1. Introduction
Among the varieties belonging to
Zea mays L., popcorn (
Zea mays L. var. everta) occupies a prominent position due to containing 583 Kcal/100 g of energy intake and 49% saturated fats, 45% carbohydrates and 7% proteins, while the average price charged by the bag is three times higher than common corn [
1,
2]. From an agronomic and economic point of view, the primary purpose of crop improvement over the last century has been yield [
3]. The enhancing in the crop yield was because of the plant breeding techniques and extensive use of fertilizers. Among these fertilizers, nitrogen (N) is a major factor in agricultural production [
4,
5].
Nitrogen is important for life on the planet, and it is the most essential nutrient for obtaining high agricultural production. However, nitrogen fertilizers applied in agriculture are not used efficiently by high-yield crops, such as wheat, maize and rice, in which only 33% of the applied N is used by the plant [
6]. The use of N leads to the release of nitrous oxide with a global warming potential of 296 times greater than the CO
2 molecule. The damaged caused by excess use of N in Europe was found to be 91–466 billion US dollars annually, hence, reducing N use in agriculture is a big challenge. Approximately 2.3 billion US dollars could be saved in annual costs of N fertilizer by improving only 1% in N uptake efficiency [
7,
8].
Demand for the most efficient cultivar in the use of N is desirable and might be possible through the selection of superior genotypes [
6,
9,
10,
11]. The idea is to develop N efficient maize cultivars that produce more or the same amount of grains, with less demand for N, and therefore, with lower production costs and environmental impact [
12,
13]. However, the nutrient use efficiency of popcorn is largely unknown [
14]. Exploring the genetic variation for nutrient efficiency should be useful to developed cultivars of popcorn with high N use efficiency (NUE). This can lead to a higher yield, and consequently, reduce the demand for fertilizers [
6].
The background on NUE and its components of uptake and utilization based on grain weight was first proposed by Moll et al. [
15], who showed that NUE is the ratio of grain weight to N available in the soil or the product of N uptake efficiency (NUpE: the ratio of total N in the plant to N available in the soil) and N utilization efficiency (NUtE: the ratio of grain weight to total N in the plant). Then, Good et al. [
16] described, based on the dry weight, that NUE is the ratio of shoot dry weight to N applied, NUpE (ratio of N in the plant to N applied) and NUtE (ratio of shoot dry weight to N in the plant). However, until now, it has been uncertain whether to include the root N content (RNC) or not while measuring the uptake and utilization efficiency, because most of the researchers dealing with these aspects have different conducting methods to include only the shoot N content (N content of leaf and stem) [
17,
18] or to include the total N content in the plant (N content of shoot and root) [
11,
15,
19].
Previously, Santos et al. [
20] evaluated 29 popcorn inbred lines from the germplasm bank of Popcorn Research Breeding Program of Darcy Ribeiro North Fluminense State University (UENF) and performed a field experiment for two different N level at two different locations. In the experiment, based on grain yield, 12, 12 and 5 inbred lines were found to be efficient and responsive, inefficient and non-responsive and intermediate efficient and responsive for N use, respectively. In the current study, two of the most contrasting popcorn inbred lines and their F
1 hybrid were evaluated under two different pot experiment and harvested at two different plant vegetative stages, with the following objectives: (i) To evaluate the performance of these genotypes for root, shoot dry weight and N content. (ii) To provide the most reliable and specific information about the N use, uptake and utilization efficiency (with the inclusion and exclusion of root N content) based on the dry weight. (iii) To find the contrasting N levels and vegetative stage that effect NUE for further comparative proteomics and molecular study. (iv) To understand the relationship between the traits related to NUE. (v) To evaluate the perspective of genetic gains for NUE by the estimate of genetic parameters.
3. Discussions
One of the primary concerns of plant breeders and agronomists is to investigate the effectiveness of plants under biotic and abiotic stress conditions [
23]. Thus, for this purpose, popcorn genotypes were grown under different N levels and harvested at different vegetative stages. We found, that at both of the stages (V6 and VT), the F
1 hybrid and inbred line P2 were performing better than inbred line L80 for the SDW, RDW and TDW (
Table 2 and
Table 3). It is interesting to mention that at both of the stages L80 showed more percentage of change for SNC and TNC from N10% to N100% compared to P2 and hybrid but insignificant change for the SDW, RDW and TDW, which is clear evidence that L80 does not utilize the available N well, and comparatively demonstrated less and insignificant changes for the shoot, root and total dry weight (
Table 2 and
Table 3). The N content in the shoot or root directly impacts the uptake and inversely impacts the utilization efficiency [
17,
18]. From
Table 1, it was detected that with the increasing N supply, the SNC and RNC increased but the NUpE-wR and NUpE-w/oR decreased instead of increased (
Table 5) because the amount of N content found in shoot and root was much less than total N supply or in other words; there was a big gap between the N absorption and supply, which was also evidenced by Ciampitti et al. [
24].
It is also noteworthy that at the VT stage the hybrid showed 39.70% and 10.50% of the decrease in the RDW and TDW from N50% to N100%, respectively, which was also documented by Torres et al. [
25]. This decrease may be because of heterotic effect and also in low N (N50%) supply, the plants can increase root depth, cortical cell size [
26] and root cortical aerenchyma [
27,
28], which ultimately affect the RDW. It is noteworthy that under N100% the means of L80, P2 and hybrid are alike for the RDW and RNC but not for the SNC and SDW. Hence, it evidenced that at the VT stage, P2 and hybrid translocate and utilized N efficiently than L80 (
Table 3).
At the V6 stage, the hybrid showed high performance than P2 and L80, and P2 was found better than L80 for the NUE, NUpE-wR and NUpE-w/oR. This performance was perceived clearly under low N supply of N10% and N25% than high N supply of N75% and N100% (
Figure 3), which is because of the increasing N level [
29]. At a high concentration of N, all the genotype use and absorb N mostly the same, as evidenced by Mundim et al. [
11]. With an increasing supply of N application, the mean values of the NUE, NUpE-wR and NUpE-w/oR were decreased for the genotype as follows N10% > N25% > N50% > N75% > N100%. This reduction was associated with the ratio of SDW to N content in the plant and shoot, respectively, and because of the 25% increase of N applied in each level (
Table 2). Fu et al. [
30] also stated a 29% decrease for the NUE while studying the effect of five different N rate on sweet-waxy maize in a greenhouse experiment.
The significant differences among the genotypes for the NUtE-wR and non-significant for NUtE-w/oR at the V6 stage means (
Table 4) that it is critical to include the root N content (N in the plant) while measuring the utilization efficiency because at the early stages of the plant; the root is also a part of the N content in plants. Additionally, the shoot N converted to biomass was equally utilized by the genotypes in the provided N levels [
18]. Kant et al. [
8] reported that at the early vegetative stage, leaves and roots operate like a sink for the N uptake and utilization. The product of N uptake and utilization efficiency has a direct measure of the NUE [
11,
15,
16]; however, at the same time, if we are considering NUpE-w/oR and NUtE-w/oR [
19], we are ignoring the N content of root, which is also a part of the plant. In the case of uptake or absorption, means the N content that is absorbed by the plant from the available or applied N in the soil or solution. In N utilization the plant utilizes the absorbed N for the root, stem, leaf, growth and grain production; thus, it is noteworthy that at the early stage (V6), measuring NUpE-wR and NUtE-wR [
16,
19] is a more dependable way of the uptake and utilization efficiency.
It is also worth mentioning that, at the V6 stage, the G × N interaction was found significant for both types of the N uptake efficiency (NUpE-wR and NUpE-w/oR); in contrast, at the VT stage, this G × N interaction was found insignificant (
Table 4), which demonstrated that at early stages of the plant (V6), the genotypes were more influenced by different N levels. At the early stages (up to V8), maize plants absorb more N than later stages, which is why fertilization mostly applied up to V6 and V8 stage of the plant [
18].
Between the uptake and utilization indices, the results of the genetic parameters revealed a greater genetic variation in the NUpE-wR and NUpE-w/oR measures compared to NUtE-wR and NUtE-w/oR for the two inbred lines. It is suggested that although there is a great contrast between the inbred lines in terms of performance at different N levels, allowing very high values of CVg, VI and H
2, the indices of utilization are less efficient to reveal these differences. On the other hands, the results demonstrate that there is a strong genetic effect on the traits of dry weight and N content, in all parts of the plants, and on the NUE, NUpE-wR and NUpE-w/oR indices (
Table 6).
From the principal component (PCA)
Figure 2A,B of the biplot, it was observed the traits related to nitrogen use, uptake and utilization efficiency were well discriminated in low N levels. Thus, the genotypes could be evaluated in low N level for finding high differences for the nitrogen use, uptake and utilization efficiency. In contrast, the dry weight and N content were well distinguished in high N levels. Hence, the genotypes could be assessed in high N level for obtaining high differences of dry weight and N content. Granato et al. [
31] reported that a low level of nitrogen and phosphorous provided more chances for genotype discrimination. Hence, genotype evaluations should be performed in low nutrient availability that will lead to a better assessment of genetic variability for nitrogen and phosphorous use. In contrast, the mean of shoot dry weight was decreased by 51% under low N compared to high N. Therefore, High N level was found to be more reliable for discriminating shoot dry weight. Adu et al. [
32] documented that low N environment could be regarded as the most discriminating environment for selecting low and high N efficient genotypes.
5. Conclusions
It was concluded that at both stages, the F1 hybrid and the inbred line P2 were found to be close and better than the inbred line L80 for N use, uptake, utilization, dry weight and N contents. Measuring NUE based on dry weight is mainly affected by shoot dry weight, uptake and utilization efficiency. In both of the stages, the leaf N content was found more than the root and stem N content, and hybrid mostly demonstrated high performance for all the studied traits compare to their parents. The traits related to nitrogen use, uptake and utilization efficiency were well discriminated in low N levels. Thus, the genotypes could be evaluated in low N level for finding high differences for the nitrogen use, uptake and utilization efficiency. In contrast, the dry weight and N content were well distinguished in high N levels. Hence, the genotypes could be assessed in high N level for obtaining high differences of dry weight and N content. Regarding the N uptake and utilization efficiency, the results further suggest, at the early stage (V6), root N content (RNC) should be considered while measuring the uptake and utilization efficiency, and the plants absorb more N at the V6 than VT stage. At the later stage (VT), RNC should not be considered for uptake and utilization efficiency. The highest values of coefficient of genotypic variation, variation index and heritability for the N use, uptake, shoot dry weight and N content at both of the stages could favor the achievement of the genetic gain in advanced segregating generations.