1. Introduction
The use of grafted plants has become a common practice among tomato growers, mainly because of the search for methods that enhance crop resistance to soil-borne pathogens [
1]. Grafting usually results in vigorous plants with higher total and commercial yield than non-grafted plants [
2]. In order to sustain this higher productivity, plants must absorb larger amounts of nutrients or use them in a more efficient fashion than ungrafted plants [
3]. Identifying the mechanisms involved in higher uptake capacity or higher use efficiency of nutrients, especially nitrogen (N), will allow us to design more sustainable production systems by reducing the application of fertilizers, thereby limiting damage to the environment.
Nitrogen can be absorbed by plant roots either as nitrate (NO
3−) or ammonium (NH
4+); where tomato has a marked preference for absorbing NO
3− [
4,
5]. Five root plasma membrane NO
3− transporters have been identified in the tomato, belonging to the NRT1 and NRT2 families [
6]. Two genes (
LeNRT1.1 and
LeNRT1.2) comprise the LeNRT1 family, which encode for high-capacity, low-affinity nitrate transporters [
7]. On the other hand, the NRT2 family comprises three genes (
LeNRT2.1,
LeNRT2.2 and
LeNRT2.3) encoding for low-capacity, high-affinity transporters [
6]. The affinity for the uptake of a particular ion is described by the application of the Michaelis‒Menten relation to data obtained from depletion experiments [
8]. In this experiment, plants are subjected to different concentrations of the ion of interest, and sequential sampling at regular intervals allows for the determination of uptake rates. Then, the uptake rate (
U) and the external concentration (
C) data are fitted to the following equation:
where
Vmax is the apparent maximum uptake capacity,
Km is the apparent affinity for the ion, and
Cmin represents the minimum concentration required for uptake to occur. High
Vmax values imply roots with enhanced uptake capacity, while low
Km values denote roots with high affinity for the ion [
9].
The synthesis of these transporters in the roots depends on external NO
3− availability, but fluctuations in the expression levels have been reported according to the rate of shoot carbohydrate export to the roots or feedback regulation from N assimilates [
7,
10]. Plant internal N status regulates NO
3− uptake capacity [
10], with both processes partly controlled by the activity of the enzymes involved in the assimilatory pathway. The first enzyme acting on NO
3− assimilation is nitrate reductase (NR), and the evidence in tomatoes suggests that it is exclusively localized in the leaves [
11].
Plant N demand is ultimately controlled by the growth rate, and environmental factors such as light intensity determine biomass accumulation [
12]. Therefore, the aim of this study was to assess the metabolic adaptations involved in root NO
3− uptake between grafted and ungrafted tomato plants subjected to different growth rates. The study was conducted with the hypothesis that vigorous rootstocks modify their root metabolism in order to absorb higher NO
3− quantities than ungrafted plants when required by plant N demand. The study was conducted using
Solanum lycopersicum L. cv. Attiya (AT) and
S. lycopersicum x
S. habrochaites cv. Kaiser (KA) as the rootstock material.
3. Discussion
Total PNU increased by 71% in AT, 66% in AT-AT, and 93% in AT-KA when light increased from 400 to 800 µmol m
−2 s
−1. This increase in the radiation level has no effect on tissue N content, except in AT-KA where a 25.6% increase (
p < 0.0014) in root N content occurred. This improvement in NO
3− absorption is mediated by a higher expression of both
LeNRT1.1 and
LeNRT1.2, in the vigorous rootstock, whereas non-grafted plants rely almost exclusively on the expression of
LeNRT1.2. These results contrast those reported for cucumber (
Cucumis sativus) or tobacco (
Nicotiana tabacum) where
NRT1.1 presents a higher expression than
NRT1.2 across various NO
3− supply [
13,
14].
Both transporters,
LeNRT1.1 and
LeNRT1.2, act as symporters coupled to the uptake of H
+ into the roots [
7]. However,
LeNRT1.1 presents the capacity to absorb NO
3− in a wide range of external concentrations due to its ability to modify the protein structural flexibility by phosphorylation/dephosphorylation [
15,
16]. The only report available in tomatoes, showing no modification in the expression of
LeNRT1.1 under different growth conditions, relates this response to the colonization by mycorrhiza; the expression of
LeNRT2.3 is affected instead [
17].
The increase in the expression of
LeNRT1.1 and
LeNRT1.2 in the rootstock is accompanied by a substantial decrease in the expression of
LeNRT2.1 and
LeNRT2.3;
LeNRT2.1 encodes for a high-affinity transporter acting at low external NO
3− concentrations [
7]. On the other hand, Fu et al. suggest that
LeNRT2.3 functions as a low-affinity transporter, involved in NO
3− loading into the xylem [
6]. This activity would allow higher N-use efficiency in tomatoes [
6,
18], but our results show that rootstocks repress the synthesis of this transporter when shoot N-demand increases.
The classic approach to studying NO
3− transporters expression is by manipulating NO
3− availability in the root zone [
19,
20,
21,
22]. More recently, a systems approach has been used to evaluate the coordination between nutrient uptake and environmental signals, such as carbon dioxide and light [
23,
24]. These studies have been conducted mainly on
Arabidopsis thaliana, and no reports were found relating growth rate to the expression of NO
3− transporters in tomato roots. Here we show that the expression patterns of NO
3− transporters in the roots of tomato differ from those found in the rootstocks.
Available kinetics studies in the literature present V
max values in the range of 0.5–2.5 mmol g
−1 DW h
−1 [
25,
26]. Our results show higher uptake capacity, possibly because of the age of the plants used in our study, in comparison to the seedlings used by the previous authors. Although, no differences were found in the uptake capacity between the treatments (
Table 2), the higher RGR found in the grafted plants suggests an enhancement in N utilization efficiency in the grafted treatments.
Despite the differences in the NO
3− uptake rates between medium and high light intensity, no differences in root respiration rates were found in AT-KA. Similar root respiration rates are reported in the literature for the tomato [
27,
28,
29], but no information is available about the components of root respiration in this crop. In
Prunus spp., Toro et al. [
30] reported that rootstocks with higher growth rates present elevated root respiration rates, which is attributed exclusively to differences in the energy invested for root growth, not for nutrient uptake. Considering that NO
3− uptake and assimilation accounts for about 25% of the energy budget in roots [
31], it is possible to assume that our findings in NO
3− uptake will not allow us to measure significantly different root respiration rates. Moreover, our results show that NO
3− is exclusively assimilated in the shoot, reducing the energy consumption in the roots from 15 ATP to only 1 ATP per mole of NO
3− absorbed because of the null activity of the enzymes involved in the assimilation process.
Among the processes controlling nutrient uptake rates, the assimilatory capacity has been suggested as a target for improving nutrient acquisition in crops [
32,
33]. However, our results show no difference in leaf NRA between the treatments, as reported for other crops like watermelon grafted onto pumpkin rootstocks [
34]. No differences in the organ of assimilation were found either, which implies a higher accumulation of NO
3− in the roots of AT-KA when exposed to high light intensities.
4. Materials and Methods
4.1. Plant Material and Growth Conditions
Tomato (Solanum lycopersicum L. cv. Attiya, Rijk Zwan, De Lier, The Netherlands) plants and a vigorous interspecific hybrid (S. lycopersicum x S. habrochaites cv. Kaiser, Rijk Zwan, De Lier, The Netherlands) were obtained from seeds germinated in plastic trays placed in a growth chamber set at 25 °C air temperature. Plants were grafted at the two true leaves stage into one of these two combinations: Attiya self-grafted (AT-AT) or Attiya grafted onto Kaiser (AT-KA). A third treatment, corresponding to ungrafted Attiya (AT) plants, was used as a control. After callus formation, eight plants of each treatment were placed in a water culture system, containing 7.0 mM N-NO3, 3.0 mM K, 0.5 mM P, 2.0 mM Ca, 1.0 mM Mg and 1.0 mM S plus micronutrients. Plants were grown for 30 days under medium (400 µmol PAR m−2 s−1) or high (800 µmol PAR m−2 s−1) light intensity, using a 10 h photoperiod and 25/18 °C day/night air temperature. Within each light treatment, plants were arranged in a completely randomized design.
4.2. Growth Measurements and N Accumulation
At transplant, 10 plants of each treatment were harvested from the plastic trays, split into shoot and roots, and individually weighed. Later, plants were dried in an oven at 60 °C for 48 h to determine dry weight. The plants in the water culture were harvested 30 days after the experiment start and were weighed similarly as described before. Then, shoot and root relative growth rates (RGR, g g
−1 day
−1) were determined using the following equation:
where
DW1 and
DW2 represent dry weight, in g plant
−1, at time 1 and time 2, respectively, whereas
t corresponds to the growth period (30 days). After the dry weight was recorded, the N content in shoots and roots was determined by Kjeldhal distillation. Then, plant N uptake (PNU, mg N plant
−1) was determined as follows:
where
DWs and
DWR represent shoot and root dry weight (g plant
−1), respectively, while
NS and
NR are the shoot and root N content (%), respectively. Subscripts
1 and
2 denote the time of measurement.
4.3. Gene Expression
At harvest, four biological root samples from four different plants of each treatment were collected. Samples were immediately frozen at ‒80 °C until RNA extraction. Total RNA was extracted from 100 mg samples using RNA-Solv reagent and RNA quality was tested using electrophoresis in a 2% agarose gel stained with GelRed. Ribonucleic acid quantification was determined by spectrophotometry (model NanoDrop 2000, Thermo Scientific, Waltham, MA, USA). First strand cDNA synthesis was obtained from 1 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random primers. Real-Time PCR reactions were carried out using four biological and two technical replicates for each target gene (
LeNRT1.1,
LeNRT1.2,
LeNRT2.1,
LeNRT2.2 and
LeNRT2.3). The PCR reactions were performed using gene-specific primers [
31] (
Table 3) and PowerUp SYBRgreen master mix (Applied Biosystems, Foster City, CA, USA). The reaction was run through a Real-Time PCR System (model StepOne, Thermo Fisher, Waltham, MA, USA) programmed at 94 °C for 1 min, followed by 35 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. Relative quantification was determined using α-tubulin as a reference gene [
35].
4.4. Enzyme Activity
Nitrate reductase activity was determined in four biological leaf and root samples collected at harvest, according to the methodology described by Kaiser and Lewis [
36] and modified by Reguera et al. [
37]. One milliliter of extraction buffer (50 mM KH
2PO
4-KOH, pH 7.5, 2 mM EDTA, 2 mM dithiothreitol and 1% polyvinylpolypyrrolidone) was added to 0.1 g of frozen tissue and extracts were centrifuged at 20,000 g for 20 min at 4 °C. Then, 700 µL of reaction buffer (50 mM KH
2PO
4-KOH, pH 7.5, 10 mM KNO
3 and 0.1 mM NADH) were added to 100 µL of total soluble proteins, and samples were incubated at 28 °C for 15 min. The reaction was stopped with the addition of 1 mL 1% sulphanilamide in 1.5 M HCl and 1 mL 0.02% n-l-naphtyl-ethylenediamine dihydrochloride. After 30 min, samples were centrifuged at 500 g for 5 min to remove suspended matter and nitrite was determined by absorbance at 540 nm in a spectrophotometer (model BioTek Power Wave HT, Shimadzu, Tokyo, Japan). Duplicate aliquots of extract from each sample replicate were assayed. Protein content was determined by Bradford’s assay (Coomassie Plus kit, Thermo Fisher, Waltham, MA, USA).
4.5. NO3− Uptake Kinetic Parameters
Root NO3− uptake kinetic parameters (Vmax and Km) were determined in a set of depletion experiments. Eight plants of each treatment were placed individually in 1-L containers and randomly arranged within each of the light conditions described above. Plants were allowed to grow for 30 days as in the experiment described above, and then roots were exposed consecutively to nutrient solutions containing 0.25, 0.5, 0.75, 1.0, 2.0, and 4.0 mM of NO3−. Five-milliliter samples were collected every 15 min in a six-hour period, and NO3− content was determined by ion chromatography (model Dionex Aquion, Thermo Scientific) equipped with an anion pre-column (Dionex IonPac AG11-HC, 4 mm) and a separator column (Dionex IonPac AS11-HC, 4 mm) coupled with a self-regenerating suppressor (AERS 500, 4 mm). The eluent (30 mM KOH) was injected at a flow rate of 1 mL min−1. Nitrate uptake (U) was calculated as the difference between NO3− content in the sample at the beginning of the experiment minus the content at the time when the concentration reached a steady state (usually between 120 and 240 min). The influx data were fitted to the Michaelis‒Menten relation (Equation (1)). Both U and Vmax are expressed in mmol NO3− g−1 DW h−1, while Km and C are in mM.
4.6. Root Respiration
In a separate set of plants, root respiration was determined using an open gas-exchange system. Four plants of each treatment were placed individually in 1-L plastic containers and randomly distributed in one of the light conditions described for the experiments detailed before. Each container was connected to a pump that continuously sprayed a nutrient solution onto the roots from a reservoir. The height of the solution within the containers was kept as low as possible by placing a drainage tube that lets out into the reservoir (
Figure 6). The composition of the nutrient solution was similar to that described for the previous experiments but at a 50% dilution. A small hole was drilled in the container’s lid to allow plant suspension into the container. All connections and the lid of the container were sealed to avoid air leaks. Air was injected into each container at a 200 mL min
−1 rate, and the CO
2 concentration was monitored for two consecutive days from one hour before lights were turned on until one hour after lights were turned off (a 12-h period). This was repeated with four different sets of plants to build a dataset composed of eight replicates per treatment at each hour of measurement. The concentration of CO
2 in the air was determined using a CO
2 sensor (model QS151, Qubit) and root respiration rates were calculated as the difference between the CO
2 content measured after passing through the container minus the content measured prior to entering the container. Results are expressed on a dry weight basis.
4.7. Statistical Analysis
Differences in growth rate, N content, gene expression, NRA, as well as daily and hourly root respiration rates were analyzed by ANOVA with mean separation by Tukey´s test. Kinetic parameters (
Vmax and
Km) were estimated by non-linear regression analysis fitting a Michaelis‒Menten curve to the influx and concentration data. Estimated parameters were then analyzed by ANOVA to test the differences between treatments and light intensity. All analyses were conducted using R statistical software [
38] through the InfoStat console [
39].
