3.1. Interaction of N Forms-Water Level on Physiological Response of the Coffee Plants
In this study, under controlled conditions, it was clearly observed that water stress significantly reduces biomass accumulation (DW), photosynthesis rate (Ps), chlorophyll content, and nutrient uptake. If the buffer capacity of the soil is low without the presence of organic carbon (Trial 2 in quartz sand), the higher the participation of NO
3−, the better the physiological parameters, due to the fact that the treatments with 100% to 75% NH
4-N showed the lowest DW and Ps (
Figure 1A,B,
Figure 2A and
Figure 3A,
Table 1 and
Table 2), indicating a negative effect of NH
4+ feeding due to the toxic accumulation of NH
4+ interacting with the strong acidification of the growth medium (pH < 5.0), this being more critical under water stress.
When the growth medium was soil (Trials 1 and 3), treatments with higher proportions of nitrates (100% NO
3-N and 75% NO
3-N) showed the lowest DW and Ps (
Figure 1C,D,
Figure 3B and
Figure 4,
Table 1 and
Table 2), mainly due to the higher accumulation of nitrates in the soil generated by the nutrient solution plus the soil nitrification (
Table 3), with this being most critical under water-stress conditions.
In the case of quartz sand, the low response displayed by the physiological parameters could be related to the low buffer capacity of the soil, significantly influencing the strong acidification in the treatment, with NH
4-N accounting for between 100% and 50% of the total N (
Figure 2A), meaning that acidification likely reduced the nitrification, as well as increasing the NH
4-N concentrations in the growth medium for these treatments (
Table 3). Meanwhile, in the trial using soil as the growth medium (Trial 3), the pH increases linearly with the NO
3-N concentration, promoting nitrification in NH
4-N-rich treatments and generating higher NO
3-N concentration in 100% NO
3-fed treatments (
Table 3).
Several studies clearly indicate that, in NH
4+-fed plants, some NH
4+ is translocated from the roots to the shoots, but this usually accounts for a small proportion of the total nitrogen moving in the xylem. Some of the reduced nitrogen compounds may have originally been formed in the leaves and been transferred to the roots, but a considerable amount taken up by plants is assimilated in the roots in the form of amino acids like asparagine and glutamine [
39], and coffee is no exception; according to Mazzafera and Gonzalves [
33], 37.5% of the total N and 90% of the N in the amino acid fraction in coffee sap is present in the form of asparagine and glutamine. When nitrates are the nitrogen source, the proportion of nitrogenous compounds in the xylem represented by NO
3− ions is much higher, indicating that nitrates are primarily assimilated in the shoots of the plants, varying considerably between plant species [
39,
41]; in coffee, 51.9% of the total N transported in the sap is NO
3− [
33].
When the highest content of nitrates is available in the soil, excessive NO
3− uptake reduces N assimilation and uptake. The response to excessive NO
3− feeding is species-dependent; for example, Duan et al. [
43] found in blackberry plants that NO
3−-fed plants were more likely to display the formation of reactive oxygen species (ROS) and malondialdehyde (MDA) than NH
4+ (or urea)-fed plants, leading to oxidative stress, an imbalance between oxidants and antioxidants, and the inhibition of root cell division and elongation. Meanwhile, when NO
3− was used as a sole N source, the levels of ascorbic acid (AsA) and reduced glutathione (GSH) increased significantly, which may be because the roots produced a large number of free radicals with the NO
3−-N treatment.
In terms of abiotic stress, Pissolato et al. [
46] found that plants grown in a 100% NO
3− nutrient solution were more tolerant to water deficit, and this response was associated with increase nitric oxide (NO) production and high NR activity in roots, increasing the activity of antioxidant enzymes, photosynthesis, stomatal conductance, and root growth.
When plants are supplied with NO3−, PEP carboxylase activity in the leaves is high but low in plants supplied with NH4+; this difference is less obvious at pH 6.0 than at pH 4.0. In the roots, PEP carboxylase activity becomes greater in plants supplied with NH4+ at pH 4.0 than in plants supplied with NO3− at the same pH, and the enzyme activity becomes higher in plants supplied with either ammonium or nitrates at pH 6.0. The PEP carboxylase fulfils an anaplerotic function, allowing for the synthesis of carbon skeletons that are required for amino acid synthesis during the assimilation of both nitrates and ammonium. The higher activity of PEP carboxylase in the roots of NH4+-grown plants reflects the need for more carbon skeletons to be available in the roots for amino acid synthesis when NH4+ is the nitrogen source.
When the nitrogen source is NO
3−, the activity of the enzyme in the leaves should be higher, as the assimilation of nitrates occurs mostly in the shoots of the plants [
39]. This was corroborated by Carr et al. [
47], who found significantly lower amino acid contents in coffee leaves when these plants were feed with a nutrient solution containing 100% NO
3−, while in the treatments with a balance between NO
3− and NH
4+, the amino acid content in the leaves increased. Amino acids are important N compounds responsible for long-distance N distribution in plants.
The excessive NH
4-N level in the soil observed in Trial 2, and the excessive NO
3-N level observed in Trial 3 (
Table 3), potentially interfered with the PEP carboxylase activity, NH
4+ assimilation, and, finally, with the formation and translocation of amino acids and proteins. Carr et al. [
47] found that coffee plants fed with 100% NH
4-N or 100% NO
3-N are less efficient in incorporating inorganic N into proteins compared with those fed with 50% to 12.5% NH
4-N/50% to 87.5% NO
3-N, suggesting that most of the inorganic N is accumulated as a soluble amino acid.
Nitrates (NO
3−) are readily mobile in the xylem and can also be stored in the vacuoles of roots, shoots, and storage organs. For the N in NO
3− to be incorporated into organic structures, nitrates must be reduced to NH
4+. Most of the ammonium, whether originating from nitrate reduction or from direct uptake from the soil solution, is normally incorporated into organic compounds through the roots, although some NH
4+ may also be translocated to the shoots, even if the plants receive nitrate as the sole N form [
35].
The reduction of nitrate to ammonium is mediated by two enzymes: nitrate reductase (NR), which catalyzes the two-electron reduction of nitrates to nitrites (NO
2−), and nitrite reductase, which transforms nitrites to ammonium in a six-electron transfer process [
35,
48]. To prevent the accumulation of nitrites, which are toxic to plant cells, NR activity is regulated by several mechanisms, including enzyme synthesis, degradation, and reversible inactivation, as well as the regulation of effectors and the concentration of the substrate. The concentration of NR is increased by light, sucrose, and cytokinin, whereas glutamine, a primary product of N assimilation, represses the NR [
35,
49]. Carr et al. [
47] found a lower abundance of glutamine in coffee leaves in plants fed with 100% NO
3-N compared with those fed with 50% NH
4-N/50%NO
3-N. The low DW and Ps rates registered in Trial 3 could be associated with NR inactivation due to the high NO
3− content in the soil, being almost 10 times higher than for the same treatment in Trial 2 (
Table 3). Meanwhile, the low physiological performance of the plants undergoing treatment with 100% NH
4-N/0% NO
3-N in Trials 1, 2, and 3 could be directly linked to a reduction in the NR activity due to the high NH
4+ content in the soil solution, as was reported by Carr et al. [
47] and Wang et al. [
50].
Independently of the source of the NH
4+ (nitrate reduction, photorespiration, lignin biosynthesis, N
2 fixation in legumes, or senescence inducing N remobilization) and the organ in which it is assimilated (roots, root nodules, and leaves), the key enzymes involved in the assimilation are glutamine synthetase (GS) and glutamate synthase (GOGAT; glutamine-oxoglutarate aminotransferase), which are present in the roots, shoots, and N
2-fixing organ [
35,
48,
49]. Wang et al. [
50] found 42% lower GS activity in wheat fed with NH
4+ in acidic conditions (pH = 5.0) compared with those in more alkaline conditions (pH = 6.5). In coffee plants, Carr et al. [
47] reported that the nitrogen form significantly influences the amino acid profile, with some of these amino acids being directly involved in stress signaling, like cysteine, which is very reactive and, therefore, toxic if it accumulates at a high proportion (>35% of the total intracellular content), with a gradual increase in cysteine concentration according to the NH
4+ levels in the nutrient solution. Similar results were reported by the authors with regard to arginine, the levels of which were 1122% higher in plants subjected to the treatment with 100% NH
4-N than with 100% NO
3-N.
Regarding the influence of the interaction of N forms and water level on Ps, in coffee plants growing in a nutrient solution, the decrease in the QA reduction for plants grown in 0% NO
3-N/100% NH
4-N and 100% NO
3-N/0% NH
4-N was related to “closed” PSII centers, reflecting an accumulation of reduced QA and also non-photochemical energy dissipation. At the same time, these treatments showed lower photosynthesis rates, indicating higher non-photochemical dissipation as a result of the non-utilized energy, while the other treatments, with NO
3/NH
4 ratios of 50% NH
4-N/50% NO
3-N and 12.5%/NH
4-N/87.5% NO
3-N, exhibited greater efficiency in employing the absorbed light in the photochemistry process [
47].
Under water-stress conditions, N application in
Coffea canephora Pierre brought about an increase in cell-wall rigidity and osmotic adjustment, improving water extraction from drying soil in addition to avoiding the excessive loss of cell volume, thus leading to some degree of drought tolerance [
20]. Moreover, N increases the long-term WUE through changes in photosynthesis [
20]. Nitrates play a key role in root hydraulic resistance; for instance, root hydraulic resistance increases when NO
3− availability is low and decreases when NO
3− supply is high [
18,
51]. NO
3− uptake and assimilation involve a net consumption of protons, raising the possibility of direct feedback between NO
3− assimilation and the regulation of aquaporins [
18,
19,
51]. Despite the existence of biochemical and biophysical mechanisms for pH homeostasis, blocking NR can lead to a measurable change in cytosolic pH, and a decrease in cytosolic pH has also been shown to reversibly alter root hydraulic properties due to the protonation of a tyrosine residue on the cytosolic side of the majority of plasma membrane intrinsic proteins (PIP), resulting in a dramatic increase of root hydraulic resistance [
51,
52]. This gating mechanism raises the possibility that changes in cytosolic pH due to NO
3− assimilation could be involved in triggering nitrate-induced changes in the permeability of the roots to water [
51]. Meanwhile, a high NH
4+ (3 mM) supply induced more apoplastic barrier formation and decreased root hydraulic conductivity when compared with low NH
4+ supply (0.03 mM) in rice seedlings [
19].
Proline (Pro) has been recognized as an essential amino acid derived from nitrogen synthesis, with glutamate as one of the main precursors. Pro accumulation is believed to play adaptative roles in plant-stress tolerance; it has been reported as a compatible osmolyte and a way to store carbon and nitrogen. Pro can be an ROS scavenger and has been proposed to function as a molecular chaperone, stabilizing the structure of proteins, and its accumulation can provide a way to buffer cytosolic pH and to balance cell redox status. Finally, Pro accumulation may be part of the stress signal influencing adaptative responses [
53]. In coffee, Pro accumulation during stress conditions has been highly documented as a stress-tolerance mechanism [
54,
55]. The total content of amino acids, including Pro, increases gradually with an increase in nitrogen rates, while a higher NO
3− concentration in soils suppresses the synthesis of amino acids, including Pro, in plants exclusively fed with NO
3− fed compared with those with fed with a balance of NH
4+ and NO
3− [
56].
In well-created agricultural soils, mineral N and especially NO
3− are the most abundant forms of available N, while NH
4+ dominates in soils which nitrification is inhibited (Von Wiren et al. [
37]). In a short greenhouse trial (4 months) with coffee seedlings, using a high-organic-matter soil (160 g kg
−1 of organic matter) as the growth medium, and using
15N-labeled urea, Salamanca et al. [
57] did not find any impact of the interaction between soil N and moisture factors on the N uptake and recovery, likely because the moisture levels evaluated were 122, 100, 80, and 61% of the FC, indicating relatively high soil moisture levels that potentially inhibit the nitrification process. This is completely different to the findings of our study, where the effect of the interaction between the water level and N forms was significant for the N uptake in both growth media, namely quartz sand and soil (
Table 2).
3.2. Synergy between Ammonium and Nitrate Nutrition
Ammonium is preferentially taken up by many plants when supplied in an equimolar concentration with nitrates, particularly when the N supply is low, and only in some low-temperature conditions can the NH
4+ uptake continue while the NO
3− uptake is suppressed. Ammonium is assimilated through the roots, imposing a direct demand for carbon skeletons, which is reflected in higher activity levels of PEP carboxylase [
35,
39]. The increased carbon consumption caused by increased NH
4+ assimilation in the roots also partially explains NH
4+-induced growth inhibition [
42]. Compared with NH
4+, NO
3− has the advantage of allowing the more flexible distribution of assimilation between roots and shoots and can be stored in higher amounts than NH
4+ in the vacuoles [
35]. On the other hand, NH
4+ and NO
3− comprise about 80% of the total cations and anions taken up by plants, and the form of N has a strong impact on the uptake of other cations and anions, as well as on cellular pH regulation and on rhizosphere pH [
35].
The assimilation of NH
4+ through the roots produces about one proton per molecule of NH
4+, and the generated protons are to a large extent excreted into the external medium to maintain cellular pH and electro-neutrality, with the latter compensating for the excess uptake of cation equivalents over anion equivalents, which are generally associated with NH
4+ nutrition [
35,
42]. Wang et al. [
50] found that the growth of wheat seedlings was seriously inhibited by NH
4+ feeding, and that this inhibitory effect was enhanced by soil-acidification stress. Excessive NH
4+ accumulation has an energy cost related to providing energy for H+ pumping, which is expressed by a higher ATPase activity [
42,
58,
59], as was observed in coffee plants treated with NH
4+, which showed significantly higher ATP when subjected to treatment with 100% NH
4/0% NO
3 compared to the others with less ammonium and more nitrates [
47]. Under mixed N nutrition, the protons generated by NH
4+ assimilation can be used for NO
3− reduction; therefore, it is easier for plants to regulate their intracellular pH when both forms of nitrogen are supplied [
35].
As reported by Vaast et al. [
38], the concurrent uptake of NH
4+ and NO
3− in coffee plants helps in the maintenance of the cation–anion balance within the root cells, thus minimizing the plant’s need for organic acid synthesis to regulate its intracellular pH. Direct NH
4+ uptake from the solution decreases the energy cost involved in NO
3− reduction and increases the supply of reduced N for protein synthesis [
60,
61]. The current absorption of NH
4+ and NO
3− also prevents acidification or alkalinization in the rhizosphere, which can in return affect the uptake [
62].
In coffee, the low DW accumulation was not related to the low pH and was more related to the inhibition generated by the higher NH4-N or NO3-N content in the soil, because in both trials using quartz sand and soil, the soil pH increased linearly with the share of NO3-N in the nutrient solution, but the DW and Ps in Trials 1 and 3 were higher in the treatments with 75% NH4-N/25% NO3-N and 50% NH4-N/50% NO3-N, while in quartz sand, better physiological performance was achieved in the treatments with 100% NO3-N, regardless of the water level. This means that the concentrations of NO3− and NH4+ in the soil are more important, and soil pH indirectly influences the nitrification rates, with a direct influence on the NH4+–NO3− balance, which has a significant effect on nutrient uptake, and nitrogen assimilation.
3.3. Effect of the Interaction between N Forms and Water Level on Nutrient Uptake
In all three greenhouse trials, the highest nitrogen uptakes were observed in the treatments that had a balance of nitrogen forms (50% NH
4-N/50% NO
3-N) without water stress; under water stress, high N uptake was achieved by the treatments with more nitrates (25% NH
4-N/75% NO
3-N) in quartz sand and in the treatment with more NH
4+ (100% NH
4-N/0% NO
3-N) in organic soil. Vaast et al. [
38] reported similar NH
4+ and NO
3− uptake rates in a solution culture with a pH range of 4.25 to 5.75 and noted that the total N uptake at any NH
4/NO
3 ratio was higher than that of plants fed solely with either NH
4+ or NO
3−.
As was reported by Carr et al. [
47], the differences in growth and nutrient absorption displayed by the coffee plants in the greenhouse trials were directly correlated with the influence of the N forms, and varying the NH
4-N/NO
3−N ratios directly affected the charge balance of young coffee plants. A higher content of NH
4-N in the nutrient solution increases the NH
4+ content in the soil, with a stronger effect under water-stress conditions directly affecting the nutrient uptake. Excessive NH
4+ feeding results in excessive acidification of the growth medium or nutrient solution, nutrient imbalance, and impaired plant growth.
The growth medium (quartz sand and soil) and the water level (with and without water stress) affect the contents of different N forms in the medium (
Table 3), directly affecting the DW accumulation, Ps, and chlorophyll content in the leaves (
Table 1 and
Table 2; and
Figure 3 and
Figure 4). Under water-stress conditions, the NH
4+ and NO
3− content in the soil was higher compared with that with the same treatments without water stress (
Table 3). Regardless of the soil moisture content, the highest N and cation uptake was achieved when the concentration of NO
3− in the soil ranged between 1.5 and 3.5 mg 100 g
−1 and that of NH
4 was <0.3 mg 100 g
−1.
According to Pilbeam and Kirkby [
39], a variety of factors are involved in differences in the uptake and distribution of inorganic anions and cations in plants supplied with two forms of nitrogen. One of the most important of these factors is the alkalinization or acidification of the rhizosphere, as under acid conditions, phosphate becomes less available in the soil, the concentration of aluminum and manganese increases in the soil, and the uptake of calcium, magnesium, and potassium is depressed by H
+ ions in the rhizosphere.
For plants grown at the same pH but treated with either NO
3-N or NH
4-N, the uptake of calcium, magnesium, and potassium is higher for the NO
3-fed plants [
47], as was observed in this research (
Table 3). In the case of plants with a high calcium demand like coffee, several disorders are generated by the shortage of calcium, namely reducing calcium translocation and mobility to new growth tissues, generating a reduction in calcium accumulation in mesophyll cells, with several effects on leaf physiology, and, together with a shortage of potassium and magnesium, significantly reducing plant growth and productivity [
15].
The detrimental effects of NH
4+ in reducing the uptake of potassium and calcium are likely to be more common when the pH of the growth medium is very low [
36,
43]. This indirect suppression of calcium and magnesium cation uptake indirectly reduces the nitrogen assimilation via low NR activity [
63,
64].
3.4. Influence of the N Forms on Productivity and Chlorophyll Content at Field Level
The differences in productivity effects between ammonium nitrate (AN) base fertilizers and urea are not always consistent, as different authors have reported that there does not exist significant differences on productivity between urea and AN base fertilizer in coffee [
23,
30,
65], as we found during the first 2 years of harvest and after 3 years of treatment (
Figure 5).
In perennial crops, it is common not to find a response to mineral N fertilization during the first few years; for example, in citrus in Brazil, Cantarella et al. [
66] compared two N forms and did not find any response to mineral N fertilization during the first 3 years when comparing urea and AN. This lack of response was attributed by the authors to the high N reserves associated with the previous years of crop growth, but, after the 4th year of the trial, the citrus plantation responded to the mineral N fertilization, and differences between N sources were observed, with a significant higher yield for the treatments with AN compared with urea at the same N rate.
The low yield provided by the urea treatments is also related to the low efficiency of this N form. Of the total amount of mineral N fertilizer applied, only 25–60% of the mineral N is effectively taken up by coffee plants [
67,
68,
69]. In coffee, for example, 20% of the mineral nitrogen applied was found in the shoots or aerial parts, 12% was found in the roots, and 26% was exported during harvest, while about 15% remained in the soil [
68]. The rest is lost from the soil–plant system through mechanisms such as denitrification, leaching from the root zone, and volatilization. [
5,
22,
30,
70,
71].
Without any soil or environmental limitations, the ammonification and nitrification of the nitrogen fertilizers take place in short periods of time [
72], meaning that the nitrogen lixiviation of urea or AN base fertilizer in coffee is the same under field conditions [
30,
31].
In coffee-production systems in Colombia, Leal et al. [
29] found a mean nitrogen volatilization (NH
3-N) of 30% to 35% of the total N applied using urea. In a long-term trial in coffee during four crop seasons in Brazil, de Souza et al. [
23] found that the NH
3-N volatilization was significantly influenced by the nitrogen rate and form, where urea led to the highest values of NH
3-N, ranging between 9% and 25%, varying with the rate and application time, while the losses associated with ammonium nitrate represented less than 1% of the doses applied in the four crop seasons.
The incorporation of fertilizer into the soil solution and exchange system is directly influenced by the rainfall volume and intensity; higher rainfall events are usually associated with better fertilizer incorporation and, consequently, lower losses by volatilization. A lower precipitation volume immediately after the application of the fertilizer to the soil hinders the incorporation of N and favors NH
3-N losses [
30]. Moreover, the architecture of coffee plants obstructs the direct incidence of rainfall that would incorporate the applied N fertilizer into the soil under the canopy [
73,
74,
75]. Additionally, the presence of plant residues in the soil also acts as a barrier to fertilizer incorporation and creates a favorable environment for volatilization due to the high concentration and activity of urease [
30,
76].
Thus, the use of ammonium nitrate (AN) as a N source contributes the most to increasing soil N stocks and supplying the demands of the vegetative and reproductive stages of coffee plants, without any interference in the soil’s microbial and enzymatic activity [
30]. The lower NH
3-N losses from the use of AN can increase the average mineral N content by 50% when compared to urea and urea with inhibitors [
77].
It is common to find in short-term field studies, not only in coffee, that the yields may not be significantly affected, despite the NH
3-N losses [
24,
31], as was also found in this study during 3 years of treatment application and two of harvest (
Figure 5). This confuses agronomists and farmers, but the logic of these results is well explained by Cantarella, [
31]: “Soil is the major supplier of nutrients for plants, including N. A substantial part of the N that plants absorb during the production cycle come from the soil; fertilizers supplement the needs of crops. Thus, losses are canceled by soil supply and are not always reflected in yields in the short term. The N losses by volatilization is dispersed in the atmosphere and does not recharge the soil stock, the one that provides the nutrient for the plant. Over time, the soil becomes impoverished and N runs out. Under these conditions, the differences between sources subject to or not subject to volatilization become apparent”.
When the N and C stocks in the soil are low or do not exist, differences between N forms are rapidly observed, as was demonstrated by Chagas et al. [
28] in coffee seedlings grown in an acidic Red Latosol soil collected in the B horizon with a very low level of organic matter (0.16%), who found significantly higher agronomic efficiency in plants fed with AN compared with urea and urea with inhibitors like formaldehyde or polyurethane.
In the present research, the differences in productivity effects between N forms could not be directly associated with soil N or C depletion due to the fact that the foliar N content after 4 years of treatment application was not significantly different among the N treatments (
Table 4). The significant effect of a higher NO
3− share on productivity could be more closely related to the better nitrogen and cation uptake compared with NH
4+-rich treatments including urea. Urea, as an organic N form, must be catalyzed into NH
4+ before it can be absorbed by plants [
78]. In other words, NH
4+ and urea share a common metabolic pathway [
43]. In our research, after 5-to-8 months in the greenhouse and 4 years in the field, the results indicate that coffee growth, Ps, nitrogen assimilation, nutrient uptake (N and cations), chlorophyll content, and productivity are affected by NH
4+ and urea application, and the main effects of long-term NH
4+ and urea feeding could be linked to the moderated inhibition of NO
3− uptake by NH
4+ [
38] and the disruption of the ion balance that strongly limits the N and cation uptake, with a subsequent influence on Ps, chlorophyll accumulation, and finally on productivity.