Signal molecules controlling nitrate uptake by roots

ABSTRACT The efficiency of nitrate uptake is a key point to decrease the waste of fertilizer and protect the environment. There are some signal substances participating in the nitrate control system. This paper reviewed the signals, including positive feed-forward and negative feedback signals, controlling nitrate uptake by roots. Firstly, sugar signals are related to photosynthesis and constitute a positive feed-forward signal. Sucrose had been identified as the positive feed-forward signal, and its nature was elucidated. Secondly, reduced N signals are related more generally to growth and constitute negative feedback. It is difficult to pinpoint an amino acid controlling nitrate uptake since they can convert each other. Thirdly, the interaction of two kinds of signal was analyzed. Finally, the current studies of a regulatory network of the signal molecules that control nitrate uptake and nitrate transporters in rice were reviewed. It was beneficial to understand N metabolism control.


Introduction
Except for water, nutrients are the environmental factor that most strongly constrains productivity (Körner 2021;Lambers and Oliveira 2019). To ensure future agricultural sustainability, the control of nutrient uptake was required comprehensive understanding to enhance N use efficiency (Hu et al. 2019;Wu et al. 2020). In plants, N is the most needed basic mineral element. Worldwide N use efficiency for cereal production is approximately 33%. The unaccounted 67% represents a $15.9 billion annual loss of N fertilizer (Raun and Johnson 1999). Loss of N fertilizer resulted in serious environmental problems, such as greenhouse warming and pollution of drinking water resources (Tilman 1999). Improving N use efficiency in cereal production is important in sustainable agriculture.
Nitrate is the major inorganic N form in aerobic agricultural soils and the main N source taken up by plants. Nitrate uptake is thermodynamically uphill and thus dependent on metabolism. The nitrate assimilation pathway starts with nitrate uptake followed by nitrate reduction resulting in ammonium which is fixed into the amino acids Gln and Glu in most plants. Metabolism in plants is very sensitive to N source and there are some precise mechanisms to regulate the process of metabolisms. Organisms can adapt the environment and their metabolism can not be separated from the inside and outside environment. If the conditions change, an organism can adjust the metabolisms process until developing a new balance (Xu 2002). At present, many researches have been done on the control of nitrate uptake (Liu et al. 2015;O'brien et al. 2016;Ruffel et al. 2014;Wang et al. 2014;Wang, Cheng et al. 2018). It is known that the rate of root growth is correlated with the root sugar content. It is concluded that the changes of carbon allocation could contribute to the changes in shoot and root growth (Scheible et al. 1997). There is evidence that shoot demand is linked to NO 3 − uptake of the root. The N demand of a crop depends on the increase of dry mass and might not be linear. These relationships can be described by mathematical models (Schenk 1996).
Nitrate uptake is a strongly regulated process adapted to the N demand of the whole plant. Pre-requisites for an integrating regulatory system are signal substances communicating the N demand of the shoots to the roots. N requirements are regulated by the integrated perception of signals from nitrate, light, sugars, amino acids, and hormones, etc. Studies on the nature and integration of these signals have revealed a complex network of controls (Foyer et al. 2003;Kant 2018;Takahashi and Shinozaki 2019). The work to date suggests that there are at least two signal systems, one involving sugars and the other involving reduced N compounds. Our general hypothesis is that the sugar signals are related to photosynthesis and constitute a positive feed-forward signal, while reduced N signals are related more generally to growth and constitute negative feedback. Signaling substances communicating the N demand of the shoots to the roots are required in an integrated regulatory system. Beside phloem mobility, such signal compounds must have the potential to decrease or increase nitrate uptake either at the transcriptional or post-transcriptional level. Particular amino compounds exert transcriptional and post-transcriptional control over nitrate uptake by roots (Geßler et al. 2004).
In addition, it is known that auxins, abscisic acid (ABA), cytokinins (CTKS), gibberellins (GAS), ethylene (ETH), and jasmonic acid have effects on the control of N sensing and acquisition. Firstly, they have effects on root growth and development. For example, auxin can promote the growth and development of lateral roots (Bhalerao et al. 2002) and exogenous application of physiological concentration of CTKS can reverse the effect of auxins (Debi et al. 2005). Secondly, auxins, CTKS, ABA, GAS, and ETH also play a role in the positive feed-forward and negative feedback control of nitrate uptake.
In a word, the control of nitrate transport, the most important inorganic uptake system in plants, is important for understanding N mechanism. This paper mainly reviewed the signal of the control of nitrate uptake by rice roots. It will also have implications for increasing the growth efficiency of crop plants, ultimately the grain protein yield per unit of N.
2. The positive feed-forward signal 2.1. Positive feed-forward control The finding that feeding sugars (sucrose, glucose, and fructose, but not sugar derivatives) to the roots suggested that new photosynthate, moving to the roots in the phloem, acted as a signal (Forde 2000;Franklin et al. 2017;Lejay et al. 2003;Walch-Liu et al. 2005). This would be positive feed-forward control. The feed-forward control on nitrate uptake is mainly constituted by carbohydrates and light.
Some carbohydrates can stimulate nitrate uptake, from which a signal molecule controlling it can be distinguished. N metabolism is regulated by signals that are derived from C metabolism (Stitt and Krapp 1999). Lejay et al. (1999) indicated that the decrease in nitrate uptake at night was reversed by feeding with sucrose. Delhon et al. (1996) noted that glucose-stimulated nitrate reduction in the dark. Sugar as a signaling molecule possibly coordinated the uptake and metabolism of N (Sheen et al. 1999). An increased supply of sugars, like glucose, fructose, and sucrose can increase nitrate uptake (Lejay et al. 2003;Stitt and Krapp 1999).
Light is the foremost regulator of plant growth and development and is required for carbohydrates synthesis. The critical role of light signaling in the promotion of nutrient uptake was clarified in recent decades (Albornoz and Lieth 2015;Alexandre et al. 2016;Gommers and Monte 2018;Sakuraba and Yanagisawa 2018;Xu et al. 2021). It has been reported that light received by shoots can lead to a change in nitrate uptake by roots. Nitrate uptake increases 20-40% in the light (Aslam et al. 1979;Matt et al. 2001) and decreases 30-50% in the dark (Cárdenas-Navarro et al. 2017;Delhon et al. 1996;Rufty et al. 1989). After prolonged darkness, nitrate uptake rate declines to a very low level (Cárdenas-Navarro et al. 1998;Macduff and Jackson 1992;Peuke and Jeschke 1998). Cárdenas-Navarro et al. (1998) found that nitrate uptake increased continuously in the light and decreased in the darkness. Macduff and Wild (1988) noted that darkness affected nitrate utilization in plants rather than nitrate uptake. Nitrate uptake is affected by light/dark and carbohydrates, which may attribute to the positive effect of sugars transported to the roots by phloem (Delhon et al. 1996;Forde 2002;Geßler et al. 2004;Larbat et al. 2016;Lejay et al. 2003;Li et al. 2013;Li et al. 2019;Rufty et al. 1989;Walch-Liu et al. 2005).
Except for light and carbohydrates, it's known that hormones, including auxins, CTKS, ABA, GAS, and ETH, can influence nitrate uptake. Among these, auxin and GAS were found to have a positive effect on nitrate uptake. Researches showed that nitrate transporters, as auxin transporters, can regulate the accumulation and transport of auxins in lateral roots (Chai et al. 2020), and GAS also can affect nitrate uptake rate (Sugiura et al. 2007). Yoon et al. (2021) pointed out that sucrose controls various metabolic processes in plants. Sucrose, and not any other individual sugar nor group of sugars, is a primary signal to nitrate uptake. Based on ten selected carbohydrates, sucrose was identified as a positive feed-forward signal controlling nitrate uptake (Li et al. 2013). Sucrose, which can be synthesized in the light or provided by external addition, can stimulate shoot growth. To match supply to demand, the root must take up nitrate faster. Sucrose is a pinpoint signal, which is definitely different from other sugars that affect nitrate uptake (e.g. glucose, fructose, and raffinose, see Li et al. 2013). The speed of nitrate uptake responds to external sucrose addition is rapid and the nitrate uptake rate is proportional to the sucrose concentration in the cytoplasm, so sucrose possesses the characteristics of a feed-forward signal. The positive feed-forward signal is rapid, but easily perturbed, therefore negative feedback via amino acid concentration compensates for these.

The nature of the signal carried by sucrose
Since an increase in sucrose concentration results in an increase in nitrate influx, it is clearly acting as a positive feed-forward signal (Li et al. 2013). In the case of sucrose carrying a signal from the shoot to speed nitrate uptake by the root system, the time taken will depend on the accumulation of higher sucrose concentration in the leaf cells, loading the phloem to a higher concentration, the time for the higher concentration to reach the root cells, to unload into the cytoplasm of root cells and have its effect on the nitrate transport system. Hence a positive feed-forward signal has the positive characteristic of speed. However, it also has the negative characteristics of not giving a definite outcome and always being susceptible to perturbations.

The amino acids involved in feedback control
The negative feedback control involves amino acids and CTKS, etc. Ivashikina and Sokolov (1997) thought that negative feedback regulation of nitrate uptake was related to the nitrate metabolites in the shoot. It is known that C metabolism and N metabolism are coordinated (O'brien et al. 2016). If photosynthesis and growth speed up, N arriving from the root will be used up faster, the concentration of free amino acids and other N compounds would fall, and the flow of reduced N to the root system would also fall and act as a signal to increase the nitrate uptake and transport rates (Forde 2000;Glass et al. 2002;Nazoa et al. 2003;Walch-Liu et al. 2005). This would be negative feedback control. It's known that Glu, Asp, Asn, Gln, Arg, Lys, and Ser have an effect on nitrate uptake (Li et al. 2010). Nitrate influx is subject to metabolic co-regulation by nitrate and amino acids levels in the cytoplasmic compartment of the roots (Macduff and Bakken 2003). Beuve et al. (2004) provided the evidence that γ-aminobutyric acid in phloem exudate may act as a putative long-distance inter-organ signal molecule in plants in conjunction with negative control exerted by Gln.
Some work on the identification of a signaling molecule has suggested that a specific amino acid(s) is involved. Gln has been implicated several times (Beuve et al. 2004;Glass et al. 2002;Nazoa et al. 2003;Pal'ove-Balang and Mistrik 2002;Thornton 2004). However, this claim is made based on examining only four amino compounds -Gln, Glu, Asn, and Aspand every one of these has been found to cause inhibition of nitrate uptake to some extent (Aslam et al. 2001;Beuve et al. 2004;Thornton 2004). It has also been shown that there is inter-conversion of amino acids going on in the root, as would be expected, so these experiments are ambiguous (Thornton 2004;Vidmar et al. 2000). Treating roots with 1 mM Gln increased cytosolic nitrate activity from 3 mM to 7 mM and this change peaked after 2 hr of treatment (Fan et al. 2006). The addition of Gln increased its concentrations in root tissue. However, the results cannot be attributed to changes in Gln alone, as its addition also resulted in the increased concentrations of other amino acids (Thornton 2004). Vidmar et al. (2000) noted that there was substantial inter-conversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants should be separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of NR, Gln synthetase, and Glu synthase, respectively). Padgett and Leonard (1996) proposed that neither Gln nor any of the remaining 19 proteinaceous amino acids appeared to be solely responsible for the regulation of nitrate uptake. The ability of amino acids to regulate nitrate uptake and assimilation appears to be more related to their overall levels in the cell rather than to an accumulation of specific amino acids. The level of amino acid in plants may indicate N status and provide a signal that can regulate nitrate uptake.

Hormones involved in feedback control
Except the auxins and GAS have a positive effect on nitrate uptake, both ETH and CTKS have some negative effects. Low N level can induce ETH biosynthesis and signal transduction, further reduce NRT2.1 expression, thereby reducing nitrate uptake in plants (Zheng et al. 2013). In addition, ABA affects the function of nitrate transporters when plants are in stress environment (Léran et al. 2015).
In the current study, CTKS has a significant impact on N uptake, transport, and metabolism in plants and can improve N utilization efficiency (Gu et al. 2017). It was also shown that an additional atmospheric N source resulted in activation of CTKS in the shoot, and increased shoot-to-root transport of CTKS in the phloem (Dluzniewska et al. 2006). Phloem mobility and basipetal transport of CTKS were also demonstrated by feeding zeatin riboside into the phloem. The resulting enrichment of CTKS in the roots caused an increased expression of a high-affinity nitrate transporter, the enrichment of amino compounds (Glu, Val, Lys) in the roots, and a significant decrease in net nitrate uptake (Dluzniewska et al. 2006). These results provided experimental evidence that, in addition to amino compounds, CTKS that were known to cycle within the plant were also communicating changes in N metabolism from the shoots to the roots.
The influences of exogenously applied amino acids and CTKS on the physiological and molecular aspects of N metabolism in poplar trees were investigated. In a shortterm feeding experiment, NO 3 − net uptake declined significantly after Gln or trans-zeatin riboside was added directly to the nutrient solution (Dluzniewska et al. 2006). The results indicated that both particular amino acids and active CTKS were involved in the feedback regulation of N uptake and metabolism in poplar. It was proposed that inhibition of N uptake by CTKS in poplar was more complex than that mediated by amino compounds, and other effector molecules were involved in this regulation (Dluzniewska et al. 2006). CTKS and amino acids, which cycling within the plants, are signal substances communicating changes in N metabolism (Collier et al. 2003). Ferreira and Kieber (2005) also pointed out that CTKS modulate the response to varying nitrate concentrations. Ivashikina and Sokolov (1997) noted that there may be the occurrence of different feedback regulatory mechanisms.
4. The interaction between the positive feedforward signal and negative feedback signal 4.1. Are the effects additive?
A higher sucrose concentration would also mean that the effect of any compound involved in negative feedback control of nitrate uptake (Glu attracting the major vote) would be different. Specifically, in the presence of higher sucrose in the root, a given change in Glu concentration would have a proportionately smaller effect on nitrate influx. In the case of Cl uptake, abscisic acid appears to affect the system by raising the set point for accumulation (Cram 1983a(Cram , 1983b. If sucrose acts in the same way on nitrate uptake, its increased concentration would stimulate uptake. Once the setpoint is fixed, a negative feedback signal would be resistant to other perturbations such as changes in temperature or in external nitrate concentration itself. When 25 mM sucrose was added together with 10 mM Gln, the effect on NR activity and the estimated rate of nitrate assimilation was antagonistic to the effect of Gln (Moecuende et al. 1998). Therefore, the sucrose and amino acids effect should not be additive.

Signals transported by phloem
Amino acids can be exported to the phloem independently of the export of sugars (Caputo and Barneix 1999). In terrestrial higher plants, phloem transport delivers most nutrients required for growth and storage processes. Some 90% of plant biomass, transported as sugars and amino N compounds in a bulk flow of the solution is propelled through the phloem by osmotically generated hydro-static pressure differences between the source (net nutrient export) and sink (net nutrient import) end of phloem paths. Source loading and sink unloading of sugars, amino N compounds, and potassium largely account for phloem sap osmotic concentrations and hence pressure differences. Sucrose, and probably certain amino acids, are loaded into minor veins from source leaf apoplasms by proton symporters localized to plasma membranes. Effluxers that release sucrose and amino acids to the surrounding apoplasms in phloem loading and unloading are yet to be cloned. Roles of sucrose and amino acid transporters in phloem unloading remain to be discovered, along with mechanisms regulating symplasmic transport (Lalonde et al. 2003).
The relationship between amino acid and sugar export to the phloem was studied in young wheat plants using the EDTA-phloem collection technique. Plants grown with a 16 hr photoperiod showed a rapid decrease in the concentration of sugars and amino acids in the phloem exudate from the beginning of the dark period. When plants grown with a 16 hr photoperiod were kept in the dark for longer than 8 hr the free amino acid content in leaves and exudate (on a dry weight basis) increased continually throughout the 72 hr of darkness. During the first 24 hr of darkness, the sugars in the phloem exudate decreased to 30% of the initial value and returned to the control level when plants were returned to light (Caputo and Barneix 1999). Stitt et al. (2002) discussed how nitrate assimilation was integrated with nitrate uptake, amino acid synthesis, and the sugar supply in tobacco leaves. Cooper et al. (1986) reported that about 30% of recently absorbed N was in the reduced form in wheat. Most of the reduction seemed to occur in shoots since, in parallel experiments, detached shoots reduced nearly as much of the previously absorbed NO 3 − as intact cultures. If the rate of nitrate uptake changes, the transcription, and activity of nitrate reductase (NR) should change, which can be regulated by light, sugars, etc. (Dennis and Blakeley 2000). In roots, NR transcript levels seem not to change but NR activity increases in the light (Matt et al. 2001); this suggests that light plays an indirect role (via photosynthesis) in regulating NR gene expression. In higher plants, NR is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. The changes in NR activity of leaves or roots after light/dark modulation or feeding sugars are all more rapid than the NR-protein turnover rates (minutes versus hours) (Taiz and Zeiger 2002), therefore sucrose is more likely to regulate the NR activity rather than NR synthesis because nitrate uptake responds immediately to sucrose supply.

Nitrate reductase regulated by signals
NR turnover in roots is controlled by several factors. NR synthesis appears to depend on sugar availability. The degradation and half-life of the NR-protein appear to be affected by NR phosphorylation (Kaiser and Huber 2001). There are multiple effects of sugars on NR expression, NR activity, and NR degradation. The NR activation state is sensitive to carbohydrates (Kaiser et al. 1999). High sugar concentration can increase NR synthesis, block the protein kinase(s) that phosphorylates and inactivates NR, thereby activating NR and inhibiting NR degradation. Sucrose, glucose, and fructose increase NR level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Lactose exerts only a slight influence on the enzymes (Sahulka and Lisá 1980). At a low nitrate supply, the inhibition of induction of NR activity by the amino acids (Glu, Asp, Gln, and Asn) is a result of the lack of substrate availability due to inhibition of the nitrate uptake system (Aslam et al. 2001). When 10 mM Gln was supplied for 8 hr to detached tobacco leaves in low light, the estimated rate of nitrate assimilation decreased, and this was accompanied by a decrease in NR activity and NR activation.

Nitrate transport system and signaling mechanism
Understanding the molecular mechanisms of how plants absorb nitrate and assimilate it is a critical step to improve N use efficiency and reduce N loss. The mechanism of nitrate uptake has been shown to be by proton co-transport systems; the thermodynamics and kinetics of these transport systems are reasonably well characterized (Forde 2000;Walch-Liu et al. 2005). In higher plants, there are two kinetically distinct nitrate uptake systems, i.e. high-affinity transport system (HATS) and a low-affinity transport system (LATS). HATS appears to play a major role in nitrate uptake when nitrate concentrations in the soil are very low (<250 μmol/L) (Crawford and Glass 1998). LATS generally has a larger capacity than HATS does and shows linear kinetics above 0.5 mM. Nitrate transporter genes have been identified in fungi, algae, and higher plants (Forde 2000;Ruffel et al. 2021;Walch-Liu et al. 2005;Zhao et al. 2004). The evidence for these signaling mechanisms is based on correlations of gene expression and transport, but the kinetics have not been examined in adequate detail. Consequently, the results are only suggestive and fall far short of confirmation.
Coordination between the activity of ion transport systems in the root and photosynthesis in the shoot is the main feature of the integration of ion uptake in the whole plant. However, the mechanisms that ensure this coordination are largely unknown at the molecular level. After light/dark or carbohydrates treatment, nitrate uptake should change by changing the nitrate transport system. Lejay et al. (2003) showed that the expression of the gene that encoded root NO 3 − transporters in Arabidopsis was regulated diurnally and stimulated by sugar supply. Sucrose, glucose, and fructose are able to induce the expression of the ion transporter gene. Induction by light and sucrose are strongly correlated, indicating that they reflect the same regulatory mechanism (i.e. stimulation by photosynthates). The functional importance of this control is highlighted by the phenotype of the atnrt2 mutant of Arabidopsis. In this mutant, the deletion of the sugar-inducible NO 3 − transporter gene AtNrt2.1 is associated with the loss of the regulation of high-affinity root NO 3 − influx by light and sugar. However, the stimulation of AtNrt2.1 transcript accumulation by sucrose and glucose is abolished in an antisense AtHXK1 line, suggesting that HXK catalytic activity and C metabolism downstream of the HXK step are crucial for the sugar regulation of AtNrt2.1 expression (Lejay et al. 2003).
Work on the mechanism of signaling by reduced N compounds suggests that it is via gene transcription (Glass et al. 2002;Ruffel et al. 2021;Walch-Liu et al. 2005). Forde (2000) introduced nitrate transporter in plants and discussed feedback repression of the nitrate transport and assimilation pathway in higher plants. Nitrate transport systems can be regulated by Gln (Glass et al. 2002). Gln (and not Glu) is responsible for down-regulating HvNRT2 expression (Vidmar et al. 2000). Repression of AtNRT2.1 is specifically related to increased internal Gln, suggesting a role for this particular amino acid in N signaling responsible for nitrate uptake regulation (Nazoa et al. 2003). Meanwhile, Collier et al. (2003) found that significant enrichment in amino acidsaccompanied by decreased nitrate net uptake but not by increased expression of the high-affinity nitrate transporterwas also observed when Gln the major long-distance  transport form of N in beech was fed into the phloem. Anyway, the molecular mechanism has not been clear and needs to be further investigated.
In this paper, the current studies of a regulatory network diagram of the signal molecules that control nitrate uptake and nitrate transporters in rice were summarized (Figure 1). The regulatory factors include the positive feed-forward (e g. nitrate, light, and sugars, in which sucrose is identified as the signal) and negative feedback (e.g. amino acids and CTKS). Besides sugars and amino acids, hormone signaling also have impacts on the regulation of genes related to nitrate transport activity. The genes localized in roots may involve in nitrate uptake control; the genes localized in vascular tissue and leaf may involve in nitrate transport, utilization, and redistribution; the genes localized in grain may involve in grain yield. Moreover, some genes can be expressed in a variety of tissues. The small font represents the other functions except the functions shown in the dashed squares.

Author Contributions
All authors contributed to the study's conception and design. Material preparation, data collection and analysis were performed by JZ Li and JM Gao. The first draft of the manuscript was written by JZ Li. All authors have read and agreed to the published version of the manuscript.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Notes on contributors
Jin-zhi Li, an associate professor of biology, is specialized in plant physiology and molecular biology, especially plant N metabolism. In her recent paper, detailed dynamics analysis of net nitrate uptake by wheat roots after sucrose signal molecule treatment was investigated by the non-invasive technique (Li et al. 2021 Brazilian Journal of Botany).
Bing Li, a postgraduate student of biology, researches on plant nutrition and molecular biology. Recent studies "Detailed dynamics change of net nitrate uptake by wheat after sucrose treatment" (Jinzhi Li & Bing Li) were published in the form of conference abstract (Proceedings of the 13th second member congress and 2021 academic annual meeting of Jiangxi Botanical Society).
Qian Guan, an undergraduate student majoring in Biological Science (normal), does some research in plant nutrition in the plant laboratory led by Jinzhi Li. The current research on the signal molecules regulating nitrate uptake by roots and their regulation mechanism were investigated, which provides a theoretical basis for improving crop quality and yield.
Jian-ming Gao, an associate researcher of biology, is specialized in plant nutrition and pest control of vegetable. New technology of insect prevention net developed by him was listed as the main technology in 2021 by Hainan Province, China. The key technical points are: the insect prevention nets are chosen according to the types of crops and pests in the shed, planting and harvesting at the same time and drying makes insects starve to death.