Blocking the Expression of the Nitrate Reductase Gene OsNia3 Confers Early Maturation in Rice

Early flowering is a major contributing factor to early crop maturation and is a prerequisite for the expansion of crop production to high latitudes with short growing seasons as well as for annual double/triple cropping systems. Nitrogen is the mineral element most limiting to plant growth and, consequently, also to crop yield. Nitrogen also plays a role in the regulation of flowering time. Here, we show that OsNia3 is a member of the rice nitrate reductase (NR) gene family. OsNia3 was found to be expressed in stem nodes of rice, while the loss of its function led to a reduction in plant height mainly due to disrupted nitrate metabolism at the site of OsNia3 expression. The expression levels of genes related to nitrate transport, nitrate assimilation, and ammonium assimilation, such as OsNia1, OsNRT2.3A, and OsNGS1, increased in the leaves of osnia3 mutant plants, concomitant with an increase in the activity of the nitrogen metabolism pathway. The ammonium and amino acid contents in the leaves also increased in the osnia3 mutant, as did the chlorophyll content. Phenotypic analysis showed that the osnia3 mutant exhibited a shorter growth period compared with wild-type plants; nevertheless, yield was not significantly affected in mutant plants. The deletion of the OsNia3 gene led to the upregulation of genes related to the photoperiod regulation network (OsGI, OsHd1, and OsHd3a), which likely promoted early flowering. These results indicated that OsNia3 is involved in the floral inhibition pathway (OsGI-OsHd1-OsHd3a) in rice under long-day conditions. Our findings provide a theoretical and material basis for the coordination and improvement of early maturity and high-yield traits in rice breeding.


Background
Flowering time is a key trait determining the ability of higher plants to adapt to different environmental conditions.There is a fundamental contradiction between early maturity and high crop yields.The shorter the growth period, the fewer the nutrients absorbed by the plant, the shorter the photosynthesis time, the less organic matter is synthesized and accumulated, and the lower the probability of a high yield.Nitrogen is the element most limiting to plant growth and, thus, also to crop yield.Nitrogen also plays a role in the regulation of flowering time (Dickens and Staden 1988;Bernier et al. 1993;Yuan et al. 2016).Reducing maturity time while ensuring nitrogen uptake and utilization is a key goal in crop breeding.The overexpression of OsNRT1.1A has been shown to significantly upregulate the expression of nitrogen utilization-related genes and that of key flowering genes (FT, Hd3a, and Ehd1) (Wang et al. 2018).Meanwhile, Ef-cd, a long noncoding RNA that positively regulates H3K36me3 enrichment and gene expression at the OsSOC1 locus, was reported to promote flowering, thereby shortening the growth period of rice.Moreover, Ef-cd can also positively regulate the expression of nitrogen assimilation-and photosynthesis-related genes, thus improving nitrogen use efficiency (NUE) and photosynthesis (Fang et al. 2019).Accordingly, it is of great practical significance to further investigate the molecular mechanism underlying the roles of nitrogen metabolism-related pathways associated with the regulation of rice flowering.Over recent years, we have employed CRISPR/Cas9 technology to uncover the function of genes involved in the nitrogen metabolism pathway.We found that flowering in the osnia3 mutant, with deletion of the NIA1-like (LOC4345798, OsNia3) gene, occurred 10 ~ 15 days earlier than that of the wild type (WT) (Nanchang, Jiangxi), without significantly affecting yield.However, the function of the OsNia3 gene has not been previously reported.In this study, we found that OsNia3 participates in both the molecular network involved in the control of rice flowering and the nitrogen metabolism regulation pathway.Our findings provide a possible solution for overcoming the seasonal contradiction between double-cropping rice production and the problem of "late maturing" caused by high nitrogen utilization in agricultural production.

Plant Material and Growth Conditions
The rice (Oryza sativa L.) variety Kitaake (WT) was used in this study.The homozygous osnia1 and osnia3 knockout mutant lines (Kitaake background; generated by CRISPR/ Cas9), an OsNia3 overexpression (OE) line, and an osnia3 mutant line with restored OsNia3 expression (RG) were identified and screened in Jiangxi Agricultural University.The specific mutation for each line is shown in Table S1.In the OE and RG lines, exogenous OsNIA3 was constitutively expressed using the CamV35S promoter in both WT and osnia3 knockout mutant plants.Construction of the carrier and vector transformation was undertaken 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 and Rice Research Institute of Jiangxi Academy of Agriculture, Nanchang, Jiangxi, P.R. China.Field phenotypic identification was mainly conducted in the experimental field of Jiangxi Agricultural University (28°76′N, 115°83′E).Prior to the experiment, soil from the upper 15 cm depth contained 23.23 g kg −1 organic matter, 1.42 g kg −1 total N, 0.31 g kg −1 total P, 3.52 g kg −1 total K, 272.03 mg kg −1 available N (alkaline hydrolyzable-N), 5.96 mg kg −1 available P (Olsen-P), 46.37 mg kg −1 NH 4 OAc extractable K, and 17.0% clay (< 0.002 mm) with an initial pH of 5.06.Seedlings were grown in a seedbed from pre-germinated seeds, and were transplanted at the seedling age of 30 days.The seedlings were transplanted on 25 April, in 2018.Transplanting was performed at a spacing of 16.6 cm × 16.6 cm with one seedling per hill.The field was maintained under flooded conditions with a water depth of about 3 cm from transplanting until midseason drainage.A one-week midseason drainage was conducted about 28 days and 18 days following transplanting the early and late rice, respectively.The field was then intermittently irrigated but without water logging until one week before maturity.Pesticides and herbicides were applied according to standard commercial practice.
A hydroponic experiment was used to analyze the expression characteristics of the OsNia3 gene and the effects of the osnia3 and osnia1 mutations on nitrogen utilization and the responses of the two mutant lines to different types of nitrogen in rice seedlings.Seedlings were cultured in a hydroponic nutrient solution prepared according to the formula of the International Rice Research Institute.After 5 days of growth, rice seedlings were cultured in nutrient solution with potassium nitrate, ammonium nitrate, or ammonium sulfate as nitrogen sources (the nitrogen content was identical in the three nutrient solutions; the ammonium: nitrogen concentration ratio was 0:1:2, respectively) (Table S2).Three repetitions were performed for the different forms of nitrogen application.Growth conditions in the incubator were 12 h/12 h (day/night), 28 °C/26 °C (day/night), 600/0 μmol m −2 s −1 (day/night), and 75% relative humidity.Samples were taken after 15 days of culture in nutrient solution.Two rows (16 plants) were randomly mixed for each sampling and 3 samples were repeatedly obtained per condition for storage at − 80 °C.In the phenotype identification test, the materials were planted in the field with conventional fertilization.

Phylogenetic Analysis and Comparison of Gene Structures and Protein Sequences
AtNia1/2 and OsNia1/2/3 gene sequences were blasted against the NCBI database (https:// blast.ncbi.nlm.nih.gov/Blast.cgi).Primers were designed for polymerase chain reaction (PCR) amplification and the amplification products was sequenced by Tsingke Biotechnology Co., Ltd (Wuhan, China).Phylogenetic analysis of the amino acid sequences of AtNIA1/2 and OsNIA1/2/3 was undertaken in Mega 6.0 using a maximum likelihood method.The OsNIA1 and OsNIA3 protein sequences were compared using Geneious software (Biomatters Ltd, Auckland, New Zealand).All primers were designed using primer-BLAST at NCBI (https:// www.ncbi.nlm.nih.gov/ tools/ primer-blast/) (Table S3).

Determination of Nitrogen Reductase (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 in 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 (2 mL total volume) contained 50 mM HEPES-KOH (pH 7.5), 100 mM NADH, and 5 mM KNO 3 with 2 mM EDTA.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.The NR activation state (% active NR) can be defined as NR activity 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 non-phosphorylated, active form.Assays were run at 25 °C.

Determination of Nitrate, Ammonium, and Free Amino Acid Contents
Rice leaves (0.5 g) were weighed and added to 10 mL of 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.The nitrate content in rice leaves was determined using the nitrosalicylic acid colorimetric method (Cataldo et al. 1975).The ammonium content in rice leaves was determined using indophenol blue colorimetry (Hachiya et al. 2012).The amino acid content in rice leaves was determined using the ninhydrin method (Xu et al. 2017).

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

Determination of Grain Yield and Yield Components
At maturity, 5 hills were sampled diagonally from a 5 m 2 harvest area to determine the yield components following the method of Xu et al. (2010).The filled spikelets were separated from unfilled spikelets by submerging them in tap water.Grain yield was determined from per plant and then was adjusted to a moisture content of 0.14 g H 2 O g −1 fresh weight.

Assay of Subcellular Localization
To investigate the subcellular localization of OsNIA3, 35S:OsNia3-GFP fusion constructs were generated by inserting the open reading frame of OsNia3 into the pCAM-BIA1390-35S:GFP vector.Plasmids were extracted and purified using the Plasmid Midi Kit (No. 12143) (Qiagen, Hilden, Germany) following the manufacturer's manual.The plasmid was sent to Biorun Biotechnology Co., Ltd (Wuhan, China) for protoplast transformation of green seedlings of rice.Confocal microscopy was used for observation of subcellular localization with chloroplast autofluorescence (red) serving as the control.

Statistical Analysis
Means and standard errors of the means were calculated from independent samples in Microsoft Excel 2007 (Microsoft, Washington, USA).SPSS Statistics 22 (IBM SPSS Inc, New York, USA) was used for statistical and correlation analysis and the least significant difference (LSD) method was used to determine whether differences between means were significant.P-values < 0.05 were considered significant.

OsNia3 is Predominantly Expressed in Stem Nodes and its Expression is Nitrate-Inducible
Rice has three known NR gene homologs-OsNia1, OsNia2, and OsNia3.Here, we performed a comparative analysis of Arabidopsis thaliana and rice NR gene homology.We found that the coding regions of the OsNia1 and OsNia3 genes in rice shared 98.66% homology, with only 37 base and 18 amino acid residue differences between them (Fig. 1A and Figure S1).Both genes are located on chromosome 8, suggesting that they have similar functions and participate in NR synthesis and the regulation of NR activity.Additionally, both OsNia1 and OsNia3 exhibited nitrate-inducible expression (Fig. 1B).These findings implied that they are likely to be involved in nitrate utilization, which is particularly important for rice, given that nitrate is not only the major nitrogen form in the paddy field, but also induces cytokinin synthesis and promotes floral transition (Sakakibara et al. 1998;Takei et al. 2004;Corbesier et al. 2003).OsNIA1 protein is mainly found in root epidermal and cortical cells and the cytoplasm of mesophyll cells (Rufty et al. 1986;Vaughn and Campbell 1988).Here, we found that OsNia3 and OsNia1 have widely differing tissue expression patterns.As seen in OsNia3 promoter :GUS transgenic plants, OsNia3 is preferentially expressed in stem nodes, except that the change in OsNIA3 activity regulates internode elongation and the developmental dynamics of the rice panicle, and ultimately affects the height and maturity of rice plants (Fig. 2A-G).The prediction results of related websites indicated that OsNIA3 is mainly localized to chloroplasts (http:// www.csbio.sjtu.edu.cn/ bioinf/ Cell-PLoc/).However, OsNIA3 localization analysis in green seedlings of rice showed that OsNIA3 was not expressed in chloroplasts, but likely in the nucleus and cytoplasm, similar to that seen for OsNIA1 (Fig. 3).

Loss of Function of OsNia3 Improves Nitrogen Utilization
Differences in nitrogen utilization in rice seedlings were investigated using OsNia1 and OsNia3 knockout mutants.We found that the loss of function of OsNia1 and OsNia3 resulted in a decrease in NR activity and an increase in nitrate accumulation (Fig. 4A, B).The content of ammonium in osnia1 mutant was not significantly changed and the content of amino acid was significantly decreased, while the content of ammonium and amino acid was significantly increased in osnia3 mutant (Fig. 4C, D).The reduction in NR activity was markedly lower in the osnia3 mutant than in the osnia1 mutant while nitrate accumulation in the whole osnia3 plant was lower than that in the whole osnia1 plant (Fig. 4A, B).These results indicated that OsNia1 is the main gene regulating nitrate metabolism in rice, with the abovedescribed OsNia3 gene expression pattern suggesting that its gene function mainly acts on stem nodes.Whether it also influences the leaves, stems, and roots remained unknown.

The Loss of Function of OsNia3 Enhances the Activity of other Nitrogen Metabolism Pathway
To further investigate the function of OsNia3 in rice, we next characterized the osnia3 homozygous loss-of-function mutant (japonica variety, a Kitaake plant) (Fig. 5A, B).Compared with the WT, seedlings of the osnia3 mutant exhibited significant growth retardation when grown in Fig. 2 GUS staining of OsNia3 promoter :GUS transgenic plants.A leaf sheath, B leaf blade, C root, D spikes, E germinated seed, F stem node, and G Cross section of stem node.Bars = 1 cm, 1 cm, 1 cm, 1 cm, 1 cm, 1 cm, and 0.1 cm in (A-G), respectively hydroponic culture with nitrate or ammonium nitrate supplementation (Fig. 5A); however, no differences in seedling growth were observed when the osnia3 mutant was grown with ammonium supplementation or without nitrogen (Fig. 5A).Meanwhile, WT and osnia1 mutant rice seedlings were significantly taller than osnia3 mutant seedlings when the nutrient solution contained nitrate/ammonium nitrate.
NR-deficient genotypes accumulate high levels of nitrate, which leads to a reduction in amino acid concentrations as well as the contents of other nitrogen-containing metabolites, resulting in low protein content and a low growth rate in plants (Wang et al. 2004).Conversely, the contents of ammonium and amino acid were increased in osnia3 mutant.Therefore, we analyzed the expression of key genes involved in nitrogen metabolism in osnia3 mutant.Under normal hydroponic cultivation with nitrate and ammonium supply, the expression of genes involved in nitrate uptake and transport, such as OsNRT2.1 and OsNRT2.3a,as well as that of genes functioning in nitrate assimilation, such as OsNia1 and OsNiR, was significantly up-regulated in the osnia3 mutant (Fig. 5B).Interestingly, the expression of genes with roles in ammonium uptake and assimilation, such as OsAMT1.1,OsGS, and OsNGS2, was also significantly up-regulated in the osnia3 line (Fig. 5B).In contrast, the expression of these nitrogen utilization-related genes was not up-regulated in the osnia1 mutant, while that of some genes, such as OsNiR, OsAMT1.1, and OsNGS2, was even repressed (Fig. 5B).These results revealed that the loss of function of OsNia3 plays a fundamental role in maintaining N utilization at high rates, not only for nitrate but also for ammonium.Our findings further indicate that OsNia3 and OsNia1 exist in a redundant system of nitrogen utilization, in that the loss of function of OsNia3 induces the upregulation of the OsNia1 gene.Together, the differences in the patterns of nitrogen-responsive gene expression, phenotypes, and expression patterns between OsNia1 and OsNia3 indicated that functional divergence has occurred between the two genes and that OsNia3 is mainly involved in regulating nitrogen utilization in stem nodes.

The Osnia3 Mutant Displays High Yield Stability and Early Flowering
In the field, osnia3 mutant plants displayed a substantial decrease in height, an advance in the flowering time, and an increase in chlorophyll content (Fig. 6A-E).However, the grain yield of osnia3 mutants was not significantly different from that of WT (Fig. 6F-M), which was likely closely related to the enhancement of the expression of genes related to the absorption, transport and metabolism of nitrate and ammonium induced by OsNia3 mutation.Although no differences in height and flowering time were detected between osnia1 mutant and WT plants, grain yield was lower (~ 13% less) and the seed setting rate was significantly reduced in the osnia1 mutant (Figure S2).
All the growth defects and the early flowering phenotypes were completely rescued by the introduction of OsNia3 into the osnia3 mutant background (RG line), and the role of OsNia3 in nitrogen metabolic pathway was further verified in OE and RG lines.(Figure S3).We also observed that the expression of GIGANTEA (OsGI), Heading date 3a (OsHd3a), Early heading date 1 (OsEhd1), and RICE FLOWERING LOCUS T1 (OsRFT1), genes known to be critical for promoting flowering in rice (Doi et al. 2004;Komiya et al. 2009;Itohet al. 2010), was significantly upregulated in osnia3 mutants (Fig. 7).The early flowering phenotype of the osnia3 line was observed when osnia3 was planted as early season rice but not as late-season rice (a reduction of 3 ~ 5 days when planted as early season rice), suggesting that the regulatory effect of OsNia3 on early flowering was more strongly influenced by long-day conditions.NR activity is regulated by photoperiod and is high under long-day conditions, which may affect flowering and plant height.These findings also indicated that OsNia3 is also involved in nitrogen-regulated flowering.

Discussion
Nitrogen application is one of the most effective means for crop yield improvement.However, nitrogen fertilization can also delay flowering, thus prolonging the maturation times of crops, which significantly increases the risk of yield losses, especially in high-latitude regions where late-season low temperatures are likely to severely restrict grain filling (Castro Marín et al. 2011;Li et al. 2017).NR is the key enzyme responsible for the assimilation of nitrogen, in the form of nitrate, in plants.Given the central role of NR  in nitrate utilization, in this study, we further explored the function and putative application of OsNia1 homologs in NUE improvement, especially in crops.Studies have shown that there are two NR genes in the rice genome, OsNia1 and OsNia2, encoding NIA1 and NIA2 proteins, respectively.OsNia1 is the main gene regulating NR activity in rice (Choi et al. 1989;Tian et al. 2009;Fan et al. 2007), which was also confirmed in our study.Sequence alignment revealed that the OsNia3 gene is highly homologous to OsNia1, and both are located on chromosome 8 of the rice genome.Here, we found that OsNIA1 and OsNIA3 had a similar nitrateinducible expression pattern, and both were expressed in the same location in cells.The biggest difference in tissue localization between the two proteins was that OsNIA3 was highly expressed in rice stem nodes, which may explain the functional divergence between OsNia1 and OsNia3 that results in reduced plant height and earlier flowering in the osnia3 mutant.OsNia1 seems to play a more fundamental role in managing nitrogen utilization, while OsNia3 has a strong functional redundancy effect, and its loss of function induces the expression of genes related to nitrogen metabolism.This is also the main reason for the increase in leaf chlorophyll content and the stability of rice yield seen in OsNia3 deletion mutants.
Flowering is a very important trait in higher plants and a key determinant of their ability to adapt to different environments.Flowering time is primarily determined by the transition time from vegetative to reproductive growth, and the transition time is regulated both by environmental signals and the developmental stage of the plant.Over recent years, knowledge of the mechanisms involved in the induction of rice flowering has gradually increased from the identification of a single gene to the systematization of multiple gene interaction networks and induction pathways.Under short-day conditions, rice flowering is promoted by two pathways, namely, the OsGI-Hd1-Hd3a regulatory pathway, with Hd1 as the core (Hayama et al. 2003), and the Ehd1-Hd3a regulatory pathway, which has Ehd1 as the core (Xue et al. 2009).Under long-day conditions, flowering in rice is known to involve one inductive pathway-the Ehd1-RFT1 pathway (Wei et al. 2010)-and two inhibitory pathways, namely, the OsGI-Hd1-Hd3a (Doi et al. 2016) and Ghd7-Ehd1-RFT1 pathways (Saito et al. 2009).In addition to the above-mentioned Hd1-and Ehd1-mediated pathways, others also exist in rice, such as those involving OsCO3 (Kim et al. 2008) and OsDof12 (Li et al. 2009).In conclusion, rice heading (flowering) consists of a complex molecular regulatory network requiring the coordination of the activity of multiple genes (Figure S4).In our study, the flowering time of the osnia3 mutant was 10 ~ 15 days earlier than that of the WT under long-day conditions (early rice).Meanwhile, with the loss of OsNia3 gene function, the expression levels of the OsGI, OsHd1, and OsHd3a genes increased, which promoted the flowering process in rice.These results indicate that the OsNia3 gene is involved in the OsGI-Hd1-Hd3a floral inhibition pathway in rice under long-day conditions (Figure S4).
Many attempts have been made to improve NUE and grain yield by regulating the expression of nitrogen utilization genes (Fang et al. 2013;Chen et al. 2016;Fan et al. 2016).All such efforts have required consideration of the influence of nitrogen in delaying maturation times.Our results demonstrated that the knockout of OsNia3 can confer improvements in nitrogen utilization and stabilize grain yield, while simultaneously shortening maturation times, suggesting that OsNia3 may provide a solution to the conflict between nitrogen nutrition and maturation time.A high nitrogen supply can greatly delay flowering, and our results were in agreement with this.The loss of function of the OsNia3 gene resulted in decreased NR activity and, consequently, reduced nitrogen accumulation at the site where OsNia3 is expressed, which promoted early flowering and reduced the height of rice plants.However, due to the specificity of OsNia3 tissue expression, nitrogen assimilation in other parts of the plant was not affected; instead, nitrogen metabolism was enhanced, thus ensuring the stability of rice yield.In contrast, the overexpression of the OsNia3 gene delayed rice flowering.In this study, we have identified the function of this gene via loss-and gain-of-function experiments; however, how OsNia3 regulates the flowering of rice under natural conditions remains unclear.Studies have shown that NR activity is induced by light, while NR is rapidly inactivated with increasing darkness.This posttranslation level regulation reduces nitrogen absorption and assimilation (as nitrate) in plants (Harris et al. 2000;Huber et al. 1996).Under long-day conditions, the duration of high-intensity NR activity is prolonged, which can promote nitrogen absorption and transformation.Our results showed that OsNia3 is involved in regulating the flowering inhibition pathway under natural, long-day conditions, and we speculate that this regulatory effect of OsNia3 may involve autophosphorylation.

Conclusion
Mutation of the OsNia3 gene can reduce nitrogen metabolism in stem nodes and alleviate the inhibitory activity of the 'OsGI-Hd1-Hd3a' flowering inhibition pathway, thereby leading to the early flowering of rice under long-day conditions.Meanwhile, the loss of function of OsNia3 can promote an improvement in nitrogen metabolism, enhance nitrogen absorption and utilization, increase chlorophyll content, and maintain yield stability.However, our understanding of the mechanism underlying the effects of OsNia3 lags far behind the recognition of its vital contribution in determining nitrogen utilization and agronomic performance in rice.Further studies concentrating on identifying OsNia3-interacting proteins will be of particular importance to obtaining new insight into how OsNia3 regulates these processes.

Fig. 1
Fig.1OsNia3 displays nitrateinducible expression.A Phylogenetic analysis of the amino acid sequences of AtNIA1/2 and OsNIA1/2/3 using a maximum likelihood method in Mega 6.0.Bootstrap = 1,000.B Analysis of OsNia1 and OsNia3 expression under different nitrogen sources (ammonium or nitrate) in rice seedlings was assessed using qPCR.Columns represent means, error bars represent mean squared deviation; Means were compared using the least square difference (LSD) procedure; different letters indicate that the means were significantly different (P < 0.05), n = 3. NN: no nitrogen; A: ammonium; N: nitrate; AN: ammonium and nitrate

Fig. 4
Fig. 4 Regulation of nitrogen utilization in osnia1 and osnia3 mutant rice seedlings.A Nitrate reductase (NR) activity, B nitrate content, C ammonium content, and D amino acid content in osnia1 and osnia3 seedlings.Columns represent means, error bars represent mean squared deviation; Means were compared using the least square difference (LSD) procedure; different letters indicate that the means were significantly different (P < 0.05), n = 3

Fig. 5 Fig. 6
Fig. 5 OsNia3 displays functional divergence with OsNia1.A Growth of wild-type Kitaake (WT) and osnia3 and osnia1 mutant seedlings under different nitrogen sources.NN, no nitrogen; A, ammonium (2 mM); N, nitrate (2 mM); AN, ammonium and nitrate (1 mM each).Scale bars = 8 cm.B RT-qPCR-based analysis of the expression of genes involved in the utilization of nitrate and ammo-

Fig. 7
Fig. 7 qPCR-based analysis of the expression of flowering-promoting genes in the leaves of wild-type Kitaake and osnia1 and osnia3 mutant rice plants.Values are presented as means ± SD of 3 rep-