Overexpression of Nitrate Transporter 1/Peptide Gene OsNPF7.6 Increases Rice Yield and Nitrogen Use Efficiency

Overuse of nitrogen fertilizer in fields has raised production costs, and caused environmental problems. Improving nitrogen use efficiency (NUE) of rice is essential for sustainable agriculture. Here we report the cloning, characterization and roles for rice of OsNPF7.6, a member of the nitrate transporter 1/peptide transporter family (NPF). The OsNPF7.6 protein is located in the plasma membrane, expressed in each tissue at all stages and is significantly regulated by nitrate in rice. Our study shows that the overexpression of OsNPF7.6 can increase the nitrate uptake rate of rice. Additionally, field experiments showed that OsNPF7.6 overexpression increased the total tiller number per plant and the grain weight per panicle, thereby improving grain yield and agronomic NUE in rice. Thus, OsNPF7.6 can be applied to be a novel target gene for breeding rice varieties with high NUE, and provide a reference for breeding higher yielding rice.


Introduction
Nitrogen is one of the most crucial macronutrients for crops and a sufficient nitrogen supply can ensure plant development and high yield [1][2][3][4]. Nitrogen fertilization is the key factor to improve crop yield and reduce hunger worldwide [5]. In an effort to boost productivity, farmers typically apply an excessive amount of nitrogen fertilizer, but less than 50% of it is actually absorbed by the plants, and the remnant ends up polluting the environment [6]. Improving rice (Oryza sativa L.) nitrogen use efficiency (NUE) offers an effective and practical way to increase grain yield and solve the environmental issues caused by excessive nitrogen application. NO 3 − and NH 4 + are two main sources of inorganic nitrogen during crop growth. Rice makes up about 25% of all calories consumed globally [7][8][9]. Conventionally grown rice is submerged in water, which inhibits nitrification and makes NH 4 + the most significant inorganic nitrogen in rhizosphere soil. Based on this, rice is regarded as an ammoniumpreferring plant [10]. On the other hand, rice can release some oxygen produced by photosynthesis to the rhizosphere through roots due to its mature aerial tissue [11]. Because oxygen promotes the growth and propagation of nitrifying bacteria, partial NH 4 + in the rhizosphere can be converted to NO 3 − [12]. Consequently, during rice growth, its root system is under the mixed nutrition of NO 3 − and NH 4 + [13]. Previous studies have established that the prominent families of transporters involved in nitrate-nitrogen uptake and transport in plants include nitrate transporter1/peptide transporter family (NPF) and nitrate transporter 2 (NRT2). The NRT2 family encodes high-affinity NO 3 − transport proteins, which have essential functions in response to lownitrogen (NO 3 − ) environments [14]. Among the members of NRT2s, OsNRT2.1, OsNRT2.2 and OsNRT2.3a require the assistance of OsNAR2.1 to complete the transport of nitrate [15]. The interaction of OsNAR2.1 with OsNIT1 and OsNIT2, affects the uptake of nitrate and ammonium by rice roots [16]. OsNRT2.3 yields two transcripts including OsNRT2.3a and OsNRT2.3b [17]. OsNRT2.3a plays a role in the long-distance transport of nitrate from roots to aerial parts [18], while OsNRT2.3b can elevate the pH buffering ability of rice [19]. Compared with all other NRT2 genes, OsNRT2.4 is a dual-affinity nitrate transporter, which is necessary for nitrate-regulation of root and shoot growth of rice [20].
The NPF family includes two genes, NRT1 (nitrate transporter1) and PTR (peptide transporter). The former is generally believed to encode the low-affinity NO 3 − transporter, and the latter encodes the oligopeptide transporter [21]. Due to the high sequence similarity of the two members, in the same branch of evolutionary relationship, the NRT1/PTR gene is uniformly named NPF (NRT1/PTR family) [22]. There are many members of the NPF family, and greater focus and thorough investigation have been given to the NPF genes' roles. Numerous studies have demonstrated that NPF genes are widely implicated in the uptake and utilization of nitrogen in plants and have essential roles and applications in improving nitrogen utilization and enhancing yield-related traits [23][24][25][26][27][28]. With the use of the homologous sequence of Arabidopsis AtNRT1.1, OsNRT1.1 has been cloned in rice [29]. Two varying splicing forms including OsNRT1.1a and OsNRT1.1b are shown in OsNRT1.1. OsNRT1.1a only acts under high-nitrogen situations, while OsNRT1.1b is found to facilitate nitrogen uptake under low-nitrogen situations [30]. Moreover, overexpression of OsNRT1.1A could increase NUE [25]. A single-base variation of NRT1.1B explains the difference in NUE between indica and japonica subspecies [26]. The NADH/NADPH-dependent nitrate reductase gene OsNR2 can interact with OsNRT1.1b, and promote the uptake of nitrate in indica rice [31]. OsNPF6.1 serves as a dual-affinity nitrate transporter, the transcription factor OsNAC42 could activate OsNPF6.1, which can thus elevate rice NUE [32].
Here, we report that the nitrate transporter OsNPF7.6 is induced by nitrate, and overexpression of OsNPF7.6 increases nitrate uptake rate, yield, as well as NUE in rice.

The Construction of pUbi: OsNPF7.6 Transgenic Rice
The ORF of OsNPF7.6 (LOC_Os04g50930) was amplified from the full-length cDNA of rice japonica cv. 'Nipponbare', primer pairs were shown in Supplementary Table S1. PrimeSTAR HS DNA Polymerase (TaKaRa Biotechnology Co., Ltd., Dalian, China) was employed during the polymerase chain reaction (PCR). The parameters for PCR were 95 • C for 5 min, 94 • C for 30 s, 56 • C for 1 min (30 cycles), and 72 • C for 10 min. The PCR products were then ligated into the pMD19-T vector independently. The correct sequence fragment was ligated to the expression vector pTCK303 to obtain the pUbi:OsNPF7.6 fusion vector. According to the previous description, the vector was introduced into the Agrobacterium tumefaciens strain, EHA105 via electroporation and subsequently transformed into Zhonghua 11 rice (WT) [3].

Identification of Positive Seedlings of Transgenic Lines
Through the application of the CATB method, the genomic DNA of T 2 generation transgenic and wild-type plants was extracted [3] for PCR amplification of hygromycin fragments to identify positive seedlings, with primer pairs shown in Supplementary Table S2. The parameters for PCR were 95 • C for 3 min, 95 • C for 30 s, 55 • C for 30 s (30 cycles), and 72 • C for 10 min. Gel electrophoresis was used to show PCR products.

qRT-PCR
Following the previously reported method, plant genomic RNA extraction and gene expression analysis was conducted [3]. The primers for qRT-PCR were demonstrated in Table S3.

Subcellular Localization of OsNPF7.6
The coding sequence of OsNPF7.6 for subcellular localization was amplified using the pMD19-T vector and ligated into pSAT6A-GFP [33] with the correct direction. The OsNPF7.6-GFP vector was transformed into Arabidopsis mesophyll protoplasts and identified by a laser scanning microscope (LSM410; Carl Zeiss, Jena, Germany). Plasma membrane dye FMTM4-64FX (Invitrogen, Life Technologies, Carlsbad, CA, USA) was used as marker.

Plant Growth Conditions
The pUbi:OsNPF7.6 transgenic plants and WT were planted in three plots, which had 180 kg N/ha. The plot size was 2 m × 2.5 m, and seedlings were planted in a 10 × 10 array. During the stage of flowering and maturation, the samples of T 3 generation were collected for further analysis.
The total nitrogen was analyzed as stated by Chen et al. [3]. According to our previously reported method, total nitrogen accumulation at the anthesis stage, total nitrogen accumulation at maturity stage, grain nitrogen accumulation at maturity, nitrogen translocation, nitrogen translocation efficiency, post-anthesis nitrogen uptake, nitrogen harvest index, as well as agronomic NUE were calculated [3]. Briefly, the calculation formulae are as follows. Nitrogen translocation (g/m 2 ) = total nitrogen accumulation at anthesis − (total nitrogen accumulation at maturity − grain nitrogen accumulation at maturity); nitrogen translocation efficiency (%) = (nitrogen translocation/total nitrogen accumulation at anthesis) × 100%; post-anthesis nitrogen uptake (g/m 2 ) = total nitrogen accumulation at maturity − total nitrogen accumulation at anthesis; the nitrogen harvest index (%) = grain nitrogen accumulation at maturity/total nitrogen accumulation at maturity. In addition, agronomic NUE (g/g) = grain yield/N supply.

Determination of 15 N Influx Rates in Roots
Rice seedlings were planted in 1 mM NH 4 + within a period of 3 weeks and subsequently subject to nitrogen starvation for 3 days for the root 15 N uptake experiment. Initially, the plants were rinsed with 0.1 mM CaSO 4 for 1 min, which were later transferred to either 0.5 mM 15 NO 3 − or 2 mM 15 NO 3 − (atom % 15 N: 99%) solution for 5 min. Then rinsed the plants once more with 0.1 mM CaSO 4 for 1 min. Based on our previously reported method, 15 N influx rates were calculated [3].

Statistical Analysis
In order to perform statistical analysis, we used the single factor analysis of variance (ANOVA) and Tukey's test in this study. The IBM SPSS Statistics 20 software (SPSS Inc., Chicago, IL, USA) was adopted for all statistical analysis.

OsNPF7.6 Was Induced to Be Expressed by Nitrate and Localizes in the Plasma Membrane
The OsNPF7.6 was localized on chromosome 4 with six exons and five introns ( Figure S1). Under different nitrogen source treatments, the OsNPF7.6 had the highest expression under 2.5 mM NO 3 − conditions, followed by 0.5 mM NO 3 − conditions, and the weakest expression under 2.5 mM NH 4 + conditions ( Figure 1A). The expression pattern of different sites at different fertility stages revealed that OsNPF7.6 was constitutively expressed in rice at all sites at all times ( Figure 1B). OsNPF7.6 had 10 transmembrane structures ( Figure S2). In order to detect the subcellular localization of the OsNPF7.6 protein, an expression vector containing the OsNPF7.6-GFP fusion gene initiated by cauliflower mosaic virus 35S promoter was Life 2022, 12, 1981 4 of 10 constructed and transformed into Arabidopsis thaliana protoplasts. After overnight transformation, the expression of OsNPF7.6-GFP in protoplasts was observed by confocal microscopy. OsNPF7.6-GFP fluorescence was completely merged with plasma membrane dye FM TM 4-64FX ( Figure 1C), and OsNPF7.6 was identified as a plasma membrane localization protein.
under 2.5 mM NO3 − conditions, followed by 0.5 mM NO3 − conditions, and the weakest expression under 2.5 mM NH4 + conditions ( Figure 1A). The expression pattern of different sites at different fertility stages revealed that OsNPF7.6 was constitutively expressed in rice at all sites at all times ( Figure 1B). OsNPF7.6 had 10 transmembrane structures ( Figure  S2). In order to detect the subcellular localization of the OsNPF7.6 protein, an expression vector containing the OsNPF7.6-GFP fusion gene initiated by cauliflower mosaic virus 35S promoter was constructed and transformed into Arabidopsis thaliana protoplasts. After overnight transformation, the expression of OsNPF7.6-GFP in protoplasts was observed by confocal microscopy. OsNPF7.6-GFP fluorescence was completely merged with plasma membrane dye FM TM 4-64FX ( Figure 1C), and OsNPF7.6 was identified as a plasma membrane localization protein.

Acquisition of OsNPF7.6 Overexpression Lines
The pUbi: OsNPF7.6 fusion vector was introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and subsequently transformed into Zhonghua 11. A total of 72 transgenic seedlings from 10 strains were available, and 32 transgenic seedlings from eight strains survived after 7 days of screening with 200 mg/L hygromycin solution. The DNA of these 32 strains was extracted, the hygromycin fragments were amplified by

Acquisition of OsNPF7.6 Overexpression Lines
The pUbi: OsNPF7.6 fusion vector was introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and subsequently transformed into Zhonghua 11. A total of 72 transgenic seedlings from 10 strains were available, and 32 transgenic seedlings from eight strains survived after 7 days of screening with 200 mg/L hygromycin solution. The DNA of these 32 strains was extracted, the hygromycin fragments were amplified by PCR, and the target bands were detectable in all 32 transgenic seedlings. The positive rate of transgenic seedlings was 44.4%.
Three transgenic strains were selected for T2 generation (Figure 2A), and then positive seedlings were verified by PCR amplification of hygromycin fragments ( Figure 2B). qRT-PCR revealed that the expression level of OsNPF7.6 increased about 7-fold in the shoot and root of transgenic lines relative to WT ( Figure 2C). PCR, and the target bands were detectable in all 32 transgenic seedlings. The positive rate of transgenic seedlings was 44.4%.
Three transgenic strains were selected for T2 generation (Figure 2A), and then positive seedlings were verified by PCR amplification of hygromycin fragments ( Figure 2B). qRT-PCR revealed that the expression level of OsNPF7.6 increased about 7-fold in the shoot and root of transgenic lines relative to WT ( Figure 2C).

Expression of OsNPF7.6 Increased 15 NO3 − Influx Rates in Rice
To evaluate the effect of pUbi: OsNPF7.6 expression on root NO3 − influx into intact plants, seedlings of transgenic lines and WT were exposed to 0.5 mM 15 NO3 − or 2.5 mM 15 NO3 − for 5 min for short-term NO3 − and NH4 + absorption. The influx rates of 15

Expression of OsNPF7.6 Increased 15 NO 3 − Influx Rates in Rice
To evaluate the effect of pUbi: OsNPF7.6 expression on root NO 3 − influx into intact plants, seedlings of transgenic lines and WT were exposed to 0.5 mM 15

Effects on Agronomic Traits of Rice after Overexpression of OsNPF7.6
In order to analyze the effects of OsNPF7.6 overexpression on rice growth and yield the current work attempted to investigate the agronomic traits of the transgenic strains The plant height, seed setting rate, and 1000-grain weight of the OsNPF7.6 overexpression

Effects on Agronomic Traits of Rice after Overexpression of OsNPF7.6
In order to analyze the effects of OsNPF7.6 overexpression on rice growth and yield, the current work attempted to investigate the agronomic traits of the transgenic strains. The plant height, seed setting rate, and 1000-grain weight of the OsNPF7.6 overexpression strains were not substantially different from WT (p > 0.05, Figure 4A,D,F). Compared to WT, the total tiller number per plant, grain weight per panicle, as well as grain number per panicle of OsNPF7.6 overexpression strains increased by 14.8%, 13.3%, and 10.9%, respectively ( Figure 4B,C,E). Ultimately, the OsNPF7.6 overexpression strains improved grain production and dry weight by 21.0% and 21.7%, respectively ( Figure 4G,H). WT and transgenic seedlings were grown in 1 mM NH4 + for 3 weeks and then were treated with nitrogen starvation for 3 days. 15 N influx rates were measured at (A) 0.5 mM 15 NO3 − or (B) 2.5 mM NO3 − for 5 min. DW, dry weight. Error bars: SE (n = 4). The varying letters (a, b) suggest an obvious change between the transgenic line and the WT. (p < 0.05, one-way ANOVA).

Effects on Agronomic Traits of Rice after Overexpression of OsNPF7.6
In order to analyze the effects of OsNPF7.6 overexpression on rice growth and yield, the current work attempted to investigate the agronomic traits of the transgenic strains. The plant height, seed setting rate, and 1000-grain weight of the OsNPF7.6 overexpression strains were not substantially different from WT (p > 0.05, Figure 4A,D,F). Compared to WT, the total tiller number per plant, grain weight per panicle, as well as grain number per panicle of OsNPF7.6 overexpression strains increased by 14.8%, 13.3%, and 10.9%, respectively ( Figure 4B,C,E). Ultimately, the OsNPF7.6 overexpression strains improved grain production and dry weight by 21.0% and 21.7%, respectively ( Figure 4G,H).

Effects on Nitrogen Utilization in Rice after Expression of OsNPF7.6
The dry matter production in plants per unit of applied nitrogen is also known as "nutrient utilization efficiency" and refers to the transfer of nitrogen to plants organs and

Effects on Nitrogen Utilization in Rice after Expression of OsNPF7.6
The dry matter production in plants per unit of applied nitrogen is also known as "nutrient utilization efficiency" and refers to the transfer of nitrogen to plants organs and yield [5]. In this study, we tested the total nitrogen content of the T 3 generation transgenic lines during the anthesis and maturity stages. At the anthesis stage, total nitrogen accumulated mostly in the leaf and culm. Compared with WT, we found that at the anthesis stage, the total nitrogen content of panicles, leaves, and culms in transgenic lines increased by 16.9%, 13.2%, and 16.3%, respectively ( Figure 5A). With the reproductive growth, the proportion of nitrogen content in spike to total nitrogen content of plant begins to increase. The results showed that the total nitrogen content of panicles, leaves, and culms in transgenic lines elevated by 18.7%%, 21.1%, and 24.1%, separately at maturity stage ( Figure 5B).
For the NUE, no obvious difference in nitrogen translocation efficiency between transgenic lines and WT was found ( Figure 5C). Compared with WT, the post-anthesis nitrogen uptake of transgenic lines increased by about 59.9% ( Figure 5D). There was no evident divergence in the nitrogen harvest index between transgenic lines and WT ( Figure 6A). The agronomic NUE of transgenic lines elevated by about 20.9% ( Figure 6B).
Life 2022, 12, 1981 7 of 10 mulated mostly in the leaf and culm. Compared with WT, we found that at the anthesis stage, the total nitrogen content of panicles, leaves, and culms in transgenic lines increased by 16.9%, 13.2%, and 16.3%, respectively ( Figure 5A). With the reproductive growth, the proportion of nitrogen content in spike to total nitrogen content of plant begins to increase. The results showed that the total nitrogen content of panicles, leaves, and culms in transgenic lines elevated by 18.7%%, 21.1%, and 24.1%, separately at maturity stage ( Figure  5B). For the NUE, no obvious difference in nitrogen translocation efficiency between transgenic lines and WT was found ( Figure 5C). Compared with WT, the post-anthesis nitrogen uptake of transgenic lines increased by about 59.9% ( Figure 5D). There was no evident divergence in the nitrogen harvest index between transgenic lines and WT ( Figure  6A). The agronomic NUE of transgenic lines elevated by about 20.9% ( Figure 6B).

Discussion
Rice contains 93 NPF family members [22]. As the necessary macronutrient for plant growth and development, nitrogen can limit crop productivity when it is scarce in the field [5]. Increasing the uptake and accumulation of nitrogen is the main way to improve

Discussion
Rice contains 93 NPF family members [22]. As the necessary macronutrient for plant growth and development, nitrogen can limit crop productivity when it is scarce in the field [5]. Increasing the uptake and accumulation of nitrogen is the main way to improve the NUE and yield of rice.
Nitrogen is crucial for crop growth and development, notably for the creation of tillers, which belongs to one of the most crucial elements determining grain yield in rice [25]. OsNPF7.1, OsNPF7.2, OsNPF7.3, OsNPF7.4, and OsNPF7.7 are all members of the NFP7 subfamily that contribute to the development of rice tiller buds, which in turn influences rice tillering. OsNPF7.1 and OsNPF7.4 are two of them that, in response to different nitrogen supply levels, exhibit opposing expression patterns in tiller buds and have enhanced and inhibited effects on tiller bud growth and development, respectively. [27]. OsNPF7.2 could control tiller bud growth and root development through the regulation of cytokinin and cell cycle in plants [25,35,36]. OsNPF7.3 overexpression promotes growth, tiller number, grain yield, and grain nitrogen content [37,38]. We also found that overexpression of OsNPF7.6 increased the total tiller number per plant in rice ( Figure 4B). Compared with WT, the grain weight and grain number of per panicle increased by 13.3% and 10.9%, respectively, in the OsNPF7.6 overexpression strains ( Figure 4C,E). Consequently, the grain yield and dry weight of the OsNPF7.6 overexpression strains increased by 21.0% and 21.7%, respectively ( Figure 4G,H).
A series of studies have shown that nitrate is necessary for nitrogen nutrition in rice. It is found that nitrate allocation and the divergent nitrate use efficiency between indica and japonica rice can be mediated by the nitrate transporter OsNPF7.9 [39]. OsNPF6.1 HapB is the rare natural allele, which can control nitrate uptake and NUE [32]. OsNRT1.1A/OsNPF6.3 exerts the vital function in nitrogen uptake, transport, and assimilation [25]. In addition, the overexpression of OsNAR2.1 is shown to improve rice yield and NUE [2]. Following the introgression of both the indica OsNRT1.1B and OsNR2 alleles into japonica accessions, the grain yield and NUE could be deeply enhanced [31]. We also found that overexpression of OsNPF7.6 can increase the agronomic NUE in rice ( Figure 6B). Although the nitrogen accumulation of OsNPF7.6 overexpression lines increased significantly ( Figure 5A,B), there existed no obvious difference in nitrogen translocation efficiency and nitrogen harvest index between transgenic lines and WT (Figures 5C and 6A). Thus, the overexpression of OsNPF7.6 does not affect nitrogen distribution in rice.
In conclusion, OsNPF7.6 is a nitrate transporter located in the plasma membrane. Overexpression of OsNPF7.6 can increase the nitrate uptake rate of rice. Field experiments showed that overexpression of OsNPF7.6 elevated the total tiller number per plant and Life 2022, 12, 1981 9 of 10 the grain weight per panicle, which could thus enhance grain yield and agronomic NUE in rice.