HAN Rui-cai ,LI Chen-yan ,Adnan RASHEED ,PAN Xiao-hua ,SHI Qing-hua ,WU Zi-ming

1 Key Laboratory of Crop Physiology,Ecology and Genetic Breeding,Ministry of Education/College of Agronomy,Jiangxi Agricultural University,Nanchang 330045,P.R.China

2 Rice Research Institute,Jiangxi Academy of Agricultural Sciences/Jiangxi Provincial Key Laboratory for Physiology and Genetics of Rice,Nanchang 330200,P.R.China

Abstract Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants,and post-translational phosphorylation regulates NR activity. To evaluate the impact of the dephosphorylation of nitrate reductase 1 (NIA1) protein on NR activity,nitrogen metabolism and plant growth,NIA1 phosphorylation site directed mutant lines (S532D and S532A) and an OsNia1 over-expression line (OE) were constructed,and the phenotype,NIA1 protein and its phosphorylation level,NR activity,nitrate metabolism and reactive oxygen metabolism of the transgenic lines were analysed. Exogenous NIA1 protein was not phosphorylated in S532D and S532A mutant lines,and their NR activities,activity states of NR and assimilation efficiencies of NO3--N were higher than those in Kitaake (WT) and OE. The changes in these physiological and biochemical indexes in the OE line were less than in S532D and S532A compared to WT. These results suggest that the removal of transcriptional level control had little effect on nitrogen metabolism,but the removal of post-translational modification had a profound effect on it. With the removal of NIA1 phosphorylation and the improvement in the nitrate assimilation efficiency,the plant height and chlorophyll content of S532D and S532A decreased and the hydrogen peroxide and malondialdehyde contents of rice seedlings increased,which may be related to the excessive accumulation of nitrite as an intermediate metabolite. These results indicated that the phosphorylation of NR may be a self-protection mechanism of rice. The reduced phosphorylation level of nitrate reductase improved the assimilation of nitrate,and the increased phosphorylation level reduced the accumulation of nitrite and prevented the toxic effects of reactive oxygen species in rice.

Keywords:rice (Oryza sativa L.),nitrate reductase,phosphorylation,directed mutation,nitrogen metabolism

1.Introduction

Nitrogen is the second only to water as the main component of rice production costs (Zhang et al.2018). China’s ricegrowing area accounts for 22% of the world’s rice-growing area,and the use of nitrogen fertilizer in Chinese rice accounts for 37% of the world’s total use of nitrogen fertilizer in rice (Zhu et al.2015). The uptake rate of nitrogen fertilizer

in Chinese rice fields is 30-35% (Wang et al.2013). If the nitrogen fertilizer is not absorbed by the rice after soil application,it is lost and causes a series of environmental hazards (Li and Li 2001;Wang et al.2004). The absorption and utilization efficiency of nitrogen in rice is obviously a key factor. Nitrate reductase (NR) is a rate-limiting enzyme for nitrogen assimilation in higher plants,and it directly regulates the reduction of nitrate (Chamizo-Ampudia et al.2017). Light induces NR phosphorylation which leads to a rapid decline of NR activity from light to dark and reduces the uptake and assimilation of nitrate nitrogen in plants(Heidari et al.2011;Nemie-Feyissa et al.2013;Creighton et al.2017). Therefore,it is of great practical significance to study the phosphorylation regulation mechanism of NR activity in rice.

Using genetic transformation to study the regulation of NR activity in tobacco,the NR gene coding region was fused with a constitutive expression promoter to relieve the transcriptional control of NR. The phosphorylation site Ser521 was mutated to an aspartic acid residue (Asp,D),which was not phosphorylated (Lea et al.2006). Nontranscriptional level control had no significant effect on nitrogen metabolism,but post-translational modification removal had a profound effect on nitrogen metabolism.The wild-type NR was rapidly inactivated from light to dark,and the phosphorylated site directed mutant NR (S521D)showed constitutive activity (Lillo et al.2003). Methionine(Met,M) on the hinge I conserved motif (RSVpSTPFM) of Arabidopsis NIA2 protein may be oxidized and coupled with oxidative signals to prevent the phosphorylation of peptides,which makes it possible to regulate NR activity using Met residues in conserved motifs (Hardin et al.2009).

The gene AtNia2 primarily governs NR activity in Arabidopsis,and mutation of the AtNia2 gene leads to a 90% loss of NR activity (Yu et al.1988). The NR gene OsNia1 in the rice genome has the highest homology with AtNia2,and NR activity decreased 80 and 95% in roots and leaves,respectively,after interference with the OsNia1 gene,which indicates that the OsNia1 gene primarily controls NR activity (Fan et al.2007;Tian et al.2009).The phosphorylation sites of NR are serine residues (Ser,S) at the 534,543,521,and 528th amino acids of NR in Arabidopsis,spinach,tobacco,and potato,respectively(Lea et al.2006;Chi et al.2015;Xu et al.2016;Creighton et al.2017). We hypothesized that the phosphorylation of Ser at the 532nd amino acid of NIA1 would regulate NR activity in rice via the alignment of amino acid sequences.

The current study cloned the NR gene OsNia1(Os08g0468100) from the rice genome,according to the biochemical characteristics of the NIA1 phosphorylation site,using directed mutations of amino acid residues to simulate permanent phosphorylation and non-permanent phosphorylation.Several transgenic lines with directed mutations of the NIA1 phosphorylation site were constructed.The present study used the NIA1 phosphorylation site directed mutation lines (S532D and S532A),the OsNia1 over-expression line (OE) and Kitaake to study the effects of NIA1 phosphorylation site directed mutations on the rice seedling phenotype,NR activity,NR phosphorylation level,nitrogen metabolism,and reactive oxygen species(ROS) level,and explore the regulatory mechanisms of NR phosphorylation and activity in rice. A theoretical basis is provided for the potential of efficient utilization of nitrogen in rice.

2.Materials and methods

2.1.Plant materials and growth conditions

NIA1 phosphorylation site directed mutant lines (S532D and S532A),the OE and Kitaake (Oryza sativa L.) were used. S532D and S532A were obtained by transferring the exogenous OsNia1 genes with Ser-532 mutated into aspartic acid (Asp,D) or alanine (Ala,A) into Kitaake.OE was similar to S532D and S532A,except that the expressed rice NIA1 protein was intact. Exogenous NR was constitutively expressed in S532D,S532A and OE using the Ubi promoter. The construction of the carrier and genetic transformation of the vectors were completed in the laboratory of Prof.Wan Jianmin,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences.

The experiments were performed at the Key Laboratory of Crop Physiology,Ecology,and Genetics Breeding of Jiangxi Agricultural University,Nanchang,Jiangxi Province,China.The test materials were grown in a walk-in climate chamber.The seeds were surface sterilized for 15 min using a 1%formalin solution (40% formaldehyde),and the buds were accelerated to 3 d. Well-germinated seeds were placed in 96-well PCR plates. Seeds were cultured in a hydroponic nutrient solution that was formulated according to the International Rice Research Institute (IRRI) (114.3 mg L-1NH4NO3,50.4 mg L-1NaH2PO4·2H2O,89.3 mg L-1K2SO4,158.2 mg L-1CaCl2,405 mg L-1MgSO4·7H2O,1.875 mg L-1MnCl2·4H2O,92.5 μg L-1(NH4)6Mo7O24·2H2O,1.1675 mg L-1H3BO3,43.75 μg L-1ZnSO4·7H2O,38.75 μg L-1CuSO4·5H2O,97.125 mg L-1FeCl3·6H2O,and 14.875 mg L-1citric acid).Growth conditions in the incubator were 12 h/12 h (day/night),28°C/26°C (day/night),600/0 μmol m-2s-1(day/night),and 75% relative humidity.

The seeds of test materials were sown in the seedling tray on March 10,2019. The soil used to raise the seedlings was taken from fields. The seedlings were transplanted to the field at 25 d,and reached the heading stage about 40 d after transplantation. All transgenic lines and the wildtype had similar growth stages.

2.2.Sampling and measurements

Leaves of rice in hydroponics were collected at 07:00 (after illumination),08:00,10:00,13:00,16:00,19:00,22:00,01:00,04:00,and 07:00 (before illumination) after the rice seedlings had grown for 20 d,and were stored at -80°C.These samples were used to analyse the diurnal variations of NR activity,NR phosphorylation level and the contents of nitrate,ammonium and amino acids. Rice leaves were also taken at 10:00 a.m.and 22:00 p.m.on days 11,17 and 20 for the detection of nitrite,hydrogen peroxide (H2O2)and malondialdehyde (MDA) contents. Plant height and chlorophyll content were measured after the seedlings were grown for 20 d.

The penultimate leaves of rice in the field were taken at the heading stage for the detection of chlorophyll content,and plant height was measured at the same time.

2.3.Levels of NIA1 protein and phosphorylation

Fresh leaves (0.5 g) were ground into a powder in liquid nitrogen,homogenized with 1.5 mL of protein extract and centrifuged at 12 000 r min-1for 10 min. The supernatant was the protein extract. The protein concentration was determined using Coomassie bright blue colorimetry,and the protein concentration was adjusted to be consistent with the protein extract. SDS-PAGE sample loading buffer (5×)was added to the protein extract in a ratio of 4:1 between the loading buffer and the protein,and the mixtures were denatured at 105°C for 10 min. Western blot (WB) was used to analyse the protein content and phosphorylation level of NIA1. The NIA1 protein antibody and its phosphorylated antibody were prepared by ABclonal Technology Co.,Ltd.(Wuhan,China). Quantitative analyses were performed via scanning of the formazan bands in the gel using a computing laser densitometer (Image J2× Software,USA).

2.4.Analysis of OsNia1 expression levels

Total RNA was isolated from the leaves of seedlings using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa,China). Approximately 0.5 μg total RNA was reversetranscribed to the first-strand cDNAs using the PrimeScript TM RT Master Mix (TaKaRa,China). QRT-PCR detection was performed on a CFX96 Real-Time PCR Detection System using SYBR®Premix Ex Taq TM II (TaKaRa,China).During qRT-PCR,OsNia1 transcripts were co-amplified with Ubiquitin (LOC4327162) mRNA as an internal control. All primers were designed using NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/).

2.5.NR activity analysis

NR activity was determined according to the method described in Lea et al.(2006). Leaves (0.5 g) were homogenized with 4 mL of 0.1 mol L-1HEPES-KOH,pH 7.5,3% (w/v) PVP,1 mmol L-1EDTA,and 7 mmol L-1Cys. The assay mixture contained 50 mmol L-1HEPES-KOH,pH 7.5,100 mmol L-1NADH and 5 mmol L-1KNO3with 2 mmol L-1EDTA or 6 mmol L-1MgCl2. The assay volume was 2 mL.Activity was measured in crude extracts by determining NO2-formation by the addition of 1% sulfanilamide and 0.2% N-(1-naphtyl)-ethylene-diamine dihydrochloride in 3 mol L-1HCl.Activity state was defined as NR assayed in the presence of Mg2+(and 14-3-3 proteins) as a percentage of NR activity measured in the presence of EDTA,which reflected the quantity of the enzyme in the non-phosphorylated active form. All assays were run at 25°C.

2.6.Plant height and chlorophyll content

For each of the three replicates of each material,10 plants were selected for plant height measurement,and the average plant height was calculated.

Chlorophyll content was determined spectrophotometrically after extraction of shoots with 80% (v/v) acetone (Lattanzio et al.2009).

2.7.Nitrate,ammonium and free amino acid content

Rice leaves (0.5 g) were weighed,and 10 mL ddH2O was added. Nitrate,ammonium and free amino acids were extracted in boiling water for 30 min. Three samples from each treatment group were tested in each replicate experiment. Nitrate content in rice leaves was determined using nitrosalicylic acid colorimetry (Cataldo et al.1975).Ammonium content was determined using indiophenol blue colorimetry (Hachiya et al.2012). Amino acid content was determined by the hydrinhydrin method (Xu et al.2017).

2.8.Nitrite content

Nitrite content was determined according to the method described in Hachiya et al.(2016). Leaves (0.5 g) were homogenized in 4 mL of 0.1 mol L-1HEPES-KOH,pH 7.5,3% (w/v) PVP,1 mmol L-1EDTA and 7 mmol L-1Cys,and centrifuged at 20 400 r min-1for 10 min. A total of 1 mL of 1% sulfanilamide and 2 mL of 0.2% N-(1-naphtyl)-ethylenediamine dihydrochloride were added to 1 mL extract,and the solution was mixed and reacted for 20 min at room temperature. The mixture was centrifuged at 4 000 r min-1for 5 min. The absorbance value was determined at 540 nm.Nitrite contents in the leaves were calculated according to the regression equation obtained from the standard curve.

2.9.H2O2 and MDA contents

H2O2content was determined using the titanium sulphate method (Qi et al.2006). MDA content was measured by the thiobarbital carboxylic acid method (He et al.2011).

2.10.Statistical analysis

Individual means and standard errors of the means were calculated using the data from independent samples in Microsoft Excel 2007 (Microsoft,USA). IBM SPSS Statistics 22 (SPSS 22,SPSS Inc.,USA) was used for statistical analyses,and the least significant difference method (LSD)was used for the inter-processing significance test. The level of significance was checked at P＜0.05 and was indicated by different letters.

3.Results

3.1.Construction,generation and confirmation of rice transgenic lines

The OsNia1 gene with directed mutation was obtained using fusion PCR and connected to a pCUBi1390 over-expression vector. The vector was transferred to the rice variety Kitaake to obtain S532D,S532A and OE. The base sequence encoding Ser (S) at position 532 of the NIA1 protein was replaced with base sequences encoding Asp (D) and Ala(A) in S532D and S532A,respectively (Fig.1-A). Using the molecular identification of T0,T1and T2transgenic plants,homozygous directed mutation and unmutated OsNia1 over-expression lines were obtained. Western blot analysis of selected T2transgenic lines demonstrated that S532D,S532A and OE transgenic plants expressed exogenous NIA1 protein. The exogenous NIA1 proteins expressed in S532D and S532A were not phosphorylated,but NIA1 was phosphorylated in OE (Fig.1-B). Homozygous transgenic lines were selected for semiquantitative PCR analysis.Expression of OsNia1 in all selected transgenic lines was higher than in WT (Fig.1-C). Total NR enzyme activity varied between the selected transgenic lines,with S532D and S532A showing approximately 1.5-fold and OE showing almost 0.5-fold higher activities compared to WT (Fig.1-D).

Fig.1 Construction of vectors and confirmation of transgenic lines. A,a simplified schematic diagram of the vectors used in the transgenic process. S532D and S532A,NIA1 phosphorylation site directed mutant lines. OE,OsNia1 over-expression line. B,the protein expression of ACTIN,GFP,exogenous NIA1 (NR protein 1),and phosphorylated NIA1 (p-NR) in transgenic lines were detected. C,expression levels of the OsNia1 gene in transgenic lines. The ubiqutin gene was used as a reference gene. D,nitrate reductase (NR) activity in transgenic lines. WT,wild type. S532D(1) and S532D(2),NIA1 phosphorylation site directed mutant lines with Ser (S) mutated to Asp (D);S532A(1) and S532A(2),NIA1 phosphorylation site directed mutant lines with Ser(S) mutated to Ala (A);OE(1) and OE(2),OsNia1 over-expression lines. Bars mean SD (n=3). Different letters indicate that the means are significantly different at P＜0.05.

3.2.Phenotypes of transgenic lines at the seedling stage and heading stages

NIA1 phosphorylation site S532 directed mutation plants were weaker than the WT,which was indicated by the lower plant heights and chlorophyll contents of S532D,S532A and OE than WT. We also found that the phosphorylated site directed mutant lines were also weaker than OE,and the weak growth was primarily manifested in plant height.The plant height of S532A was significantly lower than OE(Fig.2). The growth of experimental materials was slightly different at the seedling and heading stages,which may be due to the cultural environment.

Fig.2 Phenotypes of transgenic lines at the seedling and heading stages. A,representative phenotype images of transgenic lines at the seedling and heading stages. B,the plant height at the seedling stage. C,the chlorophyll content at the seedling stage. D,the plant height at the heading stage. E,the chlorophyll content at the heading stage. WT,wild type;S532D and S532A,NIA1 phosphorylation site directed mutant lines;OE,OsNia1 over-expression line. Bars mean SD (n=3). Different letters indicate that the means are significantly different at P＜0.05.

3.3.Diurnal variations of NR activity in leaves of transgenic lines

Total NR and actual NR activities exhibited a circadian rhythm that first increased and then decreased in all materials,and they were higher in S532D,S532A and OE than the WT throughout the day. When it was dark,the total and actual NR activities of S532D and S532A were higher than those in WT and OE,and WT was the lowest. These results indicated that a directional mutation at the phosphorylation sites may also act positively on the translation process or the stability of the NR protein. Posttranslational phosphorylation contributed strongly to the diurnal variations of NR activity in WT and OE plants,which was evident when NR activity was measured in the presence of Mg2+(Fig.3-A and B). Even in the presence of Mg2+,in situ inactivation of NR may be underestimated because some activation due to dephosphorylation likely occurred during the extraction and assay of NR,but this activation is not relevant for the mutated NR from S521D plants (Mackintosh et al.1992). NR activity in OE and WT plants may be even lower,especially in the dark,which would widen the gap between these plants and the phosphorylation site-directed mutant lines (Fig.3-A). Actual NR activity increased 83.1 and 181.2%in the WT and OE plants in the light,respectively,compared to the dark conditions,but the increases were very modest in the S532D and S532A plants (30.2 and 53.6%,respectively).The activity state (Mg2+NR/EDTA NR) was always high(75-85%) in S532D and S532A throughout the day and night,as expected (Fig.3-C). For WT and OE,the activity states varied between 55 and 85%. The phosphorylation site S532 is a targeted mutation that participates in the regulation of phosphorylation of the NR protein,and it plays a positive role in maintaining the stability and activity of NR.

Fig.3 Diurnal variations of nitrate reductase (NR) activity in the leaves of transgenic lines. A,EDTA NR (total NR activity).B,Mg2+ NR (actual NR activity). C,activity state,percentage of active NR activity out of total NR activity. WT,wild type;S532D and S532A,NIA1 phosphorylation site directed mutant lines;OE,OsNia1 over-expression line. The white and black bar at the top shows changes in the light supply to the leaves,with the dark period shaded. Bars are SE (n=3).

3.4.Diurnal variations in NIA1 protein and phosphorylation level

To investigate whether the stabilities of NIA1 were regulated via the transcriptional and directed mutations of S532,NIA1 protein and its phosphorylation levels in WT,S532D,S532A,and OE were assessed using immunoblotting with antibodies to NR and phospho-NR (p-NR) (Fig.4). The NR protein level showed a trend of first increasing then decreasing in all test materials,and it was higher during the day compared to the night. The NR protein levels were higher in S532D,S532A and OE than WT.Similar levels appeared in S532D,S532A and OE. No phosphorylation of NR protein was detected when the amino acid residue S532 was mutated to Asp (D) or Ala (A). The phosphorylation levels of NR protein in WT and OE at night were much higher than the daytime. Increased phosphorylation and reduced NR activity were observed in leaves at night.

Fig.4 Diurnal variations of nitrate reductase (NR) protein and phosphorylation levels in leaves of transgenic lines. Soluble protein was extracted from leaves of 2-wk-old transgenic plant seedlings. Each lane in the gel was loaded with equal contents of soluble protein. NR,exogenous NIA1;p-NR,phosphorylated NIA1;NR and p-NR were analysed using immunoblotting with anti-NR and anti-p-NR antibodies,respectively. Numbers above the lanes indicate relative intensity obtained via scanning of the formazan bands using a computing laser densitometer (Image J 2× Software). WT,wild type;S532D and S532A,NIA1 phosphorylation site directed mutant lines;OE,OsNia1 over-expression line.

3.5.Diurnal variations in the contents of nitrate,ammonium and free amino acids

NR assimilates nitrate into ammonium and further transforms it into amino acids. The contents of these metabolites reflect the effects of NR activity on nitrogen uptake and utilization in plants. Diurnal variations in the nitrate contents of leaves are shown in Fig.5-A. For WT and OE plants,a sustained reduction in nitrate content was observed during the day,but this reduction was not observed in S521D. These findings may be explained by the large changes in actual NR activity in WT and OE plants. After entering the dark for 3 h,the nitrate contents in WT and OE increased continuously then decreased rapidly after 6 h. They were also related to the activity change of NR. A remarkable difference in the level of nitrate throughout the day and night was observed between the NIA1 phosphorylation site directed mutant lines (S532D and S532A) and the other lines (WT and OE) contained less than one-half the nitrate of WT and OE. Because NR in the S532D and S532A mutants was constantly active,a high nitrate reduction was expected and a filling of the nitrate pool may not be possible. Diurnal variations in the ammonium contents of leaves are shown in Fig.5-B. For WT and OE plants,the contents of ammonium increased,decreased,then increased throughout the day,which confirmed the changes in NR activity. On average,the ammonium contents of S532D and S532A were approximately 40 and 20% higher than WT,and OE was similar to WT. Diurnal variations in the free amino acid contents of leaves are shown in Fig.5-C. The free amino acid contents of S532D and S532A were higher than WT and OE,and the trend in the variation of all lines was consistent.

Fig.5 Diurnal variations of nitrogen contents in the leaves of transgenic lines. A,nitrate content. B,ammonium content. C,free amino acid content. WT,wild type;S532D and S532A,NIA1 phosphorylation site directed mutant lines;OE,OsNia1 over-expression line. The white and black bar at the top shows changes in the light supply to the leaves,with the dark period shaded. Bars are SE (n=3).

3.6.Content of nitrite in transgenic lines

Nitrite (NO3-) is the direct product of nitrate reduction by NR.Nitrite must be reduced immediately after its uptake because of its high toxicity to the plant (Hachiya et al.2016). The nitrite content was measured at 10:00 a.m.and 22:00 p.m.when the seedlings had grown to 11,17 and 20 d. According to Fig.6,the nitrite contents in WT and OE leaves were lower at 22:00 p.m.compared with 10:00 a.m.,which maybe due to the decreased NR activity at night. However,S532D and S532A showed the opposite trend,with much higher nitrite contents at 22:00 p.m.than at 10:00 a.m.The accumulation of nitrite at night was lower than in the daytime,and the increase in NR activity led to the rapid accumulation of nitrite at night. The contents of nitrite in the leaves of S532D,S532A and OE increased approximately 1-fold at 10:00 a.m.compared to WT,but they increased approximately 6-,6-and 1.5-fold at 22:00 p.m.,respectively.These results showed that the removal of phosphorylation of NIA1 protein had a great effect on NR activity and nitrate accumulation.

Fig.6 Nitrite content in the leaves of transgenic lines. WT,wild type;S532D and S532A,NIA1 phosphorylation site directed mutant lines;OE,OsNia1 over-expression line. Bars are SD (n=3). Different letters indicate that the means are significantly different (P＜0.05).

3.7.Contents of hydrogen peroxide and malondialdehyde in transgenic lines

The accumulation of nitrite produces toxic reactions and changes the content of ROS in plants. Changes in ROS may also lead to different degrees of plant damage. The research here showed that the contents of H2O2and MDA in S532D,S532A and OE were higher than WT (Fig.7).As shown by the content of MDA,the degrees of damage to leaves of S532D and S532A were also higher than OE.The contents of H2O2and MDA in leaves continued to increase over time.

Fig.7 Hydrogen peroxide (H2O2,A) and malondialdehyde (MDA,B) contents in leaves of transgenic lines. WT,wild type;S532D and S532A,NIA1 phosphorylation site directed mutant lines;OE,OsNia1 over-expression line. Bars are SD (n=3). Different letters indicate that the means are significantly different (P＜0.05).

4.Discussion

4.1.The directed mutation at site S532 of NIA1 blocked the photoperiodic changes of NR activity

Phosphorylation is the main form of NR chemical modification,and it provides a regulatory mechanism for nitrogen metabolism in eukaryotes. Light induced the phosphorylation of NR and the phosphorylation level from light to dark was enhanced,which sharply reduced the activity of NR. To reveal the effect of protein phosphorylation on NR activity,nitrogen utilization and rice growth,we constructed transgenic lines with directional mutations of the phosphorylation site S532 of NIA1. The diurnal changes of total NR activity (EDTA NR),actual NR activity (Mg2+NR),the NIA1 protein and its phosphorylation level,and the contents of nitrate,ammonium and free amino acids in the seedling leaves of transgenic lines were analysed in the present study. Phospho-NR in leaf extracts (which occurred in the dark) is sensitive to Mg2+inhibition,and dephospho-NR (which occurs in the light) is completely unaffected by Mg2+. Therefore,assaying the enzyme in the absence of Mg2+represents the total potential NR activity,and assays in the presence of Mg2+reflect the alterations related to the phosphorylation/dephosphorylation mechanism(Mackintosh et al.1995;Fan et al.2007). The NR activity states (EDTA NR/Mg2+NR) of S532D and S532A were stable at approximately 80%,and the NIA1 protein could not be phosphorylated. The NR activity state of OE ranged from 55 to 85%,and the NIA1 protein could be phosphorylated,which indicated that the serine residue at position 532 of NIA1 was the phosphorylation site of NR. Directed mutation of S532 led to the failure of light/dark conversion and other treatments that regulate the activation of exogenous NR,and the directed mutation had positive effects on the stability and activity of the NR protein. Phospho-NR remained active,and its inactivation was due to the conformational change of the NIA protein caused by binding 14-3-3 protein (Lambeck et al.2012;Chi et al.2015). Previous studies showed that Asp simulated phosphorylated serine residues due to its phosphate (Wang et al.1992). The phosphate is present in the amino acid itself,so it would not be removed by the phosphatase,which leaves the protein in a simulated state of phosphorylation forever. When Ser was mutated to Ala,the amino acid never becomes attached to the phosphate group,which mimics the permanent non-phosphorylated state of NIA1. These two types of mutations were consistent in their regulation of NR phosphorylation and activity,and all of the mutations were beneficial to the stabilization of the NR activity state. These results indicated that Asp,similar to Ala,did not have the function of a phosphorylated serine and could not regulate the activity of NR via binding 14-3-3 protein. The phosphorylation sites S521 and S534 of the NIA2 protein of tobacco and Arabidopsis were mutated into Asp,and the NR in the transgenic plants showed a stable activity state (Lea et al.2004;Lillo et al.2004;Tian et al.2009). Although the NR activity states in S532D and S532A were relatively stable,the total NR and actual NR activities in the leaves of these plants still showed a certain circadian rhythm (Aitken 1996). The main reason may be that the endogenous protein and NR complementary proteins,such as NIA2 in S532D and S532A,regulate the activity of NR. The exogenous NR protein may have other phosphorylation sites in Arabidopsis,such as S627,S43 and S72,to regulate the phosphorylation level of the NR protein (Wang et al.2010,2011;Swatek et al.2014).

4.2.The directed mutations at site S532 of NIA1 improved NR activity and nitrogen utilization in rice

The comparative analysis of the activities of S532D,S532A,OE and WT showed that the total activity,actual activity and activity states of S532D,S532A and OE improved significantly compared to the WT. When entering the dark environment,the total activity,actual activity and activity state of OE decreased rapidly to a level similar to WT and significantly lower than S532D and S532A. This result showed that light may also act positively on the translation process or the stability of the NR protein. The diurnal variations of nitrate,ammonium and amino acid contents in OE were similar to WT. The average levels of nitrate in WT and OE were significantly higher than in S532D and S532A,but the the ammonium and amino acid contents in WT and OE were significantly lower than in S532D and S532A. Therefore,deregulation of NR at the transcriptional level did not have much influence on metabolite levels,but (additional) deregulation at the posttranslational level resulted in profound changes in nitrogen metabolite levels(Tian et al.2009).

4.3.Phosphorylation of NR may be a self-protective mechanism in rice

Rice seedlings of S532D and S532A grew weakly compared to WT and OE. The contents of H2O2and MDA in S532D and S532A were higher than in WT and OE. H2O2and MDA are metabolic substances produced by plants in response to adverse environmental conditions. However,the differences between S532D,S532A and WT,OE were primarily in the activity of NR and the content of nitrogen metabolites(Figs.3 and 5). Previous studies showed that nitrite and ammonium had damaging effects in plants (Britto et al.2002;Lillo et al.2003;Lea et al.2004). A mixed nutrient solution containing 20 mg L-1NO3--N and 20 mg L-1NH4+-N was used to grow rice in the present study. Excessive transformation of NO3--N was the reason for the weak growth of S532D and S532A. The analysis of nitrogen utilization showed that S532D and S532A efficiently used NO3--N to generate NH4+-N and amino acids,and excessive accumulations of nitrite and ammonium occurred in leaves (Figs.5 and 6).The contents of nitrite in S532D and S532A were much higher than in OE and WT,especially at night. The ammonium contents of S532D and S532A were also higher than in WT and OE. The increase in NR activity led to an excessive accumulation of nitrite in the dark,which caused tobacco S521 seedlings to grow weaker (Lillo et al.2003).Nitrite accumulation may be the reason for the weak growth of transgenic lines with mutated phosphorylation sites,but this result must be further verified. Previous studies showed that the phosphorylation of NR may be a self-protective regulatory mechanism to maintain the balance of supply and demand of the nitrogen metabolites in rice.

5.Conclusion

The serine residue at position 532 of NIA1 was the phosphorylation site of NR. The directed mutation at site S532 of NIA1 blocked the photoperiodic changes of NR activity and improved NR activity and nitrogen utilization in rice. The phosphorylation of NR may be a self-protective regulatory mechanism of rice. A reduction of the NR phosphorylation level may improve the utilization efficiency of nitrate,and an increase in the phosphorylation level was beneficial for reducing the accumulation of toxic metabolites,such as nitrite.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31560350 and 31760350)and the Key Research and Development Program of Jiangxi Province,China (20171ACF60018 and 20192ACB60003).

Declaration of competing interest

The authors declare that they have no conflict of interest.