Dephosphorylation of Nitrate Reductase Protein Regulates Growth of Rice and Adaptability to Low Temperature

Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and its activity is regulated by post-translational phosphorylation. The change of nitrogen uptake affects the response of rice to low temperature and its growth. To investigate the effect of NIA1 protein dephosphorylation on the growth of rice and its adaptability to low temperature, we analyzed phenotype, chlorophyll content, nitrogen utilization, and antioxidant capacity at low temperature in lines with a mutated NIA1 phosphorylation site (S532D and S532A), an OsNia1 over-expression line (OE), and wild-type Kitaake rice (WT). Plant height, dry matter weight, and chlorophyll content of S532D and S532A were lower than those of WT and OE under normal growth conditions but were higher than those of WT and OE at low temperature. Compared with WT and OE, the nitrite, H2O2, and MDA contents of S532D and S532A leaves were higher under normal growth conditions. The difference in leaf nitrite content between transgenic lines and WT was narrower at low temperature, especially in S532D and S532A, while H2O2 and MDA contents of S532D and S532A leaves were lower than those in WT and OE leaves. The NH4+-N and amino acid contents of S532D and S532A leaves were higher than those of WT and OE leaves under normal or low temperature. qRT-PCR results revealed that transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were positively correlated with those of OsNia1, and the transcription levels of OsNrt2.4, OsNia2, and OsNADH-GOGAT were significantly higher in transgenic lines than in WT under both normal and low temperature. Phosphorylation of NR is a steady-state regulatory mechanism of nitrogen metabolism, and dephosphorylation of NIA1 protein improved NR activity and nitrogen utilization efficiency in rice. Excessive accumulation of nitrite under normal growth conditions inhibits the growth of rice; however, accumulation of nitrite is reduced at low temperature, enhancing the cold tolerance of rice. These results provide a new insight for improving cold tolerance of rice.


Introduction
Rice (Oryza sativa L.) is a thermophilic crop widely cultivated in most areas of China.Low temperature causes retardation of rice growth and development, and reduction in tillering and fertility, which eventually affect rice yield.Nitrogen is one of the most important and active nutrient factors affecting plant growth and yield (Mi et al. 2017).Studies on the interaction between cold stress and nitrogen fertilizer showed that applying nitrogen fertilizer significantly improves the resistance of rice to low temperature, but high nitrogen levels aggravate the damage caused by low temperature (Cao et al. 2018).Abscisic acid and indole acetic acid levels in rice leaves are improved after treatment with high nitrogen and low temperature (Cai 2014).Higher abscisic acid and cytokinin content and lower gibberellic Handling Editor: Mikihisa Umehara.
RuiCai Han, YuPeng Wang, ChenYan Li and ZiMing Wu have contributed equally to this work and share first authorship.
acid content slow down the cold injury of plants (Li et al. 2015).Excessive application of nitrogen fertilizer results in tender stems and leaves of rice and susceptibility to cold damage (Cao et al. 2020).Microspore sterility caused by low temperature is aggravated by high nitrogen application during the panicle primordium differentiation stage of rice, reducing seed setting rate and yield (Hayashi et al. 2000(Hayashi et al. , 2006)).In production years with occurrence of low-temperature chilling injury, the amount of water and fertilizer applied affects the yield of rice; application of large amounts of nitrogen fertilizer causes especially serious panicle neck phenomenon in the field, and the setting rate and yield of rice panicles is obviously decreased.Therefore, regulation of nitrogen uptake and utilization efficiency affects the tolerance of rice to cold stress, and rice cold tolerance is not consistent under different environmental and nitrogen supply conditions.There are few studies on improving nitrogen assimilation and utilization of rice in response to low temperature stress by molecular methods.Such research might provide a means of enhancing the cold tolerance of rice.
Nitrate reductase (NR) is a rate-limiting enzyme for nitrogen assimilation in higher plants that directly regulates the reduction of nitrate (Crawford 1995).NR activity in plants is inhibited by phosphorylation at a conserved serine residue (Ser, S) in hinge I when plants are transferred from light to darkness (Muslin et al. 1996;Nemie-Feyissa et al. 2013) and ultimately affects plant growth and development (Steven et al. 1996;Kaiser et al. 1999;Harris et al. 2000).Genetic transformation was used to study the regulation of NR activity in tobacco (Nicotiana plumbaginifolia Viv.) by fusing the coding region of the Nia gene with a promoter driving constitutive expression, thereby relieving transcriptional control of NR.Residues at the phosphorylation site Ser521 were also mutated to aspartic acid residues (Asp, D) and were not phosphorylated (Bachmann et al. 1996;Lea et al. 2006).Release of transcriptional-level control had no significant effect on nitrogen metabolism, while removal of post-translational modification had a profound effect.Wildtype NR was rapidly inactivated when plants were moved from light to dark, while NR with mutated phosphorylation site (S521D) showed constitutive activity (Lillo et al. 2003).The phosphorylation sites of NR are serine residues at amino acid (aa) positions 534, 543, 521, and 528 in Arabidopsis (Arabidopsis thaliana L.), spinach (Spinacia oleracea L.), tobacco (Nicotiana plumbaginifolia Viv.), and potato (Solanum tuberosum L.), respectively (Bachmann et al. 1996;Steven et al. 1996;Harris et al. 2000;Lea et al. 2006).Through comparative analysis of amino acid sequences, we speculated that phosphorylation of Ser at aa 532 of NIA1 might regulate NR activity in rice.On this basis, we constructed rice lines with a mutated NIA1 phosphorylation site (S532D and S532A).Phosphorylation level tests revealed that the exogenous NIA1 proteins could not be phosphorylated, and the expression of endogenous NIA1 protein was inhibited (Figure S1).
Studies on the phosphorylation of NR protein have mainly focused on the influence of nucleotide sequence changes on phosphorylation level and the regulation of NR activity by phosphorylation modification.We therefore investigated the effect of deactivating NIA1 phosphorylation on NR activity, nitrogen metabolism, reactive oxygen metabolism, and plant growth in rice using lines with a mutated NIA1 phosphorylation site (S532D and S532A), an OsNia1 over-expression line (OE), and wild-type Kitaake (WT) at low temperature.Our findings suggest novel methods for improving the cold tolerance of rice by regulating nitrogen metabolism.

Plant Material and Growth Conditions
Rice lines with a mutated NIA1 phosphorylation site (S532D and S532A), an OsNia1 over-expression line (OE), and wild-type Kitaake (WT) were used.S532D and S532A were obtained by transferring exogenous OsNia1 (LOC4345795) with Ser-532 mutated into Asp or alanine (Ala, A) into Kitaake; exogenous OsNIA1 protein was the fusion protein of endogenous NIA1 and GFP protein (28KD), exogenous OsNIA1 expressed from either construct could not be phosphorylated, endogenous OsNIA1 protein expression was inhibited and its protein level could not be detected (Figure S1B).OE plants were similar to S532D and S532A with the exception that the rice NIA1 protein was intact.In S532D, S532A, and OE, exogenous NR was constitutively expressed using the CamV35S promoter.All transgenic lines used in this study were homozygous T3 plants.Construction of the carrier and genetic transformation of vectors were completed in the laboratory of Wan Jianmin, Institute of Crop Science, Chinese Academy of Agricultural Sciences.
Experiments were performed at the Key Laboratory of Crop Physiology, Ecology, and Genetics Breeding of Jiangxi Agricultural University, Nanchang, Jiangxi, P.R. China.Test materials were grown in a walk-in climatecontrolled chamber.Seeds were sterilized for 15 min using 1% (v/v) formalin solution (40%, v/v, formaldehyde) and allowed to germinate for 3 d; well-germinated seeds were then placed into individual wells of 96-well PCR plates.Seedlings were cultured in hydroponic nutrient solution formulated according to the formula of the International Rice Research Institute (IRRI) (114.3

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 (50 mM Tris HCl, 150 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 10% (v/v) glycerol, 20 mM NaF, 50 mM DTT, 10 mM PMSF), and centrifuged at 12,000 rpm (10,200 g) for 10 min.The supernatant was retained as the protein extract.Soluble protein content in protein extract was determined using Coomassie bright blue colorimetry (Han et al. 2018).The protein concentrations were adjusted to the same level by adding protein extract, and the same loading volume was used to detect phosphorylated protein as non-phosphorylated protein.SDS (5 ×) sample loading buffer (sample:buffer = 4:1) was added, and proteins were denatured at 105 ℃ for 10 min.Immunoblotting was used to analyze the protein content and phosphorylation level of NIA1.Antibodies against the NIA1 protein and its phosphorylated form were prepared by ABclonal Technology Co., LTD (Wuhan, China).Quantitative analyses were performed by scanning formazan bands in the gel using a computing laser densitometer (Molecular Dynamics) using Image J2x software (USA).

NR Activity
NR activity was determined according to the method described by Lea et al. (2006).Three samples were tested for each treatment group in each replicate experiment.Leaves (0.5 g) were homogenized with 4 mL of 0.1 M HEPES-KOH, pH 7.5, 3% (w/v) PVP, 1 mM EDTA, and 7 mM Cys.The assay mixture contained 50 mM HEPES-KOH, pH 7.5, 100 mM NADH, and 5 mM KNO 3 with 2 mM EDTA or 6 mM MgCl 2 .Assay volume was 2 mL.Activity was measured in crude extracts by determining NO 2 − formation following the addition of 1% (w/v) sulfanilamide and 0.2% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride in 3 M HCl.Activity state is defined as NR assayed in the presence of Mg 2+ (and 14-3-3 proteins) as a percentage of NR activity measured in the presence of EDTA and reflects how much of the enzyme is in the nonphosphorylated active form.Assays were run at 25 ℃.
Nitrate, Ammonium, Free Amino Acid, and Soluble Protein Contents Rice leaves (0.5 g) were ground into a powder and added to 10 mL double-distilled water.Nitrate, ammonium, and amino acids were extracted using boiling water for 30 min.Three samples were tested for each treatment group in each replicate experiment.Nitrate content in rice leaves was determined using nitrosalicylic acid colorimetry (Cataldo et al. 1975).Ammonium content in rice leaves was determined using indophenol blue colorimetry (Hachiya et al. 2012).Amino acid content in rice leaves was determined using the ninhydrin method (Xu et al. 2017).
Rice leaves (0.5 g) were ground into a homogenate with 2 mL of 50 mmol/L phosphoric acid buffer (pH7.0)(6.8 g/L KH 2 PO 4 , 1.164 g/L NaOH), washed with 6 mL doubledistilled water and the sample volume adjusted to 10 mL.Three samples were tested for each treatment group in each replicate experiment.Soluble protein content in rice leaves was determined using Coomassie bright blue colorimetry (Han et al. 2018).

Chlorophyll Content
Chlorophyll content was determined spectrophotometrically after extraction of shoots with 80% (v/v) acetone (Lattanzio et al. 2009).Three samples were tested for each treatment group in each replicate experiment.

Nitrite Content
Nitrite content was determined according to the method described by Hachiya et al. (2016).Three samples were tested for each treatment group in each replicate experiment.Leaves (0.5 g) were homogenized with 4 mL of 0.1 M HEPES-KOH, pH 7.5, 3% (w/v) PVP, 1 mM EDTA, and 7 mM Cys and centrifuged at 20,400 rpm (29,500 g) for 10 min.The supernatant was retained, and 1 mL 1% (w/v) sulfanilamide and 2 mL 0.2% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride were added to 1 mL extract, mixed, and incubated for 20 min at 23 C before centrifuging at 4000 rpm (1133 g) for 5 min.Absorbance was determined at 540 nm.Nitrite contents in leaves were calculated according to the regression equation obtained from a standard curve.

Hydrogen Peroxide (H 2 O 2 ) and Malondialdehyde (MDA) Contents
H 2 O 2 content was determined using the titanium sulfate method (Qi et al. 2006).MDA content was measured using the thiobarbituric acid (TBA) method (Qi et al. 2006).

Statistical Analysis
Individual means and standard errors of the means were calculated using data from independent samples in Microsoft Excel 2007 (Microsoft, USA).IBM SPSS Statistics 22 (SPSS 22, SPSS Inc, USA) was used for statistical analysis and correlation analysis, and the least significant difference method (LSD) was used to determine significant differences between means.Level of significance was checked at P < 0.05 and indicated by different letters.Geneious R10 (Biomatters, NewZealand) was used for protein sequence alignment analysis.

Dephosphorylation of NIA1 Protein Enhances the Rice Tolerance to Low Temperature
We observed that growth of lines with a mutated NIA1 phosphorylation site was weaker than that of WT at normal temperature (26 °C), but the situation was opposite at low temperature (8 °C).We hypothesized that phosphorylation of NIA1 protein might affect the stress response of rice to low temperature.To test our hypothesis, we performed a low-temperature test for WT, S532D, S532A, and OE (Fig. 1).Rice seedlings were treated with low temperature at 20 d old, before the different lines showed any major differences in phenotype.Rice seedlings were transferred to low-temperature treatment at 8 °C for 6 d, then transferred back to normal temperature (26 °C) for 3 d; controls were maintained at 26 C. Plant height and dry matter weight of S532D and S532A plants were lower than those of WT and OE at normal temperature (NT6d and NT9d), while these two indicators were higher in S532D and S532A than in WT after low-temperature treatment for 6 d followed by normal-temperature treatment for 3 d (LTR3d) (Fig. 1B, C).Except in S532A, chlorophyll content remained the same at NT6d as before treatment (BT), being lower in S532D, S532A, and OE than in WT at NT9d (Fig. 1D).Compared with levels at NT6d, chlorophyll contents in all test lines were lower at LT6d, although levels in S532D and S532A were significantly (P < 0.05) higher than those in WT and OE.The chlorophyll content of all lines was increased at LTR3d compared with levels at LT6d, and was still significantly higher in S532D and S532A plants than in WT and OE plants.In general, S532D and S532A showed decreased growth capacity under normal conditions and increased cold tolerance under low temperature, indicating that regulating the dephosphorylation of NIA1 protein might change the stress response of rice seedlings to low temperature.

Dephosphorylation of NIA1 Protein Increases the Ratio of Active NR Protein
To investigate the effect of low temperature on NR phosphorylation, we assessed NIA1 protein and its phosphorylation levels in WT, S532D, S532A, and OE using immunoblotting with antibodies against NR and phospho-NR (p-NR) (Fig. 2).We adjusted the soluble protein concentration to similar levels in samples from the same line at different time The ratio of p-NR/NR for S532D and S532A was always 0, indicating that the exogenous NR protein could not be phosphorylated in these two lines due to the mutation of S532 to Asp or Ala, and the NR protein was always in a highly active state.Similar to WT, OE also showed high activity of NR at low temperature and low activity at room temperature, and its exogenous NR protein activity was regulated by phosphorylation modification.

Dephosphorylation of NIA1 Protein Increases NR Activity in Rice Leaves
We observed different stress responses of the test materials to low temperature, which we speculated to be due to the regulation of NR activity by low temperature.As shown in Fig. 3, NR activity was decreased under low temperature, but the reduction in NR activity was smaller in the lines with a mutated NIA1 phosphorylation site (S532D and S532A) than in the other lines.The NR activity in S532D and S532A was higher than that in WT or OE under normal or low temperature, with WT having the lowest NR activity.Significant differences (P < 0.05) in NR activity were caused by replacement of nutrient solutions with fresh nutrients at normal temperature.However, since nutrient solutions were all replaced at the same time, the impact of this on data analysis was relatively small.After returning to normal temperature for 3 d, the NR activity in all lines subjected

Dephosphorylation of NIA1 Protein Increases Nitrogen Utilization in Rice
As can be seen from Fig. 4A, the content of nitrate in leaves of S532D, S532A, and OE was lower than that in WT under normal or low temperature.Nitrate contents after low-temperature treatment for 6 d were higher than those during treatment periods at normal temperature.After normal-temperature recovery growth for 3 d, the content of nitrate increased in leaves of WT and OE plants and decreased in those of S532D and S532A.
According to Fig. 4B-D, contents of ammonium, amino acids, and soluble protein in S532D and S532A were higher than those in WT and OE under normal or low temperature, with WT having the lowest contents.After 6 d of low temperature, the ammonium and amino acid contents of leaves of all lines increased, while the contents decreased again after normal-temperature recovery growth for 3 d.

Differences in Nitrite Accumulation Between Transgenic Lines and WT Decreased at Low Temperature
Nitrite is the direct product of nitrate reduction by NR.Nitrite must be reduced immediately after its uptake because of its high toxicity to plants (Hachiya et al. 2016).The nitrite content in WT and OE was lower at 22:00 pm than at 10:00 am (Fig. 5), which may be due to decreased NR activity at night.However, S532D and S532A showed the opposite trend, with nitrite content much higher at 22:00 pm than 10:00 am.This indicated that the nitrite reduction rate at night is lower than that during the daytime, and an increase in NR activity leads to rapid accumulation of nitrite at night.The content of nitrite in leaves of S532D, S532A, and OE was approximately one fold higher than that in WT leaves at 10:00 am under normal temperature, while it was 6.1, 6.3, and 1.5 times higher, respectively, at 22:00 pm.
Compared with normal-temperature treatment for 6 d, the content of nitrite in leaves of S532D, S532A, and OE was significantly lower after low-temperature treatment for 6 d, while the WT nitrite content remained similar.The content of nitrite in leaves of S532D, S532A, and OE was 0.59, 0.31, and 0.35 times greater, respectively, than that in WT at 10:00 am under low-temperature treatment, while it was 1.2, 1.3, and 0.19 times greater, respectively, at 22:00 pm.When normal temperature growth resumed for 3 d, the nitrite content of S532D and S532A increased, especially at 22:00 pm.Compared with WT, the content of nitrite in leaves of S532D, S532A, and OE was 0.42, 0.44, and 0.08 times higher, respectively, at 10:00 am, while it was 3.4, 2.9, and 0.24 times higher at 22:00 pm, respectively.This indicated that low temperature can reduce the accumulation of nitrite, narrowing the gap between transgenic lines and WT.Contents of H 2 O 2 and MDA in S532D, S532A, and OE were higher than those in WT under normal temperature (Fig. 6A, B).H 2 O 2 and MDA contents of all lines increased after low-temperature treatment for 6 d, but decreased again after normal-temperature recovery growth for 3 d.Contents of H 2 O 2 and MDA in leaves of S532D and S532A were lower than those in leaves of WT and OE after low-temperature treatment for 6 d and recovery for 3 d.These results showed that the antioxidant capacity of S532D and S532A at normal temperature was lower than that of WT and OE, while the antioxidant capacity of S532D and S532A at low temperature was higher than that of WT and OE, improving cold tolerance.

Transcript Levels of Genes Involved in Nitrogen Catabolism Upstream and Downstream of OsNia1
Two Nia1 genes have been identified in rice, designated as OsNia1 and OsNia2, respectively (Chen et al. 2013).OsNRT2.4 is a dual-affinity transporter that functions in nitrate-regulated root growth and nitrate distribution in rice (Wei et al. 2018).Nitrite reductase (NiR) is the control enzyme for plant nitrite (NO 2 − ) assimilation (Wang et al. 2007).Glutamic acid is one of the basic amino acids of nitrogen metabolism in organism; ammonium is reduced to glutamate (Glu) by glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway enzymes, and Glu serves as the precursor for the amino acid cycle (Sathee et al. 2021).Insertion of exogenous OsNia1 or mutant OsNia1 genes into rice produced significantly higher transcript levels of OsNia1 in S532D, S532A, and OE than in WT (Fig. 7A, B).Transcript levels of the NR gene (OsNia2), nitrate transporter gene (OsNrt2.4),NiR gene (OsNiR), and glutamate synthase gene (OsNADH-GOGAT ) in these transgenic lines all increased as OsNia1 expression level increased at normal temperature.Transcript levels of OsNia2, OsNrt2.4,and OsNADH-GOGAT also increased while those of OsNiR decreased as OsNia1 expression increased at low temperature.Correlation analysis of these gene transcript levels showed that except for the extremely significant negative correlation between the transcript levels of OsNiR and OsNia1 under low-temperature treatment for 4 d, expression of all other genes was positively correlated with that of OsNia1 throughout the experiment (Fig. 7C).

Low Temperature Reduces Nitrite Accumulation in Rice to Reduce Growth Inhibition
We observed that rice seedlings of S532D and S532A showed weaker growth than those of WT and OE at normal temperature, while the situation was reversed under low temperature.Plant height, dry weight, and chlorophyll content of S532D and S532A were lower than those of WT and OE at normal temperature, but higher than those of WT and OE following low-temperature treatment for 6 d and normal-temperature recovery for 3 d (Fig. 1).Meanwhile, H 2 O 2 and MDA contents of S532D, S532A and OE were higher than those in WT under normal temperature.H 2 O 2 and MDA are metabolic substances produced by plants in response to adverse environmental conditions.According to the biological radical injury theory, damage is caused by an imbalance between production and elimination of free radicals in cells under stress; excessive reactive oxygen species can lead to membrane lipid peroxidation, which causes  (Mchdy 1994;Gill and Tuteja 2010).However, the difference between our transgenic lines and WT mainly lies in the activity of NR and the accumulation of nitrogen metabolites (Figs. 2, 3).Previous studies have shown that excessive accumulation of nitrite and ammonium in plants is self-damaging (Britto et al. 2002;Lillo et al. 2003;Lea et al. 2004).In this study, the nutrient solution used in the test contained 20 mg/L NO 3 − -N and 20 mg/L NH 4 + -N.Excessive transformation of NO 3 − -N was the reason for weak growth of S532D and S532A.Analysis of nitrogen utilization showed that S532D and S532A could efficiently utilize NO 3 − -N to generate NH 4 + -N and amino acids, while excessive accumulation of nitrite and ammonium occurred in leaves (Figs.3B, 4).The nitrite content in S532D and S532A was much higher than that in OE and WT at normal temperature, especially at night.Meanwhile, the content of nitrite in S532D and S532A decreased significantly under low temperature, and the ammonium content of S532D and S532A was higher than that of WT or OE.Low temperature promoted the accumulation of ammonium in rice leaves, and the ammonium content in S532D and S532A was higher than that in WT and OE.Studies of tobacco NR protein phosphorylation showed that an increase in NR activity leads to excessive accumulation of nitrite in the dark, which might cause tobacco S521 seedlings to grow weakly (Lillo et al. 2003).This indicates that the weak growth of S532D and S532A was mainly due to the accumulation of nitrite at normal temperature.Meanwhile, as NR activity decreased, the accumulation of nitrite decreased accordingly during lowtemperature treatment.In addition, the difference in nitrite content between transgenic lines and WT was reduced under low temperature, decreasing the damage to S532D and S532A caused by nitrite.The accumulation and reduction of nitrite is quite rapid, and the key enzyme involved in this process is nitrite reductase (NiR).Studies have found that there is coupling regulation between NR and NiR, and NiR activity has a similar circadian rhythm to NR activity in plants (Faure et al. 1991).Our qRT-PCR results showed that the transcription level of OsNiR was positively correlated with that of OsNia1 at normal temperature (Fig. 7).These results indicate that despite increased transcriptional level or enzyme activity of NiR, increased NR enzyme activity still disrupted the balance between the production and reduction of nitrite.Expression of OsNiR was significantly negatively correlated with that of OsNia1 at low temperature.However, compared with those at normal temperature, the contents of nitrite in S532D and S532A leaves were significantly lower

Dephosphorylation of NIA1 Protein Enhances Nitrogen Utilization and Plant Growth in Rice at Low Temperature
Previous studies have demonstrated that S532 is an important phosphorylation site of NIA1 protein and that directional mutation of phosphorylation site S532 improves NR activity and the assimilation of nitrate in rice (Han et al. 2020).The same result is shown in Figs. 2, 3, 4; NR activity and the nitrate assimilation rate of lines with a mutated NIA1 phosphorylation site were enhanced under low or normal temperature, while low temperature inhibited NR activity and increased nitrate nitrogen, ammonium nitrogen, and free amino acid contents in all lines tested.All phenotypic indicators of S532D and S532A, including plant height, dry matter weight, and chlorophyll content, were higher than those of WT and OE at low temperature, while the contents of H 2 O 2 and MDA in S532D and S532A were lower than those in WT and OE, indicating that increased NR activity enhances the tolerance of rice to low-temperature stress.The utilization efficiency of NO 3 − -N in S532D and S532A during low-temperature treatment was much higher than that in WT and OE, and the accumulation of amino acids in the leaves was also higher, enhancing the ability of rice to resist low-temperature stress.Amino acids are the building blocks of proteins.Low temperature promotes the accumulation of soluble protein, and cold-tolerant varieties have higher soluble protein content (Chung et al. 2006;Deng et al. 2011;Sun et al. 2014).Meanwhile, our qRT-PCR results showed that transcription levels of OsNia2, OsNrt2.4,and OsNADH-GOGAT were positively correlated with those of OsNia1, indicating that an increase in NR activity improves nitrate transport efficiency and ammonium assimilation efficiency.In general, S532D and S532A had higher nutrient levels and stronger antioxidant capacity than WT at low temperature.
Phosphorylation of NIA1 protein can effectively change NR activity and regulate nitrogen utilization, making it a hotspot of NR research.We determined that the S532 phosphorylation site of NIA1 is important in rice, regulating the circadian rhythm of NR, and mutation of S532 confers a stable active state on NR.At normal temperature, directed mutation of NIA1 S532 intensifies the toxicity of nitrite accumulation in rice leaves.Under low temperature, however, directed mutation of NIA1 S532 improves the assimilation ability of nitrate to meet the plants' nutritional needs, reduces the accumulation of nitrite, and enhances the tolerance of rice to low temperature (Fig. 8).This study on the regulatory mechanisms of NR phosphorylation not only provides new resources for introducing efficient utilization of nitrogen and cold tolerance in rice, but also provides new clues for the physiological mechanism of NR phosphorylation regulating rice growth and development.

Fig. 1
Fig.1 Opposing regulation of rice seedling growth at different temperatures.Representative pictures are shown A and plant height B dry weight C and chlorophyll content D were measured.WT, wild type.S532D and S532A, NIA1 phosphorylation site directed mutant lines.OE, OsNia1 over-expression line.BT, before low-temperature treatment (i.e., age of rice seedlings was 20 d).NT6d, normal-temperature (26 ℃) treatment for 6 d.LT6d, low-temperature treatment

Fig. 6
Fig.6 H 2 O 2 and MDA contents in transgenic seedlings under low temperature.A H 2 O 2 content.B MDA content.WT, wild type.S532D and S532A, NIA1 phosphorylation site directed mutant lines.OE, OsNia1 over-expression line.BT, before low-temperature treatment (i.e., age of rice seedlings was 20 d).NT6d, normal-temperature treatment for 6 d.NT9d, normal-temperature treatment for 9 d.LT6d, low-temperature treatment for 6 d.LTR3d, normal-temperature treatment for 3 d after low-temperature treatment for 6 d.Column data are means, and short lines represent mean square deviation; values in the same column show means via the LSD, and different letters indicate that the means were statistically different (P < 0.05), n = 3

Fig. 7
Fig.7 Analysis of gene expression related to nitrogen metabolism.A Expression analysis of nitrogen metabolism-related genes at normal temperature for 4 d.B Expression analysis of nitrogen metabolismrelated genes at low temperature for 4 d.Column data are means, and short lines represent mean square deviation, values in the same