Soluble fractions and kinetics parameters of nitrate and ammonium uptake in sunflower ( “ Neon ” Hybrid ) 1

Sunflower plants are source of oilseeds with great importance for agroenergy. Nitrogen deficiency is considered one of the factors limiting growth and yield. The objective of this study was to assess the nitrogen uptake (high and low supply of nitrate and ammonium) and to determine the uptake kinetics parameters (Km and Vmax) of these ions in sunflower plants (Neon hybrid). The experiment was carried out in a growth chamber using a hydroponic system. The following treatments were applied: 0.2 and 2.0 mmol L -1 of N-NO3 and N-NH4 +. The two kinetics parameters (Km and Vmax) involved in the uptake of nitrate and ammonium were determined, as well as the soluble fractions (N-NO3 -, N-NH4 +, amino-N and sugars) and total N, in different plant tissues. The plants grown with ammonium showed higher Vmax than those cultivated with nitrate. High ammonium rates caused reduction of root volume and the plants supplied with 2.0 mmol L-1 of N-NO3 produced more biomass from the roots and aerial part. The highest concentrations of NNO3 were found in the petioles. Analyzed together, the data suggest a greater potential for use of nitric-N as a nitrogen source for fertilization in the cultivation of sunflower.


INTRODUCTION
The sunflower (Helianthus annuus L.) belongs to the family Asteraceae.It is one of the main plant species in the world for production of vegetable oil (FERNÁNDEZ-MARTÍNEZ; PÉREZ-VICH; VELASCO, 2009).Nitrogen (N) deficiency is one of the main factors limiting the growth and development of plants.For the sunflower, this nutrient is crucial for production of seeds and oil.Various studies have demonstrated that the oil yield can increase or decrease according to the N dose applied.Zagonel and Mundstock (1991) found that the cultivars tested responded differently to nitrogen fertilization: for the Contisol 711 cultivar, the oil yield decreased at the highest N rate applied (120 kg ha -1 ), while for the DK180 cultivar the effect was the opposite.Rocha (2013) verified that at low N doses (from 30 to 50 kg ha -1 ), the BRS324 genotype presented greater oil production, but this declined with higher doses.
Nitrogen can be found in various forms in the soil, but the main forms absorbed by plant roots are nitrate (NO 3 -) and ammonium (NH 4 + ) (MARSCHNER, 2012).The transport of these ions into the cells is carried out by two types of systems, the High Affinity Transport System (HATS), which operates at low nutrient concentrations, and the Low Affinity Transport System (LATS), which operates at concentrations higher than 1 mmol L -1 .The high affinity transport system is divided into two subsystems, constitutive (cHATS) and induced (iHATS), activated by low external nutrient concentrations (ASLAM; TRAVIS; HUFFAKER, 1993;OKAMOTO et al., 2006;SIDDIQI et al., 1990).The study of the nutrient absorption kinetics aims to elucidate the roles of these transport systems in the uptake of nutrients by plants.For this purpose, two parameters are used, V max , which describes the maximum absorption velocity, i.e., when all the transporter sites are loaded; and K m , which describes the affinity of the carrier protein for the nutrient.
Sunflower genotypes can respond differently to nitrogen fertilization (ROCHA, 2013;ZAGONEL;MUNDSTOCK, 1991), so it is important to perform studies to characterize the particularities of the N uptake system of the different genotypes used to produce seeds and oil.Therefore, the objective of this study was to assess the behavior of the "Neon" sunflower hybrid regarding nitrogen uptake under high and low supply of nitrate and ammonium and to determine the absorption kinetics parameters of these nutrients (K m and V max ).

MATERIAL AND METHODS
The experiment was conducted in a hydroponic system in a growth chamber of the Soils Department of Universidade Federal Rural do Rio de Janeiro, located in the city of Seropédica.The plants were submitted to a photoperiod of 14h/10h (light/dark) with luminosity of 500 µmol m -2 s -1 (photosynthetic photon flux), relative air humidity of 50% and temperature of 28 ºC in the daytime and 26 ºC at night.Sunflower seeds of the Neon hybrid were germinated in washed and autoclaved sand.Ten days after germination, the plantlets were transferred to pots with capacity of 3.0 L (two plantlets per pot) and were supplied with 2.8 L of nutritive solution formulated according to Hoagland and Arnon (1950) at ¼ of the ionic strength, modified with 1.0 mmol L -1 of N-NO 3 -and 0.5 mmol L -1 of N-NH 4 + .Three days later, the plants were transferred to a solution at ½ ionic strength, with the solution exchanged every three days and maintenance of the pH at 5.8.Thirty days after germination, the plants received a modified Hoagland-Arnon solution without N for 72 hours.After this period, the treatments were applied, consisting of: 0.2 or 2.0 mmol L -1 of N-NO 3 -and 0.2 or 2.0 mmol L -1 of N-NH 4 + .During five hours, samples of the nutritive solution were collected every 30 minutes from all the treatments, after which the samples were collected every 60 minutes until exhaustion either of the N-NO 3 -or N-NH 4 + from the solution.In the treatments with 0.2 mmol L -1 of NO 3 or NH 4 + , 22 collections were performed, while in the treatments with 2.0 mmol L -1 of NO 3 -or NH 4 + there were 29 collections.At every collection an aliquot of 1 mL of the solution was taken to measure the concentrations of N-NH 4 + (FELKER, 1977) and N-NO 3 - (CATALDO et al., 1975) and Miranda, Espey and Wink (2001) with modification: An aliquot of 30 µL of sample was mixed with 60 µL of Griess reagent (1:1:2), followed by incubation at 40 ºC for 50 minutes.After the addition of 110 µL ultrapure water (MilliQ), the spectrophotometric reading was taken at 540 nm using KNO 3 (10,25,50,100,150,200 and 250 mmol L -1 NO 3 -) as standard.Readings were taken on microplate spectrophotometer (ThermoFisher Scientific) using 96-well microplates.The results were used to construct depletion curves of these ions during the period analyzed.
The plants were then harvested and separated into leaf blades, petiole, stem and root, and the fresh mass and root volume were determined.Samples of fresh material of each plant part, weighing 0.5 g, were separated and stored in ethanol (80%), and after the alcoholic extraction, chloroform partitioning was carried out.The extracts obtained were analyzed to measure the following soluble fractions: free amino-N (YEMM; COCKING, 1955) Soluble fractions and kinetics parameters of nitrate and ammonium uptake in sunflower ("Neon" Hybrid) WILLIS, 1954).The remaining plant material was dried, ground and used to measure the total nitrogen content (TEDESCO et al., 1995).

RESULTS AND DISCUSSION
When the plants were submitted to 0.2 mmol L -1 of NO 3 -, the pH declined over time (Figure 1A), but with 2.0 mmol L -1 the pH increased until about 26 hours after application of the treatment, after which it gradually fell (Figure 1B).The ammonium nutrition promoted a gradual increase in the acidity of the nutritive solution in both treatments (Figures 1A and 1B).Increased acidity of the solution was also observed by (SILVA; COUTO; SANTOS, 2010), who reported a reduction of the nutritive solution's pH with time when studying sunflower nutrition.) of N assimilated in the cytosol, or it can be an indication that this genotype contains a larger number of NH 4 + carriers in the roots (Table 1).
The plants cultivated with 0.2 mmol L -1 of NO 3 -or NH 4 + presented similar root volumes.However, the root volume of the plants supplied with 2.0 mmol L -1 of NO 3 -was greater than that of the plants given the same concentration of N-NO 3 -(Table 2).Since NH 4 + is basically assimilated in the roots, the plants use insoluble carbohydrates from the roots, causing a reduction in the root system's biomass (BORGES et al., 2009).Cometti et al. (2004), working with lettuce plants grown hydroponically, also found lower biomass of the roots with supply of ammonium nutrition.The root/aerial part ratio reflects the plant's capacity to develop the root system in relation to the aerial part.In this respect, the plants supplied with a high ammonium dose presented a lower root/aerial part ratio, a finding that corroborates the observation of Rocha (2013).The architecture and morphology of the root system is influenced by the forms of available N, and L. S. Alves et al.
nitrate promotes elongation of the roots, while ammonium mainly stimulates the emergence of lateral roots (XU; FAN; MILLER, 2012).
In comparing the treatments with high dose of N, the plants grown with NH 4 + presented a higher V max value (Table 1) and also a lower root/aerial part ratio (Table 3), suggesting the existence of more sites for uptake of this ion.
The application of the highest N-NO 3 -rate (2.0 mmol L -1 ) resulted in a significant increase in the fresh mass yield of the leaves when compared with the lowest dose (Table 3).This difference can be explained by the fact that with low supply of N-NO 3 -, there is low utilization of carbon skeletons (C), directly affecting the production of biomass.Brito et al. (2013), working with crambe (Abyssinian kale) in a hydroponic system, also observed this increase in biomass of the leaves when the plants were supplied with 2.0 mmol L -1 of N-NO 3 -.
In the treatments with low dose, from both sources, there was no significant difference in biomass of the different plant compartments (Table 3).These results indicate there is no source preference when the N supply is low.In general in relation to the treatments, the plants preferentially accumulated N in the leaves (Table 4).

Analyzed together, the data on root volume and fresh mass accumulation indicate that resupply
The treatment with 2.0 mmol L -1 of NO 3 -led to the highest N concentrations in all four plant parts (Table 3), possibly because of the greater biomass yield, as shown by the values of root volume (Table 2) and fresh mass (Table 3), demonstrating better development when submitted to this treatment.
Higher concentrations of N-NO 3 -were found in the petiole when the plants were submitted to the treatment with the lowest nutrient level, with both N sources, suggesting that the petiole is a preferential organ for storing N-NO 3 -(Figure 2a).Rocha et al. (2014), also studying sunflower plants in a hydroponic system, found the stem to be the compartment with highest storage of NO 3 -.However, in that study the author did not analyze the quantity of the nutrient in the petioles.Santos et al. (2009) concluded that in rice, flag leaf and leaf 2 act as preferential sites of N storage.Concentration analysis of N-NO 3 -per pot (Figure 3a, 4b) showed a reduction of the dilution effect and allows stating that the petiole really was the main organ for storing N, for both forms of the nutrient, when supplied at a dose of 0.2 mmol L -1 .Thus, petiole seems act as reserve organ for sunflower plant, suggesting greater potential for translocation of N stored in these tissues.
Soluble fractions and kinetics parameters of nitrate and ammonium uptake in sunflower ("Neon" Hybrid) According Crawford and Glass et al. (1998), excess of nitrate uptake by the plant can be stored in the vacuole serving as mineral nitrogen source when the supply from the soil becomes.In maize, the yield increase in genotypes was due to their ability to accumulate NO 3 -in their leaves during vegetative growth and its efficient remobilization during the grain filling (HIREL et al., 2001).
In the plants provided with resupply of the highest NO 3 -rate, the greatest level of this nutrient was found in the roots, while at the highest dose of NH 4 + the greatest concentration was in the petiole (Figure 2b).
In the plants grown with NH 4 + as the only Nsource, in both treatments, concentrations of NO 3 -were surprisingly found in the tissues (Figures 2a,2b,3a,3b), possibly due to the fact that plants were grown with NO 3 and NH 4 + before the N deprivation step.Therefore, the time during which the plants were deprived of N may not have been long enough for complete exhaustion of the NO 3 -from the "reserve pool" stored in the vacuoles for subsequent remobilization.
In the treatments with 0.2 mmol L -1 of NO 3 -and NH 4 + , concentrations of N-NH 4 + were only found in the roots of the plants cultivated with com N-NO 3 -(Figure 2c), because at a low dose of N-NH 4 + , the nutrient taken up by the roots was quickly assimilated.On the other hand, the appearance of concentrations of N-NH 4 + only in the roots (Figures 2c and 2d) can also be explained by the greater capacity to assimilate this ion in the aerial part in function of the greater availability of carbon skeletons (Figures 2g and 2h) from photosynthesis.
The resupply with 2 mmol L -1 of NO 3 -and NH 4 + caused accumulation of N-NH 4 + in the roots, although  2d and 3d).The assimilation of NH 4 + occurs basically in the roots, due to its toxicity to the plant, so the nutrient tends to be quickly assimilated (SOUZA;FERNANDES, 2006).
When plants were submitted to high doses of both NO 3 -and NH 4 + , there was an increase of N-amino levels in the tissues, except for the petioles, in which the concentration declined (Figure 2f).The fact that the petioles presented lower concentrations of N-amino can indicate lower assimilation of the nutrient in this compartment (Figures 2f, 3e and 3f).It was observed that when there is NO 3 -reduction or remobilization from roots or shoots occurs free amino-N production in such compartments (Figure 3).The same was observed by Santos et al. (2007) in rice plants grown under nitrate fertilizer.
The treatment with the high dose of NH 4 + compared with the high dose of NO 3 -produced a greater concentration of N-amino in the roots (Figure 2f).This can be explained by the fact that NH 4 + needs to be rapidly assimilated, which occurs preferentially in the roots.Sperandio et al. (2011) also observed a greater concentration of N-amino in the roots of rice plants grown with NH 4 + compared to plants supplied with NO 3 -.
The stem was the compartment containing the highest levels of soluble sugars, followed by the petiole, in all the treatments (Figures 2g and 2h).These results indicate that the Neon hybrid might have better potential for storing N in these compartments.In analyzing the concentration of soluble sugars, the stem stood out from the other compartments by presenting , after N deprivation for 72 h.The bars represent the mean standard error of the repetitions higher values, so this region appears to be the main one for accumulation of sugars (Figures 3g and 3h).Rocha et al. (2014) also found the stem to be the place of greatest accumulation of sugars in the sunflower BRS324 variety cultivated in a hydroponic system.

CONCLUSIONS
1.The Neon sunflower genotype, when grown with N-NH 4 + , presented higher V max in comparison with the plants supplied with N-NO 3 -; 2. Although presenting the lowest V max value, the plants supplied with 2.0 mmol L -1 of N-NO 3 -presented higher biomass than those supplied with the same dose of N-NH 4 + , which can indicate a greater potential for use of nitric N; 3.There was no difference in accumulation of biomass in the plants grown with the lowest N dose (0.2 mmol L - 1 ), suggesting that in the conditions of this experiment, there is no preference for the N source; 4. The petiole is the preferential organ for accumulation of N-NO 3 -.

Figure 2 -Figure 3 -
Figure 2 -Concentrations of N-NO 3 -(a, b), N-NH 4 + (c, d), free N-amino (e, f) and soluble sugars (g, h) in different parts of sunflower plants of the Neon hybrid submitted to resupply of 0.2 and 2.0 mmol L -1 of NO 3 -or NH 4 + , after N deprivation for 72 h.The bars represent the mean standard error of the repetitions

Table 1 -
Kinetics parameters of Neon hybrid sunflower plants grown with different concentrations (0.2 and 2.0 mmol L -1 ) and sources *Equal letters in the line do not differ between each other at 5% probability by the Tukey test

Table 2 -
Root system volume of Neon hybrid sunflower plants submitted to resupply of 0.2 and 2.0 mmol L -1 of NO 3 *Equal letters in the line do not differ between each other at 5% probability by the Tukey test

Table 3 -
Fresh Equal letters in the column do not differ between each other at 5% probability by the Tukey test *

Table 4 -
Total N and N content in the leaves, petioles, stems and roots of Neon hybrid sunflower plants Equal letters lowercase in the column and uppercase letter in the line do not differ between each other at 5% probability by the Tukey test the same was not observed in the aerial parts (Figures *