Tolerance to Nitrogen ions enrichment in a Gulf of Mexico's freshwater submerged grass

Tolerance to the enrichment of ionic nitrogen (N) with ammonium (NH 4 ) and nitrate to be distinguished in a submerged grass ecotype-species of freshwater wetlands. Concentrations of total Nitrogen (TN: 500 to 2000 µgL -1 ) and three N sources (NS: NH 4 , NO 3 , 1:1 NH 4 :NO 3 ratio) in an overlaying aqueous phase, using local tap water as control (174 µgL -1 TN), were evaluated in rooted autotrophic juvenile Vallisneria americana established in vitro in two-phase culture medium. The treatments ranged from 7 to 111 µM TN. Phenotypes changes, pH and ionic strength in the aqueous phase were registered at 15 days. In addition, biomass of lyophilized leaf and root as well as N contents (gas and high-performance liquid chromatography) were analysed to estimate accumulations of N-NH 4 , N-NO 3 and N-NO 2 . accumulations The results provide direct evidence of the tolerance to N enrichment with three different N sources in the focal ecotype at the seedling stage, based mainly on its cumulative capacity of N-NO 3 and N-NO 2 under eutrophic conditions. The findings imply that NO 2 is a relevant ion in the tolerance to N enrichment in overlaying water with NH 4 and NO 3 ions. Future studies of N cumulative effect with an in vitro approximation will be to support stress predictions by N enrichment.

approximation. Under the overall premise that the tolerance of submerged grasses varies ontogenetically [5,13]. The hypothesis being tested is that tolerance by N-centred eutrophication in the juvenile stages of V. americana species is expected to be similar with the three sources of N. This response implies increasing their survival by assimilating N in environments subject to gradual enrichment of N. The study offers knowledge about the local species tolerance due the N enrichment by its implications for the resettlement of freshwater wetlands of the Gulf of Mexico.

Results
All seedling of Vallisneria americana, including those of the control, survived; showing homogeneous phenotype of leaves totally green and abundant root (Additional file 1: Fig. S1). Variations of total biomass, likewise pH and ionic strength were insignificant ( N-NH 4 contents were negligible, due to the low chromatographic detection limit. Hence, TN content (N-NO 3 plus N-NO 2 ) was of 42.99 µgg − 1 . N accumulation was similar in leaf (df = 50, t = 0.23, p > 0.05) and root (df = 50, t = 0.02, p > 0.05), ranging from 2.2 to 3.5 µmolg − 1 of N endogenous. Direct effect of N source and N concentration influenced the cumulative capacity of N ions in both tissues; expressly, NO 3 and NO 2 accumulations were highly significant (Table 1, Additional file 2: Table S1).
Leaf and root accumulated low N-NO 3 and N-NO 2 with NH 4 sources, but showed high accumulation with the NO 3 source. Regarding to NH 4 :NO 3 , the accumulations of N-NO 3 and N-NO 2 was high in leaf and low in root (Fig. 1). Conversely, N accumulations with NH 4 :NO 3 source were inverted to those of the NO 3 , and similar only in N-NO 2 of the root with NH 4 . Cumulative differences of N between concentrations of TN are indicated in the same Fig. 1. At 500 µgL − 1 TN, N-NO 3 accumulation in root was doubled; whilst the rest of the concentrations maintained accumulations close to each other in both biomass. Similarly, N-NO 2 accumulation is highlighted in the first N concentration; in the root this was quadrupled with NO 3 source and doubled with NH 4 :NO 3 . Under the interactive effect, N source and N concentration on the cumulative capacity of N-NO 3 and N-NO 2 were significant in root and leaf (Table 1, Additional file 2: Table S1); the results are explained through the significant variation between treatments, in the same tables. In general, N accumulation curves showed a negative trend to the increment of micromolar, but gradient depended on the N concentration of treatments (Fig. 2). In the two tissues, all the revealed N accumulations were identified in the first concentrations of NO 3 , NH 4  In the leaf, the maximum accumulation was observed at 18 µM, followed by 8 and 32 µM that formed the consecutive statistic level (Fig. 2). The order of descent of this ion was irregular with respect to NO 3 ion.

Discussion
Submerged grasses have been found sensitive to inorganic N excesses into the water column in short-time, under both natural and semi-controlled environmental conditions [4,14,28,29]. In this contribution, a controlled environment (i.e. in vitro) suggests that local ecotype Vallisneria americana at a juvenile age was tolerant to enrichments of NH 4 and NO 3 , including co-supply and within the meso-and eu-trophic range. The above supports the hypothesis that juvenile plants can tolerate very high ionized N concentration from any source of N [18,30,31], to the extent that under in vitro conditions, both variation phenotypic of V. americana and their total biomass showed similarities.
In addition, the in vitro approximation allows to control factors of the exogenous environment that can cause confounding effects in the results, like osmotic stress or pH abrupt changes.
Correspondingly, in a buffered medium the Arabidopsis species presented similar total biomass by different N ionic supplied sources (NH 4 vs. NO 3 ) related to the high adaptability of the non-aquatic plant [32]. The ability to tolerate high levels of NH 4 , and by extension NO 3 and NH 4 :NO 3 implies an efficient metabolic system to assimilate excess of N ions during the early development of American Wild Celery.
Both N ions (hereinafter any source of NH 4 and NO 3 ) caused maximum accumulations of NO 3 and NO 2 at 500 µgL − 1 TN ( Fig. 2) which correspond to the oligotrophic environmental limit, and were found to be similar to the control experimental of local tap water at the root with NO 3 . This maximum cumulative response coincides with N deficit in aquatic plants roots [33] to the extent of limiting the growth of aquatic grasses by N in the oligotrophic environment, supporting the need to develop an endogenous pool of N [29]. Under oligotrophy conditions, the mean of NO 3 accumulation registered in local Vallisneria seedling was approximate to those reported on submerged species (root plus leaf), such as Berula erecta, Potamogeton coloratus and Elodea canadensis; and lower compared to some emerging hydrophytes [34].
Toxidrome by NH 4 has resulted in freshwater plants [5]. In this contribution, the avoidance of a degradative phase during the process of N extraction in tissues [35,36] may be the explanation of tolerance to NH 4 , maintaining the concentration of free cytosolic NH 4 at sub-millimolar levels attributed to NH 4 detoxifying [5,23,37]. This analytical precaution was used in the preparation of the shoot and root of Oryza sativa (rice) juvenile plants, registering micromolar NH 4 accumulation [38] and showing the ability to assimilate this ion by means of the transaminases glutamate synthases ferredoxin dependents in leaf, and reduced nicotinamide adenine dinucleotide in root [39]. Although, submerged plants can present dual assimilation of NO 3 in leaf and NH 4 in root, they require less energy for uptake and assimilation NH 4 mainly because NO 3 has to be reduced prior to assimilation [30]. The previous may explain the accumulations of NO 2 in the leaf and root in the present study by NO 3 and NH 4 :NO 3 supplies. Similar to rice [40], NO 3 accumulations in the root, and NO 2 in the leaf, were found to have three times more NH 4 than NO 3 ; coinciding too with studies that have evaluated the transportation of these two ions in the roots of diverse plant species [41].
Nevertheless, the NO 2 accumulation, by NO 3 supplied, occurred with and without NH 4 . Furthermore, NO 2 is a product of nitrate reduction within the N assimilation pathway and its concentrations in the cytosol are maintained at a micromolar range to prevent NO 2 toxicity in the cell [42,43]. This ion in addition to being channelled through the ammonium assimilation pathway is capable of being reduced in nitric oxide, which is a by-product of hypoxic N metabolism in the root. The nitric oxide is recently recognized as important signalling molecule in plants, influencing their growth, development and responses to stress [44,45]. The experimental approximation of rice root against NH 4 and NO 3 enrichments in the early stages of water stress suggest its protective role in the antioxidant defence system, since nitric oxide can also serve as a source of reactive nitrogen species [46]. However, more research is required to explain NO 2 accumulations to NH 4 regimens to detail their metabolic function.
Nonetheless, it is possible that the high accumulation of NO 2 in Vallisneria americana associated to a deficit of N, NO 3 in the root and NH 4 :NO 3 in the leaf, has been a metabolic signal [47]. In accordance to the observed N accumulations curves in our study, it was evident that the cumulative capacity NO 3 and NO 2 in seedlings were independent of the N source. On the contrary, showed a relationship with the N atoms concentration present in the external solution. The above alludes to the utility of revealing the cumulative effect of N according to the stoichiometric homeostasis extended to include other bioelements [48]. Additionally, stress by NO 3 has been pronounced under N enrichment at high relations N/P provoking inhibition of foliar growth [12].
Under In vitro plant culture The procedure of in vitro culture requires the asepsis of seeds and preparation of two-phase medium [12] and regeneration of the seedlings (complete and functional) in a controlled environment. The two phases of the culture medium were supplemented with synthetic freshwater (SF) at pH 7.5 [52] dissolved in deionized water (grade HPLC). Support phase was prepared with 20 ml of 0.4% agar-agar, while the overlaying phase contained either 40 or 60 ml. First, each phase was sterilized in glass containers (5 cm Ø x 7 cm height) with Magenta® covers. Asepsis was realized with 10% bleach (Cloralex®, México) for 10 min, followed by three washes with water sterile type 2 pure Mexican Standards. Before cultivating the seeds, the culture container was prepared with the overlaying phase, SF for local tap water, slowly poured over the support phase containers, using the horizontal laminar flow cabinet (Veco, E-5750). Between 40 and 50 seeds were cultivated for culture unit.  [52]. The analysis of these five parameter was performed according to standard methods (Additional file 3: Table S2).
Environmental conditions were the same as those of the regeneration stage, except for lighting which changed to 20 µmolm − 2 s − 1 in static non-renewal tests, over 15 days.
At the end, total dry biomass and N accumulations in root and leaf tissues (n = 26 each tissue) were registered. Previously, phenotypic stress was analysed in each plant with qualitative markers (leaf colour: no colour, partially green and totally green and root proliferation: null, scarce, abundant), as well as pH (Corning 240) and either conductivity or ionic strength (Hanna HI 8033) on the spent aqueous medium (n = 5).

Processing of samples and analytical
The plants from the control and treatments were removed during the support phase and were cut with scissors flush with the gelled surface to separate the leaf biomass (including dry coleoptile) and root biomass. After, each one was manipulated over a glass plate, hydrated with three drops of the aqueous medium of origin. The seeds or shells and gel were removed with a metallic rod without damaging the root. Aqueous phase was drained from the samples in the sloped glass plate for 1 min and the rest was adsorbed with a soft paper for 10 secs. The sample was frozen in liquid N 2 for 30 min, lyophilized at -40° C and 133 × 10 − 3 mBar (Labconco 6 L Benchtop Freeze Dry System), ground in a mortar and preserved in a desiccator. Each sample was weighed before and after lyophilizing on a four-decimal place gram balance (Ohaus Explorer Balance). Samples were then reweighed to obtain the biomass in dry weight (D.W.). Extractions of three N inorganic ions, N-NH 4, N-NO 3 y N-NO 2 in tissue samples were carried out in mortar with methanol (HPLC grade) at 60% dissolved in deionized water. This extraction medium was used to avoid the degradation of N metabolites during the extractive procedure [35,36]. Biomass to extractive solution was 1 mgml − 1 .
Methanolic extract was sonicated for 15 min and the supernatant was filtered in paper Whatman # 1.
In the three N determinations the same methanolic extract was used.
The N-NH 4  accumulation (µmolg − 1 ) of leaf and root by ion (N-NH 4 , N-NO 3 and N-NO 2 ) was calculated using N contents (µgg − 1 ) [34]. The N accumulation curves were obtained with each ion by tissue (N endogenous, µmolg − 1 ) versus increment of concentrations of the N source supplied (N exogenous, µM).

Statistical analysis
The data analysis software system was Statistica V8 (Stat Soft, Inc. Availability of data and materials.
The datasets generated and/or analysed during the current study are available in Ruiz-Carrera, Violeta. Tolerance to Nitrogen ions enrichment in a Gulf of Mexico's freshwater submerged grass.

Competing interests
The authors declare that they have no competing interests

Supplementary Files
This is a list of supplementary files associated with this preprint. Click to download.  Table S1.docx Table S2.docx