Response of Root Development and Nutrient Uptake of Two Chinese Cultivars of Hybrid Rice to N and P Fertilization in Sichuan Province, China

Chemical fertilization helped modern agriculture in enhancing grain yield to overcome food security. The response of chemical fertilization for higher hybrid rice production is highly dependent on optimal fertilization management in paddy elds. To assess such responses, the current research examined the yield, root growth, and expression of related genes responsible for stress metabolism of N and P in two hybrid-rice cultivars Deyou4727 (D47) and Yixiangyou2115 (Y21). The experiment followed four N (N 0 , N 60 , N 120, and N 180 kg/ha) and P (P 0 , P 60 , P 90 , and P 120 kg/ha) fertilizer levels. The D47 was more sensitive toward nitrogen application, which resulted in comparatively higher biomass and yield, while Y21 was more sensitive toward phosphorus application. The grain yield was more sensitive to nitrogen application in D47 but not in Y21. Our ndings were corroborated by gene expression studies of glutamine synthetase OsGS1;1 and OsGS1;2 and phosphate starvation-related genes PHR1 and SPX, which also concluded D47 response sensitivity towards N application and Y21 response sensitivity toward P application. In the case of root numbers, D47 was less sensitive in low nitrogen application N 0 -N 60 , but the overall nutrient response difference was signicantly higher due to the deep rooting system as compare to Y21. The higher yield, low N and P uptake, and versatile root growth of D47 make it suitable to reduce unproductive usage of N and P from paddy elds, improving hybrid rice productivity, and environmental safety in the Sichuan basin area of China.


Introduction
The extensive use of chemical fertilizers in current agricultural practice is detrimental to the environment and reduces soil quality and crop production in the long run. Thus, sustainable solutions to the improvement of crop production are an important current challenge for global agriculture (Iqbal et al., 2019). According to FAO (2019), the annual world consumption of the three main fertilizer nutrients, nitrogen (N), phosphorus applied as phosphate (P 2 O 5 ), and potassium applied as potash (K 2 O), reached 185 million tons (total N, P 2 O 5 , and K 2 O) in 2016 and is expected to top 200 million tons annually by 2022. As the consumption increases, it will prompt the production of commercial fertilizers, intermediates, and raw materials, which will signi cantly contribute to global carbon emissions. China is among the top ve markets of commercial fertilizer production and consumption; according to FAO (2015), China consumed 27.3 million tons N and 15 million tons P fertilizer for rice and wheat fertilization in 2014.
It has been estimated that only about 1/3 of the applied nitrogen fertilizer is used by crops, with the remaining 2/3 stay behind in the environment, accounting for ~ 16 billion USD worth loss of N fertilizer worldwide on annual basis (Raun and Johnson, 1999). Unassimilated N fertilizer leaches from farming and creates environmental damage, including nitrate accumulation, water pollution and eutrophication (Tahir and Rasheed, 2008), and soil deterioration (Iqbal et al., 2019). In order to avoid excessive N fertilizer usage, current research in agronomy is focused on developing strategies for enhancing the nitrogen use e ciency of crop plants (Fageria and Raun and Johnson, 1999;Sharma et al., 2018). In cereal crops, enhancing grain yield without increasing fertilizer input remains one of the most important challenges (Mueller et al., 2012).
Problems related to the use of phosphate fertilizer include both leaching of excess phosphate into the environment (Chen et al., 2008), and the prospective depletion of nite reserves of phosphate rock, which represents the raw material for the production of fertilizer (Baker et al., 2015). Additionally, cereals and some other food crops convert a signi cant part of assimilated phosphate into phytic acid, which has no nutritive value and even acts as an antinutrient (Gupta et al., 2015). Agronomic measures for the prevention of phosphate leaks into the environment have proven relatively ine cient and transgenic approaches have been suggested for improving phosphate uptake and limiting the synthesis of phytic acid for improved phosphate use e ciency (Kopriva and Chu, 2018).
Recently, an array of various N and P management strategies have been launched for gradual or spatially targeted release of commercial fertilizers (Iqbal et al., 2019), they are combining with organic manure (Ye et al., 2020) and vermicomposting (Mondal et al., 2017), the use of biochar (Ayaz et al., 2021) or delivery of fertilizers to target plants through the use of nanoparticles (Kah et al., 2019). The use of these strategies has however been so far limited because they are either insu ciently e cient, costly, or labor-intensive (Iqbal et al., 2019).
According to FAO data for 2019 (http://www.fao.org/faostat/en/#data/QC), rice (Oryza sativa L.) is the world's third most important crop after maize and wheat, in terms of both harvested area (162 million hectares worldwide) and annual production (755 million tonnes). Furthermore, for rice fertilization in China, a 75% higher (180 kg/ha) nitrogen input is used than the world average, whereas in the Guangdong province of Southern China fertilization rates as high as 250 kg/ha are applied (Wehmeyer et al., 2020). Among the major food crops, rice accounts for the highest amounts of wasted nitrogen fertilizer (Anas et al., 2020). Furthermore, optimizing the right measure of phosphate fertilizer to deliver su cient phosphorus to rice crops without risking environmental damage has proven as a major challenge for rice cultivation, for instance in West African countries (Dogbe et al., 2015). More than 3/4 of the applied phosphate fertilizer is wasted and remains in the environment, as only about 1/4 is taken up by rice crops . Fertilization of rice paddy elds represents an important target for decreasing large-scale consumption of commercial fertilizers, reducing the environmental damage from fertilizer use, and at the same time cutting the costs for fertilizer purchase and transport. Thus, hybrid rice varieties, which show enhanced tolerance to abiotic stresses and produce high yields in low nitrogen and phosphorus fertilization, represent a promising perspective for reducing the use of chemical fertilizers for rice cultivation (Chaturvedi, 2005). Like other cereal crops hybrid rice varieties have different fertilization patterns, highly depending on their genetic nature (Sharma et al., 2018;Xie, X et al., 2019). For such hybrid rice types, effective fertilization management includes selecting the proportion of fertilizer, the source of fertilizer, the timing of fertilizer application, and the combination of fertilizers that match the needs of the crop to maximize fertilizer utilization e ciency, optimize crop production, and minimize the negative impact of fertilizer on the environment (Malhi et al., 2001).
This study's main objectives were to estimate the impact of low and high nitrogen and phosphorus input on the yield, nutrient uptake, use e ciency and root growth, of hybrid rice varieties Deyou4727 and Yixiangyou2115 under different N and P levels. Also, the expression levels of N and P metabolism-related genes OsGS1;1, OsGS1;2, PHR1, and SPX were quanti ed to assess the molecular response of the cultivars to different nutrient concentrations. The results provide a basis for planning measures to reduce the losses of N and P in paddy elds and for choosing a well-performing variety to improve rice yield in the Sichuan Basin area of China and other regions with similar environments.

Study Area
The experiments were carried out in the laboratory and eld at the South West University of Science & Technology in Mianyang, Sichuan Province, China, in 2019. The soil of this region is clay-loamy soil, with a bulk density of 1.29 g/cm 3 and organic matter content of 28.6 g/kg, while total Nitrogen, Phosphorus, and Potassium content were 1.68, 0.37, and 1.86 g/kg respectively. The major food crops cultivated in this region are rice and oilseed rape, which form a paddy-dryland rotation system in this area, with oilseed rape cultivation occurring from late September to April, and rice cultivation from April to September.

Experimental Design
Field experiment treatment sets were carried out for two rice varieties Deyou4727 (D47) and Yixiangyou2115 (Y21).
Rice seeds were cultivated from March-September 2019. Field design was carried out as a randomized complete block design, each plot was 3 m×3.5 m with 0.5 m corridors. When the seedlings were around 7-8 cm long, eld plots were treated. For the N application, four N application levels were used, i.e., N 0 , N 60 , N 120 , N 180 [kg/ha]. To ensure that nutrients other than N would not limit rice growth, 90 kg P 2 O 5 kg/ha was applied to each plot. For the P application, four P application levels were used, i.e., P 0 , P 60 , P 90 , P 120 [kg/ha]. To ensure that nutrients other than P would not limit rice growth, 120 kg N kg/ha was applied to each plot. Each treatment was replicated three times. The plant sampling was done at four time points after treatments, wrapped in aluminum foil, frozen in liquid nitrogen, and stored at -80°C until further analysis.

Samples Collection
Plant samples of all treatments were collected at four time points (10, 25, 40, 55 days after treatment) in 2019. Fresh samples were used for morphological measurements and gene expression studies. The soil samples for N and P uptake calculation were transported to the lab in ice boxes. Fresh samples were used to determine initial nutrient concentrations.

Measurements
At maturity, plant samples were separated into straw, leaves, and panicles for dry weight determination after ovendrying to constant weight at 70°C. Plant roots were counted according to Gu, et al., (2017). Grain yield was measured from a 5 m 2 area from the center of each plot at the maturity stage. Panicles were placed into bags and labeled to determine yield and yield components (Qi et al., 2020). Grain and panicles were separated. Filled and empty panicles were separated and counted, grains were weighed. The seed setting rate was calculated according to Xiang et al., (2019). Yield component data (1000-grain weight, the number of panicles, and percentage of lled grains) from all treatments were determined according to Fageria, (2009).
Total nitrogen content in the soil was determined by the Kjeldahl method as described by Okalebo et al., (2002). Available phosphorus was determined by the Olsen method following the procedure described by Juo, (1978).
Crop Response to N and P Inputs N and P Use E ciencies N and P use e ciency parameters were calculated based on the grain yield and N/P accumulation in plots treated at different N and P rates. The de nitions are as follows: Where GY +N/P is the grain yield of the plots that received N and P fertilizer, GY N/P0 is the grain yield in the N0 and P0 plots, FN is the amount of N and P fertilizer applied, TN +N/P is the total N and P accumulation in the plots that received N and P fertilizer at maturity, and TN N/P0 is the total N and P accumulation in the N0 and P0 plots at maturity.

qPCR Analysis
Total RNA from ag leaves of mature rice plants was isolated using the RNeasy Plant Mini Kit (Qiagen) and reversely transcribed using Omniscript RT Kit (Qiagen). Quantitative real-time PCR (qPCR) was performed using the SYBR Premix Ex Taq (TaKaRa, Dalian, China) and the CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR mix was composed of 5μl SYBR Premix Ex Taq, cDNA corresponding to 30 ng RNA, 0.3 μl of each F and R primer (10 mM), and PCR grade water up to the nal volume of 10 μl. The primer sequences are available in Supplementary Table S1. The incubation temperature of the reaction was as follows: 1) denaturation at a cycle of 95°C for 3:00 min; 2) ampli cation: 40 cycles at 95°C for 30 sec, 60°C for 30 sec, 72°C for 30 sec; 3) nal elongation 72°C for 3 min, and 4) melting curve analysis (65 to 95°C). Each sample was analyzed in triplicate, and the relative expression levels were calculated relative to control (N 0 or P 0 treatment) using the 2-ΔΔCt comparative CT method (Schmittgen and Livak, 2008).

Statistical Analysis
Origin 19.0 and Excel 2016 statistical software were used to analyze the experimental data. The correlation analysis between grain yield and N/P accumukation was done using ggpubr R package. Each treatment value is expressed as mean ± standard deviation (SD) of 3-6 biological replicates. The differences were determined by Student's T-test at P< 0.05(*), 0.01(**), or 0.001(***).

Yield and Physiological Traits Responses
The effect of nutrient and genotype on hybrid rice grain yield and effective panicles were signi cant (P > 0.05). Both rice cultivars D47 and Y21 showed a maximum yield of rice grains in high N treatment N 180 and the lowest yield observed in no-N treatment N 0 . The cultivar D47 showed a maximum yield of 9.17 t/ha, in high N treatment N 180 that was 5% higher than Y21. The response of both cultivars toward N application was signi cantly different. D47 was more sensitive to N application as compare to Y21, as it showed a signi cantly higher difference of yield response between N 0 -N 60 , N 60 -N 120 , N 120 -N 180 (Table 1). Yield response was positively correlated with effective panicles in D47 and Y21, which resulted in yield difference. Y21 was less sensitive to N application from N 0 -N 60 and N 120 -N 180 .
The rice type D47 overall showed signi cantly higher grain yield in all treatments, more speci cally under different nitrogen treatments it showed 3%, 3%, 4%, and 5% higher grain yield in N 0 , N 60 , N 120 , and N 180 treatments respectively, as compared to Y21.
Among different P treatments, both rice types favored moderate phosphorus concentration P 90 . Likewise, in different phosphorus treatments, D47 showed, 6%, 10%, 4%, and 1% higher grain yield in P 0 , P 60 , P 90, and P 120 treatments respectively. The response of P application for both cultivar yields were different. D47 showed higher sensitivity of yield response from P 0 -P 60 , P 60 -P 90 and P 90 -P 120 , as compare to Y21, which showed maximum sensitivity between P 60 -P 90 . The P application response was equally observed for effective panicles that resulted in yield differences respectively.
Similarly, effective panicle signi cantly increased with increasing N and P treatments. Higher nutrient input caused higher effective panicles in both rice types, and they followed N 180 >N 120 > N 60 >N 0 , and P 120 >P 90 >P 60 >P 0 trend for Nitrogen and phosphorus respectively. Unlike EP, seed setting rate, grains per panicle, and 1000 grain weight were less affected by N and P supply (Table 1).

Correlation Analyses of Grain Yield and N/P Accumulation
A Pearson's correlation was computed to assess the relationship between grain yield and nutrient accumulation for both rice types (Fig. 1). Mostly negative correlations between low or high nutrient accumulation and yield were found in different nitrogen and phosphorus treatments. A high increase in nutrient uptake was negatively correlated with the amount of yield output. More speci cally in the D47 in N 180 treatment, was positively correlated (R = 0.69) while in Y21 N 60 and N 120 were more positively correlated with R values of (R = 0.76) and (R = 1) respectively.
Similarly in the case of phosphorus accumulation D47 showed a positive correlation in all treatments up to P 120 .
The correlation was highest up to P 60 (R=1), while it slightly decreased in P 60 and P 90 with R values near 0.94 and 0.97 respectively. Unlike D47, Y21 showed a negative correlation (R= -0.76), (R= -0.94) at P 60 and P 90 respectively, however, it had a good positive correlation at P 120 with R-value near 0.99.

Plant Biomass and Nutrient Accumulation in Hybrid Rice
Total biomass at maturity signi cantly (P < 0.05) increased with N application rate and differed in two hybrid rice cultivars ( Table 2). The highest plant biomass, N, and P accumulation were found in high N and P treatments for both hybrid rice varieties. Overall D47 showed 4%, 9%, 3%, and 6% higher biomass production than Y21 in N 0 , N 60 , N 120 , and N 180 treatments respectively. A similar trend was found for different P treatments, in which D47 showed 10%, 7%, 3%, and 7% higher biomass production than Y21 in P 0 , P 60 , P 90, and P 120 treatments respectively. Like biomass, N and P also followed higher accumulation in high treatments and low accumulation in low treatments. N accumulation followed N 180 >N 120 > N 60 >N 0 , and phosphorus accumulation followed the P 120 >P 90 >P 60 >P 0 trend in both rice types, at constant environmental factors. D47 accumulated 5-10% greater total biomass compared to Y21 at all fertilizer treatments ( Table 2). Accumulation of nitrogen biomass was similar for both cultivars at all treatments, however, D47 accumulated 10-15% more phosphorus biomass on most treatments (Table 2). N/P accumulation, biomass and N/P concentration in both cultivars were sensitive to N and P application. Like yield and yield parameters, N/P accumulation response and biomass were higher in D47. It showed a higher sensitive response between N 0 -N 60 , N 60 -N 120 , N 120 -N 180 . Unlike N/P accumulation and biomass, D47 showed less sensitivity in N/P concentration. That makes it more nitrogen e cient, as compared to Y21. While Y21 showed less response sensitivity in biomass and N/P concentration while N/P accumulation was signi cantly more sensitive between N 0 -N 60 , N 60 -N 120 , N 120 -N 180 .
Nutrient Use E ciency Nutrient uptake and nutrient use e ciency varied among treatments and hybrid rice varieties. The AE, PE, and RE signi cantly (P < 0.05) increased with a nutrient application, such as the highest values were found in N 120 and P 90 treatment for both hybrid rice types ( Table 3). The AE for N 120 treatment in D47 showed 6%, 65%, and 2% higher AE, PE, and RE respectively as compared to Y21. Unlike N 120 , in the case of P 90 , Y21 showed 6%, 15%, and 5% higher AE, PE, and RE in P 90 treatment as compared to D47. The response of nutrient use e ciency of both cultivars was highly sensitive to N application and showed a signi cant difference of AE and PE. A higher sensitive response was found between N 60 -N 120, while Y21 showed less sensitivity for these values as compared to D47. In case of NRE most sensitive difference was found between N 120 -N 180 for both cultivars, with the less signi cant difference (P <0.05). Similarly, the P application resulted in a high response difference of AE and PE between P 60 -P 90 for both cultivars, however, D47 was more sensitive for P application as compare to Y21. In case of RE, the D47 showed a high response difference between P 60 -P 90 while Y21 showed more response difference between P 90 -P 120 and no signi cant difference found between P 60 -P 90 . These ndings suggest that D47 had a more sensitive response difference for N and P applications in each treatment, while Y21 was less sensitive to nutrient application. The above results indicate D47 is the more nitrogen utilizing e cient rice type, while Y21 was the more Phosphorus utilizing e cient rice type.

Impact of Nutrient Input on Root Number
The number of roots increased progressively from day 10 to day 55 in all treatments, but the number of roots and dynamics of root appearance differed between treatments (Fig. 2), which indicates nutrient (N, P) application had a low response difference in initial days. Among the nitrogen treatments, the highest number of roots on day 55 was recorded in N 120 (Fig. 2a). Similarly, in the case of different phosphorus concentrations, D47 and Y21 favored P 90 phosphorus concentration (Fig. 2b). As compared with Y21, D47 collectively showed 12.4% higher root numbers in N 120 P 90 (478 for D47, as compared to 425 for Y21). D47 showed better development of root architecture and higher root numbers in all treatments of N and P, as compared to Y21 (Fig. 2c). For N application higher sensitive difference was found between N 60 -N 120 for both cultivars. While in case of P application higher sensitive difference was found between P 60 -P 120 in both cultivars. However, D47 was less sensitive in low nitrogen application N 0 -N 60 , but the overall nutrient response difference was signi cantly higher due to the deep rooting system as compare to Y21.
Expression of Glutamine Synthetase Genes OsGS1;1 and OsGS1;2 with Nitrogen Fertilization The expression levels of two genes of the glutamine synthetase family, OsGS1;1 and OsGS1;2, which are considered important markers of nitrogen metabolism, have been analyzed in response to nitrogen availability in hybrid rice cultivars D47 and Y21.
Interestingly, OsGS1;1 was upregulated in both low (N 0 , N 60 ) and high (N 180 ) nitrogen compared to moderate nitrogen treatment (N 120 ), where its expression in both cultivars was relatively low (Fig. 3A). The expression of OsGS1;2 followed the opposite pattern in D47, being strongly upregulated on moderate nitrogen treatment compared to low and high nitrogen treatments. However, in Y21, OsGS1;2 was upregulated at low nitrogen and downregulated on moderate and high nitrogen (Fig. 3B).

Expression of PHR1 and SPX Gene with Phosphorus Fertilization
The expression levels of two genes that are considered important markers of phosphorus availability, PHR1, and SPX, have been analyzed in response to nitrogen availability in hybrid rice cultivars D47 and Y21.
The gene PHR1 was strongly upregulated on the no-phosphorus (P 0 ) treatment compared to the other three treatments for the cultivar D47, while in Y21 its expression was also higher on moderate (P 90 ) than on high (P 120 ) phosphorus treatment (Fig. 4A). On the other hand, the expression of SPX was remarkably upregulated at P 90 compared to other treatments, while in Y21 it was downregulated on higher P concentrations compared to low P treatments (Fig. 4B).

Yield and Yield-Related Parameters and Correlation Analysis
Our results showed that grain yield progressively increased with nitrogen fertilization in both cultivars, although in Y21 the yield difference between N 120 and N 180 was not statistically signi cant ( Table 1). The underperformance of Y21 with high nitrogen fertilization is in concordance with its low nitrogen e ciency as compared to other rice cultivars, as has been previously reported Li, J. et al., (2020), and demonstrated further by comparing the nitrogen use e ciency of the two cultivars in this study.
Both cultivars performed better at moderate (P 90 ) than at high (P 120 ) phosphorus fertilization (Table 1). It was reported before that high phosphorus fertilization is ine cient in promoting the agronomic performance of rice, as no signi cant differences in grain yield were found between phosphorus applied at 90 and 135 kg/ha in both upland and paddy rice (Zhang, Y. et al., 2012).
Effective panicle was positively affected by both high N and P fertilization in both cultivars, but neither high N nor high P fertilization could signi cantly enhance the seed-setting rate, the number of grains per panicle, or 1,000-grain weight in cultivars (Table 1). Overall, it can be concluded that while high N fertilizer dosage contributed to higher yield in D47 (but not Y21), a high dosage of P fertilizer does not confer any signi cant bene t to the most important agronomic parameters of either D47 or Y21 output.
Hybrid rice varieties possess different nutrient uptake abilities, the low and high N and P application rate determines plant physiological needs. In our study, we assessed 2 rice types for yield and nutrient uptake correlation. We found D47 had a positive correlation with N application only in N 180 treatment (R=0.69), while Y21 on other hand had a positive correlation at N 60 and N 120 with R-values of 0.76 and 1 in respective treatments. In our study D47 ndings are positively consistent with (Cazetta et al., 2008), in which they found increasing N application positively correlated yield uptake up to N 180 , but in the case of Y21, the results were different. It showed a positive correlation of R=1 in the N 120 treatment. Similarly, many studies have shown that rice yield increases with the increase of nitrogen application within a certain range, but the yield decrease when the nitrogen application is too high (Pan et al., 2017;.
In the case of P accumulation and yield correlation, the hybrid variety D47 showed a positive correlation up to P 120 .
The correlation was highest up to P 60 (R=1), while it slightly decreased in P 90 and P 120 with R-values near 0.94 and 0.97 respectively. Unlike D47, Y21 showed a negative correlation at P 60 and P 90 respectively, however, it had a good positive correlation at P 120 with R-value near 0.99. The above ndings suggest that D47 is a more phosphorus e cient type and its yield highly depends on phosphorus input, while in the case of Y21 the low P input resulted in a negative correlation, which makes it less phosphorus e cient type. Similar results were stated by Zhang et al., (2012), in which they reported that P accumulation highly depends on rice variety used and P input. High input resulted in high yield (positive correlation), while between varieties the P accumulation and yield output differed greatly.

Nitrogen and Phosphorus Uptake and Use E ciency
In our study, high N and P fertilization positively affected both total plant biomass, and accumulation of N or P, respectively, in the plant biomass (Table 2). It is well known that N is the limiting nutrient for plant growth because it is at the same time both largely required for the synthesis of proteins and nucleic acids, and limitedly available to The genotypes D47 and Y21 differed in N and P uptake ( Table 2) and use e ciency (Table 3) at different fertilizer dosages. The genotype D47 was superior (~5-10%) at total biomass compared to Y21 at all fertilizer treatments (Table 2). It showed similar N uptake rates as Y21 (Table 2) but 1.5-3-fold higher nitrogen use e ciency on all fertilizer treatments (Table 3). It is already known that nitrogen use e ciency can considerably vary between lowland rice genotypes (Fageria, 2007). Superior nitrogen use e ciency of D47 compared to Y21 is likely accounting for its more successful total biomass accumulation, as these two parameters are known to greatly coincide ( Shan Li et al., 2018).
When it comes to phosphate assimilation, D47 accumulated slightly higher phosphate biomass than Y21, but this was probably only because of its greater total biomass, as its percentage of phosphorus biomass was similar to Y21 (Table 2). However, Y21 showed considerably higher values for phosphorus use e ciency compared to D47 on all P fertilizer treatments, especially for physiological P use e ciency (Table 3). Similar to nitrogen, phosphate use e ciency has also been proven to considerably vary between lowland rice genotypes, and it also importantly correlates with rice grain yield (Fageria, 2014).
Both nitrogen and phosphate use e ciency are reported to have relatively low values in rice -NUE typically ranges from 30-50% (Raun and Johnson, 1999) while PUE is typically less than 30% (López-Arredondo et al., 2014). Therefore, breeding the rice varieties with improved nutrient use e ciency -is the most promising strategy to raise both grain yield and nutritional quality with limited fertilizer input (Zhang, Z. et al., 2020). The nutrient use e ciency parameters such as agronomic use e ciency (AE), physiological use e ciency (PE), and recovery e ciency (RE) were studied in this experiment. AE, PE, and RE signi cantly (P < 0.05) increased with the nutrient application, but both AE and PE dropped at the highest fertilizer dosage of both nitrogen and phosphate. An important exception is the PE value for nitrogen fertilization of the Y21 cultivar, which was extremely nitrogen-ine cient at low and moderate doses and continued to grow even at N 180 (Table 3). We conclude that the values of nitrogen and phosphate use e ciency of rice cultivars D47 and Y21 argue in favor of moderate fertilizer application to achieve better nutrient use e ciency.

Root Growth of D47 and Y21 Genotypes at Different Fertilizer Treatments
In our ndings, root growth depended on both genotype, and fertilizer dosage (Fig. 2). In most treatments, root growth was more pronounced in D47, which is a cultivar known for drought tolerance and a well-developed root system (Wang et al., 2019). It has been suggested before, that root growth is strongly related to nitrogen use Both nitrogen and phosphorus fertilization had a positive impact on root growth up to moderate doses (N 120 , or P 90 ), whereas high fertilizer doses of either N or P (N 180 , or P 120 , respectively) had an inhibitory effect on root growth, resulting in signi cantly lesser number of roots compared to moderate doses starting from day 40 (Fig. 2). Ammonium toxicity from excess N fertilization can negatively affect root growth (Britto and Kronzucker, 2002;Lips et al., 1990). Furthermore, on day 10, both D47 and Y21 had developed a greater number of roots when grown at N 60 compared to N 120 and N 180 treatments, whereas since day 25 this relationship was reversed to favor higher doses of N fertilizer application (Fig. 2). It has been recently reported that urea from the N fertilizers inhibits the early stages of a root, but does not shoot growth (Sharma et al., 2018). When it comes to the effect of phosphorus fertilizer on root growth, it has been shown that the number of roots in upland rice is positively affected by growing amounts of P fertilizer up to a certain limit (De Bauw et al., 2020), which was con rmed by our results.

Expression of Nitrogen and Phosphate Homeostasis-Related Genes
The need for improving the nutrient use e ciency of large-scale cultivated crops has pushed the research towards the identi cation of target genes for genetic modi cations (Vinod and Heuer, 2012).
The key step in nitrogen assimilation in higher plants is the incorporation of the inorganic ammonium ion (NH 4 + ) into organic compounds. The initial step in this pathway is the synthesis of the amino acid glutamine through the activity of the enzymes glutamine synthetases (GS) (James et al., 2018). Thus, the genes encoding glutamine synthetases are important genetic markers for nitrogen assimilation and were proven related to the nitrogen use e ciency in crop species such as maize (Hirel et al., 2001) and tobacco (Oliveira et al., 2002). In this study, we investigated the expression of two cytosolic GS1 (OsGS1;1 and OsGS1;2) genes in rice cultivars D47 and Y21 to assess their role in response to different N fertilization treatments. The two GS1 genes have different roles in rice, with OsGS1:1 being responsible for the initiation of synthesis of a broader range of metabolites (Kusano et al., 2011;Kusano et al., 2020), whereas OsGS1;2 has diverse functions, like the primary assimilation of ammonium ions in roots (Funayama et al., 2013) and cross-talk with other signaling pathways, like cytokinins which affect the outgrowth of axillary buds in the rice shoot (Ohashi et al., 2017). Previous research has shown that expression pro les of OsGS1;1 and OsGS1;2 at different N fertilizer treatments can vary between rice cultivars, with expression peaks coinciding with optimal levels of N fertilization for each cultivar (Gaur et al., 2012). Our results for OsGS1;2 expressions suggest that moderate N levels (N 120 ) are optimal for the cultivar D47, whereas in Y21 which is a less nitrogen-e cient cultivar, OsGS1;2 has relatively lower expression and is more active at lower doses of nitrogen fertilization (Fig. 3).
Members of the PHOSPHATE STARVATION RESPONSE (PHR) gene family have been identi ed as MYB transcription factors with a regulatory role in response to phosphorus nutrient de ciency (Rubio et al., 2001;Sega and Pacak, 2019). The PHR1 family has 12 members in rice, designated OsPHR1-OsPHR12 (Xu, Y. et al., 2018). The role in phosphate homeostasis was con rmed for OsPHR2 Zhou et al., 2008) and OsPHR4 (Ruan et al., 2017). Our results show that in both D47 and Y21 OsPHR1 has high transcriptional activity when P fertilizer is not applied, whereas their activity is downregulated with the growing application of P fertilization (Fig.  4), con rming its role in regulating plant metabolism at low P nutrition.
Members of the PHR family are subject to negative regulation by members of another family of transcription factors, SPX (Jung et al., 2018;Liu et al., 2018). The SPX genes might have a role in a broader array of physiological processes, but just like PHR, they might serve as genetic targets for improving phosphorus use e ciency . The expression pro les of the OsSPX gene studied in our work revealed a similar pattern as for OsPHR, with high transcript levels when P fertilizer is not applied, but being downregulated at higher P treatments. Expression levels for both OsPHR and OsSPX were similar for both cultivars D47 and Y21 (Fig. 4).

Conclusion
In this research, we investigated an array of agronomic traits related to yield and nutrient use e ciency of the rice cultivars Deyou4727 (D47) and Yixiangyou2115 (Y21) at low, moderate and high levels of nitrogen and phosphorus fertilization. D47 was more sensitive toward nitrogen application, the response of total biomass and yield was greater than Y21, but Y21 was more sensitive toward phosphorus application. We con rmed our outcomes by gene expression studies of glutamine synthetase OsGS1;1 and OsGS1;2 and phosphate starvation-related genes PHR1 and SPX, which also concluded D47 response sensitivity towards N application and Y21 response sensitivity toward P application.
Similarly, root numbers of D47 were less sensitive in low nitrogen application N 0 and N 60 , but the overall nutrient response difference was signi cantly higher due to deep rooting system as compare to Y21. Our results suggest that large amounts of N fertilizer have limited bene ts for D47, whereas P fertilization with more than 90 kg/ha brings no signi cant agronomic bene t for the cultivation of D47 or Y21 rice. We further recommend research should be undertaken in the area of optimal fertilization management based on hybrid rice type, soil properties, and regional conditions to solve agronomic needs and environmental concerns.

Declarations
Acknowledgment Special thanks are extended to the staff of the agricultural station of SWUST for their irreplaceable work to manage the experiment eld and to collect the samples. We would also like to thank the reviewers and editor who provided valuable suggestions to improve this paper.

Con ict of Interest
The authors declare there are no con icts of interest. from each other at P < 0.05.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. TableS1.docx