Total RNA of the transgenic and wild-type rice plantlets was extracted with TRIzol reagent (Invitrogen, Waltham, MA, USA) for the qRT-PCR experiment. RNA and cDNA were obtained referring to the method of the previous study [23].
The plantlets of the other group were cultivated in the planting pots (one seedling per pot). The pot sizes were 0.40 m in both diameter and depth, with eight 1.0 cm diameter holes located at the bottom. Each pot was filled with 10 kg of low N soil (pH 5.0 to 5.5) with an organic matter content of 7.09 g/kg, alkali-hydrolyzable N of 0.04 g/kg, effective phosphorus of 0.02 g/kg, rapidly available potassium of 0.10 g/kg, total N of 0.38 g/kg, total phosphorus of 0.28 g/kg, and total potassium of 20.1 g/kg. All planting pots were provided with the same amount of water and under the same cultivation management. Grain number and grain yield per plant were recorded at harvest time.

RNA-Seq Data Analysis and qRT-PCR Experiment
Based on RNA-Seq in the previous study [23], five differentially expressed (padj < 0.05 and a fold change ≥ 2) AMT family genes (AMT1.1, AMT1.2, AMT2.1, AMT3.2, and AMT3.3) were identified in the two contrasting NUE sugarcane cultivars (ROC22 and Badila) under low N condition. The expression profiles (in log 2 Fold change) of the mentioned five genes in the leaves and roots were detected, respectively.
The qRT-PCR experiment was further used to examine the expression profiles of the five AMT family genes in the leaves and the roots of ROC22 and Badila under low N stress. In addition, the relative expression levels of ammonium assimilation key genes, e.g., GS1.1, GS1.2, GDH, ferredoxin-dependent glutamate synthase (Fd-GOGAT), NADH-GOGAT1, and NADH-GOGAT2 were assessed in the transgenic rice. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [30] and eukaryotic elongation factor1-alpha (eEF-1a) [31,32] were used as the reference genes in sugarcane and rice, respectively. The 2 − Ct method [33] was used to calculate the relative gene expression of the samples. The experiments were done in three biological replicates, and SYBR Green was used for training. The primer sequences of qRT-PCR in sugarcane and rice are shown in Supplementary Table S1 and  Supplementary Table S2, respectively.

Gene Cloning and Bioinformatics Analysis of Sugarcane ScAMT1.1 Gene
The samples of RNA and cDNA from the sugarcane cultivar ROC22 were obtained from the previous study [23]. A pair of cloning primers, ScAMT1.1-F (5 -GCCACACCCTC CCAATCC-3 ) and ScAMT1.1-R (5 -ACACACTGAAAAAGGCAAGCAC-3 ), was designed according to the unigene template from the transcriptome sequencing data. The PCR amplification procedure was consistent with the previous study [23]. The PCR product was ligated to pMD20-T cloning vector and transformed into Escherichia coli cells for proliferation. The plasmid was extracted and sequenced. NCBI

Generation of the Transgenic Rice Overexpressed ScAMT1.1
The ORF of the ScAMT1.1 gene was subcloned into the Bsa I site of the binary plant expressed vector pBWA(V)BU, which was used to construct the overexpressed vector pBWA(V)BU-ScAMT1.1. The recombinant expression vector pBWA(V)BU-ScAMT1.1 was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. Then, the genetic transformation of rice (O. sativa L. sp. Japonica cv. Nipponbare) was carried out to obtain the T0 generation transgenic plants. These works were entrusted to BioRun Co., Ltd. (Wuhan, China).
From the generations T0 to T3, an assay for the transgenic rice with Basta resistance was as follows. Three hundred seeds of each transgenic line were taken and soaked in 0.1% Basta solution for germination. The rice seedlings germinated normally were planted in the seedling tray. When the seedlings were at the three-leaf stage, 0.2% Basta solution was used for the second spray screening. The plants with normal growth were transplanted into pots for soil culture to obtain seeds. The screening procedure was repeated. The seeds were firstly soaked with 0.1% Basta solution for screening, followed by spraying the seedlings with 0.2% Basta solution for further screening until the homozygous transgenic lines of the T3 generation were screened. The transgenic-positive plants were confirmed by PCR using ScAMT1.1F (5 -TGGGTTCATGCTCAAGTCCG-3 ) and ScAMT1.1R (5 -ATAACAGGGTAATGCGGCCC-3 ) primers.

Determination of Enzyme Activities Involved in Ammonium Assimilation
The activities of the ammonium assimilation key enzymes GS, GOGAT, and GDH were measured in the transgenic and wild-type rice plantlets under low N stress for 16 days. The GS activity was measured according to the method of Wang et al. [34]. The activities of GOGAT and GDH enzymes were determined according to the experimental methods of Groat and Vance [35].

Conclusions
We characterized a sugarcane ammonium transporter gene, ScAMT1.1, with a significantly higher expression level in the low N-tolerant cultivar ROC22 than that in the low N-sensitive cultivar Badila under low N stress. The activity of ammonium assimilation key enzymes (GS and GDH) and the gene expression levels of ammonium assimilation key genes (GS1.1, GS1.2, GDH, Fd-GOGAT, and NADH-GOGAT2) in the transgenic rice were all significantly higher than those in the wild-type under low ammonium condition, indicating that the ScAMT1.1 has the ability to improve ammonium assimilation. Transgenic rice lines overexpressed ScAMT1.1 show superior growth and significantly higher grain yield under low N stress in the pot experiment. Taken together, it is suggested that ScAMT1.1 has the potential for improving ammonium assimilation, plant growth, and grain yield under low N fertilizer conditions; however, the potential for increasing production needs to be further verified in the field.