Supplementary calcium ameliorates ammonium toxicity by improving water status in agriculturally important species

Fertilization of plants with ammonium is highly desirable because it is less susceptible to leaching than nitrate, with associated reductions in environmental pollution and risk to human health; however, important agricultural species exhibit a reduction in growth when fertilized with high levels of ammonium. The present study proved that tolerance to ammonium can be increased in bell pepper plants when calcium is incorporated at higher concentrations; calcium should thus be integrated into crop management to enhance the tolerance of agricultural plants to excess ammonium.

a number of important agricultural species exhibit a reduction in growth when fertilized with NH 4 + , and increasing the tolerance to NH 4 + may be of importance for the establishment of sustainable agricultural systems. The present study explored the feasibility of using calcium (Ca) to increase the tolerance of bell pepper (Capsicum annuum) to NH 4 + fertilization. Although NH 4 + at proportions ≥25 % of total nitrogen (N) decreased leaf dry mass (DM), supplementary Ca ameliorated this decrease. Increasing NH 4 + resulted in decreased root hydraulic conductance (L o ) and root water content (RWC), suggesting that water uptake by roots was impaired. The NH 4 + -induced reductions in L o and RWC were mitigated by supplementary Ca. Ammonium induced increased damage to the cell membranes through lipid peroxidation, causing increased electrolyte leakage; Ca did not reduce lipid peroxidation and resulted in increased electrolyte leakage, suggesting that the beneficial effects of Ca on the tolerance to NH 4 + may be more of a reflection on its effect on the water status of the plant. Bell pepper plants that received NO 3 − -N had a low concentration of NH 4 + in the roots but a high concentration in the leaves, probably due to the high nitrate reductase activity observed. Ammonium nutrition depressed the uptake of potassium, Ca and magnesium, while increasing that of phosphorus. The results obtained in the present study indicate that NH 4 + caused growth reduction, nutrient imbalance, membrane integrity impairment, increased activity of antioxidant enzymes and affected water relations. Supplementary Ca partially restored growth of leaves by improving root L o and water relations, and our results suggest that it may be used as a tool to increase the tolerance to NH 4 + fertilization.

Introduction
Nitrate (NO 3 − ) and ammonium (NH 4 + ) are the predominant forms in which nitrogen (N), a critical nutrient for the growth of agricultural species (Berger et al. 2013), is absorbed; nonetheless, the growth of cultivated plants may be affected by the predominant form of N present in the soil or nutrient solution. The use of NH 4 + as the N source for crop production is particularly attractive when compared with NO 3 − as it is less susceptible to leaching from the soil profile (Ferná ndez- Escobar et al. 2004), potentially increasing N use efficiency and reducing environmental pollution and the risk of human diseases, such as methemoglobinemia, cancers of the digestive tract, thyroid problems and diabetes (Knobeloch et al. 2000). Economic issues must also be considered as the production of NH 4 + -based fertilizers is less expensive than that of NO 3 − -based fertilizers because a higher energy input is needed to oxidize NH 4 + into NO 3 − (Havlin et al. 2013).
Plants may also benefit from NH 4 + nutrition when provided at limited concentrations as its assimilation represents a lower energy cost due to its redox state, eliminating the requirement for reduction (Britto and Kronzucker 2002), which may result in increased plant growth and yield. However, high concentration of NH 4 + may be toxic to some agricultural species (Borgognone et al. 2013), mainly due to a depletion of carbon supply caused by damage to the chloroplast ultrastructure (Takács and Técsi 1992), decreased photosynthesis rate (Setién et al. 2013) and an energydemanding transmembrane NH 4 + cycle in order to maintain low intracellular NH 4 + concentrations (Britto et al. 2001).
Other plant responses to high concentration of NH 4 + include increased intracellular pH (Bittsánszky et al. 2015), damage to the cell membrane permeability (M'rah-Helali et al. 2010) and decrease in the uptake of essential cations such as potassium (K), calcium (Ca) and magnesium (Mg) (Hawkesford et al. 2012). Ammonium at high concentrations induce a reduction in K uptake in several species; however, enhancing the external supply of K has been proposed as a tool for the alleviation of NH 4 + toxicity ( The objective of the present study was to determine the effect of NH 4 + nutrition on growth, photosynthetic parameters, root hydraulic conductance (L o ), leaf water potential (c w ), antioxidant enzymes, nitrate reductase activity (NRA) and the nutrient status of Capsicum annuum cv. Darsena (bell pepper) plants in soilless culture, a species particularly sensitive to NH 4 + and widely cultivated in protected agriculture and soilless systems in the southern portion of North America, and to explore the feasibility of using Ca to increase the tolerance to high NH 4 + concentration.

Cultural conditions and plant material
The study was conducted under greenhouse conditions in Northeast México (25827 ′ LN, 101802 ′ LW, 1610 m above sea level); average minimum/maximum temperature and relative humidity for experiment duration were 28 8C/15 8C and 45 %/75 %, respectively, and average photosynthetically active radiation (PAR) measured at solar noon was 501 mmol m 22 s 21 . Thirty-three-day-old bell pepper plants (15 cm height) were transplanted to a 39-L rigid plastic container with a drainage hole for retrieval of the nutrient solution. The container was filled with horticultural grade perlite [33 % (v/v) water holding capacity, 64 % air-filled pore space and 0.25 g cm 23 apparent density], with each container being considered as an experimental unit.

Nutrient solutions
Nutrient solutions with 14 meq L 21 total N were prepared with distilled water and varying proportions of NH 4 + : 0 % (control), 25% and 50 %; the remaining N was applied as NO 3 − . Potassium, phosphorus (P), Ca, Mg and sulfate were supplied at 6, 1, 10, 4 and 4 meq L 21 , respectively. Nutrient solution with 50 % of N as NH 4 + was prepared with two concentrations of Ca: 9 and 14 meq L 21 . The pH of the nutrient solutions ranged from 5.8 to 6.0 and electrical conductivity (EC) from 2.0 to 2.4 dS m 21 . Fertigation was applied through a drip irrigation system (six emitters per experimental unit), designed to collect the leachate for reuse. Plants were irrigated for 10 min ( 3.6 L) six times a day. Evapotranspirated water was replenished daily to each stock tank of nutrient solution with distilled water and the nutrient solutions were replaced every other day.

Gas exchange parameters and leaf water potential
Net photosynthetic rate, transpiration rate and stomatal conductance were measured (LI-COR Inc., LI-6200, Lincoln, NE, USA) at noon 40 days after planting on young leaves (the third fully developed leaf from top to bottom). Average PAR, CO 2 concentration and temperature were maintained at 400 mmol m 22 s 21 , 355 p.p.m. and 25.5 8C, respectively. Three measurements on each leaf from one plant per experimental unit were recorded. Water potential of two young leaves per plant was measured 30 days after planting (Scholander Pressure Chamber, Soil Moisture Equipment Corp., Santa Barbara, CA, USA).

Assessment of plant growth and nutrient status
Growth measurements were recorded at harvest (67 days after planting), including leaf area (LI-COR Inc., LI-3100) and dry mass (

Lipid peroxidation, antioxidant enzymes and NRA
Lipid peroxidation was measured in leaves sampled 60 days after planting by quantifying the concentration of malondialdehyde using the thiobarbituric acid method (Kuk et al. 2003), whereas the superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were determined according to Bonnet et al. (2000) and Kuk et al. (2003), respectively. Nitrate reductase activity was determined 60 days after planting using the procedure described by Ventura et al. (2010) with minor modifications. Briefly, fresh leaf samples (0.25 g) were collected at noon and vacuum infiltrated (pressure ¼ 50 cmHg) for 1 min in a mixture containing 0.1 M KNO 3 and 0.1 % n-propanol in 50 mM KH 2 PO 4 buffer (pH 7.0), in 1 : 20 (w : v) ratio. The reaction was allowed to proceed for 40 min at 30 8C in the dark. Samples were collected at the beginning and end of the assay, and the level of nitrite was determined with a spectrophotometer (Shimadzu UV-150-02) after the addition of 1 % sulfanilamide in 3 M HCl and 0.02 % N-1-naphthyl ethylenediamine mixture 1 : 1 (v : v) at 540 nm.

Electrolyte leakage and root hydraulic conductance
Electrolyte leakage was measured in 10 young leaf segments (1 cm 2 ) at 30 and 60 days after planting. The leaf segments were washed with distilled water to remove surface contamination and placed in stoppered vials containing 10 mL of distilled water. The vials were placed in a shaker at room temperature (27 8C) for 24 h at 100 r.p.m. Electrical conductivity (EC 1 ) of the bathing solution was read after the incubation period and then the samples were placed in an autoclave at 120 8C for 20 min; EC 2 was measured after cooling the bathing solution. Electrolyte leakage was calculated using the formula: 100 × (EC 1 /EC 2 ).
Root L o was measured 40 and 62 days after planting using a modified procedure as described by Joly (1989). Each plant was placed into a 100-mL beaker with distilled water. Shoots of plants were removed 1 cm above the root insertion and enclosed in a pressure chamber. A rubber tube was attached to the stump and protruded through a rubber gasket. The pressure in the chamber was increased gradually to 0.8 MPa and the sap flow captured in a glass vial for 30 min and weighed. The flow rate of sap exudate was determined as the average exudate weight per second (mg s 21 ), and L o was estimated as the exudate flow rate versus applied pressure per gram of root (mg g 21 s 21 MPa 21 ).

Statistical design
Six replicates of each experimental unit (container with six plants) were distributed in a complete randomized block design. Significant effects of NH 4 + and Ca were determined using ANOVA and Duncan's multiple mean comparison test (P , 0.05) with SAS (SAS v. 8.0, SAS Institute).

Results
Increasing NH 4 + in the nutrient solution reduced leaf area and DM of stems and leaves, but increased DM of roots when NH 4 + was at 50 % of total N (Table 1). Supplementary Ca at 14 meq L 21 partially reversed the toxicity in plants irrigated with 50 % of total N as NH 4 + when compared with Ca at 9 meq L 21 as leaf DM was higher than that of plants with Ca at 9 meq L 21 . Photosynthesis, transpiration rate and stomatal conductance were unaffected by the proportion of NH 4 + in the nutrient solution (Table 2). Leaf c w and RWC decreased as the proportion of NH 4 + increased in the nutrient solution (Table 3) in the 30-and 60-day measurements (Table 4). Increasing NH 4 + caused an increase in lipid peroxidation and the activities of APX and SOD (Table 4), but reduced that of NRA (Table 5). However, when Ca was provided at higher concentration, the activity of the SOD was significantly decreased (Table 4). Increasing proportions of NH 4 + had no effect on NH 4 + in the leaf tissue, while the root tissues exhibited increased NH 4 + concentration (Table 5). Nitrate concentration was significantly decreased in both leaf and root tissues when 50 % of total N was provided as NH 4 + (Table 5).
Total leaf N concentration was increased as the proportion of NH 4 + in the nutrient solution was increased, but root N concentration was unaffected ( Calcium is reported to increase the tolerance of plants to abiotic stresses by regulating the response reactions and developmental processes (Steinhorst and Kudla 2013). Kaya et al. (2002) and Tuna et al. (2007) reported that increased Ca concentrations resulted in enhanced growth and yield of strawberry (Fragaria × ananassa) and tomato plants exposed to high salinity; however, to our knowledge, there is no information as to the potential role of Ca in enhancing the tolerance of agricultural species to high NH 4 + stress. The present study demonstrates that supplementary Ca ameliorated to some extent the stress caused by high NH 4 + proportions as suggested by the higher leaf DM than that of plants with typical Ca concentrations. The observed reduction in leaf c w , leaf expansion and shoot growth when NH 4 + was .25 % may have been due to affected water transport to the shoot as demonstrated by the decrease in root L o and RWC. Supplementary Ca improved to some extent root L o , resulting in increased RWC and leaf c w , and partially restored leaf DM accumulation. As L o is largely determined by the activity of aquaporins (Li et al. 2015), probably the level of NH 4 + used in the present study may have affected these water root channels, whereas Ca could have played a role in restoring it. Gao et al. (2010) reported that aquaporins activity in rice was higher when seedlings were supplied with N as NH 4 + and indicated that the increase in L o may be a consequence of the effect of NH 4 + on root morphology (less development in the Casparian strip), growth, number of tips and surface area. However, our results showed that bell pepper plants responded differently and we have no evidence of modifications in root morphology (root DM of NO 3 − -fed plants was comparable with that of plants irrigated with 50 % of total N as NH 4 + ), suggesting that the increase in L o may be the effect of the supplementary Ca. Surprisingly, the restoring effect of Ca on L o did not affect gas exchange parameters. However, our data showed that the decrease in transpiration rate and stomatal conductance was associated with an increase in root Ca and N concentration. Rothwell and Dodd (2014) provided evidence that shows that Ca added to the rhizosphere can decrease stomatal conductance and photosynthesis in beans (Phaseoulus vulgaris) and pea (Pisum sativum), resulting in increased xylem sap Ca concentration in beans. However, the higher Ca concentration in the roots observed in our study in plants with supplementary Ca suggests that Ca translocation rate was maintained as in control plants as leaf Ca concentration was not increased, which suggests that this nutrient was not affecting gas exchange parameters. Nonetheless, the decrease in stomatal conductance in plants supplemented with Ca was associated with an increase in total N in leaves, which in turn was associated with a significant increase in leaf NH 4 + and NO 3 − concentrations. Similar effects of N nutrition of gas exchange parameters were reported by Hu et al. (2015) as stomatal conductance in Chinese cabbage (Brassica pekinensis) was decreased at proportions of 25 % of N in NH 4 + form, compared with plants provided with lower NH 4 + , and by Du et al. (2015), that reported similar deleterious effects in NH 4 + -fed tea plants (Camellia sinensis) compared with NO 3 − -fed plants. Therefore, the no relationship between stomatal conductance and the increased root hydraulic conductance in plants supplemented with increased Ca could have been related to the increase in leaf and root NH 4 + and total N concentration.
Abiotic and biotic stresses are reported to primarily target the cell membranes, so that the maintenance of the integrity and stability of the plasmalemma are of foremost importance to stress tolerance (Bajji et al.  supplementary Ca was associated with a reduction in SOD activity, suggesting that ROS were causing less damage. Calcium has been associated with increased tolerance to stress by alleviating the damage caused by ROS to cell membranes in tomato plants exposed to salinity (Tuna et al. 2007). Bell pepper plants that received exclusively N in NO 3 − form exhibited a low concentration of NH 4 + in the roots but a high concentration in the leaves, probably associated with the high reduction rate of NO 3 − as a result of the high NRA activity observed in the leaves. The decreased leaf and root NO 3 − concentration observed may have been caused by the lower availability of NO 3 − when external NH 4 + was increased and due to its rapid reduction, as suggested by the limited effect observed on NRA activity under high NH 4 + .
Leaf and root NH 4 + concentrations were not increased even when external NH 4 + was at 50 %, suggesting that bell pepper plants may regulate internal NH 4 + content, probably through efflux mechanisms across the plasma membrane (Britto et al. 2001). The higher leaf and root NH 4 + concentrations when plants were exposed to high NH 4 + and higher concentrations of Ca suggest that bell pepper was able to take up higher amounts of N in NH 4 + form from the external solution and/or that the higher NRA activity rapidly reduced NO 3 − to NH 4 + .
Nitrogen nutrition with high levels of NH 4 + is reported to cause nutrient imbalance as it depresses the uptake of K, Ca and Mg, while increases that of phosphate and sulfate (Roosta and Schjoerring 2007), which was confirmed in our study. The decrease in internal K concentration observed in our study was probably caused by competition with high levels of NH 4 + (ten Hoopen et al. 2010), which in turn may be the cause of the decrease in leaf c w , affecting leaf expansion and DM. Supplementary Ca in plants exposed to high NH 4 + resulted in increased root Ca and decreased leaf Mg, probably due to the competition between Ca and Mg for uptake sites (Marschner 1995). The nutrient imbalance also explains the more reduced growth of plants with 25 % of N in NH 4 + compared with plants with 50 % NH 4 + , as a higher root concentration of NH 4 + directly affected leaf, stem and shoot growth, whereas a higher root NO 3 − affected root growth.

Conclusions
The results obtained in the present study indicate that NH 4 + caused growth reduction, nutrient imbalance, membrane integrity impairment, increased activity of antioxidant enzymes and affected water relations in bell pepper plants; however, as supplementary Ca partially restored the growth of leaves by improving root L o and water relations, it may be part of an integral management to enhance the tolerance of important agricultural species to excess NH 4 + .

Sources of Funding
Our work was funded by Universidad Autó noma Agraria Antonio Narro.