Introduction

Nitrogen (N) is a key essential mineral element that is required for various life processes of plants such as biosynthesis of amino acids, nucleic acids, chlorophylls, cofactors, several secondary metabolites, and so on (Crawford 1995; Scheible et al. 1997; Stitt 1999). Plants are able to acquire both inorganic as well as organic N from the soil in the form of nitrate (NO3−), ammonium (NH4+), urea, amino acids, and so on depending on their species and the physico-chemical properties of soil (Masclaux-Daubresse et al. 2010; Miller et al. 2007). Among the different forms of nitrogen, nitrate is the predominant form in aerobic soil (Forde 2000) and hence the most preferred form of nitrogen for cultivation of wheat. Owing to the mobile nature of nitrate in the soil, the percentage of the applied nitrogenous fertilizer actually used by the wheat crop is very low and consequently a majority of it is lost to the environment by processes such as leaching, denitrification, volatilization, and so on (Good et al. 2004). The N losses lead to environmental pollution globally (Zhang et al. 2015). Further, because the lost N is not available to plants for growth, development, or yield, it causes economical losses for farmers. Being one of the staple foods of the major part of the world, enhancing nitrogen use efficiency (NUE) of wheat has now become a major concern for wheat improvement programs. Nitrate uptake efficiency (NUpE), that is, the ability of a genotype to take up nitrate from the soil, is probably the most important component trait toward improving NUE in wheat.

The first step of nitrate metabolism in plants is its uptake from the external environment into the cell. Nitrate (NO3−) molecules are actively transported by nitrate transporters located across plasma membrane of epidermal and cortical cells of the root tissues belonging to mainly two types of nitrate uptake systems, that is, high-affinity transport system (HATS) and low-affinity transport system (LATS) (Fan et al. 2017). These transport systems are further classified based on their transport kinetics into mainly three distinct nitrate transport categories (Miller et al. 2007; Tsay et al. 2007; Wang et al. 2012): (i) Constitutive high-affinity nitrate transport systems (CHATS), which are characterized by their lower values of both Km and Vmax; (ii) inducible high-affinity nitrate transport systems, which show higher values of Km and Vmax and are induced by exposure of NO3− within hours to days; (iii) constitutive low-affinity transporters that take up nitrate at higher concentrations and in contrast to HATS fail to saturate at NO3− concentration as high as 50 mM (Crawford and Glass 1998). In Arabidopsis, constitutive LATS (CLATS) and inducible LATS (ILATS) have also been reported (Tsay et al. 1993; Huang et al. 1999). LATS and HATS are encoded by the NRT1 and NRT2 gene families, which have 53 and 7 known members, respectively, in Arabidopsis thaliana (Wang et al. 2012 and references therein). The various aspects of nitrate transport, sensing, and signaling have been reviewed in recent publications (O’Brien et al. 2016; Kiba and Krapp 2016; Noguero and Lacombe 2016; Fan et al. 2017). The expression study of some of the putative low- and high-affinity nitrate transporter genes of wheat has been studied under different growth stages and external concentrations of nitrogen (Buchner and Hawkesford 2014; Guo et al. 2014; Melino et al. 2015; Taulemesse et al. 2015), however, only TaNRT2.1 was isolated and characterized in detail (Yin et al. 2007). The N status and growth stage at the time of N treatment in these studies are entirely different than the present study.

Nitrate acquisition by plants depends on several internal and external conditions such as fluctuating nitrate availability in the soil and physiological conditions of plants (for example, NO3− assimilation, acquisition of other nutrients, growth condition, etc.) (Ruffel et al. 2014). To compensate for fluctuations of external NO3− concentrations and the actual N demand of plants for normal growth and development, the plant root system undergoes continuous modulation for its NO3− uptake kinetics (Alvarez et al. 2012; Krouk et al. 2010; Vidal and Gutierrez 2008). The monocot cereals including wheat have complex root structures composed of several root types (Klepper 1992). Apart from an embryonic primary root, wheat seedlings also form variable numbers of seminal roots that emerge from the scutellar node which are also embryonic. All of these embryonic roots can form first- and second-order lateral roots. Later, they also form several crown roots which are non-embryonic in nature. All of these root systems play significant roles in nutrient and water acquisition. The effects of different nutrients on growth and development of roots in model plants have been widely studied. However, detailed root system architecture studies under the influence of external nutrients are by and large more restricted to the model plant, that is, Arabidopsis (Gruber et al. 2013; Kellermeier et al. 2014). However, the influence of nitrate on overall growth of barley root systems including lateral roots has been reported (Drew et al. 1973). Knowledge of the best root system architecture for efficient nitrate uptake will play an important role in developing strategies for root breeding for efficient nitrate uptake in its limited and heterogeneous availability condition in the soil. Nitrate uptake by roots is under the control of complex regulation by at least two different N-signaling pathways. NO3− itself locally induces the expression of several genes encoding its own transport systems, as well as the main enzymes involved in its assimilation (Wang et al. 2004; Gojon et al. 2009). The root NO3− uptake system is also under systemic feedback repression by whole-plant signals associated with high N status of the organism (Tabata et al. 2014; Ohkubo et al. 2017).

After the entry of NO3− into the root tissues, it is metabolized to ammonium by two enzymes (nitrate reductase and nitrite reductase) (Meyer and Stitt 2001), and is further assimilated to glutamate by the GS-GOGAT pathway (Lea and Miflin 1974; Lea and Forde 1994). The assimilation of ammonium requires a carbon (C) skeleton for the biosynthesis of important molecules such as amino acids, proteins, and nucleic acids. Therefore, NO3− uptake is not only determined by NO3− availability and N demands of the whole plant (Imsande and Touraine 1994; Forde 2002; Alvarez et al. 2012), but also by the C metabolites produced by photosynthesis (Delhon et al. 1996). It has been observed that plant root tissues are capable of taking up exogenously supplied carbon sources like sucrose and biochemically diverse types of amino acids (Malamy and Ryan 2001; Hirner et al. 2006; Svennerstam et al. 2007; Walch-Liu et al. 2006; Nasholm et al. 2009). Our current understanding about nitrate uptake and its regulation is more or less restricted to the model plant Arabidopsis, and not to field crops such as wheat. The number of genes that encode low- and high-affinity nitrate transport systems seems to be relatively high in wheat owing to its large genome size and the complex environment in which it grows as compared to Arabidopsis. Therefore, understanding the contribution of HATS and LATS towards overall N uptake and modulation of root system architecture under limited N conditions will be highly useful to mine the best allele among several nitrate uptake efficient wheat genotypes.

We studied nitrate uptake in seedlings of a high N-responsive wheat genotype that was subjected to induction with low to high nitrate concentrations (0.04–2 mM NO3−) and made comparisons to non-induced seedlings. We hypothesized that the presence of even a smaller amount of nitrate in the induction system would modulate root system architecture and also the activities of different nitrate transporters, which consequently change the nitrate–N content in root and shoot tissues. The carbon and nitrogen status of the plants would most likely impact different nitrate transporter activities and hence, we included sucrose, asparagines, and glutamine (as C and N sources) in one of the induction systems (2 mM NO3−) to observe their effects on these parameters. The present study would give an insight towards the relation of nitrate transport system under external NO3− induction and RSA which might play a role in nitrogen use efficiency of wheat which is conferred by the complex root system.

The objectives of the present investigation were to study the gene expression of kinetically different nitrate transporters in root tissue under different induced and non-induced conditions, changes in root morphology, and root system architecture during the induction period. The effects of inclusion of sucrose and two amino acids (glutamine and asparagines; the end products of nitrate and ammonium assimilation) in the nutrient media on these parameters were also studied.

Materials and Methods

Plant Material and Growth Conditions

In the present study, a widely grown Indian wheat variety known as HD-2967 (a highly N-responsive variety released from ICAR-Indian Agricultural Research Institute, New Delhi, India in 2011 for North Western and North Eastern Plain Zone of wheat growing zones of India with yield potential 5.4 ton/ha in timely sown irrigated condition) was used in all experiments.

Wheat seedlings were hydroponically grown after surface sterilization of uniform size seeds following the methods described by Goyal and Huffaker (1986) and Sinha et al. (2015). The modified hydroponic system was used as described by Negi et al. (2016), except for using square containers (Phyta Jar (box) from HIMEDIA, India; width × length × height = 75 mm × 74 mm × 138 mm) for growing wheat seedlings. The Phyta Jar consists of a stand supported net having perforations through which the radicle emerges and grows in liquid nutrient media. The germinated seeds were kept on the net which supports seedling growth throughout the experiment. The number of seedlings grown was 4 seedlings/box and all the experiments were carried out in controlled and aseptic conditions. The seeds were germinated by imbibing aerated de-ionized distilled water at 25 ± 1 °C in the dark. Then seedlings were kept in 0.2 mM CaSO4 solution for 10 days in the dark at 25 ± 1 °C as described previously (Aslam et al. 1992). After 10 days of growth, the 0.2 mM CaSO4 solution was replaced by 1× MS (Murashige and Skoog) media (Nitrogen minus) and grown further for 3 days at 25 ± 1 °C under 150–200 µmoles photon/m2/s light intensity, 10/14 dark/light hours, 70% relative humidity. The MS media at this stage were devoid of nitrogen just to prevent any induction of the transporters during this period.

After 13 days of growth, seedlings (four uniformly grown seedlings/box) were subjected to various pre-treatments on the 14th day with the solution (hereafter induction and non-induction media) specified in Fig. 1 for 24 h under similar growth condition as described above (Aslam et al. 1992) to observe various physiological and molecular changes as discussed below. The source of NO3− in all the experiments was Ca(NO3)2. The calcium concentration in all media was kept constant by adjusting the amount of CaCl2. During the entire period of plant growth, continuous aeration was ensured to provide ample O2 and to maintain the uniform solution concentration at the root surfaces. The complete experiment plan is explained with the help of a flow chart (Fig. 1).

Fig. 1
figure 1

Flow chart depicting the experimental plan

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To observe the effect of chronic N starvation, seedlings were also grown in another set of experiments for 15 days; however, in this case, germinated seeds were directly transferred to a box (Phyta Jar) containing MS media consisting of either 8 mM NO3− (optimum) or 0.08 mM NO3− (stress). In this case, media were refreshed after every third day. Other growing conditions were the same as described above.

For biomass and morphological studies wherever mentioned, seedlings were divided into shoot and root and then the root and shoot length and fresh weight were measured separately. Dried weight of these tissues was measured after drying them at 50 °C temperature in hot air oven.

Nitrate–Nitrogen Estimation

After pre-treatment of seedlings as described (Fig. 1), the seedlings were transferred to uptake solution containing 8 mM NO3− (1 mM Mes buffer, pH 6.0; 0.2 mM CaSO4, and 4 mM calcium nitrate). NO3−–N content was measured in aliquots (20 µL) taken at the end of uptake and expressed as µg NO3−–N/mL by following the method of Cataldo et al. (1975). The fine ground root and shoot tissues were suspended in de-ionized water and incubated at 45 °C for 1 h. Then samples were mixed and centrifuged at 5000×g for 15 min and supernatants were used for analysis. Nitrate–nitrogen was also estimated in dried root and shoot tissues by the same method. In the case of dried tissue, 100 mg of dried root/shoot tissue was ground and mixed in 10 mL of de-ionized water for nitrate extraction and for further downstream process. Uptake solution was directly used for NO3−–N content analysis.

Root System Architecture Study

Images of properly washed and well spread roots were taken immediately after pre-treatment of seedlings using a flatbed scanner (Epson Perfection V700) at a resolution of 400 dpi. The Images thus generated were then analyzed with WinRhizo software which analyses the morphology of roots in the form of link analysis and can be converted into different root classes (Regent Instruments Canada Inc., Arsenault et al. 1995). WinRhizo analyses morphology (length, area, volume etc.), topology, architecture, and color analysis of roots. The data are stored in excel files and images in tiff, jpeg, or bmp files. All roots emerging from either the radicle or the scutellar node are here referred to as seminal roots; however, among all such seminal roots the one that emerges from the radicle is referred to as the main/primary seminal root, whereas those emerging from scutellar nodes are referred to as lateral seminal roots. The post-embryonically developed lateral roots that emerged from these seminal roots are classified as 1st order lateral roots (LRs), whereas those developed from 1st order LRs are referred to as 2nd order LRs (Kellermeier et al. 2014). Out of the huge amount of data generated, some of them were processed for interpretation of five root parameters as per the given definition such as total root size (TRS: sum of path length of all types of roots described here), main root path (MRP length; primary root (seminal) that emerges from the radical, here the longest seminal root considered to be emerged from the radicle, and hence called a primary root), LRS (lateral root size; sum of path length of lateral roots (post-embryonic; 1st and 2nd order LRs) as a fraction of TRS), 1st order LR number (number of LRs emerging from primary and lateral seminal roots), and 2nd order LR number (number of LRs emerging from first-order LRs) were studied as described by Kellermeier et al. (2014) in the case of Arabidopsis roots with minor modification in Sinha et al. (2015) in case of wheat roots.

Nitrate Transporter Genes Expression Profile

Root tissues of induced (0.04 mM, 0.4 mM, 2 mM, and 8 mM NO3−) and non-induced seedlings were harvested and immediately snap-frozen in liquid nitrogen. Total RNA was extracted from three biological replicates following the Trizol method and treated with DNaseI as per the manufacturer’s instructions. The integrity of RNA was confirmed by agarose (1.2%) gel electrophoresis. Complementary DNA was synthesized from the DNase-treated RNA using Superscript III Reverse Transcriptase (Invitrogen) as per the manufacturer’s instructions. Results were normalized to the expression of the actin gene whose expression stability had already been validated in our several such experiments. Primers were synthesized to quantify the expression level of four nitrate transporter genes TaNRT2.1 (AF332214.1), TaNRT2.3 (AY053452.1), TaNRT2 (AF288688.1), and TaNRT1.1 (AY587265.1) (Suppl. Table 1). The quantitative real-time PCR experiments (qPCR) were conducted on an Eppendorf realplex with the SYBR Green Master Kit (Applied Biosystems). The qPCR program involved pre-incubation for 10 min at 95 °C, followed by 40 cycles of amplification, each consisting of denaturation for 15 s at 95 °C followed by annealing for 1 min at 60 °C. To ensure that single products were amplified, a melting curve was analyzed at the end of each assay. cDNA templates were diluted to 100 ng/µL and qPCR was carried out using 10 µL reaction volume containing 1 µL diluted cDNA, 5 µL SYBR green mix, 1 µL each of 10 µM forward and reverse primers, and 2 µL nuclease-free water of three replicates of each samples. Relative expression was determined by the ∆∆CT method corrected for primer efficiency (Livak and Schmittgen 2001) and compared against the non-induced treatment. In case of inclusion of sucrose, Gln, and Asn (+S, +G, and +A, respectively), the gene expressions were compared with a 2 mM induction system.

Statistical Analysis

All experiments and/or measurements reported in this study were done with five biological and three technical replicates except for the gene expression study wherein three biological replicates were taken. Mean values were presented for all the parameters with error bars (standard error of means). Least significant difference (LSD) at 5% was calculated for significance of treatment effect and further a range test was performed to show the level of significance between and among the treatments in each experiment. The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS, 2003) and MSTATC V2.1 (MSU, USA) (MSTATC 1990) software at alpha level 0.05. For comparing the means of optimum (8 mM NO3−) versus N stress (0.08 mM NO3−), we conducted a t test and wherever significant results were obtained results were presented with ‘*.’ For any given two samples, initially an F-test at alpha level 0.05 was performed to see if the variances are equal. Based on the result of F-test, an appropriate t test was performed to see if the means are equal. For all other experiments in this article, means were compared and significant differences were determined, followed by ranking according to their significant differences. Factorial analysis (one factor) of variance (ANOVA) has been performed with a complete random design using MSTAT C version 2.1. Each experiment was performed with three biological replicates and four plants in each biological replicate. Ranking has been obtained as ascending order by ‘range test’ using the same software. Error mean square, degree of freedom, number of means to be separated, and number of observations per means were obtained from the ANOVA table. The least significant different difference test was performed as a sub-program under the range test to finally obtain the range with distinct letters.

Results

Chronic N starvation (0.08 mM NO3−) to wheat seedlings (HD-2967) grown for 15 days showed a significant increase in root/shoot ratios for their length, fresh weight, and dry weight (Fig. 2a). However, the NO3−–N content in both the tissues was drastically under stress conditions reflecting the imposition of N stress conditions (Fig. 2b).

Fig. 2
figure 2

Wheat seedlings grown under optimum (8 mM) and stress (0.08 mM) NO3− conditions for 15 days. Root and shoot ratio in terms of length, fresh weight, and dry weight basis under optimum and stress condition (a). Nitrate–N content in shoot and root tissues under optimum and stress condition (b). *Above bars indicate significant differences (p < 0.05) between the treatments

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In a further study with a different induction system, we observed that 0.4 mM NO3− induced maximum uptake of NO3− (Fig. 3a). Therefore, we were interested to know what happened immediately after induction to roots with respect to their morphology, architecture, and expression of genes encoding high- and low-affinity nitrate transporters.

Fig. 3
figure 3

Nitrate–N estimation in uptake solution and shoot and root tissues: amount of nitrate–N present in nitrate depleted uptake solution (8 mM NO3−) estimated after 24 h containing pre-treated (induced/non-induced (NI)) wheat seedlings with different NO3− concentrations (0.04 mM, 0.4 mM, and 2.0 mM) (a). NO3−–N accumulation in shoot tissues (dried) immediately after induction with different NO3− concentrations (0.04 mM, 0.4 mM, 2.0 mM, and 8.0 mM) or non-induced (NI) wheat (b) and root tissue (C). NO3−–N accumulation in shoot tissues (dried) induced with 2.0 mM NO3− along with sucrose (+S), glutamine (+G), asparagines (+A), sucrose + glutamine + asparagines (+SGA), sucrose + glutamine (+SG), sucrose + asparagines (+SA) (d), and root tissues (e). Letters above bars indicate significant differences (p < 0.05) between the treatments

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Effect of Different Nitrate Induction Systems on Nitrate Uptake and Its Allocation in Roots and Shoots

We first measured depletion of nitrate by quantifying NO3−–N in the uptake solution containing induced and non-induced (NI) wheat seedlings (14 days old; 10 days in dark, 3 days in light without N, and 1 day in induced and non-induced condition as described in M and M) and observed the lowest amount of NO3−–N (463 µg NO3−–N/mL) in the uptake solution containing wheat seedlings induced by 0.4 mM NO3− as compared to non-induced, and the other two inducible systems (0.04 mM and 2.0 mM) (Fig. 3a). The uptake solution containing NI wheat seedlings contains the maximum amount of NO3−–N, that is, 873 µg NO3−–N/mL. The other two systems have intermediate amounts ranging from NI to 0.04 mM. Our results in this case demonstrated that these inducible systems induce the nitrate uptake process to different degrees and among them the 0.4 mM inducible system caused maximum nitrate uptake by wheat seedlings.

NO3−–N was measured in both root and shoot tissues of wheat seedlings after 24 h of induction compared to NI systems. We detected very low levels of NO3−–N in shoot and root tissues of NI seedlings. However, exposure of various amounts of nitrate present in the induction solution for 24 h influences NO3−–N content in shoot and root tissues. The maximum amount was found in seedlings induced with 2 mM nitrate solution (8.83 and 14.5 µg NO3−–N/mg dry tissues in both in shoots and roots, respectively). In the case of other NO3− induction systems, NO3− accumulation in shoots was on par with NI shoots in 0.04 and 0.4 mM induction, but NO3− accumulation was significantly higher in the 0.4 mM induction system but not in the case of the 0.04 mM system. Interestingly after a further increase beyond 2–8 mM NO3−, the induction system reduced NO3− accumulation significantly, however, it was still significantly higher than that of the NI system in both shoot and root tissues (Fig. 3b and c). As the 2 mM NO3− induction system caused the maximum amount of NO3−–N, we further included different C and N sources in the 2 mM NO3− induction system to observe their effect on NO3−–N content (or nitrate uptake) in shoot and root tissue. All of them influenced the allocation of NO3− in root and shoot tissues differently. The major observation was that the C and N sources were significantly inhibitory in NO3−–N accumulation/uptake in shoot and root tissues as compared to the 2 mM induction solution (8.83 and 14.5 µg NO32−–N/mg in shoot and root, respectively). Addition of 1% sucrose (w/v) and 1 mM asparagine (1.0 µg NO3−–N/mg in both cases) severely inhibits NO3−–N accumulation in shoot tissues. Similar inhibition of NO3− accumulation was observed in the case of root tissues as well. Interestingly, glutamine was less inhibitory compared to asparagines and sucrose and other combinations (4.17 µg NO32−–N/mg) in both shoot and root tissues except the +SA condition in shoots (Fig. 3d and e).

Effect of Different Induction Systems on Morphological Attributes

As far as morphological attributes of induced and non-induced wheat seedlings are concerned, we did not observe significant differences in a few parameters such as shoot length and root dry weight during both induction as well as C and N interaction; shoot dry weight was affected during the induction experiment only (Fig. 4a, c, i, j and l). However, root length showed significant changes wherein 2 mM induction system caused a maximum increase in root length, that is, 29.26 cm as compared to NI (13.33 cm) and the other induction systems. The 8 mM condition, however, had less impacts on root length than 2 mM, that is, 21.9 cm, but more impact compared to the NI, 0.04 and 0.4 mM conditions which have statistically similar effects (Fig. 4b). Sucrose, Gln, and Asn were inhibitory as far as total root length was concerned in comparison to the 2 mM condition (Fig. 4d). Fresh shoot weight was at a maximum in the case of 2 mM induction system (Fig. 4e). The other induction systems had less influence than NI. Root fresh weight in the NI condition at a maximum, that is, 41.07 mg, among all treatments followed by the 2 mM condition wherein the fresh weight was 29.33 mg (Fig. 4f). However, sucrose induction showed increased root fresh weight, that is, 36.33 mg, as compared to 2 mM, yet less than the NI condition (41.07 mg) (Fig. 4h). In the case of shoot fresh weight among all combinations, only the +SA system had a similar effect as the 2 mM system, whereas the other systems had inhibitory effects (Fig. 3g). In the case of shoot dry weight, +SA conditions showed little increase as compared to all other systems (Fig. 4k).

Fig. 4
figure 4

Variations in different morphological parameters under induced, non-induced, and different combinations of C and N metabolites in 2 mM induction conditions: length (NI and induced systems: a, b); C and N combination systems: c, d fresh weight (NI and induced systems: e, f; C and N combination systems: g, h); dry weight (NI and induced systems: i, j; C and N combination systems: k, l). Letters above bars indicate significant differences (p < 0.05) between the treatments, whereas no letters above bar shows no significant difference between treatments

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Effect of Different Induction Systems on Root System Architecture

As far as TRS is concerned, the induction system containing 8 mM nitrate was stimulatory, that is, (220.45 cm) (Fig. 5IIe), as compared to NI which was just 101.29 cm (Fig. 5IIa), whereas the other induction systems were either on par or less than the NI condition (Fig. 5Ia). Among all C and N sources, the +S and +SGA induction systems (236.44 cm, 251.97 cm, respectively) and +A (210.51 cm) had interestingly stimulatory effects on TRS when incorporated in the 2 mM (108.87 cm) induction system (Fig. 5If). Although sucrose and asparagine were stimulatory in TRS when used separately (Fig. 5IIf and h), glutamine and its combination, that is, +SG (40.81 cm) were inhibitory (47.73 cm) (Fig. 5g and j). In this case, the stimulatory effect of asparagines was reduced when combined with sucrose (+SA-39.15 cm) (Fig. 5If and IIk). In the case of the main root path length (MRP) that represents the length of the root that emerges from the radicle or primary root, it does not get induced by any of the induction systems except 2 mM (46.84 cm) as compared to NI (42.47 cm) (Fig. 5Ib and IId). Both C and N sources and their combinations were inhibitory compared to 2 mM (Fig. 5Ig and IIf–k). Another RSA parameter, that is LRS, which reflects the fraction of lateral root length to TRS, in the cases of 0.04 mM (0.58), 0.4 mM (0.52), and 2 mM (0.53) were interestingly on par with NI (0.54) except the 8 mM (0.19) induction system which was inhibitory in nature (Fig. 5Ic and IIe). However, in this case, +G (0.619) imparted a significant increase as compared to 2 mM (0.53), whereas other incorporations were inhibitory in nature (Fig. 5Ih and IIg). As far as lateral root numbers are concerned, the 1st order lateral root numbers increased in 0.4 mM (18.5) and 8 mM (23.66) conditions as compared to NI (12.33) (Fig. 5Id and IIc and e). However, the 2nd order lateral root numbers in the NI and 2 mM conditions were on par, whereas both lower concentration (0.04 mM and 0.4 mM) and higher concentration (8.0 mM) conditions had lower numbers of second-order lateral roots (Fig. 5Ie). Incorporation of sucrose in the 2 mM induction system, that is +S, induced the number of first-order lateral root (Fig. 5II). As far as 2nd order lateral root numbers were concerned, neither C or N metabolites had any significant impact on this parameter (Fig. 5Ij).

Fig. 5
figure 5

Variations in root system architecture parameters under induced, non-induced (upper panel I: a–e), and different combinations of C and N metabolites in 2 mM induction conditions (lower panel I: f–j): TRS (total root size), MRP (main root size) LRS (lateral root size), and 1°—LRN and 2°—LRN (1st and 2nd lateral root number). Root morphology of non-induced (II:a), 0.04 mM (II:b), 0.4 mM (II:c), 2.0 mM (II:d), 8.0 mM (II:e), +S(II:f), +G(II:g), +A(II:h), +SGA(II:i), +SG(j), and +SA(i) studied using flatbed scanner (Epson Perfection V700) at a resolution of 400 dpi

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Effect of Different Induction Systems on Expression of Nitrate Transporter Genes

All high-affinity nitrate transporter genes (TaNRT2.1, TaNRT2.3, and TaNRT2) were up-regulated in root tissues ranging from 4 to 40-fold, which were exposed with lower external nitrate concentrations (0.04 mM and 0.4 mM) compared to NI root tissues. Except in the case of TaNRT2.3 (down-regulated by almost eight times), the 2 mM condition induced the expression of two other high-affinity nitrate transporter genes as compared to NI root tissues. The 8 mM induction system always down-regulated gene expression of all transporters except TaNRT2.1. As far as C and N metabolite inclusion in the 2 mM induction system is concerned, +S and +SGA caused up-regulation of TaNRT2.3 as compared to 2 mM root tissues. +G, +A and their combinations with sucrose down-regulated the expression of TaNRT2.3. In the case of TaNRT2.1, all N and C combinations cause down-regulation of the gene when included in the 2 mM induction system but independently these sources have positive regulations. These systems, however, down-regulated TaNRT2, except +G and +S, when included in the 2 mM induction system. The low-affinity nitrate transporter TaNRT1.1 was down-regulated in 0.04 mM, 2 mM, and 8 mM root tissues, but interestingly in the case of the 0.4 mM induction system it was up-regulated. In this case except +A and +SG, other C and N systems caused up-regulation of TaNRT1.1 expression when included in the 2 mM induction system (Fig. 6).

Fig. 6
figure 6

Real-time gene expression profile under induced, non-induced (a) and different combinations of C and N metabolites in 2 mM induction conditions. b Genes are shown by their names, for example, TaNRT2.1, TaNRT2.3, TaNRT2, and TaNRT1.1

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Discussion

In this study, we observed reduction in biomass in terms of the shoot and root length, fresh, and dry weight in N stressed wheat seedlings undergoing 15 days of chronic N starvation. At the whole-plant level, the ratios of root versus shoot in terms of length, fresh weight, and dry weight significantly increased under N stress conditions indicating partitioning of sources towards roots to support their growth so that they can forage for N sources from the external media under N-limited conditions. Previous studies also suggest that mild nitrogen unavailability to the plant suppresses shoot growth and stimulates root growth; however, extreme conditions lead to an overall inhibition of plant growth, but still the inhibition is more prominent in shoots than root growth (Agren and Ingestad 1987; Fichtner and Schulze 1992).

Although NO3− uptake capacity varies with the nitrate concentration in the induction media it is always more than that of the condition wherein roots were never exposed to external nitrogen. Because nitrate uptake is absolutely a root physiological phenomenon, it seems quite likely that nitrate available in the induction system could have modulated the root systems vis-a-vis nitrate uptake system activity which enables them to take up considerably higher amounts of nitrate from uptake solution than the NI system. Considering these assumptions, we focus on the physiological and molecular changes that have been induced in wheat seedlings during the nitrate induction period. We observed varied levels of nitrate–N both in root and shoot tissues irrespective of the initial concentration of N in the induction systems indicating tight regulation and dynamic allocation of nitrate in both roots and shoots for its further metabolism depending on C and N demand (Fig. 3b and c). The lower amount of NO3−–N in the 8 mM condition in both roots and shoots as compared to the 2 mM condition may be because of further metabolism of nitrate by nitrate reductase (NR) and/or nitrite reductase (NiR) in these tissues. At low external nitrate concentrations, NR is found primarily in epidermal cells and cortical cells of the root, whereas at higher external nitrate concentrations, activity is also detected in cells of the cortex and vascular system. We speculated that at higher nitrate concentrations, that is 8 mM, both NR concentration and activity might be more as compared to the 2 mM condition, therefore it is metabolized faster in this case and consequently nitrate was found in lesser amounts. NR activity has also been found to be inducible by nitrate itself (Kronzucker et al. 1995). We also observed that both external C and N sources inhibit nitrate uptake, however, surprisingly the degree of inhibition of nitrate uptake differs with the kind of N source, for example, Gln and Asn, which suggests their distinct direct or indirect regulatory roles (Fig. 3d and e). It has been observed in several studies that the downstream metabolites of ammonium assimilation such as amino acids translocated from the shoot to the roots via the phloem may act as signal molecules which mediate the down-regulation of root N uptake (Cooper and Clarkson 1989; Muller and Touraine 1992). Inclusion of sucrose, Gln, and Asn in the 2 mM induction system reduced the nitrate content in both root and shoot tissues. This might have happened either because of further assimilation of nitrate to ammonium during this period or the absence of accelerating activity of sucrose as a signal molecule for the nitrate uptake process because of a longer lag time in this case (Zhou et al. 2009). However, the degree of decreased concentration varies with the C and N sources used indicating their diverse role apart from acting just as a C or N source. The negative feedback relationship of external amino acids or tissue concentration of amino acids on nitrate uptake by plants has been observed in crops as well as model plants (Lee et al. 1992; Muller et al. 1995) with some degree of deviation (for example, Brassica napus; Laine et al. 1995). Biomass results indicate that under such circumstances roots are more plastic in nature than shoots to adjust the N requirement of wheat seedlings by changing length and fresh weight without any fluctuations in dry weight (Fig. 4).

To understand the possible role of different components of the root system in nitrate uptake in a changing external N concentration, we measured the major five root system architecture (RSA) parameters of wheat seedlings immediately after the induction period together with effects of external C and N sources. We observed variation in TRS under induced and non-induced conditions which has been resolved further into its component parameters (Fig. 5I and II). Our results show the significant influence of either lower or complete absence of external nitrate on lateral root parameters (LRS and 1st order LR numbers) than to main roots (MR) with some degree of variation. Within the LR parameters, the number (1st order LR numbers) and length might have played some role in nitrate sensing and acquisition. The variation in RSA parameters under different conditions indicates their probable role in nitrate sensing and acquisition under different external N status. Both the nutritional status of the plant and the external nutrient availability can induce changes in the overall root morphology (Giehl et al. 2014). Severe nitrogen starvation in the external media decreases total root length, whereas its availability even in a milder concentration induces its increase (Gruber et al. 2013). Such RSA alterations may reflect different strategies used by plants in response to the amount of available nutrient (Giehl and Wiren 2014). We observed that mild external NO3− concentrations in the induction media inhibit MRP significantly as compared to complete N starvation. It has been observed that supply of nitrate to nitrogen-deficient plants inhibits primary root elongation and stimulates LR growth by regulating auxin activity by a mechanism that involves the auxin receptor AUXIN SIGNALING F-BOX3 (AFB3; Vidal et al. 2010). However, the TRS in the case of NI is considerably less than that of 8.0 mM. We observed that the addition of C and N sources separately or in combinations in the induction media causes a specific impact on these RSA parameters. These observations indicate the possibility of their roles in either cell division or elongation of root cells, however, this needs further study.

As far as wheat is concerned, except TaNRT2.1, no other nitrate transporters are characterized in detail. Based on sequence homology these genes have been categorized in respective groups. Sequences available in the public domain were used in this study for gene expression. To understand the possible reasons for variation in nitrate concentrations in roots and shoots under inducible and non-inducible conditions, we examined the expression pattern of the three high- and one low-affinity putative nitrate transporter genes in root tissues. We observed that all three high-affinity nitrate transporter genes were up-regulated in root tissues, which were induced with nitrate present at low concentrations (0.04 mM and 0.4 mM) in the induction media as compared to non-induced root tissue (Fig. 6). Their degree of up-regulation differs with the external nitrate concentration indicating the possibility of their activation at specific external N concentrations. However, the pattern of expression of these transporters varies at two concentration levels, that is 2 mM and 8 mM. Although TaNRT2.3 and TaNRT2.1 were down- and up-regulated, respectively, at these concentrations, TaNRT2 was up-regulated at the 2 mM condition but down-regulated at the 8 mM condition. Under different growth conditions, TaNRT2.3 has been found to be up-regulated in root tissues after exposure with different concentrations of nitrate (Yin et al. 2007; Guo et al. 2014). Melino et al. (2015) have also found the up-regulation of high-affinity nitrate transporter genes by lower concentrations of nitrate, but did not observe any changes in low-affinity nitrate transporter gene expression (TaNRT1.1 in this case), though the N status of wheat plants at the time of gene expression was different than in the present study. Because in this study seedlings exhausted almost all stored food during first 10 days of growth in the dark, exposure to different concentrations of nitrate during 24 h of induction causes changes in expression of different high- and low-affinity nitrate transporters as compared to non-induced tissues. Our results corroborate the existence of kinetically different nitrate transport systems within each category of HATS and LATS in wheat too. The down-regulation of TaNRT2.1 and TaNRT2.3 gene expression under the +G condition together with the up-regulation of TaNRT1.1 as compared to 2 mM wheat seedlings which suggests that TaNRT1.1 probably suppresses IHATS (TaNRT2.1 and TaNRT2.3 in this case) activity when high concentrations of alternative N sources (Gln in this case) are present (Munos et al. 2004; Krouk et al. 2006). Several groups (Quesada et al. 1997; Krapp et al. 1998; Vidmar et al. 2000) also demonstrated a role for Gln in inhibiting the expression of inducible high-affinity nitrate transporters. Both nitrate-induced influx and transporter transcript abundance decrease simultaneously in root tissue when treated with exogenously applied amino acids (Vidmar et al. 2000). NRT1.1 has been shown to act as a NO3− tranceptor (transporter as well as receptor) (Gojon et al. 2011), which controls the expression of the high-affinity NO3− transporter gene NRT2.1, and the development of lateral roots (Krouk et al. 2006; Remans et al. 2006; Ho et al. 2009; Wang et al. 2009). In our TaNRT1.1 expression study, we observed the up-regulation of TaNRT1.1 in the 0.4 mM induction system and down-regulation at the other two lower concentrations. However, even at the higher concentrations, that is 8 mM and 2 mM, there was lower expression as compared to the non-induced. Under high nitrate concentration, NRT1.1 switches to a low-affinity function and NRT2.1 gene expression is strongly induced which is evident in our TaNRT2.1 expression. In one of the cases (TaNRT2.3), we found that the +S condition induced gene expression which is similar to the condition in which NRT2.4 expression was found to be induced by N starvation and C (Kiba et al. 2012). Further, all levels of NRT2.1 expression (promoter activity, transport level, transport activity) have been shown to be repressed by downstream N metabolites and induced by NO3− (Ruffel et al. 2014).

Overall in the present studies, we observed that wheat seedlings modulate their root system architecture more specifically its lateral root traits (LRS and 1st order LR numbers), under either complete absence of nitrate or presence of smaller amounts of it (Fig. 5c and d). Further, these smaller amounts of nitrate (0.04, 0.4, and 2 mM nitrate) in the external media cause higher amounts of nitrate uptake as compared to the non-induced root (Fig. 3a) probably because of activation of different high-affinity nitrate transporters which result in acquisition and translocation of nitrate into roots and shoots, respectively (Fig. 3b and c). The presence of smaller amounts of nitrate probably in this case passes a sense of availability of nitrogen in the external media to the plant which allows specific root growth to forage for nitrogen sources and concomitantly activates the nitrate uptake system. On the other hand, the presence of externally available sucrose has been found to induce TRS and 1st order LR No. of root systems (Fig. 5f and i) and probably thereby influencing higher activity of HATS and LATS (Fig. 6b) in root tissue. However, the reduced nitrogenous end product of nitrate and ammonium assimilation, that is Gln and Asn, have been found to be inhibitory in nitrate–N content in both roots and shoots (Fig. 3d and e), whereas they have been found to both up- and down-regulate the HATS and LATS genes. The result indicates the involvement of potential post-translational regulation of nitrate transporters and feedback control by reduced N compounds in triggering nitrate uptake. Amino acids including Gln and Asn have been considered as feedback inhibitors of nitrate uptake (Muller and Touraine 1992). However, studies in this direction are needed to find explanations for these complex regulative parameters.

Conclusion

The results show that inducible nitrate transporters in roots contribute more in higher amounts of nitrate uptake from the external media specifically under nitrogen-limited conditions in wheat seedlings than that of non-inducible conditions. The lower external nitrate concentrations modulate the root system architecture, more precisely the lateral root parameters, both in terms of their number as well as length which probably contributes in nitrate uptake by increasing the expression of all high-affinity nitrate transporters gene in coordination with LATS thereby causing more uptake of NO3−–N from the uptake solution. The data also suggest that root tissues have different types of high-affinity nitrate transporters which seem to be working at different concentrations of external nitrate. Interestingly, C and N sources impact wheat seedlings both positively and negatively for its morphological as well as RSA attributes indicating C and N crosstalk in N sensing and signaling which ultimately impacts the overall growth and development, RSA, and also activation of nitrate transporter genes. Therefore, from the present studies, we can conclude that wheat seedlings utilize a specific and selective nitrate transport system which works in coordination with a network of regulatory steps of the nitrate assimilation pathway together with carbon assimilation to maintain a nutritional balance for optimal growth of shoots and roots including root architecture. Therefore, RSA traits, that is LRS and LR numbers, together with identification of efficient allele(s) for inducible high-affinity nitrate transporters will pave the way towards improving nitrogen use efficiency in general or nitrate uptake efficiency in particular in wheat.