Abstract
Key message
Mixed N fertilization with NO3−:NH4+ ratio of 0.50:0.50 mitigates negative water stress effects on growth in young Eucalyptus urophylla plants.
Abstract
Plant tolerance to water deficit can be influenced by several factors, including the available ionic forms of fertilization. The goal of this study was to assess the effects of nitrate (NO3−) and ammonium (NH4+) ratios in N fertilization on growth-related morphophysiological and biochemical traits, with the aim of mitigating water stress in young Eucalyptus urophylla plants. A greenhouse experiment was arranged in a completely randomized design and factorial scheme 5 × 2, with different NO3−:NH4+ ratios (0.0:1.0 × 0.25:0.75 × 0.50:0.50 × 0.75:0.25 × 1.0:0.0) and water regimes based on irrigation at 90 and 30% of pot capacity. The results showed that water deficit inhibited plant growth, resulting in lower plant height, and smaller stem diameter, total leaf area, and leaf, stem and root dry mass. Decreases in stomatal conductance, net photosynthesis, and the content of reducing sugar and starch are involved in growth inhibition. Increasing the NO3−:NH4+ ratio can mitigate negative water deficit effects on leaf water potential, stomatal conductance, and photosynthesis. Furthermore, leaf nitrate reductase activity improves under mixed NO3−:NH4+ fertilization, compared with NO3– or NH4+ fertilization alone. Despite this, variations in N sources proved to be ineffective in preventing growth inhibition under water deficit. However, the NO3−:NH4+ ratio of 0.50:0.50 provided the best performance of morphophysiological traits, regardless of the water regime. This was particularly relevant at irrigation levels under 30% since fertilization with an NO3− ratio equal to NH4+ can mitigate water stress effects on plant growth, despite not preventing damage to morphophysiological traits.
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Introduction
The increasing demand for wood in Brazil has led to the expansion of Eucalyptus cultivation throughout the country. However, in many regions low and/or unstable rainfall distribution can limit the establishment of new forest stands. Water deficit negatively affects seedling growth, and can threaten their survival in the field. Water deficit-tolerant genotypes are usually recommended for cultivation in these regions, but this strategy alone may not be sufficient to prevent the harmful physiological effects of low soil water availability, especially in the early growth stages.
Plant nutritional status is one of the determining factors for growth under water deficit conditions, and nitrogen is the most essential nutrient. Inorganic nitrogen is absorbed as NO3− and NH4+ ionic forms (Hawkesford et al. 2012) although it is now acknowledged that organic N forms (e.g., amino acids) are also used by plants (Warren 2006, 2009). Nitrate absorption and assimilation both demand energy, either for the influx from the apoplast to the cytosol across the plasma membrane or for the subsequent reduction to nitrite (NO2−) and NH4+, mediated by nitrate reductase (NR) and nitrite reductase (NiR), respectively (Taiz et al. 2017). In contrast, ammonium absorption requires less energy and can occur passively (Li et al. 2013), although it can become toxic when it accumulates in plant tissues.
Previous studies have reported that NO3− and NH4+ have different effects on the performance of some growth-related morphophysiological and biochemical traits that improve tolerance to water deficit, such as root dry mass (Holzschuh et al. 2011), water uptake (Gao et al. 2010; Faustino et al. 2015), photosynthesis rate (Cao et al. 2018), osmoregulator content such as reducing sugar and proline, and antioxidant activity (Zhang et al. 2011b; Fernández-Crespo et al. 2012). The beneficial effects of mixed NO3−:NH4+ fertilization have been attributed to such factors as the maintenance of intracellular pH stability, lower energy demand for N assimilation, regulation in the uptake of other cations, rational use of carbon skeletons, and mitigation of NH4+ toxicity (Li et al. 2013; Hachiya and Sakakibara 2016).
Previous studies have shown that the highest growth rate is reached in Eucalyptus urophylla seedlings under hydroponic medium with mixed NO3−:NH4+ fertilization with a 0.75:0.25 ratio (Guimarães et al. 2014). Mixed NO3−:NH4+ fertilization improves the tolerance of water deficit in corn (Zhang et al. 2011a) and rice (Cao et al. 2018; Wang et al. 2018) found that mixed NO3−:NH4+ fertilization in Camellia oleifera seedlings, regardless of water regime, increased leaf N, chlorophyll, soluble sugar and protein content, and promoted higher enzymatic activity.
Mixed NO3−:NH4+ fertilization can also improve the absorption efficiency of other nutrients (Holzschuh et al. 2011; Wang et al. 2018), thereby mitigating the negative effects of water deficit (Ashraf et al. 2011; Waraich et al. 2011). Moreover, it can promote auxin accumulation in the roots (Fu et al. 2020; Meier et al. 2020), favoring expansion and deepening (Hachiya and Sakakibara 2016; Liu et al. 2019). The increase in root/shoot ratio improves the water absorption capacity, as observed in Populus deltoides (Woolfolk and Friend 2003) and Oryza sativa L. (Holzschuh et al. 2011) under mixed NO3−:NH4+ fertilization. Despite the positive effects on some species, there is still a knowledge gap regarding the ideal NO3−:NH4+ ratio to provide more tolerance to water stress in young Eucalyptus plants.
We hypothesized that a high NO3−:NH4+ ratio could reduce water deficits in Eucalyptus seedlings through changes in stomatal conductance, improvements in photosynthesis rate and osmoregulation, higher root/shoot ratio and increased water and nutrient absorption. Thus, the goal of this study was to assess the effects of different NO3−:NH4+ ratios in N fertilizer on growth-related morphophysiological and biochemical traits, with the aim to mitigate water stress in young Eucalyptus urophylla plants.
Materials and methods
Site description and experimental design
A greenhouse experiment was carried out at State University of Southwest Bahia, in Vitória da Conquista, Bahia, Brazil (14º53′08′′-S, 40º48′02′′-W, 881 m asl), from October to December 2019. The local climate is Cwb type (dry-winter subtropical highland climate), according to the Köppen-Geiger classification. During the experimental period, the average temperature and relative humidity inside the greenhouse were 24 ± 2 °C and 61.5 % ± 5 %, respectively.
The experiment was arranged in a completely randomized design and factorial scheme 5 × 2, four replicates (one plant per pot), with five different NO3–:NH4+ ratios (0.0:1.0 × 0.25:0.75 × 0.50:0.50 × 0.75:0.25 × 1.0:0.0) and two water regimes based on irrigation at 90 and 30 % of pot capacity.
Plant material and growing conditions
The seedlings of Eucalyptus urophylla clone AEC 144 used for this study were produced in small tubes (54 cm3) containing the following substrates: bovine manure (40%), vermiculite (40%) and coconut husk powder (20%) (Oliveira Júnior et al. 2011). Seedlings 120-days-old, with 6‒8 fully-expanded leaves on average, and approximately 20 cm in height were planted in 15 dm3 pots using sand previously washed with deionized water as a substrate which was fertilized in two stages, using a nutrient solution proposed by Hoagland and Arnon (1952) (Table 1).
The first fertilization was carried out immediately after the seedlings were planted in the pots, and the ionic strength of the nutrient solution was gradually increased (30%, 60 and 100%) every 3 days, to aid in seedling adaptation to the new substrate. Twenty-one days after planting, the second fertilization was conducted using only the nutrient solution at its maximum ionic strength. For both fertilizations, the volume of nutrient solution supplied was sufficient to maintain the substrate moisture at 90% of the pot capacity, which was measured by the gravimetric method. The electrical conductivity of the solution was kept below 1.5 mS cm–1, and the pH was 5.5 ± 0.2, using HCl 0.1 M solution. Water regimes were established 45 days after planting. Fifteen days after water regimes started, we carried out measurements of morphology, plant water status, gas exchange, and biochemical and metabolic traits.
Plant water status
Plant water status was assessed at predawn in fully expanded leaves (n = 4) from the middle part of the canopy. A pressure chamber (Model 1000, PMS, Albany, USA) was used for leaf water potential measurements, according to Scholander et al. (1965). Relative water content (RWC) measurements were based on fresh (FM), turgid (TM), and dry mass (DM) from 10 leaf disc samples, and data were used in this equation (Weatherley 1950):
Gas exchange
Net photosynthesis (A), transpiration (E) and stomatal conductance (gs) were measured from 8:00 to 10:00 in fully expanded leaves (n = 4) from the middle part of the canopy, using an infrared gas analyzer (IRGA LCPro, ADC, UK). Measurements were performed under a photon irradiance of 900 mmol m−2 s−1 and a CO2 air concentration of 375 mmol mol−1. The intrinsic water use efficiency (iWUE) was calculated as the ratio between A and gs (A/gs).
Biochemical and metabolic traits
Fully expanded leaf samples (n = 4) were oven-dried at 70 °C for 2‒3 days prior to analysis of reducing sugar, proline and starch content. Reducing sugar was extracted from 200 mg of dry leaves after immersion in 15 mL of KH2PO4 0.1 M buffer solution, 3 × centrifuged at 2500g for 45 min. The supernatant was taken as the extract, and the reducing sugar content was determined using a spectrophotometer (Miller 1959). Starch was extracted from 250 mg of dry leaves previously subjected to fat removal by hexane, followed by immersion in 5 mL of H2SO4 0.5 M at 100 ºC for 1 h. Starch content was determined using a spectrophotometer, according to Normative Instruction No 20 (Brasil 1999). Proline was extracted from 200 mg of fresh leaves, after immersion in 6 mL of 3 % (w/v) sulfosalicylic acid, 3 × centrifuged at 7500 rpm for 10 min. The supernatant was taken as the extract, and proline content was determined in a spectrophotometer (Bates et al. 1973).
Leaf nitrate reductase activity in vivo was assessed as described by Guimarães et al. (2014). The enzymatic assay was based on a 500 mg sample of fresh fragmented leaves (n = 4), after immersion in 5 mL of KH2PO4 0.1 M buffer, 3 % (v/v) npropanol, and KNO3 (0.1 M) at pH 7.5, and a water bath at 30 °C in the dark. After 1 h, a 1 mL aliquot from the assay was added to a medium consisting of 1 mL of 1 % (w/v) sulfanilamide in HCl (1.5 M), 1 mL of 0.02 % (w/v) n-1-naphthylethylenediamine di-HCl, and 1 mL of deionized water. Nitrite content was determined using a spectrophotometer.
Morphophysiological traits
Measurements of plant height and stem diameter (measured at the root collar) were measured using a graduated ruler and digital caliper, respectively. For total leaf area, a leaf area meter (LICOR, LI-3100) was used. The dry mass of leaves, stem and roots was obtained after drying in an oven at 70 ± 5 ºC, followed by weighing on a scale.
Statistical analysis
The data were subjected to an analysis of variance. Mean values from the water regimes were compared using Tukey’s test. Regression models were adjusted for data from the NO3−:NH4+ ratios. When traits were not adjusted to the regression models, data from NO3−:NH4+ ratios were compared using Tukey’s test, using the SISVAR statistical program (Ferreira 2011).
Results
Plant water relations
The results only showed a significant interaction (p < 0.005) between NO3−:NH4+ ratios and water regimes in relation to leaf water potential (Ψw). As an isolated factor, the NO3–:NH4+ ratios influenced both Ψw and relative water content (p < 0.001). Regarding Ψw, there was a quadratic effect on plants under 90 % irrigation in response to increasing NO3−:NH4+ ratio (Fig. 1). There was an increase in Ψw, followed by a decrease from an estimated 0.58:0.42 ratio. However, under 30 % irrigation, increasing the NO3−:NH4+ ratio caused a linear increase. The water regime effect was significant only at the 1.0:0.0 ratio, where Ψw was higher under 30 % irrigation (Fig. 1A). Regarding relative water content, there was a significant decrease at 1.0:0.0, irrespective of water regime, although this data was similar to that observed for the 0.25:0.75 ratio (Fig. 1B).
Gas exchange
The results showed a significant interaction (p < 0.001) between the NO3−:NH4+ ratios and water regimes for all gas exchange traits. The increase in NO3−:NH4+ ratio under 30 % irrigation caused linear increases in A, E and gs, and a linear decrease in iWUE. Conversely, under 90 % irrigation, these traits were not influenced by the different NO3−:NH4+ ratios. Results from A, E, and gs were higher under 90 % irrigation than under 30% irrigation, for the majority of the NO3−:NH4+ ratios, except at 1.0:0.0, where there was no difference between water regimes. The performance of iWUE, in turn, was consistently higher under 30 % irrigation, irrespective of the NO3−:NH4+ ratio (Fig. 2).
Biochemical and metabolic traits
The results showed a significant interaction between NO3−:NH4+ ratios and water regimes for reducing sugar (p < 0.001), starch (p < 0.005), and proline (p < 0.005) contents, as well as nitrate reductase (NR) activity (p < 0.001).
Regarding reducing sugar, there was no difference between the water regimes when the NO3−:NH4+ ratio was 0.0:1.0. However, the other ratios showed a lower reducing sugar under 30 % irrigation than under 90%. With 30% irrigation, reducing sugar decreased linearly in response to an increased NO3−:NH4+ ratio (Fig. 3A). Starch content, in turn, was lower under irrigation 30 % than under 90%, regardless of the NO3−:NH4+ ratio. The increased NO3−:NH4+ ratio caused a slight quadratic effect at 30%, with starch content decreasing up to an estimated 0.61:0.39 ratio (Fig. 3B). Proline content increased under 30 % irrigation at most of the NO3−:NH4+ ratios, except at 0.75:0.25, where it did not differ between water regimes. This increase was more pronounced in fertilization with only one ionic N form than with mixed NO3−:NH4+ fertilization (Fig. 3C).
Leaf nitrate reductase activity was not influenced by water regimes, fertilization with NO3− or NH4+, or mixed N fertilization at 0.75:0.25. Enzymatic activity at 0.25:0.75 and 0.50:0.50 was higher under 30 % irrigation. At 90 %, RN activity showed a slight decline, in response to an increased NO3−:NH4+ ratio, whereas with 30 % irrigation, the increase in NO3−:NH4+ ratio caused a quadratic effect, with enzymatic activity increasing to an estimated 0.46:0.54 ratio (Fig. 3D).
Morphophysiological traits
The results showed no significant interaction between NO3−:NH4+ ratios and water regimes in relation to plant height, stem diameter, total leaf area, and leaf, stem and root dry mass. Nevertheless, both NO3−:NH4+ ratios and water regimes showed significant differences for all traits (p < 0.001). In general, increasing the NO3−:NH4+ ratio had positive effects on growth-related morphophysiological traits, which reached the best performance at 0.50:0.50, despite some similarities with higher NO3−:NH4+ ratios, depending on the variable (Table 2). As for water regimes, 30 % irrigation negatively affected all morphophysiological traits, especially leaf and stem dry mass, which decreased 28.96 and 32.30 %, respectively (Table 3).
Fifteen days after starting treatments with mixed N fertilization, typical symptoms of NH4+ toxicity were observed in plants at 0.0:1.0 and 0.25:0.75. The first symptoms appeared at stage B (Silva et al. 2020) of leaf development, when a purple to reddish color became noticeable at leaf edges, followed by upper edge curling (Fig. 4A). These symptoms evolved to necrosis at stage C of leaf development (Fig. 4B).
Discussion
The results showed that increasing the NO3−:NH4+ ratio under water deficit was not effective in preventing significant decreases in growth-related morphophysiological traits such as plant height, stem diameter, total leaf area, and leaf, stem and root dry mass (Table 3). Regarding dry mass, these effects were more pronounced on leaf and stem, which reduced in size by 28.96 and 32.30 %, respectively, when compared to well-watered plants. Due to the smaller decrease in root dry mass (18.83 %), there was likely a higher source-to-sink assimilate partitioning for this organ, which favors water uptake, thus providing higher tolerance to water deficit. The best morphophysiological traits were achieved at NO3−:NH4+ ratios of 0.50:0.50, irrespective of water regime (Table 2). This effect is especially interesting for plants under 30 % irrigation since it helps to mitigate the effects of water stress on plant growth.
For gas exchange, gs was one of the most important factors, showing a strong correlation with E and A. Under 30 % irrigation, gs, E, and A values were lower at low NO3−:NH4+ ratios (Fig. 2). These results agree with those of Cramer and Lewis (1993) in wheat, and Lopes et al. (2004) in barley, who determined that gs and A values were significantly lower in NH4+- than in NO3–-fertilized plants.
Water deficit usually promotes partial stomatal closure, reducing gs, E, and A (Sharma et al. 2020). In our study, the results revealed that increasing the NO3−:NH4+ ratio attenuated the water deficit effect on gas exchange affected by water potential (Fig. 2). The increase in A, in response to increased NO3− supply, partly relates to the concomitant decrease in NH4+ supply, thus mitigating the negative effects of this cation on gas exchange. A high NH4+ content may also be involved in stomatal closure (Foyer et al. 2003), as gs and A are decreased. These deleterious effects of high NH4+ content on gas exchange corroborate other studies on strawberry, sugar cane, and fir, even under well-watered conditions (Rothstein and Cregg 2005; Tabatabaei et al. 2006; Pissolato et al. 2019). In contrast, in our study NO3−:NH4+ ratios did not cause significant changes in gas exchange under 90% irrigation, and this result verifies that of Guimarães et al. (2014), also using Eucalyptus urophylla.
The reasons for NH4+-supplemented plants inducing decreased gs remains unclear, but a more restricted osmotic adjustment is assumed to be involved. Rhizosphere acidification due to NH4+ assimilation restricts cation absorption compared to fertilization mainly with NO3− sources (Basra and Goyal 2002). Decreased gs values may reflect a low NO3− supply, which is not only a relevant osmolyte (Mcintyre 1997; Lopes and Araus 2006), but also an essential anion for cation translocation through the xylem (Guo et al. 2003; Wang et al. 2012). According to Marschner (2012), plants mainly-fertilized with NH4+- reveal less Ca, Mg, and K content than those with NO3− as the main agent. In this regard, it should be noted that a lower K content may restrict stomatal function (Laporte et al. 2002). In our study, some slight signs of root senescence were observed in plants that were mainly NH4+-fertilized, which may cause decreased gs (Basra and Goyal 2002; Britto and Kronzucker 2013).
NO3−:NH4+ ratios less than 0.50:0.50 caused a decrease in root dry mass (Table 2), which may be related to the limiting effect of high NH4+ content on water uptake, leading to a hardening water stress. Therefore, it is assumed that under 30 % irrigation a decrease in NH4+ supply due to an increase in the NO3−:NH4+ ratio must have contributed to improved water uptake, thus increasing leaf Ψw. Moreover, the increase in NO3–:NH4+ ratio up to 0.75:0.25 may have contributed to maintaining leaf relative water content, in whole or in part, despite the increase in E (Fig. 1).
Increasing Ψw concomitant with increased NO3−:NH4+ ratio under 30 % irrigation corroborates Faustino et al. (2015) with Pinus taeda. According to those authors, increasing NO3– supply induces changes in root dry mass and hydraulic conductance, providing more tolerance to water deficit. However, the increase in Ψw may also relate to a decreased NH4+ supply, as evidenced in studies on other species under water stress, in which NH4+ fertilization complicates water uptake making it further even more difficult, thus reducing Ψw (Pill and Lambeth 1977; Wu et al. 2017). Conversely, under 90 % irrigation, NO3−:NH4+ ratios higher than an estimated 0.58:0.42 caused a decrease in Ψw, which reached the lowest value at 1.0:0.0.
The decrease in reducing sugar and starch content, as observed in plants under 30 % irrigation (Fig. 3), may be another consequence of stomatal closure under water deficit, which impedes CO2 absorption, negatively affecting carbohydrate synthesis (Hartmann et al. 2020). Under 30% irrigation, increasing the NO3−:NH4+ ratio decreased the reducing sugar content (Fig. 3A) concomitantly with an increase in A. This suggests a short-term conversion from reducing sugar to sucrose, and subsequent source-to-sink assimilate partitioning, corroborated by a similar positive effect on plant height, stem diameter, and leaf, stem, and root dry mass (Table 2).
With 30% irrigation, increasing the NO3−:NH4+ ratio up to an estimated 0.61:0.39 caused a slight decline in starch content (Fig. 3B). As the NO3−:NH4+ ratio increases, so does the demand for NO3− reduction, which is energetically expensive (Nunes-Nesi et al. 2010) and is avoided when NH4+ is the main N source (MacNeill et al. 2017). In tobacco (Nicotiana tabacum L.) and soybean (Glycine max L.) leaves with increasing NO3− supply, the starch content decreases as the carbon is reallocated to metabolites of the Krebs cycle, organic acids, and amino acids (Veau et al. 1992; Scheible et al. 1997). The increasing NO3−:NH4+ ratio in Arabidopsis results in a decline in starch content and an increase in metabolites associated with tricarboxylic acids (Hachiya et al. 2012; Sato and Yanagisawa 2014).
Proline content was higher in water-stressed plants, particularly in those fertilized solely with NO3−- or NH4+-over those receiving mixed N (Fig. 3C). The increase in leaf proline content usually occurs both in water deficit conditions (Hossain and Fujita 2010) and fertilization with only NH4+ fertilization (Fernández-Crespo et al. 2012; Kováčik and Klejdus 2014; Ravazzolo et al. 2020). In our study, it suggested that either NO3− or NH4+ root accumulation may be involved in a pronounced increase in proline content. In Arabidopsis thaliana L. mutants under water stress, increased proline content is related to NO3− accumulation in the roots (Chen et al. 2012). In Triticum durum L. (var. Amilcar), however, the increase in proline content was seen as a response to NH4+ toxicity (Torralbo et al. 2019).
Different NO3−:NH4+ ratios influenced leaf NR activity, but only in plants under 30 % irrigation. This finding disagrees with other studies that suggest water deficit as an inhibitory factor of NR activity (Zahoor et al. 2017; Huang et al. 2018). Leaf NR activity increased up to an estimated 0.46:0.54 ratio with 30 % irrigation. This response may be due to both the positive effect of increased NO3− supply on enzymatic kinetics (Sauro 2011) and to an attenuation of the inhibitory effect of NH4+ on NR activity (Botella et al. 1993). However, this result was reversed under higher NO3−:NH4+ ratios, suggesting that a high NO3− content may inhibit leaf NR activity (Fig. 3D). Similar findings were reported by Wang et al. (2018) and Zhang et al. (2019).
Increasing NO3−:NH4+ from the 0.5:0.5 ratio attenuated leaf NH4+ toxicity occurred with the 0.0:1.0 and 0.25:0.75 ratios (Fig. 4). Symptoms of NH4+ toxicity also occur in other species (Guo et al. 2007; Helali et al. 2010; Wang et al. 2018), and can lead to a disruption in hormonal homeostasis (Walch-Liu et al. 2000), reduced photosynthesis (Pissolato et al. 2019), oxidative stress (Wang et al. 2010), acidification of cellular organelles, and photophosphorylation inhibition (Bittsánszky et al. 2015).
The results also showed that mixed N fertilization favored NR activity more than only NO3− or NH4+ supply. Underwater stress, NR activity maintenance is necessary because this enzyme acts in the synthesis of nitric oxide (Pissolato et al. 2020), which plays an important role as in mitigating water stress, as observed in other species (Cai et al. 2015; Pissolato et al. 2020).
The positive effect of increasing NO3−:NH4+ ratio on morphophysiological traits under water deficit can be attributed, to some extent, to a close relationship among NO3− ratio, NR activity, and A. Carbon skeletons derived from CO2 assimilation are required for amino acid synthesis, after NO3−–NH4+ reduction. In this process, NR acts as a key enzyme that provides metabolic regulation in NO3− reduction to ensure that NH4+ production does not exceed the availability of carbon skeletons for amino acid synthesis (Heldt and Piechulla 2011).
Conclusions
The growth of young Eucalyptus urophylla plants is negatively affected by water deficit, resulting in lower plant height, less stem diameter, total leaf area, and dry mass of leaves, stems, and roots. Reduction in turgor pressure, associated with decreases in stomatal conductance, net photosynthesis, and reducing sugar and starch contents are the main physiological and biochemical changes that lead to growth inhibition.
Mixed N fertilization with increased NO3−:NH4+ ratio can mitigate negative water deficit effects on leaf water potential, stomatal conductance, and photosynthesis. Furthermore, leaf nitrate reductase activity improved with mixed NO3−:NH4+, compared with only fertilizing with NO3− or NH4+. Despite this, variations in N sources proved to be ineffective in preventing growth inhibition in young plants under water deficit.
The best performance of growth-related morphophysiological traits was achieved at a NO3−:NH4+ ratio of 0.50:0.50, regardless of the water regime. These data can be seen as particularly relevant for plants under 30 % irrigation, since fertilization with an NO3− ratio equal to NH4+, despite not preventing damage to morphophysiological traits, can mitigate water stress effects on plant growth.
Author contributions statement
Conceptualization: PC and PS. Greenhouse plant cultivation and data collection: PS, RB, MB, MS, SM, and NM; laboratory chemical analyses: PS, RB, MB, LS, MS, and MA; data analyses and interpretation: PS, PC, and LS; original writing and English language translation: PC and PS. All authors have provided consent for the publication of the final version of the manuscript.
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Soares, P.P.d., Cairo, P.A.R., da Silva, L.D. et al. Mitigating water stress by increasing NO3−: NH4+ ratio in young Eucalyptus urophylla plants. Trees 36, 81–91 (2022). https://doi.org/10.1007/s00468-021-02183-y
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DOI: https://doi.org/10.1007/s00468-021-02183-y