The improved phosphorus utilization and reduced phosphorus consumption of ppk-expressing transgenic rice

Field Crops Research 248 (2020) 107715

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The improved phosphorus utilization and reduced phosphorus consumption of ppk-expressing transgenic rice

T

Ruping Weia, Xin Wangb, Wen Zhanga, Jianing Shena, Huifen Zhanga, Yan Gaoa, Liuyan Yanga,* a b

State Key Laboratory of Pollution Control and Resources Reuse, School of the Environment, Nanjing University, Nanjing, 210023, PR China State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210023, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Polyphosphate kinase Transgenic rice Phosphorus fertilizer Grain yield

To reduce the phosphorus (P) environmental resources consume, it is imperative to cultivate new elite rice varieties with a higher utilization efficiency of P fertilizer. The expression of the polyphosphate kinase gene (ppk) can mediate the synthesis of polyphosphate by free inorganic phosphate (Pi), and mimicking the Pi deficiency signal in plants, thereby enhancing plant Pi uptake of Pi by plants. A ppk from Microcystis aeruginosa had been expressed in Nipponbare, and the ppk-expressing transgenic rice (ETR) had been successfully constructed. In this research, two field experiments were carried out from 2018 to 2019 using different P fertilizer (superphosphate) treatments with high P (HP, 450 kg/ha), medium P (MP, 225 kg/ha), low P (LP, 112.5 kg/ha) and no P (NP, 0 kg/ ha) at test sites in Changxing County in the Yangtze Delta and in Sanya, an island city in southeastern China’s Hainan province. Unlike the increase in grain yield of wild type Nipponbare (WT), coincident with the increase in P fertilizer, the grain yield of ETR with a single-copy line (ETRS) reached the maximum in MP treatment in Changxing and in LP treatment in Sanya. More importantly, when P fertilizer was insufficient, the grain yield of ETRS was significantly higher than WT. Correlation analysis showed that the increase in ETRS production can be the result of more tillers. At the same time, a quantitative Real-Time PCR (qRT-PCR) was performed and the results revealed altered expressions of some key genes involved in the P metabolism and ETRS. As a result, changes in genes expressions increased the number of tillers and thus improved grain yield when the P fertilizer was inadequate. Our study provided a strategy for constructing a high P utilization rice, which will make a strong contribution toward reducing P fertilizer use in rice production in China.

1. Introduction Phosphorus (P) is one of the most required mineral nutrients for plant growth, and it can only be assimilated by plant roots in the form of inorganic phosphate (Pi). However, most of the Pi in the soil combines with metal cations to form insoluble precipitates and binds to some plate silicates, which cannot be absorbed by plants. Thus, nearly 70 % of the world's cultivated land faces the problem of P deficiency (Lopez-Arredondo et al., 2014; Xu et al., 2019). The P fertilizer application is a common method to improve nutrients in farmland and maintain high crop yields. However, the plants have a low utilization efficiency of P fertilizer(the ratio of P absorbed by the rice plant to the P in the applied fertilizer, accounting for only 10%–20% of the total fertilizer applied in the first year(Sattaria et al., 2012; Li et al., 2011). Therefore, the amount of applied P fertilizer often exceeds the amount

absorbed by the crops (Hou et al., 2013). As a result, the high application of P fertilizer will cause a shortage of global P resources (Cordell et al., 2009; Van Vuuren et al., 2010). Simultaneously, rice (Oryza sativa L.) is a food staple for more than half of the world's population (Xu et al., 2018). On a global scale, its planting area reaches 167 million hectares and consumes 21.2 % of the world’s total P fertilizer supply (FAO, 2019; Liu et al., 2018). In China, the rice planting area is 31 million hectares, accounting for 1/4 to 1/3 of China's cultivated land (FAO, 2019). Therefore, it is of great significance to develop new elite rice varieties with high utilization of P fertilizer for saving P resources and ensuring considerable grain production. To improve rice crops’ P utilization, studies about the key regulators underlying P signals and P homeostasis in rice have focused on the Phosphate Starvation Response Regulator 2 (OsPHR2), the SPX-domain containing proteins, and Oryza sativa Pi transporters (OsPTs) (Wu et al.,

Abbreviations: ETR, ppk-expressing transgenic rice; ETRS, ppk-expressing transgenic rice with single-copy line; ETRD, ppk-expressing transgenic rice with doublecopy line; WT, wild type Nipponbare; HP, high phosphorus; MP, medium phosphorus; LP, low phosphorus; NP, none phosphorus ⁎ Corresponding author. E-mail address: [email protected] (L. Yang). https://doi.org/10.1016/j.fcr.2020.107715 Received 13 October 2019; Received in revised form 2 January 2020; Accepted 2 January 2020 0378-4290/ © 2020 Published by Elsevier B.V.

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(H2PO4)2·H2O), which was applied as base fertilizer before seedling transplantations. According to the rice production practices of Chinese farmers, four P fertilizer treatments were designed with a high P (HP, 450 kg/ha), medium P (MP, 225 kg/ha), low P (LP, 112.5 kg/ha) and no P (NP, 0 kg/ha). The four experiment plots were relatively closed—unable to exchange nutrients. The N and K fertilizers were normally supplied in the form of urea (CO(NH2)2) and potassium sulphate (K2SO4), respectively. The N fertilizer, 450 kg/ha, was applied in a ratio of 5:3:2 at basal, tillering and panicle initiation. The K fertilizer was applied twice with 112.5 kg/ha for the base fertilizer and panicle fertilizer, which totaled 225 kg/ha. The fertilizers were spread over the rice field in the form of solid particles to ensure uniform application. At Changxing and Sanya, rice seeds were sown on May 29, 2018 and December 2, 2018, respectively. One-month-old rice seedlings were transplanted into the paddy fields on June 30, 2018 and January 5, 2019, respectively. Each rice line was planted in a square experimental sub-plot of 2 m × 2 m at intervals of 20 cm between adjacent hills with two seedlings per hill. Three protective rows were placed around each sub-plot to prevent cross-pollination between adjacent rice lines. Meanwhile, each rice line was replicated three times in each experimental plot, and 9 square sub-plots were completely randomly distributed. In total, there were 36 small sub-plots in the four experimental plots. Groundwater was used for irrigation with pH 6.77 and 6.38, TN 3.69 and 4.45 mg/L, TP 0.086 and 0.102 mg/L, and dissolved orthophosphates 0.022 and 0.017 mg/L in paddy fields at Changxing and Sanya, respectively. Insecticides and herbicides were used to control insects, diseases and weeds during the study to ensure maximum grain yields.

2013). As the central transcription factor, OsPHR2 positively regulates the expression of downstream Pi Starvation Induced (PSI) genes such as OsPTs. In shoots of the OsPHR2-overexpressing plants, Pi concentrations were 2.0- to 2.5-fold higher than those in wild-type plants grown at normal rice culture solution with 10 mg Pi/L (Zhou et al., 2008). At the same time, the OsPHR2 expression was negatively regulated by OsSPX (Wang et al., 2009). For example, the result of solution culture experiments with transgenic plants that simultaneously over-express OsPHR2 and OsSPX1 indicated that the Pi concentrations in the shoots were reduced to the level of wild-type plants (Liu et al., 2010). In another research investigation, the shoot Pi concentration was reduced significantly in transgenic rice overexpressing OsSPX4 compared with the wild type rice (Lv et al., 2014). In addition, OsPTs are the direct operators of Pi uptake and transport in rice plant and regulated by OsPHR2 (Paszkowski et al., 2002). Previous researches have shown that overexpression of certain endogenous genes controlling the Pi metabolism, such as OsPHR2, OsPT2, OsPT8, OsPT9 and OsPT10, can increase P concentration in rice plants, but at the same time the symptom of “Pi toxicity” has not been well resolved at the same time (Zhou et al., 2008; Ai et al., 2009; Jia et al., 2011; Wang et al., 2014). In our laboratory, an exogenous gene, polyphosphate kinase (ppk) from Microcystis aeruginosa, had been expressed in Oryza sativa, ssp. Japonica, cv. Nipponbare using using the Agrobacterium tumefaciensmediated transgene process, and the ppk-expressing transgenic rice (ETR) had been constructed. With Southern Blot, we had identified a single-copy line (ETRS) and a double-copy line (ETRD). The use of ETR had alleviated the “Pi toxicity” symptoms. Hydroponic experiments have shown that the ppk expression can mediate the polyphosphate (polyP) synthesis from the free Pi, which facilitated the Pi uptake and transport in the transgenic rice plants. However, no experiments have been able to quantify the utilization efficiency of P fertilizer by using ETR in the paddy field. The purpose of our study was to investigate the relationship between the grain yield of ETR and the applied P fertilizer amount in the field by using a controlled study. We assume that ETRS has a higher P fertilizer utilization efficiency and can achieve a maximum yield under lower P fertilizer input conditions. To this end, we designed four different P fertilizer treatments. Both ETRS and ETRD were planted while wild type Nipponbare (WT) was used as a control. We conducted two seasons of field experiments in 2018 and 2019 at different places in China to determine whether the eugenic rice plants ETR could achieve a higher P utilization efficiency than WT, and if achieve a higher grain yield when the P fertilizer was proved to be inadequate.

2.2. Grain yield measurement After crop maturity, rice of one square meter in the middle of each sub-plot was selected to count the number of panicles per square meter, and six hills of plants were randomly selected in each test plot to record the agronomic traits such as tiller number, panicle number and earing rate (panicle number/tiller number×100 %). In addition, two productive panicles were randomly chosen from each selected hill of plant samples, and spikelet number per panicle was calculated based on these 12 panicle samples. The filled grains of each of the six selected hills of rice plants were separated using the water flotation method after the rice was hand-threshed, and the grain-filling rate (filled grain number/ total grain number×100 %) was calculated. Then, 1000 filled grains from each sub-plot were collected and dried at 65℃ to a moisture content of 14 % for determination of the 1000-grain weight. All the rice plants in each sub-plot were harvested, and after being threshed by machine and winnowed, the grains were adjusted to 14 % moisture content for quantifying the total weight. The grain yield per hectare was calculated according to the grain yield of each sub-plot.

2. Materials and methods 2.1. Experimental sites and design Two field experiments were carried out in paddy fields located at transgenic control farms in Changxing County (119°38′E, 30°53′N) in the Zhejiang Province of China from May 29 to September 18, 2018 and in Sanya (109°28′E, 18°19′N) in the Hainan Province of China from December 2, 2018 to March 29, 2019, respectively. Changxing and Sanya experience subtropical and tropical maritime monsoon climates, respectively. Moreover, the temperatures during the study ranged from 21 °C to 34 °C and from 19 °C to 32 °C, respectively (Additional Information (AI) Table A1). Furthermore, the paddy soils at the top 0–25 cm layer were sampled before seeding and their properties were pH 5.34 and 5.69, organic matter 38.2 and 22.5 g/kg, total nitrogen (N) 2.09 and 1.73 g/kg, total P 0.15 and 0.21 g/kg, available P 3.86 and 25.36 mg/kg, and available potassium (K) 82.3 and 96.6 mg/kg in the paddy soils from Changxing and Sanya, respectively. The ETR used in the experiments had been constructed in our laboratory, and WT was planted as a control at the same time. After three generations of breeding, the T3 generation seeds were used in both experiments. The P fertilizer used in this study was superphosphate (Ca

2.3. Determination of TP and pi concentrations of rice plant The six whole rice plants samples selected in each plot were taken back to the laboratory and then divided into roots, stems and leaves. The adhered soil and other residues were washed with tap water and rinsed three times with deionized water. After aspirating the water, a portion of the samples were dried at 65 ℃ for 48 h to constant weight after fixing at 110 ℃ for half an hour and crushed for TP determination. The other portion of the samples were quick-frozen and ground into powder in liquid nitrogen for Pi determination. Crushed dry samples were digested with concentrated sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) at 300 ℃, and the TP concentration was determined using the molybdate blue method (Chen et al., 2007). While the Pi was extracted from frozen powder by perchloric acid (HClO4), its concentration was determined by molybdate blue method as previously described (Nanamori et al., 2004). 2

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differences were found in TP concentration of roots from the different rice lines. The TP concentration in the above-ground parts (stems and leaves) of ETRD were 1.78–2.25 times and 1.95–3.66 times, which were as high as WT from Changxing and Sanya, respectively. The TP concentrations of the above-ground parts of ETRS from Changxing in the LP and MP treatments increased by 18.13 %–36.01 % compared with WT. Meanwhile, the TP concentration of ETRS stems from Sanya only increased significantly in the NP and LP treatments, about 1.7 times that of WT. The Pi concentrations of rice tissues showed a similar trend to TP concentrations. The Pi concentrations in the above-ground parts of ETRD from Changxing and Sanya were 3.24–4.34 and 1.68–4.10 times as high as WT, respectively. In addition, the Pi concentrations in the ETRS stems and leaves increased significantly in the LP, MP and HP treatments at Changxing and in the NP, LP and MP treatments at Sanya, which were 1.36–1.81 and 1.18–1.60 times as high as WT, respectively. Obviously, the ETR contained more P than WT in the above-ground parts of the rice plants.

2.4. The relative expression analysis of phosphorus and tillering related genes The relative expression levels of ppk and phosphorus, as well as tillering-related genes, such as OsPHR2, OsSPX1, OsSPX4, OsPT2, OsPT4, OsPT8, MOC1, OsTB1, OSH1, TAD1, TE and SLR1, were also analyzed in rice plants from Sanya. At the rice tillering stage, six hills of plant samples were selected in each plot; then, a piece of flag leaf was taken from each sample for expression analysis of ppk, OsPHR2, OsSPX1, OsSPX4, OsPT2, OsPT4 and OsPT8, while tiller bud was used for expression analysis of MOC1, OsTB1, OSH1, TAD1, TE and SLR1. After being washed with tap water and then deionized water, the leaves and tiller buds were quickly placed in polypropylene tubes and frozen in liquid nitrogen. They were then transported back to the laboratory packed in ice. The total RNA was extracted using the TRIzol reagent (TaKaRa) according to the manufacturer's instructions. The cDNA synthesis, quantitative Real-Time PCR (qRT-PCR) and expression data analysis were performed followed the previously described method (Wang et al., 2018). Briefly, one microgram of qualified total RNA was subjected to reverse transcription and qRT-PCR of the resulting cDNA was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA). The cell scaffolding protein gene Actin was used as a reference, and the gene-specific primers of ppk were designed using BioEdit, while the other primers were from the reference (AI Table A2).

3.3. The relative expression levels of ppk and phosphorus related genes In order to reveal the molecular mechanisms underlying grain yield changes, the expression levels of ppk were analyzed (Fig. 4a). As expected, no expression of the gene was detected in WT. The relative expression levels of ppk in ETRS and ETRD were 0.23 - 0.28 and 0.58 0.66, respectively. Due to being related to ppk expression, the expression levels of OsPHR2, the central transcription factor gene in regulating the phosphate signal, and two OsSPX domain protein genes, OsSPX1 and OsSPX4, were also determined (Fig. 4b-d). The OsPHR2 expressions in ETR were up-regulated compared with that in WT. These OsPHR2 expressions in ETRS and ETRD were 1.28–1.58 and 1.60–1.79 times as high as WT, respectively. The OsSPX1 expression in ETRS was not significantly different from that in WT, but it was dramatically downregulated in ETRD, and only 49 %–52 % of WT. Compared with WT, the OsSPX4 expressions in ETRS and ETRD were down-regulated by 24 %–26 % and 39 %–41 %, respectively. As downstream genes of OsPHR2, the OsPT family genes were quantitatively analyzed, and three of them, namely OsPT2, OsPT4 and OsPT8, significantly increased their expressions in ETR (Fig. 4e-g). The OsPT2 expression levels in ETRS and ETRD were 1.23–1.40 and 1.86–2.21 times of WT, respectively. For OsPT4, these values were 1.97–2.06 and 4.01–4.52 times that of WT, respectively. Compared to WT, the OsPT8 expression levels of ETRS and ETRD increased by 23 %–33 % and 51 %–66 %, respectively.

2.5. Statistical analysis Agronomic trait data were analyzed following the one-way analysis of variance (ANOVA) using SPSS Software (20.0). Means of rice lines were compared based on the least significant difference (LSD) test at the 0.05 probability level by Dunnett's or Tukey's multiple comparison model. The correlations between grain yield and its components, TP concentration and Pi concentration, as well as tiller number per hill and TP concentration were investigated by regression analysis. 3. Results 3.1. Grain yield and yield components After crop maturity, the phenotypes of rice plants and panicles from Sanya were photographed (Fig. 1). We observed that the tiller number of the ETRD was extremely high (AI Table A3) but its earing rate was so low compared with ETRS and WT (AI Figure A1). The grain yields and their components under four different P amounts at Changxing and Sanya were recorded (Tables 1 and 2). In Changxing experiment, the maximum yields of WT and ETRS reached 6.43 and 6.70 t/ha, respectively, while the maximum grain yield of ETRD was only 2.82 t/ha. Similar results were obtained in the Sanya experiment and the maximum grain yields were 6.91, 7.24 and 2.18 t/ha for WT, ETRS and ETRD, respectively. In the two experiments, the maximum grain yields of ETRS did not decrease compared with WT, whereas the maximum grain yields of ETRD were 56.1 % and 68.5 % less than WT. It is worth noting that the grain yield of WT reached the maximum in the HP treatment, while the grain yields of ETR reached the maximum in the MP and LP treatments at Changxing and Sanya, respectively. In terms of yield components, the number of panicles per square meter of ETR was generally higher than that of WT, among which ETRD was more obvious. Furthermore, the spikelet number per panicle, grain filling rate and 1000-grain weight of ETRD were significantly lower than those of WT and ETRS, but there were no significant differences between WT and ETRS on the whole.

3.4. The relative expression levels of tillering related genes The expressions of some key genes that regulate rice tillering such as MOC1, OsTB1, OSH1, TAD1, TE and SLR1 were also determined (Fig. 5). The expressions of MOC1 in ETR were significantly higher than that in WT, and those in ETRS and ETRD were 1.19–1.59 and 2.01–2.44 times as high as that of WT, respectively (Fig. 5a). Its downstream gene, OSH1, showed a similar trend, and its expressions in ETRS and ETRD were up-regulated by 17.02–76.55 % and 170.40–228.70 %, respectively (Fig. 5c). Except for the HP treatment, the TE expressions in ETR significantly increased, which were 1.17–1.68 times that in WT, but there was no significant difference between ETRS and ETRD (Fig. 5d). In addition, the SLR1 expression level in ETRD increased significantly compared with WT in the LP and NP treatments, up-regulated by 10.69 % and 14.67 %, respectively (Fig. 5f). 4. Discussion

3.2. TP and pi concentrations of rice plant 4.1. Contribution of ppk-expressing transgenic rice to phosphorus savings In general, the TP and Pi concentrations of rice plants from Sanya were higher than those from Changxing (Figs. 2, 3). No significant

With the rapid growing population and decreasing paddy areas, it is 3

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Fig. 1. Phenotypic photographs of plants (a) and panicles (b) of wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Sanya, Hainan Province, China in 2019. The photos of the plants were taken at the rice mature stage on 29 March 2019 before their harvest. In order to facilitate the shooting, the rice plants selected from the field plots with average phenotype were uprooted and moved to the barrels.

the number of panicles per square meter, the number of grains per panicle and the 1000-grain weight (Huo et al., 2017). The tiller number and earing rate determined the panicle number, and the grain number per panicle is determined by the spikelet number per panicle and grain filling rate. In our field experiments, the grain yields of WT and ETRS were significantly positively correlated (r2 ranged from 0.7289 to 0.9816) with the tiller number, panicle number and grain filling rate (AI Figure A3, A4). Among the three agronomic traits, no significant differences were found between the grain filling rate of ETRS and WT. When the P fertilizer was inadequate, the grain yield, tiller number and panicle number of ETRS was generally significantly higher than that of WT (Tables 1, 2). Because the earing rate of ETRS was always lower than or close to that of WT in the experiments (AI Figure A1), we inferred that the higher grain yield of ETRS under low P condition was the result of the increased tiller number. However, significant increases in ETRD tillers and panicles did not lead to a higher grain yield. On the one hand, the 1000-grain weight of ETRD was lower (Table 1, 2). Nevertheless, there were significant

imperative to improve the rice yield per unit of land area to ensure adequate food production (Jiang et al., 2016). The development of new elite rice varieties with high yield through genetical modification has been one of the main objectives of agronomic research (Huang et al., 2018). However, over-expressions of some endogenous genes related to P regulation, such as OsPHR2, OsPT2, OsPT8, OsPT9 and OsPT10, can cause ‘Pi toxicity’ and reduce grain yield (Zhou et al., 2008; Ai et al., 2009; Jia et al., 2011; Wang et al., 2014). Conversely, the constructed ETRS manifested no disadvantage in grain yield. According to previous reports, the grain yield of Nipponbare was 4.99–6.66 t/ha under sufficient fertilizer amounts (Saitoh et al., 2015; Mae, 2011; Hirooka et al., 2018). In our study, the grain yield of WT from Changxing appeared in this range, and the grain yield from Sanya was slightly higher than 6.66 t/ha, which may be due to the more fertile paddy soil in Sanya. The grain yields of ETRS from Changxing and Sanya were not lower than that of WT. More importantly, when P fertilizer was insufficient, the grain yield of ETRS was significantly higher than WT. Rice grain yield is determined by three key components, including 4

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Table 1 Grain yields of wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus fertilizer treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Changxing County, Zhejiang Province, China in 2018. P treatment HP

MP

LP

NP

Rice line WT ETRS ETRD WT ETRS ETRD WT ETRS ETRD WT ETRS ETRD

Grain yield (t ha−1) 6.93 6.66 2.02 5.31 6.70 2.82 3.50 4.92 2.04 1.66 2.18 1.02

± ± ± ± ± ± ± ± ± ± ± ±

0.45 a 0.46 a 0.22 b 0.33 b 0.53 a 0.16 c 0.39 b 0.38 a 0.15 c 0.17 b 0.23 a 0.09 c

Panicle number per m2

Spikelet number per panicle

496 ± 36 b 517 ± 41 ab 556 ± 36 a 428 ± 41 c 526 ± 35 b 631 ± 44 a 358 ± 31 c 476 ± 39 b 579 ± 32 a 256 ± 22 c 302 ± 28 b 412 ± 51 a

74 ± 8 a 67 ± 5 a 54 ± 7 b 72 ± 7 a 66 ± 8 ab 59 ± 5 b 68 ± 6 a 65 ± 4 a 52 ± 6 b 62 ± 6 a 59 ± 6 a 50 ± 4 b

Grain filling (%) 77.66 75.63 29.33 68.05 74.36 32.66 57.29 61.08 28.75 42.35 48.62 21.62

± ± ± ± ± ± ± ± ± ± ± ±

3.44 a 5.03 a 1.89 b 2.97 b 4.21 a 2.73 c 4.05 a 3.77 a 1.92 b 3.26 a 4.15 a 2.05 b

1000-Grain weight (g) 24.55 25.43 22.99 24.93 25.96 23.19 25.08 26.01 23.56 24.69 25.12 22.94

± ± ± ± ± ± ± ± ± ± ± ±

0.43 0.86 0.73 0.89 0.73 0.36 0.49 1.02 0.59 0.37 0.91 0.41

a a b a a b a a b a a b

The data consists of mean ± SD and the repetitions are 3 for grain yield, panicle number per m2 and 1000-grain weight, 12 for spikelet number per panicle and 6 for grain filling rate. Different letters indicate significant differences (P < 0.05) under the one-way ANOVA.

positive correlations (r2 ranged from 0.7143 to 0.8789) between the grain yield and the spikelet number per panicle and grain filling rate of WT and ETRD (AI Figure A5, A6), but the two agronomic traits of ETRD were significantly lower (Table 1, 2). Notably, the grain filling rate supplies the endosperm development and accumulation of nutrients, which has a great impact on grain yield (Zhou et al., 2013; Zhao et al., 2019). The decrease of the spikelet number per panicle and grain filling rate resulted in a serious decline in the grain number per panicle, which completely offset the contribution of the increased panicle number when assessing grain yield. This is consistent with the reported results that the reduced grain number per panicle led to the reduced grain yield (Fang et al., 2016; Huo et al., 2017); moreover, the decreased spikelet number was the main reason for the decrease in grain production of ETRD. The increased tiller number of rice plants can contribute momentously to agricultural production especially in China, which is a big agricultural country (Yan et al., 1998; Liang et al., 2014). China is the second largest rice planting country and the largest rice yield country in the world (FAO, 2019). As a result of the highly intensive agricultural production mode, a large amount of chemical fertilizer is consumed every year. China ranks first in the world in the P2O5 use per area of cropland, which reaches up to about 120 kg/ha (FAO, 2019). Phosphorus is a non-renewable resource and there has been no substitute for P in modern agriculture. Some researchers have estimated that global P reserves may be consumed by 40–60 % by 2100, which will pose a great threat to the sustainable development of agriculture (Cordell et al., 2009; Van Vuuren et al., 2010). Our field experiments were conducted

in Changxing and Sanya, belonging to the Middle-Lower Yangtze Plain and Hainan Island, respectively, both of which are important ricegrowing areas in China. This study’s results showed that the grain yield of WT reached the maximum when the P fertilizer was adequate, while the grain yield of ETRS reached higher levels when the P fertilizer was inadequate. The maximum grain yield of ETRS was not only maintained at a considerable level, but was also obtained with less P fertilizer. As for the difference in the P fertilizer amount applied to achieve the maximum grain yield, this may be due to the different nutrient background of the paddy soils. Notably, the available P was at a higher level in paddy soil at Sanya. Therefore, planting ETRS instead of WT will make a strong contribution to reducing P fertilizer use in rice production in China. At present, the planting area of rice in China exceed 31 million hectares and about 3.6 million tons of P fertilizer are consumed by rice cultivation every year (Wang et al., 2009). If ETRS is promoted, hundreds of thousands or even millions of tons of P fertilizer will no longer be needed every year. Undoubtedly, this could be of great significance for P resources savings. 4.2. The mechanism underlying improved phosphorus utilization and reduced phosphorus consumption of ppk-expression transgenic rice In ppk transgenic microorganism and plant, polyphosphate kinases reversibly catalyze the synthesis of polyphosphate chains by terminal Pi from ATP (Wang et al., 2018; Nagata et al., 2006). Similarly, the ppk expression in rice can mediate the synthesis of polyphosphates from Pi, leading to the reduction of free Pi concentration, which consequently

Table 2 Grain yields of wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus fertilizer treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Sanya, Hainan Province, China in 2019. P treatment HP

MP

LP

NP

Rice line WT ETRS ETRD WT ETRS ETRD WT ETRS ETRD WT ETRS ETRD

Grain yield (t ha−1) 6.91 5.39 1.17 5.65 6.95 2.15 4.68 7.24 2.18 3.05 2.90 1.11

± ± ± ± ± ± ± ± ± ± ± ±

0.38 a 0.42 b 0.18 c 0.25 b 0.40 a 0.23 c 0.18 b 0.28 a 0.21 c 0.23 a 0.15 a 0.10 b

Panicle number per m2

Spikelet number per panicle

719 ± 62 ab 654 ± 60 b 759 ± 63 a 588 ± 57 c 719 ± 49 b 806 ± 71a 583 ± 51 c 709 ± 43 b 846 ± 56 a 521 ± 42 b 496 ± 38 b 678 ± 49 a

60 ± 7 a 51 ± 6 ab 49 ± 8 b 59 ± 8 a 56 ± 6 a 51 ± 10 a 55 ± 7 ab 59 ± 8 a 47 ± 7 b 43 ± 6 a 44 ± 5 a 45 ± 7 a

Grain filling (%) 66.90 65.17 13.90 65.05 66.47 22.76 57.65 67.02 23.65 53.57 52.43 16.90

± ± ± ± ± ± ± ± ± ± ± ±

5.09 a 3.49 a 3.10 b 4.80 a 2.31 a 4.52 b 4.36 b 4.38 a 3.29 c 8.12 a 6.72 a 2.69 b

1000-Grain weight (g) 24.21 25.19 22.74 25.18 26.02 23.44 25.38 25.96 23.49 25.41 25.91 23.01

± ± ± ± ± ± ± ± ± ± ± ±

0.68 1.10 0.38 0.53 1.12 0.82 0.35 0.95 0.54 0.87 0.97 0.26

a a b a a b a a b a a b

The data consists of mean ± SD and the repetition are 3 for grain yield, panicle number per m2 and 1000-grain weight, 12 for spikelet number per panicle and 6 for grain filling rate. Different letters indicate significant differences (P < 0.05) under the one-way ANOVA. 5

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Fig. 2. TP and Pi concentration in root (a, d), stem (b, e) and leaf (c, f) of wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus fertilizer treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Changxing County, Zhejiang Province, China in 2018. The data consists of mean ± SD and the repetition is 6. Different letters indicate significant differences (P < 0.05) under the one-way ANOVA.

results. The expressions of OsPHR2, OsPT2, OsPT4 and OsPT8 in ETR leaves were up-regulated in varying degrees. OsSPX1 down-regulated its expression in ETRD and OsSPX4 down-regulated its expression in both ETRS and ETRD, among which ETRD changed more dramatically. It is known that OsPHR2 is a central P-signaling regulator gene, and its

mimic a signal of Pi deficiency in rice plant (Fig. 6). Previous studies revealed that under hydroponic conditions, the ppk expression upregulated the expressions of OsPHR2 and most genes of the OsPT family, as well as the down-regulated expressions of the OsSPX gene family in rice roots and shoots. Our field experiment at Sanya obtained similar

Fig. 3. TP and Pi concentration in root (a, d), stem (b, e) and leaf (c, f) of wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus fertilizer treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Sanya, Hainan Province, China in 2019. The data consists of mean ± SD and the repetition is 6. Different letters indicate significant differences (P < 0.05) under the one-way ANOVA. 6

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Fig. 4. Relative expression levels of ppk (a) and some genes related to regulation of phosphate starvation signal such as OsPHR2 (b), OsSPX1 (c), OsSPX4 (d), OsPT2 (e), OsPT4 (f) and OsPT8 (g) in wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus fertilizer treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Sanya, Hainan Province, China in 2019. The data consists of mean ± SD and the repetition is 6. Different letters indicate significant differences (P < 0.05) under the oneway ANOVA.

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Fig. 5. Relative expression levels of some genes controlling tillering such as MOC1 (a), OsTB1 (b), OSH1 (c), TE (d), TAD1 (e) and SLR1 (f) in wild type Nipponbare (WT) and ppk-expressing transgenic rice (ETR) with single-copy line (ETRS) and with double-copy line (ETRD) in four different phosphorus fertilizer treatments with high phosphorus (HP), medium phosphorus (MP), low phosphorus (LP) and none phosphorus (NP) from Sanya, Hainan Province, China in 2019. The data consists of mean ± SD and the repetition is 6. Different letters indicate significant differences (P < 0.05) under the one-way ANOVA.

Fig. 6. A working model of the ppk expression affecting tiller number in the ppk-expressing transgenic rice (ETR) by changing the expressions of phosphorus and tillering related genes under inadequate phosphorus fertilizer condition. The upward red arrow indicates that the gene expression is up-regulated or P concentration and tiller number increase in ETR compared to that in wild type Nipponbare (WT). The downward blue arrow indicates that the gene expression is down-regulated in ETR compared to that in WT. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

2010). Consistent results were obtained in this study. The expression of OsSPX1 in ETRD was significantly down-regulated, while the concentrations of TP and Pi in stems and leaves significantly increased. Furthermore, as a rapid turnover protein, OsSPX4 can interact with OsPHR2 and regulate its activity (Lv et al., 2014). In our study, the down-regulated expressions of OsSPX4 in ETR were the results of the

overexpression would enhance Pi deficiency signals (Wu et al., 2013; Zhou et al., 2008). In the same way, the ppk expression in rice mimics the Pi deficiency signal, which explained why the OsPHR2 expression was up-regulated in ETR in our experiment (Fig. 6). OsSPX1 is a negative regulator of OsPHR2, and a knockout of OsSPX1 will eventually lead to the increase of Pi in the above-ground part of rice (Liu et al.,

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the focus of future research.

ppk expression to simulate Pi deficiency signal. In addition, Pi transporters, regulated by OsPHR2, act directly on Pi uptake and transport in rice (Wang et al., 2009). Our previous hydroponic experiments demonstrated that the expressions of OsPT1, OsPT2, OsPT3, OsPT4, OsPT8 and OsPT10 in ETR were up-regulated, while in our field experiments, OsPT2, OsPT4 and OsPT8 showed similar trends. As a low-affinity Pi transporter, OsPT2 plays an important role in Pi transport in rice plants (Ai et al., 2009). OsPT4 was reported to be expressed in the rice root, leaf, stamen and caryopsis, and it encodes a plasmalemma-localized Pi transporter that mediates the acquisition and transport of Pi in rice plants (Ye et al., 2015; Zhang et al., 2015). OsPT8 functions in Pi-distribution between older and younger leaves, and it expresses in various tissues and organs from root to seed without being affected by Pi supply (Jia et al., 2011; Li et al., 2015). Therefore, the up-regulated expression levels of OsPHR2 and three OsPTs resulted in the increases of Pi concentrations in stems and leaves of ETR. In our experiments, the concentrations of TP and Pi in different rice tissues showed a highly consistent change trend (AI Figure A7), indicating that the increase of TP concentration in ETR under certain conditions was the result of the Pi increase. According to previous studies, P plays an important role in crop tillering, and the increase of P concentration can promote the tillering of gramineous plants, such as wheat and rice (Rodríguez et al., 1999; Takehisa and Sato, 2019). Our results showed that the tiller number was positively correlated (r2 ranged from 0.8013 to 0.9452) with TP concentration in stems and leaves (AI Figure A8). At the same time, rice tillering is a process in which many genes function together. In the past two decades, a large number of studies have been conducted on the molecular mechanism of rice tillering, and key genes such as MOC1, OsTB1, OSH1, TE and TAD1 have been found to regulate rice tillering (Liang et al., 2014). Among them, MOC1 is the first single tiller gene cloned from rice, and it is the most important regulator of the tiller number in rice (Li et al., 2003). MOC1 controls the initiation of the axillary meristem and the formation of tiller buds by affecting the downstream genes, OsTB1 and OSH1 (Takeda et al., 2003; Sato et al., 1996). At the same time, MOC1 is also regulated by TE and TAD1, which degrade the MOC1 protein by interacting with an anaphase-promoting complex (Lin et al., 2012; Xu et al., 2012). In addition, recent studies have found that the DELLA protein SLENDER RICE 1 (SLR1) can binds to MOC1 and inhibits its degradation. SLR1 also inhibits rice elongation, which explains why there is an inverse relationship between tiller number and plant height (Liao et al., 2019). Our results were highly consistent with the above conclusions. In our study, the ppk expression changed the P concentration in the stems and leaves of ETR, and then changed the nutrient status of the rice plant, thereby inducing changes in the expressions of tilling-controlled genes (Fig. 6). The expression of MOC1, OsH1 and SLR1 were up-regulated, which lead to the increase of the tiller number in ETR (Fig. 6). The tiller number is an important agronomic trait of rice and one of the determinants of grain yield (Wang and Li, 2011). The ppk expression in rice can promote P absorption by simulating the signal of Pi deficiency; it can also increase rice tillers, thereby improving the P utilization and grain yield when P fertilizer is inadequate. However, higher Pi concentration in the above-ground plant tissues of ETRD also resulted in the occurrence of ‘Pi toxicity’, which was shown as a decreased plant height, shorter panicles (AI Figure A2), sharply dropped grain-filling rate (Tables 1, 2) and earing rate (AI Figure A1) in our field experiments (Fig. 1). Those results were alike to the phenotype of over-expression of some endogenous phosphorus signal-related genes (Zhou et al., 2008). However, the mechanism of ‘Pi toxicity’ caused by high Pi concentration has not been reported in detail. It maybe because high P concentration in rice plants resulted in high expression levels of MOC1 and SLR1, which led ETRD to undergo reproductive growth without ending vegetative growth in the later stage of its life cycle and produce unlimited tillers. How to avoid ‘Pi toxicity’ while enhancing the uptake of phosphate fertilizer by rice is

5. Conclusion The ppk expression in rice can increase the plant absorbency of Pi in the soil by simulating the Pi deficiency signal and improve the utilization efficiency of the P fertilizer. When the P fertilizer was inadequate in the rice paddy soil, a higher P concentration in ETRS increased the rice plant tillers thereby improving grain yield. The maximum grain yield of ETRS was not only maintained at a considerable level, but was also achieved with less P fertilizer application. This study demonstrated the high P utilization efficiency of ETRS as new elite rice variety. ETRS promotion can make a strong contribution to reducing P fertilizer use in China’s rice production. Additional information Temperature during the study (Table A1); primers for qRT-PCR used in the study (Table A2); the tiller number per panicle (Table A3); the earing rate (Figure A1); the plant height and panicle length (Figure A2); relationship between grain yield and the yield components (Figure A36); relationship between phosphate (Pi) and total phosphorus (TP) content (Figure A7); relationship between total phosphorus (TP) and tiller number per hill (Figure A8). Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work is supported by grant from National Special Program of Water Environment (2017ZX07204002), the National Natural Science Foundation of China (41871082) and Jiangsu Provincial Natural Science Foundation of China (BK20190320). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fcr.2020.107715. References Ai, P., Sun, S., Zhao, J., Fan, X., Xin, W., Guo, Q., Yu, L., Shen, Q., Wu, P., Miller, A.J., Xu, G., 2009. Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation. Plant J. 57, 798–809. Chen, A., Hu, J., Sun, S., Xu, G., 2007. Conservation and divergence of both phosphateand mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species. New Phytol. 173, 817–831. Cordell, D., Drangert, J.-O., White, S., 2009. The story of phosphorus: global food security and food for thought. Glob. Environ. Change 19, 292–305. Fang, N., Xu, R., Huang, L., Zhang, B., Duan, P., Li, N., Luo, Y., Li, Y., 2016. Small grain 11controls grain size, grain number and grain yield in rice. Rice 9 (1), 64. FAO, 2019. Food and Agriculture Organization of the United Nations Home Page. (accessed June 27). http://www.fao.org/home/en. Hirooka, Y., Homma, K., Shiraiwa, T., Makino, Y., Liu, T.-s., Xu, Z., Tang, L., 2018. Yield and growth characteristics of erect panicle type rice (Oryza sativaL.) cultivar, Shennong265 under various crop management practices in Western Japan. Plant Prod. Sci. 21, 1–7. Hou, Y., Ma, L., Gao, Z.L., Wang, F.H., Sims, J.T., Ma, W.Q., Zhang, F.S., 2013. The driving forces for nitrogen and phosphorus flows in the food chain of china, 1980 to 2010. J. Environ. Qual. 42, 962–971. Huang, L., Sun, F., Yuan, S., Peng, S., Wang, F., 2018. Different mechanisms underlying the yield advantage of ordinary hybrid and super hybrid rice over inbred rice under low and moderate N input conditions. Field Crops Res. 216, 150–157. Huo, X., Wu, S., Zhu, Z., Liu, F., Fu, Y., Cai, H., Sun, X., Gu, P., Xie, D., Tan, L., Sun, C., 2017. NOG1 increases grain production in rice. Nat. Commun. 8, 1497. Jia, H., Ren, H., Gu, M., Zhao, J., Sun, S., Zhang, X., Chen, J., Wu, P., Xu, G., 2011. The phosphate transporter gene OsPht1;8 is involved in phosphate homeostasis in rice. Plant Physiol. 156, 1164–1175.

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