Introduction

N uptake and utilization efficiency of rice is extremely important to improve the yield, reduce N fertilizers waste and environmental pollution1,2. N available to plants can be divided into two forms: organic N and inorganic N, while NO3− being one of the main forms of inorganic N. NO3− uptake by roots is extensively investigated since it is the main N resources. Rice is the second largest cereal crop and occupies one-third of the total cereal crop area worldwide. Nitrate uptake and transport by rice is significant for its yield. While, the first step of nitrate metabolism is the regulation of nitrate uptake by roots.

It was demonstrated that the uptake of NO3− is regulated by both positive feed-forward and negative feedback3,4. It was well-known that light and photosynthesis products can increase NO3− uptake, which refers to as positive feed-forward regulation5. It has proposed that the regulation of certain molecules, such as sugars, is a key factor affecting NO3− uptake6. Moreover, it has provided evidence supporting the regulatory role of sugar in plant development and metabolism7. Thus, a hypothesis is proposed that one specific sugar is likely to be a signaling molecule that regulates the uptake of NO3−7,8.

If the signaling molecule exists, its impact is expected to be directly, and the lag would be short or even no lag exists. The response of net nitrate uptake is immediate to sucrose supplied to the solution surrounding the roots, comparing with slower responses to other non-signaling sugars. Thus, the following questions are raised: What is the sugar signal molecule regulating nitrate uptake? What are the kinetics of response of nitrate uptake to the sugar signal? What is the regulatory mechanism of this sugar signal8?

Furtherly, what is the molecular mechanism of nitrate uptake? Nitrate uptake by plants is primarily facilitated by high-affinity transport system (HATS) and low-affinity transport system (LATS)9,10. When the concentration of NO3− in the environment is within the range of 1 μM to 1 mM, it primarily absorbs by HATS. HATS mainly includes OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b11,12,13. However, when the concentration of NO3− exceeds 1 mM, it mainly absorbs by LATS such as OsNPF2.2 and OsNPF2.4. LATS mainly involves OsNPF2.2, OsNPF2.4, OsNPF4.1, OsNPF4.5, OsNPF6.1, OsNPF6.5, OsNP7.7-1 and OsNPF7.9. OsNRT2.4 are a dual-affinity nitrate transporter14. It is important to emphasize that the plants were in 1/10 Long Ashton solution containing 1.0 mM nitrate in our experiments. Therefore, nitrate uptake is mainly related to HATS. Additionally, OsNRT2.1, OsNRT2.2, OsNRT2.3a, and OsNRT2.3b genes, which coding NRT2 gene family in HATS, are the major contributors to the inducible HATS11,15,16,17.

Previously, in vitro root systems were often used for measurement. Non invasive method in this study has two major advantages. Firstly, the entire measurement process has no impact on the plant, and the plant can continue to grow intact and undamaged. Its metabolism is basically the same as under natural conditions. Secondly, real-time monitoring can be carried out, so the measurement has high accuracy and precision. In order to obtain the time courses with a high enough resolution to answer the previously stated questions, a non-invasive method was used that enables precise measurement of changes of net nitrate uptake by the root system of rice plants that are intact and normally growing.

To identify signal molecules, it is essential to monitor nitrate uptake in real time to detect whether there is a lag before the nitrate uptake changes after feeding various carbohydrates. It is worth noting that these conclusions are usually drawn from relatively less data, that is, they are measured at least once an hour18. This cannot solve the question of whether there is a lag before the nitrate uptake changes after light condition change or carbohydrates addition.

The non-invasive measurement method has high accuracy and can be measured every 10 min or even 5 min. Plants can grow normally throughout the entire experimental process. At the same time, due to the real-time and high accuracy of the measurement method, signal molecules can be identified from various compounds, and the size and mode of signal molecule action can be clarified, thereby clarifying the molecular mechanism of signal molecule action. In the molecule level, OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b can be induced expression by light or sugar treatment11,12,13. To further investigate the mechanism of signal molecule, these four genes were verified the expression by qRT-PCR to make sure that the regulation of signal molecule was in the gene level or post transcriptional level. This study has important value for improving N uptake efficiency and increasing protein content in plants.

Materials and methods

Plant materials and growth conditions

Seed germination of rice (Oryza sativa cv. Jiyou 225) was done on filter paper soaked with ddH2O for 7 days in the dark. Seedlings were transferred to hydroponic vessel in 1/4 Long Ashton nutrient solution at 25 ± 2 °C for 15 h photoperiod19. Natural light is simulated with LED light in the plant cultivation shelf (MBTZPJ-5C, MoreBetter Biological Technology Co. Ltd., China) from 6:00 to 21:00 every day, with a light flux density of 630 µmol m−2 s−1 (UT381 portable photosynthesis, Unilever Technology Co. Ltd., China).

Non-invasive measurement of net nitrate uptake

In the non-invasive system, intact rice seedlings used as the experimental materials, enabling the plants to conduct undisturbed normal metabolic activities throughout the experiment20. This method has high time resolution, enabling real-time measurements, which is essential for the experiments. The non-invasive system includes the beaker for containing the nutrient solution, constant flow pump, automatic sample collector, the ion meter and perspex tray (Fig. 1). Two 25-day-old plants were selected and transferred from the culture solution into two perspex trays. The trays were at about 10° angle. In order to reduce the impacts of transplant shock, the intact rice plants were placed in the experimental trays at least 14 h prior to the measurements.

Figure 1
figure 1

The non-invasive system for measuring nitrate uptake by plants. The system includes constant flow pump, automatic sample collector, ion meter, perspex tray and the vessel containing the nutrient solution.

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1/10 LA solution is pumped in from the junction between root and stem by constant flow pump (BT100S-1, Baoding Lead Fluid Technology Co. Ltd., China), flows slowly through the roots, and subsequently pumped out to automatic sample collector (BS-100A, Shanghai Hu Xi Analysis Instrument Factory Co. Ltd., China). The rate of the outflow was three times higher than the rate of the inflow to prevent any possible overflow. After collection, the outflow solution collected after flowing over the root system was measured for nitrate concentration (Cout/mM) by the ion meter (PXSJ-216F, Shanghai Yidian Scientific Instrument Co. Ltd., China), and the nitrate concentration (Cin/mM) in the inflow liquid was measured for calibration. The root system was cut off and weighed at the end of a series of 4-day measurements. Net nitrate uptake (Φnet) was calculated as

$$\Phi_{{{\text{net}}}} = {6}0 \cdot \left( {{\text{C}}_{{{\text{in}}}} - {\text{C}}_{{{\text{out}}}} } \right) \cdot \left( {{\text{W}}_{{\text{n}}} - {\text{W}}_{{{\text{n}} - {1}}} } \right)/\uprho {\text{t}} \cdot {\text{W}}_{{\text{R}}} \left( {\upmu {\text{mol}}\;{\text{gfwt}}^{{ - {1}}} \;{\text{h}}^{{ - {1}}} } \right)$$

ρ is equal to 1 g ml−1, Cin is nitrate concentration of the inflow solution, Cout is nitrate concentration in the outflow solution, (Wn − Wn−1) is difference between the weight of each test tube before and after collecting solution, WR is fresh weight of roots, t is time collecting each sample.

The effects of light and carbohydrates on net nitrate uptake by roots

The effects of illuminating the shoot

After transferring into the non-invasive system, shading devices were added to both of them. The control is keeping in the dark for 24 h, while the shading device in the experimental group was removed at 13:00–20:00. To ensure the consistency of plant growth conditions during the experiment, the plants were alternatively treated. 1/10 LA nutrient solution was pumped at 0.3 ml min−1 and the solution was collected every 30 min. Except for this light experiment, the solution were collected every 10 min.

The effects of carbohydrates

To identify the signal, nine kinds of carbohydrates, which includes sucrose, glucose, fructose, trehalose, raffinose, arabinose, lactose, galactose and mannitol, were selected out. Among them, sucrose, glucose and fructose are the main carbohydrates transported by phloem. Sucrose serves as the primary product of photosynthesis and is a non-reducing sugar with stable chemical properties. Moreover, sucrose is the primary substance transported via phloem due to its high water solubility and rapid transport speed21. Thus, sucrose is highly likely to function as a signaling molecule. Trehalose is a non-reducing disaccharide that is ubiquitous in plants and is essential for the plant growth and development. The remaining selected sugars are important components of the cell wall. In order to make the transport system saturated and minimize the impact of osmotic pressure, 10 mM concentration is chose8.

The effects of carbohydrates were analyzed by measuring net nitrate uptake of two plants in the light from 8:00 to 17:00. 1/10 LA solution was pumped in at a rate of 1 ml·min−1 and collected every 10 min. 10 mM carbohydrate was added to one plant from 13:00 to 15:00 and to the other from 15:00 to 17:00, so that one plant served as a control for the other.

Detailed dynamics of the effects of signal molecule

After the signal molecule was identified based on analyzing the effect of various sugars and mannitol on net nitrate uptake by rice roots, the detailed dynamics of the effects of signal molecule was investigated. Firstly, to determine the lowest concentration at which sucrose played a signal role, the effect of 10 mM, 1 mM, 0.1 mM, 0.05 mM and 0.01 mM signal molecule within 2 h on net nitrate uptake by rice roots was compared.

After that, the long-time effect of various concentrations of sucrose on net nitrate uptake was compared under different light conditions. The experiment was collected from 11:00 to 21:00. The nutrient solution was pumped at the rate of 1 ml·min-1 and collected every 10 min. Under dark or light conditions, 10 mM or 1mM signal molecule was added to one plant from 13:00 to 19:00 and to the other from 15:00 to 21:00.

Quantitative real-time polymerase chain reaction (qRT-PCR) validation experiment

In the experiment, 10 mM sucrose was supplied for 10 min, 1 h, 2 h, 4 h and 6 h respectively, and one plant was used as the control. Subsequently, the total RNA of rice root was extracted by the CTAB method, and cDNA was obtained by reverse transcription. Finally, qRT-PCR experiment was carried out.

In the experiment of the reverse transcription, 1 μL RNA, 1 μL primer (10 μM), 1 μL dNTP Mix (10 mM) and 9 μL DEPC treated water were firstly added to the sterile EP tube to obtain Mix I (total system 12 μL). Mix I was incubated at 65 °C for 5 min, immediately put it on ice for 1 min. Then, to obtain Mix II, 4 μL 5 × First-Strand Buffer, 2 μL dTT (0.1 M), 1 μL RNaseOUT (40 U/µL) and 1 μL SuperScript III RT (200 U/µL) were added to Mix I. Finally, Mix II (total system 20 μL) was treated at 25 °C for 5 min, 42 °C for 60 min, 70 °C for 15 min, and then immediately put it on ice to obtain cDNA for qRT-PCR.

Primers were designed by Sangon Biotech (Shanghai) Co. Ltd., China. qRT-PCR experiment was carried out in Henan Baijun Biotechnology Co., Ltd. In detail, the forward primer and reverse primer of OsNRT2.1 (4328051) were 5′-GGAGCACAAGGCGAAGACGATC-3′ and 5′-AGGTGGAGACGAAGCAGGAGAAG-3′, respectively; The forward primer and reverse primer of OsNRT2.2 (4328052) is 5′-GGAGCACAAGGCGAAGACGATC-3′ and 5′-AGGTGGAGACGAAGCAGGAGAAG-3′, respectively; The forward primer and reverse primer of OsNRT2.3a (AK109776.1) is 5′-GGCTACTCTCCGACTGGCTCTC-3′ and 5′-GATTCCGAGCACCACGCAGAG-3′, respectively; The forward primer and reverse primer of OsNRT2.3b (AK072215) is 5′-GGCTACTCTCCGACTGGCTCTC-3′ and 5′-GATTCCGAGCACCACGCAGAG-3′, respectively. 18sRNA of Oryza sativa was selected as the control, its forward primer and reverse primer is 5′-ATGATAACTCGACGGATCGC-3′ and 5′-CTTGGATGTGGTAGCCGTTT-3′, respectively.

15N marker measurements

After the 15N labeling experiment, the plants were removed from the non-invasive system. The rice roots was rinsed with 0.1 mM CaSO4 (sigma), dried with filter paper and then dried at 80 °C in an aluminum box in 101–18 electric constant temperature drying oven (Shangcheng Instrument Manufacturing Co., Ltd., Shaoxing, Zhejiang, China) for 12 h. Then, the dry tissue was placed in a round bottom centrifuge tube and was ground at least three times with a GenoGrinder 2010 SPFX SamplePrep high-throughput tissue grinder for 30 s each time to ensure that the powder is sufficiently uniform and into powder form. The grinder was equipped with an adapter for 6 centrifuge tubes, with 2 ml centrifuge tube on the hole.

Four groups were set up for the 15N labeling experiment, including 15N labeling for 10 min, 15N labeling + sucrose for 10 min, 15N labeling for 6 h, and 15N labeling + sucrose for 6 h. During the detection, roots and shoots were individual tested with three replicates, and totally 24 samples. In addition, plants with cut leaves as a comparison, 24 samples were tested to clarify the transport of 15N between stems and leaves. Totally 48 samples were placed in order in a 72 position centrifuge tube box.

15N labeled sample was a crushed dry sample with labeled abundance and time, and then sent to State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences for testing. Analysis of stable nitrogen isotopes was done by isotope-ratio mass spectrometers (EA-DELTA plus XP) using stable isotope ratio mass spectrometer method. Take one-fifth of each sample for measurement. Four indicators was measured to analysis 15N exchange dynamics changes, included Amount (the amount of sample weighed, not show here), N% (the percentage content of total N), δ15N Air‰ (thousand fraction of isotopes) and AT% 15N/14N (percentage of 15N atomic number/total atomic number).

Replication and statistical analysis of data

The methods for processing data are as follows: first, net nitrate uptake was expressed relatively to that in the control or relatively to a linear extrapolation of the rates before a change in light or addition of carbohydrates. The relative change of each set of data was calculated, and then statistical analysis was conducted. Statistical analysis of data based on “at least 3 experiments” are summarized in Tables. All experiments were repeated at least three times (Except for qRT-PCR validation and 15N labeling experiment which is repeated three times, there is at least four replicates).

The single factor ANOVA test and correlation analysis by IBM SPSS statistics 26 was performed for statistical analysis. Values are presented as the means ± s.d (n). Significance of differences between means was determined at the level of P < 0.05.

Ethical approval

Experimental research on plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation.

Results

The effect of illuminating the shoot on net nitrate uptake by rice roots

When the shading device was removed from the experimental group, net nitrate uptake immediately started to increase, that is to say, there was without detecting any time delay when collected every 30 min (Fig. 2a). After approximately 4.4 h of light exposure, net nitrate uptake reached a steady state, showing an increase of about 60% (Table 1). After from light to dark, net nitrate uptake started to decrease after a lag, and decreased by about 55% after about 5 h, and finally reached a steady state (Fig. 2b, Table 1). The lag before nitrate uptake change and the time of reaching a new steady state is significantly different, however, there is no difference in the change of net nitrate uptake between darkness and light. This indicates that the time course of these two responses is not simply a reversal of a single process. After determining the time of reaching a new steady state from dark to light, the effect of light on net nitrate uptake can be excluded in the following signaling identification.

Figure 2
figure 2

The real-time effects of light on net nitrate uptake by rice roots. Samples were collected every 30 min from 8:00 to 2:00 on next day, ϕ represent for net nitrate uptake, the same below. One plant served as the control under the dark, the other was transferred from dark into light at 13:00. (a) ϕ change from 8:00 to 20:00 when dark to light transition at 13:00, (b) ϕ change from 13:00 to 2:00 on next day when light to dark transition at 20.00.

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Table 1 The effects of light condition on net nitrate uptake by rice roots.
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The effects of carbohydrates on net nitrate uptake by rice roots

To identify signal molecule, the effects of carbohydrates on net nitrate uptake by rice roots were compared. The responses of carbohydrates to net nitrate uptake fall into three categories. The feeding of sucrose alone causes a rapid increase of net nitrate uptake which measured every 10 min, without any detectable lag (Fig. 3a). After the sucrose addition for 2 h, net nitrate uptake increased by about 80% (Table 2). After the addition of glucose, fructose and raffinose for 2 h, net nitrate uptake has a slight effect of about 30% with a lag of about 30 min, which is significantly different to that of sucrose (Fig. 3b–d, Table 2) (P = 0.05). Trehalose, arabinose, lactose, galactose and mannitol had no effect (Table 2).

Figure 3
figure 3

The real-time effects of 10 mM sucrose, fructose, glucose and raffinose on net nitrate uptake by rice roots in the light. Samples were collected every 10 min from 8:00 to 17:00. A sugar was supplied to one plant from 13:00 to 15:00 and to the other from 15:00 to 17:00. (a) sucrose, (b) fructose, (c) glucose and (d) raffinose.

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Table 2 The effects of nine kind of 10 mM carbohydrates on net nitrate uptake by rice roots.
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The effects of sucrose on net nitrate uptake by rice roots

Comparison of the effects of various concentrations of sucrose

The effect was immediate and significant when the concentration of sucrose at 10 mM (Fig. 4). To further determine the effective concentration, the effects of 10 mM, 1 mM, 0.1 mM, 0.05 mM and 0.01 mM sucrose were compared. The results showed that when the concentration of sucrose was 1 mM or 0.1 mM, net nitrate uptake increased by about 80% after 2 h (Table 3), which was not significantly different with that of 10 mM sucrose (P > 0.05). It indicated that within the effective concentration range, net nitrate uptake was independent to the level of sucrose concentration. However, 0.05 mM sucrose can not lead to the change of net nitrate uptake. Therefore, the minimum effective concentration of sucrose in the light was firstly verified to be 0.1 mM (Fig. 4, Table 3).

Figure 4
figure 4

The real-time effects of various concentrations of sucrose on net nitrate uptake by rice roots in the light. Samples were collected every 10 min from 8:00 to 17:00. A sugar was supplied to one plant from 13:00 to 15:00 and to the other from 15:00 to 17:00. (a) 10 mM Sucrose, (b) 1 mM Sucrose, (c) 0.1 mM Sucrose and, (d) 0.05 mM Sucrose.

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Table 3 The effects of various concentrations of sucrose addition on net nitrate uptake by rice roots in the light (P > 0.05).
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Comparison of the effects of sucrose under different light condition

Since both light and sucrose can affect net nitrate uptake, can these two factors work in coordination? To answer this question, the effects of sucrose on net nitrate uptake in different light conditions were compared. The results demonstrated that whether it's darkness or light, high concentration (10 mM) or low concentration (1 mM) of sucrose could stimulate net nitrate uptake by roots immediately (Table 4) and persistently (Fig. 5). No matter light or dark, net nitrate uptake increased dramatically after 1 mM sucrose addition. Moreover, 1 mM sucrose removal brought the dramatic decrease of net nitrate uptake (Fig. 5). Similarly, 10 mM sucrose addition and removal also resulted in the increase and decrease of net nitrate uptake (not shown here). It is suggesting that sucrose can control net nitrate uptake indeed.

Table 4 The change of net nitrate uptake by rice roots after sucrose addition under different light condition.
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Figure 5
figure 5

The real-time effects of 1 mM sucrose on net nitrate uptake by rice roots. Samples were collected every 10 min from 11:00 to 21:00. Sucrose was supplied to one plant from 13:00 to 19:00 and to the other from 15:00 to 21:00. (a) in the light and (b) in the dark.

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In the light, net nitrate uptake increased by about 2 times after 10 mM or 1 mM sucrose addition for 6 h (no difference between concentrations, which not shown here). Surprisingly, in the dark, net nitrate uptake increased by more than 6 times after 10 mM or 1 mM sucrose treatment for 6 h (no difference between concentrations, which not shown here). The effect of sucrose is significantly higher in the dark than that in the light (Table 4) (P = 0.01). Nevertheless, this effect is independent of sucrose concentration.

The changes of HATS genes expression in rice roots after sucrose addition

To make sure sucrose signal control nitrate uptake in the gene level or the post transcriptional level, OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b were selected and performed expression analysis. To examine the effects of sucrose on the changes of HATS genes expression in rice roots, rice seedlings were supplied with 10 mM sucrose for 10 min, 1 h, 2 h, 4 h and 6 h, respectively (compared to those without sucrose supply), and then these four gene expression were investigated by qRT-PCR. The results showed that these gene expression levels increased immediately after sucrose addition for 10 min. This increase continued and reached a maximum after sucrose addition for 2 h, and then had a significant decrease at 4 h. However, gene expression after sucrose supply for 4 h remained at the nearly same or a slightly higher level than that of the control. After sucrose addition for 6 h, the expression of these genes had a slight recovery (Fig. 6). Correlation analysis was conducted on the differences of gene expression after sucrose treatment for different times, Table 5 showed Spearman Rho coefficient for the expression of four genes and sucrose treatment times. The results showed that at the 0.01 level, the correlation was significant among the four genes (Table 5).

Figure 6
figure 6

The changes of HATS genes expression in rice roots after sucrose addition. In horizontal ordinate, 1–6 represent for the control, 10 min, 1 h, 2 h, 4 h and 6 h, respectively. (a) OsNRT2.1, (b) OsNRT2.2, (c) OsNRT2.3a and (d) OsNRT2.3b.

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Table 5 Correlation analysis of gene expression of OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b in rice after sucrose treatment.
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15N/14N exchange dynamics analysis in rice treated by sucrose

15N exchange dynamics changes were analyzed in shoots, included δ15N Air‰, N% and AT% 15N/14N. Regardless of control or treatment, N% remained unchanged (Fig. 7e,f), which showed that the percentage content of total N had no difference. For δ15N Air‰ and AT% 15N/14N, there is significantly different between the control and sucrose treatment if leaves was cut off during sampling (Fig. 7a,c); no significantly different between the control and sucrose treatment if leaves wasn’t cut off, but there are higher after long-term labeling than that after short-term labeling (Fig. 7b,d). Comparing the effects of defoliation on 15N exchange kinetics, it was found that δ15N Air‰ and AT% 15N/14N in defoliated stem was significant difference with that of stem with leaves after sucrose treatment. For short-term labeling, δ15N Air‰ and AT% 15N/14N in the stem after sucrose treatment were significantly increased. For long term labeling, regardless of the control or treatment, δ15N Air‰ and AT% 15N/14N in the stem were less if leaves was cut off during sampling (Fig. 7a–d). It indicates that 15N was transferred from stem to leaves after a long period of 15N/14N exchange.

Figure 7
figure 7

Comparison of δ15N Air‰, AT% 15N/14N and N% in stem only with that of the above-ground after sucrose treatment with 15N tracing for short-term and long-term. (a), (c), and (e) represent δ15N Air‰, AT% 15N/14N and N% in stem only, (b), (d) and (f) represent δ15N Air‰, AT% 15N/14N and N% in the above-ground.

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15N exchange dynamics changes were analyzed in roots, included δ15N Air‰, N% and AT% 15N/14N (Fig. 8). N% remained unchanged (Fig. 8E,F), which showed the percentage content of total N had no difference. If leaves were cut off, δ15N Air‰ and AT% 15N/14N in roots had no significant difference (Figs. 8a, 7c). If leaves weren’t cut off, δ15N Air‰ and AT% 15N/14N in roots also had no significant difference between the control and sucrose treatment, but there were higher after long-term labeling than that after short-term labeling (Figs. 8b, 7d). For short term labeling, whether for the control or treatment, δ 15N Air ‰ and AT% 15N/14N in the root is significantly higher when leaves were cut off during sampling (Fig. 8a–d). It indicated that for 15N short time marker, if leaves are cut off, more 15N will be in the roots. In addition, regardless of whether 15N is labeled or not, there is no difference in net nitrate uptake. When 15N is not marked, net nitrate uptake was shown in Tables 2, 3 and 4 (not shown here when marked with 15N).

Figure 8
figure 8

Comparison of the difference if defoliation or not during sampling, measurement of δ15N Air‰, AT% 15N/14N and N% in rice roots after sucrose treatment with 15N tracing for short-term and long-term. (a), (c) and (e) represent δ15N Air‰, AT% 15N/14N and N% in rice roots when defoliation during sampling. (b), (d) and (f) represent δ15N Air‰, AT% 15N/14N and N% in rice roots when leaves were not cut off during sampling.

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Discussions

To investigate the positive feed-forward control on nitrate uptake, the effects of light and carbohydrates on net nitrate uptake were investigated. Firstly, based on knowing how long net nitrate uptake is stable in the light (Table 1), the effect of carbohydrates can be investigated without consideration of light effect. To make sure the size and mode of sucrose effect, various concentration of sucrose in the different condition were compared. Then, qRT-PCR was used to perform expression analysis of OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b after sucrose treatment. Lastly, 15N tracer experiment was done to analyze N transport.

The signaling molecule was identified based on the comparison of real-time effects of nine carbohydrates on net nitrate uptake by rice roots. The results revealed that, among these nine carbohydrates, only sucrose can cause the biggest increase of net nitrate uptake, and without any detectable lag (Fig. 3a; Table 2). Therefore, sucrose was identified as a positive feed-forward signal molecule that promotes nitrate uptake by rice roots. Sucrose serves as the primary product of photosynthesis and is a non-reducing sugar with stable chemical properties. Moreover, sucrose is the primary substance transported via phloem due to its high water solubility and rapid transport speed21. Thus, sucrose is highly likely to function as a signaling molecule.

Both light exposure and the addition of exogenous sucrose can increase the concentration of sucrose in the cytoplasm of root cortical cells. In comparison with the response of shoot to light within 5 h (less than 55%), the response of direct supply sucrose around the roots was bigger (above 80%) within 2 h (Tables 1 and 2). The findings suggested that the sucrose concentration in the cytoplasm of root cells increased to a greater extent following direct supply of sucrose, compared to the transport of synthesized sucrose in the cytoplasm of root cells via phloem in the light. In addition, the effect of direct addition of exogenous sucrose on net nitrate uptake was more lasting than that of light (Tables 1, 4), indicating that secondary reactions may be involved.

What’s more, the effective concentration of sucrose, and the effects of the various concentration of sucrose in various light conditions were investigated. The effects of various concentration of sucrose in different light condition proved that there was a coordination effect of sucrose and light on net nitrate uptake by rice roots5. There are two sources of sucrose in the root cytoplasm. On the one hand, sucrose is synthesized through photosynthesis in the aerial parts and transported to the roots through the phloem22. On the other hand, sucrose can be directly absorbed from the external environment. When the plant is under dark conditions, the lower supply of sucrose from the above-ground parts leads to a lower concentration of sucrose in the root cytoplasm. Therefore, under dark conditions, the initial concentration of sucrose in the cytoplasm becomes lower, proportionally, the effect of adding exogenous sucrose on net nitrate uptake is relatively bigger (P < 0.05) (Table 4).

Further, it is speculated that sucrose may affect the gene expression of nitrate transporter OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b in rice roots4. The results of qRT-PCR illustrated that sucrose addition caused the increase of these genes expression immediately. Moreover, the expression of these genes was still higher than that of the control after sucrose addition for 6 h (Fig. 6), which was consistent with the continuous increase of net nitrate uptake. Moreover, since the expression of these genes was immediately up-regulated, which occurs within a few minutes, which indicated that it should not be a regulation of gene expression levels, but rather a regulation of post transcriptional levels23. In a conclusion, sucrose serves as the signaling molecule, participating in the control of nitrate uptake by roots in the post transcriptional level.

Finally, to investigate the transportation direction of N, 15N exchange dynamics changes were analyzed both in shoots and roots, included δ15N Air‰, N% and AT% 15N/14N24. To clarify the transport of 15N between stems and leaves, defoliated plants as a comparison. For short-term labeling, δ15N Air‰ and AT% 15N/14N in the aboveground with cut leaves after sucrose treatment were significantly higher and decreased over the time. The decrease indicates that 15N was transferred from stem to leaves (Fig. 7). For short term labeling, whether for the control or treatment, δ 15N Air ‰ and AT% 15N/14N in the roots was significantly higher when leaves were cut off during sampling (Fig. 8). It indicated that for 15N short time marker, more 15N would be in the roots if leaves are cut off during sampling.

Conclusions

To identify the signaling molecule of net nitrate uptake, its responses to various light and carbohydrates were compared. To ensure that plant metabolism is not affected during the measurement process and measure it in real time, a non-invasive method was used. The results showed that after the sucrose addition for 2 h, net nitrate uptake increased without a lag, while other sugars had a small effect with a lag or no effect. It provided an evidence that sucrose was a positive feed-forward signal molecule of nitrate uptake by rice roots. To further investigate its regulatory mechanism, qRT-PCR was used to further verify the effect of sucrose on the genes expression of OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b in HATS. The results showed that these genes was immediately up-regulated, which suggested that these genes were post transcriptionally regulated. Consequently, sucrose functioned as a specific positive feed-forward signal regulating net nitrate uptake, and post transcriptionally regulated some genes in HATS. Further, 15N exchange dynamics changes were analyzed both in shoots and roots. There was a difference between the control and sucrose treatment after short-term labeling and no difference after long-term labeling, which may be due to the transferring 15N from stem to leaves. The work are important for controlling nitrate uptake, increasing the protein content and improving the yield.

However, further research on transporter genes is needed, such as whether over-expression of transporter genes alters N levels, and further investigating the molecular mechanisms through gene knockout, mutant and nitrate reductase inhibitors and gene editing etc.