pOsNAR2.1:OsNAR2.1 expression enhances nitrogen uptake efficiency and grain yield in transgenic rice plants

Summary The nitrate (NO3−) transporter has been selected as an important gene maker in the process of environmental adoption in rice cultivars. In this work, we transferred another native OsNAR2.1 promoter with driving OsNAR2.1 gene into rice plants. The transgenic lines with exogenous pOsNAR2.1:OsNAR2.1 constructs showed enhanced OsNAR2.1 expression level, compared with wild type (WT), and 15N influx in roots increased 21%–32% in response to 0.2 mm and 2.5 mm 15NO3− and 1.25 mm 15 NH 4 15 NO 3. Under these three N conditions, the biomass of the pOsNAR2.1:OsNAR2.1 transgenic lines increased 143%, 129% and 51%, and total N content increased 161%, 242% and 69%, respectively, compared to WT. Furthermore in field experiments we found the grain yield, agricultural nitrogen use efficiency (ANUE), and dry matter transfer of pOsNAR2.1:OsNAR2.1 plants increased by about 21%, 22% and 21%, compared to WT. We also compared the phenotypes of pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines in the field, found that postanthesis N uptake differed significantly between them, and in comparison with the WT. Postanthesis N uptake (PANU) increased approximately 39% and 85%, in the pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines, respectively, possibly because OsNRT2.1 expression was less in the pOsNAR2.1:OsNAR2.1 lines than in the pOsNAR2.1:OsNRT2.1 lines during the late growth stage. These results show that rice NO 3 – uptake, yield and NUE were improved by increased OsNAR2.1 expression via its native promoter.


Introduction
Nitrogen (N) is an essential macronutrient for plant growth and crop productivity. NO À 3 is the main inorganic N nutrient for plants in aerobic uplands, and NH þ 4 is the main form in anaerobic paddy fields (Foyer et al., 1998;Scheible et al., 2004;Stitt, 1999). In upland cultivation system, NO À 3 is readily dissolved in soil water and very mobile in soil and therefore it was very easily lost into environment (Jin et al., 2015;Zarabi and Jalali, 2012). NO À 3 is acquired by roots through NO À 3 transporters and then transported throughout the plant, or it can be assimilated with carbon into amino acids before being redistributed (Katayama et al., 2009;Miller et al., 2007;Xu et al., 2012). In plants, seed dormancy can be broken by NO À 3 as a signalling molecule (Alboresi et al., 2005;Matakiadis et al., 2009), regulating lateral root development (Zhang and Forde, 1998;Zhang et al., 1999) and leaf growth (Hsu and Tsay, 2013;Rahayu et al., 2005), integrating the expression of nitrate-induced genes for growth and development (Dechorgnat et al., 2012;Ho and Tsay, 2010;Huang et al., 2015;O'Brien et al., 2016;Wang et al., 2012) and altering flowering time (Castro Marin et al., 2011).
As for adapting to the low and high NO À 3 concentrations in soil, the plants have developed two different absorption systems (L eran et al., 2014;Miller et al., 2007;Siddiqi et al., 1990), including the low NO À 3 affinity system (LATS) and high NO À 3 affinity system (HATS) (Crawford and Glass, 1998). As we know the NPF (NRT1/PTR) and NRT2 families contribute to LATS and HATS responding the NO À 3 uptake and translocation in plants (Fan et al., 2005;L eran et al., 2014;Miller et al., 2007;Orsel et al., 2006;Szczerba et al., 2006). Some NRT2 family members in plant are needed NAR2 partners in transporting nitrate crossing cell membrane (Galv an et al., 1996;Liu et al., 2014;Okamoto et al., 2006;Orsel et al., 2006;Quesada et al., 1994;Tong et al., 2005;Zhuo et al., 1999). In Chlamydomonas reinhardtii Quesada et al. (1994) firstly found that CrNar2 and CrNar3 can restore NO À 3 absorption of the NO À 3 uptake-defective mutants. Zhou et al. (2000) further demonstrated that CrNar2 was a partner protein of CrNRT2.1 in NO À 3 transporting cross the oocyte cell membrane. Okamoto et al. (2006) reported that, based on NAR2-type gene expression, both NAR2s and NRT2s constitute the NO À 3 inducible HATS, but not the LATS in Arabidopsis, such as AtNRT3, although the protein had no known transport activity. Yong et al. (2010) reported that in vivo NAR2.1 and NRT2.1 forming a complex on plasma membrane and played the role in absorbing low concentration of nitrate in Arabidopsis roots. Orsel et al. (2006) used oocyte expression and yeast split-ubiquitin systems to show that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS.
Two-component NRT2-NAR2 system also exists in rice NO À system to show that only OsNAR2.1, but not OsNAR2.2, interacts with OsNRT2.3a or OsNRT2.1/2.2 to promote NO À 3 uptake. Katayama et al. (2009) reported that overexpression of OsNRT2.1 improved the growth of rice seedlings, but did not increase nitrogen uptake. Tang et al. (2012) showed that rice OsNRT2.3a gene is involved in root transport of NO À 3 to shoots. The OsNRT2.3a or OsNRT2.1/2.2 and OsNAR2.1 interaction at the protein level was demonstrated using bimolecular fluorescence complementation, the yeast two-hybrid system and Western blot analysis (Liu et al., 2014;Yan et al., 2011). Furthermore Yan et al. (2011) also reported that knockdown of OsNAR2.1 by RNA interference (RNAi) can suppress expression of OsNRT2.3a, OsNRT2.2 and OsNRT2.1 in mutants roots and demonstrated that OsNAR2.1 does a key function in both high and low NO À 3 uptake. Chen et al. (2016) showed that using OsNAR2.1 promoter instead of ubiquitin promoter driving OsNRT2.1 can improve the ANUE and yield in rice. In this study, we created new construct of OsNAR2.1 promoter to drive the open reading frame (ORF) of the OsNAR2.1, investigated the transformation effects of pOs-NAR2.1:OsNAR2.1 on rice NO À 3 uptake, yield and NUE and also presented many different characteristics of pOsNAR2.1: OsNAR2.1 from pOsNAR2.1:OsNRT2.1 transgenic plants.

Results
Generation of transgenic rice expressing pOsNAR2.1: OsNAR2.1 We used the Agrobacterium tumefaciens-mediated method to introduce the pOsNAR2.1:OsNAR2.1 expression construct (Figure S1) into Wuyunjing 7 (O. sativa L. ssp. Japonica cv., the wild type for this experiment, WT), a high yield rice cultivar used in Jiangsu, China. We obtained 10 lines with increased the expression of OsNAR2.1 ( Figure S2a) and analysed biomass and yield of the transgenic plants in the T1 generation. Compared to WT, biomass and yield of the 10 lines of T1 generation increased by approximately 13% and 20%, respectively ( Figure S2b). Based on a Southern blot analysis of T2 generation and the data of RNA expression for the T1 and T2 generations (Figures 1c, S2a and 1b), we selected three independent lines of pOsNAR2.1: OsNAR2.1 designated Ox1, Ox2 and Ox3 (Figure 1a).
The expression of OsNAR2.1 in roots was increased four-to fivefold in the Ox1, Ox2 and Ox3 lines. OsNAR2.1 expression increased approximately 3.5-fold in culms and increased approximately 2.6-fold in leaf blades of the pOsNAR2.1:OsNAR2.1 transgenic plants (Figure 1b). The Western blot showed that the protein level of OsNAR2.1 was increased in shoots of Ox1, Ox2 and Ox3 lines compared with WT ( Figure 1d). The field data showed that the transgenic lines exhibited increased grain yield and dry weight, compared with the WT (Figures 1e and S2b). Field data of the T2, T3 and T4 generation lines showed that total aboveground biomass, increased by as much as 23%; yields of T3 transgenic plants grown at Sanya were enhanced by approximately 20%, and the yields of T2 and T4 plants grown at Nanjing increased by 21%-23%, relative to the WT (Table S3).
For the T4 transgenic plants at harvest, height increased 5%, total tiller number per plant increased 26%, panicle length increased approximately 12%, grain weight per panicle increased 25%, seed setting rate increased 13%, grain number per panicle increased 16%, and grain yields increased by 23% relative to the WT; however, 1000-grain weight had no difference between WT and the transgenic lines (Table 1).
Effects of pOsNAR2.1:OsNAR2.1 expression on plant seedling growth and total nitrogen content As previous data showed that knockdown of OsNAR2.1 in rice affects N uptake and growth . We further analysed the effect of pOsNAR2.1:OsNAR2.1 expression on plant seedling growth and nitrogen content by planting WT and transgenic rice seedlings in the solution containing 1 mM NH þ 4 of IRRI for 2 weeks and then in 2.5 mM NH þ 4 , 0.2 mM NO À 3 , 2.5 mM NO À 3 or 1.25 mM NH 4 NO 3 for 3 more weeks (Figure 2a (Figure 2e), they increased, respectively, by 152%, 149% and 151% in 0.2 mM NO À 3 ( Figure 2f); by 124%, 181% and 95% in 2.5 mM NO À 3 ( Figure 2g); and by 62%, 51% and 47% in 1.25 mM NH 4 NO 3 , compared with WT after harvest ( Figure 2h).
Rates of NO À 3 and NH þ 4 influx in WT and transgenic plants We analysed short-term NO À 3 and NH þ 4 uptake in same-size seedlings of the pOsNAR2.

Translocation of dry matter and nitrogen in WT and transgenic plants
Methods to measure NUE usually depend on calculating plant biomass production per unit of applied N, regardless of the crop and whether the root, leaf, fruit, or seed is measured, the transfer of N to plant organs and yield is known as "nutrient utilization efficiency" (Good et al., 2004;Xu et al., 2012). We analysed the dry matter, total nitrogen concentration and the total nitrogen content of the T4 generation of the pOsNAR2.1:OsNAR2.1 transgenic lines in the anthesis and maturity stages. The result showed that the biomass of panicles, leaves and culms in the transgenic lines increased 26%, 20% and 28%, respectively, in the anthesis stage (Figure 6b), and increased 23%, 29% and 25% in the maturity stage compared to those of WT ( Figure 6c). Total nitrogen concentration in leaves of the transgenic lines increased approximately 10% in the anthesis stage, but did not change in panicles or culms compared to those of WT. Total nitrogen concentration of panicles, leaves and culms was not different at the maturity stage compared to that in WT ( Figure 6e); total nitrogen content of panicles, leaves and culms in the transgenic lines increased by approximately 34%, 33% and 33%, respectively, during the anthesis stage (Figure 6f), and by 35%, 33% and 34% in the maturity stage, respectively, compared to those in the WT (Figure 6f).
We also calculated the harvest index (HI), dry matter translocation (DMT), dry matter translocation efficiency (DMTE) and the contribution of pre-anthesis assimilates to grain yield (CPAY), based on a method described by Chen et al. (2016). DMT increased by approximately 21%, whereas DMTE, CPAY and HI had no difference in pOsNAR2.1:OsNAR2.1 transgenic plants from WT (Table 3). We investigated nitrogen translocation (NT), contribution of pre-anthesis nitrogen to grain nitrogen accumulation (CPNGN) and NT efficiency (NTE), based on a method described by Chen et al. (2016). NTE and CPNGN did not differ pOsNAR2.1:OsNAR2.1 transgenic plants from WT, whereas NT increased by approximately 33%, relative to that in WT (Table 3).
NUE of pOsNAR2.1:OsNAR2.1 transgenic lines NUE is inherently compound and can be further defined with component parts, including NUpE, NUtE, ANR, AE NTE, NRE . Because both yield and biomass were increased in the pOsNAR2.1:OsNAR2.1 transgenic lines, in the meanwhile, we also investigated ANUE in transgenic plants of T2-T4 generations, and nitrogen recovery efficiency (NRE), PANU, nitrogen harvest index (NHI), and physiological nitrogen use efficiency (PNUE) traits in the T4 transgenic plants to determine whether nitrogen use was changed in these lines, using the method described by Chen et al. (2016). Compared to WT, the ANUE of the pOsNAR2.1: OsNAR2.1 transgenic lines was enhanced by approximately 22%  in T3 generation grown at Sanya under the tropical climate condition and by 21%-24% in the T2 and T4 plants grown at Nanjing under semi-tropical condition (Table S3, Figure 7a). NRE and PANU increased approximately 125% and 39% in the T4 generations, compared to those in WT (Figure 7b, c), but PNUE and NHI values had no different between those and WT (Table 3).

Discussion
All levels of plant function were affected by nitrogen nutrition, from metabolism to growth, development and resource allocation (Crawford, 1995;Scheible et al., 1997). NO À 3 is a main available form of nitrogen for plants and is absorbed in the roots by active transport processes and passive transport ion channels, stored in vacuoles of rice shoots Kucera, 2003;Li et al., 2008;Pouliquin et al., 2000). OsNRT2.1/2 and OsNRT2.3a need to be combined with OsNAR2.1 protein for uptake and transport of NO  GNAM (g/m 2 ) 9.11b 12.44a 11.79a 12.55a Statistical analysis of data from T4 generation; n = 3 plots for each mean. The pOsNAR2.1:OsNAR2.1 expression increases NO À 3 uptake of transgenic rice plants Feng et al. (2011) had proved that OsNAR2.1 interacts with OsNRT2.3a and OsNRT2.1/2.2 in an oocyte expression system to take up NO À 3 . The OsNAR2.1 and OsNRT2.3a or OsNRT2.1/2.2 interaction in the protein level was demonstrated using bimolecular fluorescence complementation, Western blot analysis and a yeast two-hybrid assay (Liu et al., 2014;Yan et al., 2011). Tang et al. (2012) showed that OsNRT2.3a gene is important in NO À 3 root transport to shoots. Katayama et al. (2009) reported that increased OsNRT2.1 expression slightly improved the growth of rice seedling in hydroponic condition, but did not affect the nitrogen uptake. In this study, we demonstrated that OsNAR2.1 driven by the native OsNAR2.1 promoter increased NO À 3 uptake by rice roots.
As we know, the native OsNAR2.1 was expressed in all parts in rice plant, and mainly expressed roots and leaf sheaths (Chen et al., 2016;Feng et al., 2011;Liu et al., 2014;Yan et al., 2011), but we do not know why one more native promoter driving OsNAR2.1 can increase the expression level of OsNAR2.1 more than one time and in different organs, the increase patterns were different. The possible reason about this was that the methylation level was different in the transferred homologous exogenous promoter sequence compared with the endogenous promoter sequence (Matzke et al., 1989). However more experiments are needed for this understanding.
Enhanced NO À 3 uptake promotes NH þ 4 uptake in rice Kronzucker et al. (2000) used 13 N to show that the presence of NO À 3 promotes NH þ 4 uptake, accumulation and metabolism in rice. Duan et al. (2006) found that increasing NO À 3 uptake promotes dry weight and NO À 3 accumulation and assimilation of NH þ 4 and NO À 3 by 'Nanguang', which is an N-efficient rice cultivar, during the entire growth period. Li et al. (2006) showed that supplying NH þ 4 and NO À 3 enhances OsAMT1;3, OsAMT1;2 and OsAMT1;1 expression compared with supplying only NH þ 4 or NO À 3 , thereby enhancing NH þ 4 uptake by rice. High expression of OsNRT2.3b in rice improves the pHbuffering capacity of the rice resulting in less 15 N-NH 4 15 NO 3 uptake in 5-min uptake experiment, and more 15 N-15 NH 4 NO 3 increased uptake at pH 4 and pH 6 . Our results showed that the influx rates of 15 NO À 3 and 15 NH þ 4 increased 22% and 21%, respectively, in pOsNAR2.1:OsNAR2.1 transgenic lines in 1.25 mM NH 4 15 NO 3 or 1.25 mM 15 NH 4 NO 3 ( Figure S3), and that the ratio of 15 NO À 3 to 15 NH þ 4 influx into pOsNAR2.1:OsNAR2.1 transgenic plants was not different from WT in 1.25 mM NH 4 15 NO 3 or 1.25 mM 15 NH 4 NO 3 ( Figure S3). Eventually, the biomass and total nitrogen content of pOsNAR2.1: OsNAR2.1 transgenic lines increased by 50.7% and 68.9% after 3 weeks in 1.25 mM NH 4 NO 3 (Figures 2d, 2h and 3h).
Exogenous of pOsNAR2.1:OsNAR2.1 transformation in rice enhances ANUE and NRE During recent years, NO À 3 transporter gene as a target gene was applied in crop high NUE breeding (Fan et al., 2017). For examples, the OsNRT1.1B low-affinity NO À 3 transporter can increase the indica rice NUE by approximately 30% (Hu et al., 2015). Fan et al. (2016) showed that increased OsNRT2.3b expression improved NUE and grain yield by up to 40% in Japonica cultivars. Chen et al. (2016) reported the ANUE of pOsNAR2.1:OsNRT2.1 transgenic plants increased by 28% of in the same background cultivar (Wuyunjing 7) as this experiment. Our present data show that OsNAR2.1 driven by the native OsNAR2.1 promoter can produce a relatively higher yield and ANUE in rice plants (Figure 7, Figure S2b and Table S3).
Nitrogen redistribution can be altered by the expression change of some nitrogen use relative gene, such as the autophagy gene ATG8c (Islam et al., 2016) and also presents different patterns in different genotypes (Sanchez-Bragado et al., 2017;Souza et al., 1998). During rice grain filling, 70-90% of the nitrogen was redistributed from the vegetative organs to the panicles (Yoneyama et al., 2016). Dry matter and nitrogen content of pOsNAR2.1: OsNAR2.1 lines were more than WT plants in the anthesis and maturity stages ( Figure 6). Although DMT and NT increased by approximately 21% and 33%, compared to that of WT, DMTE and NTE of pOsNAR2.1:OsNAR2.1 transgenic plants and WT were not different (Table 3), suggested that dry matter and nitrogen transfer from shoots to grains did not change significantly between pOsNAR2.1:OsNAR2.1 transgenic plants and WT; thus, the physiological NUE and NHI of pOsNAR2.1:OsNAR2.1 transgenic plants did not increase (Table 3). NRE and ANUE increased 25% and 22% due to the increase in nitrogen accumulation and grain yield, respectively, at maturity in pOsNAR2.1:OsNAR2.1 transgenic lines (Table 2; Figure 7a, b).
Designing a genetically modified crop using tissuespecific expression conferred by selected promoters Although using either the ubiquitin promoter (pUbi) or OsNAR2.1 promoter (pOsNAR2.1) to drive OsNRT2.1 expression could significantly increase total biomass and grain yield compared with those in WT, ANUE was decreased 17% by pUbi:OsNRT2.1 expression and increased 28% by pOsNAR2.1:OsNRT2. promoters driving OsNRT2.1 expression on ANUE were caused mainly by altered tissue localization and abundance of OsNRT2.1 transcripts which may be linked to postflowering transfer of dry matter into grains (Chen et al., 2016). Another transformation example of native promoter driving its ORF is pOsPTR9:OsPTR9 transgene in rice with improving on growth, grain yield and NUE (Fang et al., 2013). Fang et al. (2013) investigated the expression pattern of OsPTR9 and found that it is regulated by nitrogen sources and light. Although OsPTR9 does not appear to directly transport NO À 3 , its overexpression results in enhanced NH þ 4 uptake, increased grain yield and promoted lateral root formation (Fang et al., 2013). These results indicate that expression of genes using specific promoters may be a good approach for plant breeding.
Several phloem NO À 3 transporters, such as NPF2.13, NPF1.1 and NPF1.2, are responsible for redistributing xylem-borne NO À 3 into developing leaves to increase shoot growth Hsu and Tsay, 2013). Therefore, selecting and applying the promoters of genes specifically expressed in senescing leaves or other source organs could be used to drive phloem-expressed NO À 3 , transporters, which would decrease residual N in old vegetative organs and increase growth and NUE.
In this experiment, we demonstrated that rice NO À 3 uptake, yield and NUE of rice were ameliorated by increasing OsNAR2.1 expression using its native promoter.

qRT-PCR and Southern blot analysis
Total RNA was extracted using TRIzol reagent (Vazyme Biotech Co., Ltd, http://www.vazyme.com). DNase I-treated total RNAs were subjected to reverse transcription (RT) with HiScript Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co.). Triplicate quantitative assays were performed using the AceQ qPCR SYBR Green Master Mix kit (Vazyme Biotech Co.) and a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). The relative quantitative calculation of real-time PCR was described in Chen et al. (2016). The primers for PCR are shown in Table S2.
The Southern blot was carried to identify the T-DNA insertion. The genomic DNA exaction of T2 plant shoots, DNA digestion and hybridization were followed the previous report (Chen et al.,

Western blot
OsNAR2.1 antibody and Western blot process was described in Yan et al. (2011). The total protein of 10 g shoots were sampled and 50 lg of each protein was analysed in gel-loaded buffer and boiled in 10% SDS-PAGE. Protein transfer to PVDF membrane and incubated with OsActin (1 : 5000), or OsNAR2.1 (1 : 2000) overnight at 4°C. The membrane was then incubated with the appropriate secondary antibody (1 : 20 000; Pierce), then carries on the chemiluminescence detection (Tang et al., 2012;Yan et al., 2011).

Field experiments for harvest yield
The rice plants of T0 to T4 generations, except T3 generation, were cultivated in plots at the Experimental Site of Nanjing Agricultural University, Nanjing, with subtropical climate from May to October in a year. For T3 generation, transgenic lines were tested in plots of Experiment Site of Sanya Nanjing Agricultural University with tropical climate from December to April. Soil properties in Nanjing field experiment were described as before (Chen et al., 2016).
T2-T4 generation pOsNAR2.1:OsNAR2.1 and wild-type plants were planted in three plots with 300 kg N/ha and without nitrogen fertilizer as blank control. The plots were 2 9 2 m in size, and the seedlings were planted in a 10 9 10 array. During rice flowering and mature stages, we collected samples from each plot for further analysis. Random four replicates (each replicate with four individual plants) from each plot were selected within the plots free from the edges, and therefore, the data of total 16 individual plants were pulled into mean value of each plot (Chen et al., 2016).
The agronomic characters of T4 generation plant height, total tiller number per plant, grain weight per panicle, grain number per panicle, seed setting rate, panicle length, 1000-grain weight, yield and biomass per plant were measured at the maturity stage under 300 kg N/ha N fertilizer condition.
Dry weight, total nitrogen measurement and calculation of nitrogen use efficiency We harvested T4 generation shoot samples from the field to analyse biomass and nitrogen under 300 kg N/ha fertilizer condition according to our previous method (Chen et al., 2016) DMTE and NTE were calculated according to Chen et al. (2016). Determination of total N content, root 15 N-NO À 3 influx rate and 15 N-NH þ 4 influx rate in WT and transgenic seedlings WT and transgenic rice seedlings were grown in the solution containing 1 mM NH þ 4 in IRRI solution for 2 weeks and then transferred in different forms of nitrogen for 3 additional weeks. The nitrogen treatments in this experiment included in 2.5 mM NH þ 4 , 0.2 mM NO À 3 , 2.5 mM NO À 3 and 1.25 mM NH 4 NO 3 . The biomass and nitrogen concentration were measured for each line (n = 4 plants) under each N treatment after 3-week treatment.
For root 15 N uptake experiment, new rice seedlings were grown in 1 mM NH þ 4 for 3 weeks and then were nitrogen starved for 1 week before 15  was used, and the 15 N influx rate was calculated following the method in Tang et al. (2012).

Statistical analysis
The single-factor analysis of variance (ANOVA) and Tukey's test data analysis were applied in our data statistical analysis (Chen et al. (2016).

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article:  Figure S3 Ratio of 15 NH þ 4 to 15 NO À 3 influx in wild-type and pOsNAR2.1:OsNAR2.1 transgenic lines in 1.25 mM NH 4 NO 3 . WT and transgenic seedlings were grown in 1 mM NH þ 4 for 3 weeks and nitrogen starved for 1 week. 15 NH þ 4 or 15 NO À 3 influx was measured at (a) 1.25 mM 15 NH 4 NO 3 or (b) 1.25 mM NH 4 15 NO 3 for 5 min. DW, dry weight. (c) The 15 NH þ 4 to 15 NO À 3 influx ratios with 1.25 mM NH 4 NO 3 in the roots of wild-type and pOsNAR2.1: OsNAR2.1 lines (Ox1, Ox2, and Ox3) are presented. Error bars: SE (n = 4 plants). The different letters indicate a significant difference between the transgenic line and the WT (P < 0.05, one-way ANOVA). Figure S4 Expression ratios of OsNRT2.1 to OsNAR2.1 in culms of transgenic lines and wild type. The pOsNAR2.1:OsNRT2.1 lines (O6, O7 and O8), pOsNAR2.1:OsNAR2.1 lines (Ox1, Ox2, and Ox3) and wild type are presented. Table S1 Primers for amplification OsNAR2.1 ORF. Table S2 Primers used for qRT-PCR. Table S3 Comparison of dry weight, grain yield, and ANUE between the wild-type and pOsNAR2.1:OsNAR2.1 transgenic lines in the T2-T4 generations. n = 3 plots for each mean. The different letters indicate a significant difference between the transgenic line and the WT (P < 0.05, one-way ANOVA). Table S4 Increased nitrogen-use efficiency in pOsNAR2.1: OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines relative to wild type. Statistical analysis of data from T4 generation; n = 3 for each mean. The different letters indicate a significant difference between the transgenic line and the WT (P < 0.05, one-way ANOVA).