Characterization of Nitrogen Use by Neotropical Myrtaceae in Dry and Wet Forests of Southeast Brazil

Abstract

We hypothesized that neotropical Myrtaceae could be organized into groups that are naturally less or non-responsive to NO₃⁻, and that use other N forms, such as amino acids, for internal N transport. Ecophysiological tests were conducted to measure nitrate reductase activity (NRA), NO₃⁻ content, total N, δ¹⁵N natural abundance, the C:N ratio in leaves, free amino acid, and NO₃⁻ transport via xylem sap. We showed that Myrtaceae tree species have a relatively low NRA, in addition to little NO₃⁻ in leaves and free NO₃⁻ in the xylem sap during the wet and dry seasons. We suggested a possible compartmentalization of N use, wherein plants derive their internal N from and use their transport mechanism to move N between below-ground and above-ground parts, assimilating and transporting more N and C through amino acids such as glutamine, arginine, and citrulline. Evidence of low NO₃⁻ availability in tropical soils is important when trying to understand forest species’ N-use strategies, given their importance to plant nutrition. Differences in the responses of some Myrtaceae species to the seasonality of environmental factors suggest the need for further studies concerning N in natural forests, for example, to help understand the problem of N deposition ecosystems.

1. Introduction

The diversity and availability of nutrients in the environment influence the interaction, distribution, abundance, diversity, local composition, and individual performance of plants [1,2,3]. Nitrogen (N) is an essential nutrient and a major limiting factor for plant growth [4]. Nitrate (NO₃⁻) and ammonium (NH₄⁺) are the predominant forms of inorganic N in the soil [5].

The concentration of these ions can vary between different ecosystems; for example, in natural forest soils where NH₄⁺ is more abundant than NO₃⁻, some plants preferentially use NH₄⁺ [6,7]. However, among the inorganic nutrients, NO₃⁻ is essential given its high mobility, is disputed and used economically by organisms, and is quickly absorbed and assimilated into soluble and biologically useful compounds [8,9]. In addition, NO₃⁻ has broad spatial and seasonal variations established by biotic and abiotic factors and is a typical fertilizer, but as a free ion is easily lost by leaching [10]. There is therefore a high potential for contamination in drinking water sources, giving rise to health problems when in appearing in excess in the environment [11,12].

Plants have adapted to different environmental conditions and use either NH₄⁺ or NO₃⁻, which are often available in different proportions in the soil [6,7,13]. In natural systems, native plants use defined strategies for the acquisition and use of N, according to the diversity of N forms available together with their variation in quantity in the environment [6,13,14,15,16].

The most common form in which the plant assimilates N is NO₃^−, and plants have evolved flexible mechanisms for processing NO₃⁻, such as assimilation in different locations of the plant allied to its transport in the xylem sap, regulation of NO₃⁻ content in seeds, transport into the vacuole for storage in leaves [13], and efficient belowground systems for the use of available NO₃⁻ after environmental stress [17]. The belowground organ is responsible for absorbing and transporting water and mineral elements, constituting an efficient mechanism of N uptake, transport to aboveground parts, and utilization internally in the plant [12]. This process it is a crucial step of N metabolism and has been widely studied, especially with tree species, concerning the internal transport via xylem and reduction of NO₃⁻ in the aboveground part of the plant [18].

It is essential to determine the form of N taken up by plants. In leaves, the content of N_total has traditionally been used to estimate the status of N in plants; however, there are limits to its use as a nutritional indicator, such as the low response to high N supply in trees [19], as well as its insensitivity to seasonal demand in the use of the nutrient [20]. Instead, certain enzymatic activities have been proposed as more sensitive indicators for the nitrogen status in plants [21]. For example, in field experiments, nitrate reductase (NADH) (EC 1.7.1.1) activity (NRA) is a good indicator of NO₃⁻ uptake and use by roots and leaves [12,13,17]. Nitrate reductase (NR) is a highly stable enzyme in vitro and in vivo that can be influenced by external factors, such as NO₃⁻ concentration, light period, carbohydrate and carbon dioxide levels, in addition to other environmental factors [22].

The assimilation capacity can vary, and in this respect, plants can be organized by families, taxonomic characters, or functional traits and by ecological succession stages [7,13,17,23,24,25,26] NR is cited as an important biochemical marker for N deficiency, and measurements of its activity are used as a tool for evaluation of the nutritional status of N in plants [7,12]. Thus, depending on the level of available NO₃⁻ in the soil, one can determine which species are the most responsive to the use of available NO₃⁻, which is crucial for predicting how plants respond to the seasonality of environmental factors to maintain their internal N status for survival.

Some physiological measurements, such as the natural N isotope abundance (δ¹⁵N values) in plants, have been suggested as an effective tool for understanding N dynamics in ecosystems [27]. In particular, when NH₄⁺ is the predominant form available, plants may prefer the use of this ion rather than NO₃⁻ [28]. One way to investigate this preference using N forms is to measure leaf δ¹⁵N to characterize N in the soil and N preference of the cooccurring species [29]. For plant species or communities under the same soil N status, different uptake or preferences between NH₄⁺ and NO₃⁻ can determine the relationships between leaf N contents and δ¹⁵N values at the species or community levels [21,30].

NO₃⁻ is an N solute derived from soil mineral N that can be transported in the xylem as free NO₃⁻ or as amino acids after NO₃⁻ reduction and NH₄⁺ assimilation in roots [31]. In this sense, the amino acid composition in the xylem sap can thus help understand N utilization and transport in plants [32,33]. Furthermore, plants have mechanisms that enable them to conserve nutrients, for example, via the remobilization of previously stored nutrients such as amino acids [34,35].

Although it is clear that plants require N for the synthesis of amino acids and a variety of other metabolites [35] and that NO₃⁻ and NH₄⁺ are the primary sources of inorganic N for plant growth [21], for many neotropical tree species, there is still no clear understanding of the physiological processes regarding N use, especially when considering soil-plant relations concerning NO₃⁻ use. This discussion is essential because of the impacts caused by increases in N in natural systems (e.g., deposition and leaching) that may alter plant community structure and function by affecting the plant’s N metabolism (e.g., uptake, subsequent assimilation, and internal transport of amino acids). In the neotropical region’s tropical rainforest and savanna forest, it is unclear how variations in N availability may impact the species found there.

Among the great diversity of tropical tree species, the Myrtaceae is an ecologically important family in Brazil’s Atlantic Rainforest and Cerrado (Brazilian savanna). This botanical family has more than 5500 species and is one of the ten most species-rich angiosperm families. Members of the Myrtaceae are remarkably diverse in the tropical biomes of America, Asia, and Australia, invariably contributing to a significant proportion of the total species composition [36]. Despite the extraordinary richness of this family in the neotropical region, research concerning physiological responses related to N use is scarce, especially when considering soil–plant relations regarding NO₃⁻ use [13]. Therefore, there is a need to characterize the strategies of N use in groups of neotropical plants. In addition to this large, diverse family, many other families of tropical plants have yet to be studied, so there is little information on the functional diversity of tropical flora and the use of nutrients such as N. In this context, this study aimed to characterize and compare the N metabolism in different species—with a focus on the use of NO₃⁻ of Myrtaceae species—working on the hypothesis that Myrtaceae species present a characteristic pattern of N use with an expectation of low responsiveness to NO₃⁻ availability in the tropical soil, which is naturally variable between seasons and depends on the moisture gradient and forest type.

2. Material and Methods

2.1. Study Areas

We chose three legally protected areas in regions of the Cerrado and Atlantic Forest biomes of the State of São Paulo, Brazil. The trees were selected and ordered according to a humidity gradient of the forest types, organized in a dry-wet sequence: Cerrado (SW), located in the Estação Ecológica de Jataí (JES), an area of Semideciduous Forest (SF) in the Santa Genebra Biological Reserve (BRSG); and Dense Ombrophilous Forest (OD) in the Carlos Botelho State Park (SPCB). The JES area has an annual rainfall of 1470 mm and Dark Red Latosol soil (Oxisol—0–5 cm deep) that is dystrophic, alic, and deficient in nutrients such as phosphorus, calcium, and magnesium, with a high concentration of iron, a low content of total nitrogen, and an NH₄⁺:NO₃⁻ ratio = 3.13. The BRSG area has an annual rainfall of 1400 mm and Haplic Cambisol (Inceptisol—0–5 cm depth) that is a clayey, mesotrophic, acid soil, with a high concentration of iron and carbon and an NH₄⁺:NO₃⁻ ratio = 4.25. The SPCB area has an annual rainfall of 1800 mm, and Red Argisol soil (Ultisol—0–5 cm depth) that is acidic, dystrophic, and poor in nutrients such as phosphorus, calcium, magnesium, and potassium with an NH₄⁺:NO₃⁻ ratio = 23.82). (Appendix A).

2.2. Species Studied

For this study, eight common tree species of Myrtaceae were chosen from understory forest formations, using three individual samples of isolated and individually marked trees per species (

Table 1. List of Myrtaceae tree species chosen to study the acquisition, absorption, transport, and use of N. Acr—the species acronym, SW—Savanna Woodland, OD—Ombrophylous Dense and SF—Semideciduous are types of tropical forest. The references that support the listed species considered the information from the Virtual Herbarium of the Reflora Project (Flora do Brasil 2020). To see more, visit: https://reflora.jbrj.gov.br/reflora/PrincipalUC/PrincipalUC.do?lingua=en (accessed on 4 December 2022).

Species	Authors	Acr	n	Forest Type
Myrcia lingua	(O. Berg) Mattos & D. Legrand	Mli	3	SW
Campomanesia pubescens	(DC.) O. Berg	Cpu	3	SW
Eugenia cuprea	(O. Berg) Mattos	Ecu	3	OD
Marlierea suaveolens	Cambess.	Msu	3	OD
Myrciaria floribunda	(H. West ex Willd.) O. Berg	Mfl	3	SF
Plinia cauliflora	(DC.) Kausel	Pca	3	SF
Campomanesia guaviroba	(DC.) Kiaersk.	Cgu	3	SF
Eugenia excelsa	O. Berg	Eex	3	SF

). The sample number was restricted due to the difficulties of collecting very tall trees.

2.3. Soil Sampling and Analysis

For each area, 15 soil samples were collected from the topsoil layer (the top 5 cm, which a fertile soil layer and the layer most influenced by precipitation, biogeochemical processes (especially in savanna forest), and nutrient availability) at sites that contained most of the plants selected in each forest type. We analysed the following chemical variables: pH—Active acidity (CaCl₂), H⁺ + Al³⁺—Potential acidity (SMP buffer) and OM—Organic matter (Photometry); macronutrients: P—Phosphorus, S—Sulphur, K⁺—Potassium, Ca²⁺—Calcium and Mg²⁺—Magnesium; and soil quality variables: OC—Organic carbon, CEC—Cation exchange capacity, SB—Sum of bases and V—Base saturation.

To determine the NO₃⁻ and NH₄⁺ content in wet and dry seasons, we buried in the holes left after soil sampling bags of 200 mesh—Nytal (6.0 × 6.0 cm) containing 5.0 g of ion-exchange resin Dowex^® Marathon^® MR-3 hydrogen and hydroxide forms to collect leached inorganic N [12,14,37,38]. After one week, the bags were removed and stored at −20 °C until processing for analysis. The resin was eluted with 2 N KCl solution to determine NO₃⁻ [39] and NH₄⁺ [40]. The data were expressed as µg g⁻¹. Total nitrogen (N_total) in the leaves was only measured in the JES area, with determination performed by the Kjeldahl method.

2.4. Collection of Plant Material and Analysis

2.4.1. In Vivo Activity of Nitrate Reductase (NADH) and Xylem Sap Collection

Samples of fresh leaves were taken from healthy branches in the wet and dry seasons. The defoliated branches were attached to a manual vacuum pump by the basal cut end to extract the xylem sap. All the following analyses were performed in replicates for each individual.

Nitrate reductase (NADH) (EC 1.7.1.1) activity (NRA) was evaluated in vivo [17] using fresh subsamples of leaves of each specimen. Leaves were separated into replicates of 0.1 g aliquots of fresh material and transferred to test tubes. These samples were dark vacuum-incubated in a buffer solution (KH₂PO₄; 0.05 M, pH = 7.5; 30 °C for 60 min) containing 1-propanol 1% (v/v) and KNO₃ (0.1 M). After incubation, the assay mixture was analysed for nitrite formation by spectrophotometry (λ = 540 nm), and the absorbance readings were converted to pkat g FW⁻¹ (Fresh Weight).

2.4.2. Sample Preparation and Nitrate (NO₃⁻) Determination

The responsiveness of plants to NO₃⁻ utilization was investigated at two levels: accumulation in the leaves and transport in the xylem sap. Leaf NO₃⁻ content was determined after extraction of a 0.5 g quantity of leaves of each individual immersed in 5 mL of methanol for 24 h at room temperature followed by storage at −20 °C. The methanol leaf extract and the xylem sap were used to determine NO₃⁻ content. An aliquot of 0.2 mL was used for NO₃⁻ measurement by salicylic acid nitration [39], followed by conversion of the spectrophotometer absorbance readings (410 nm) to µg mL⁻¹ of NO₃⁻, based on a calibration curve.

2.4.3. Natural Abundance (δ¹⁵N Atom%), N_total (N%), and C_total (C%)

The fresh leaf samples were dried at 60 °C until achieving a constant weight. Subsamples were milled using a ball mill (40 mesh), encapsulated and analysed for the natural abundance of N isotopes (δ¹⁵N), nitrogen—N_total (N%) and, carbon—C_total (C%) using a continuous flow element analyser (Carlo Erba^®) connected in line with a mass spectrometer (Finnegan Delta Plus^®).

2.4.4. Free Amino Acids and N Transport

For OD and SF species, the internal transport of N as amino acids was investigated. Aliquots of 10 μL of xylem sap collected in the field were derivatized with 30 μL of OPA-borate + mercaptoethanol for separation and quantification of individual free amino acids by high-performance liquid chromatography (HPLC-LKB Bromma^® 2150, Industriële Veiling Eindhoven BV, Eindhoven, The Netherlands) [41] with modifications [42]. The concentration of amino acids was established by peak area compared with a Sigma^® AAS⁻¹8 standard to which glutamine, asparagine, and GABA were added. Citrulline was also added when necessary. N transport in the form of amino acids in the xylem sap was estimated as the proportion relative to the total amino acid concentration, considering the number of N atoms. Unfortunately, this analysis could not be performed for the JES area. All chemicals used to prepare solutions were of analytical grade with a purity of ≥99%.

2.5. Statistical Tests

The homoscedasticity and normality of the data were checked by evaluating the model residuals and applying the Shapiro–Wilk test for a normal distribution. To assess the differences in the edaphic set and plant N use variables between sites (different forest types) and seasons (wet and dry), we applied ANOVA and Tukey’s HSD post hoc test (α = 0.05). Possible outliers were examined using Dixon’s Q-test (p < 0.05). All the numeric data are presented as the mean ± standard error. We read outliers using Dixon’s Q-test (p < 0.05). XLStat software—Addinsoft© 2022 was employed for statistical analysis.

3. Results

3.1. Soil Chemical Properties

The soil pH in the three areas was acidic, with a significant difference between each location (

Table 2. Soil chemical properties in three types of tropical forest: Savanna Woodland (SW), Ombrophylous Dense (OD), and Semideciduous Forest (SF). Values are the mean ± standard error for n samples of each forest type. Means are followed by letters, where lowercase letters indicate significant differences between forest types, uppercase letters indicate significant differences between inorganic N forms (NO₃⁻ vs. NH₄⁺) in the same season, and uppercase letters in italics indicate significant differences between the same inorganic N forms by season (ANOVA and a post hoc Tukey’s HSD post hoc test, α = 0.05). Abbreviations: OM—Organic Matter, OC—Organic Carbon, CEC—Cation Exchange Capacity, SB—Base Saturation and V—Percentage of Saturation by Bases.

Soil Chemical Properties	Forest Type		SW				OD				SF
	n		11				23				12
	Unit
pH	CaCI2		3.8	±	0.02	a	3.7	±	0.03	b	4.3	±	0.34	c
K+	mmolc/dm3		1.3	±	0.13	a	3.5	±	0.31	b	2.2	±	0.77	b
Ca+2			1.4	±	0.36	a	18.2	±	2.2	b	38.2	±	18.3	b
Mg+2			1.3	±	0.27	a	8.9	±	0.92	b	10.9	±	5.0	b
H+ + Al+3			53.9	±	0.73	a	98.6	±	3.3	b	101.2	±	27.8	b
Al+3			13.9	±	0.34	a	18.2	±	1.6	b
S	mg/dm3		9.1	±	0.80	a	8.1	±	0.90	a	7.9	±	2.1	a
P			3.3	±	0.75	a	14.7	±	1.5	b	23.6	±	4.6	c
OM	g/dm3		28.6	±	1.1	a	44.3	±	3.3	b	65.3	±	5.7	c
OC			16.6	±	0.9	a	25.7	±	1.8	b	37.9	±	3.2	c
CEC	mmolc/dm3		57.8	±	1.3	a	130.3	±	5.0	b	152.4	±	28.6	b
SB	%		3.9	±	0.72	a	31.6	±	3.3	b	51.3	±	22.8	b
V			6.6	±	0.97	a	23.5	±	1.9	b	33.6	±	13.5	b
NH4+	µg/g	wet	3.0	±	0.09	aAA	2.7	±	0.08	bAA	13.3	±	1.5	cAA
		dry	1.1	±	0.07	aAB	1.4	±	0.11	bAB	8.7	±	1.6	cAB
NO3−		wet	1.0	±	0.20	aBA	1.1	±	0.34	aBA	3.1	±	0.61	bBA
		dry	0.06	±	0.01	aBB	0.03	±	0.02	aBB	10.3	±	1.5	bAB

). The OM, OC, CEC, SB, and V% and the nutrient contents in SF and OD were significantly higher than in the SW area. In detail, the soil of SW can be characterized as acidic and dystrophic (V% < 30), with a deficiency in elements such as P (Table 2). The elevated acidity is typical of tropical soil, possibly caused by higher Al³⁺ availability (m% > 50%). The content of H⁺ + Al³⁺ and the low cation exchange capacity (CEC < 190 cmolc L⁻¹) suggest a low chemical reactivity of organic matter (OM < 60 g/dm³) and, therefore, relatively low soil fertility. In the other areas (SF and OD), although dystrophic (m% < 50, V% < 30, OM < 60 g/dm³), OD soil had chemical thresholds closer to SF but was more influenced by high precipitation (Appendix A), a lower content of H⁺ + Al³⁺, and lower CEC, with a subtle influence on the concentrations of Ca²⁺, Mg²⁺, and K⁺ when compared with SF (Table 2).

3.2. Soil Nitrogen

Inorganic nitrogen concentrations differed between soil types with a strong influence of seasonal changes (wet and dry) (

Table A1. Abiotic characteristics of the protected areas studied. Savanna Woodland (SW)—Jataí Ecological Station (JES), Ombrophylous Dense Forest (OD)—State Park of Carlos Botelho (SPCB) and Semideciduous Forest (SF)—Biological Reserve of Santa Genebra (BRSG), Brazil. Coord. = Geographic coordinates, Prec. = Precipitation, Et_p = Potential evapotranspiration and T = Temperature.

Site	Forest Type	Coord.	Area	Altitude	Prec.	Etp	T	Season		Soil
			ha	m	mm	mm/year	°C	dry	wet
JES	SW	21°36′ S 47°47′ W	9075	520–642	1470	~1320	14.2–32.5	May–Oct	Nov–Apr	Dark Red Latosol (Oxisol)
SPCB	OD	24°15′ S 47°98′ W	37794	30–1003	1700–2400	~1400	17.0–31.7	Apr–Sept	Oct–Mar	Haplic Cambisol (Inceptisol)
BRSG	SF	22°44′ S 47°06′ W	252	580–610	100–1360	~1241	15.5–30	Apr–Sept	Oct–Mar	Red Argisol (Ultisol)

and Table 2). The NH₄⁺ content of the topsoil was significantly higher than to that of NO₃⁻ for all forest types except for SF in the dry season, where the difference was not significant. Indeed, NO₃⁻ levels were conspicuously low in SW and OD in the dry season. Both inorganic forms of N were much higher in SF than SW and OD in both seasons. In all forests, NH₄⁺ mineralization was higher in the wet season, whereas NO₃⁻ was higher in OD and SW but not in SF (Table 2).

3.3. Plant Nitrogen

We observed some interesting characteristics of N ecophysiology, with a focus on NO₃⁻ nutrition for the analysed Myrtaceae tree species (

Table 3. Nitrate reductase activity (NRA), nitrate concentration (NO₃⁻), natural N abundance (δ¹⁵N), total nitrogen (N_total), and carbon:nitrogen ratio (C:N) in leaves. Data are the mean ± standard error; lowercase letters indicate significant differences between seasons by species, and uppercase letters indicate significant differences between the same Myrtaceae genera for forest types (ANOVA and Tukey’s HSD post hoc test, α = 0.05). Acr—the species acronym, F—Forest type, S—Season, OD—Ombrophylous Dense, SF—Semideciduous, and SW − Savanna Woodland (SW) are types of tropical forest.

	F	S	Leaves
Acr			NRA				NO3−				δ15N				Ntotal				C:N
			pkat g−1 FW				mol g−1 FW				‰				%
Mli	SW	wet	13.5	±	1.0	a					−0.2	±	0.12	a	1.0	±	0.01	a	39.9	±	1.8	a
		dry	10.1	±	2.9	a					−0.17	±	0.12	a	1.2	±	0.1	a	37.8	±	2.7
Cpu	SW	wet	59.5	±	4.3	aB					−0.11	±	0.08	a	0.3	±	0.14	aB	46.9	±	2.2	aB
		dry	49.8	±	5.4	aA					−0.19	±	0.01	a	0.4	±	0.04	aB	43.1	±	7.1	aB
Ecu	OD	wet	98.9	±	62.3	aA	6.0	±	1.0	aA	1.1	±	1.3	aA	2.1	±	0.7	aA	22.4	±	6.6	aA
		dry	38.0	±	11.8	aA	6.9	±	0.9	aA	0.5	±	0.6	aB	1.9	±	0.2	aA	24.6	±	2.7	aA
Msu	OD	wet	104.2	±	10.8	a	5.0	±	0.9	a	0.7	±	1.2	a	1.5	±	0.1	a	30.4	±	3.1	a
		dry	87.2	±	34.0	a	4.8	±	0.9	a	1.0	±	1.1	a	2.1	±	1.0	a	25.6	±	10.2	a
Mfl	SF	wet	134.4	±	105.9	a	6.6	±	2.0	a	2.2	±	1.2	a	1.9	±	0.1	a	24.0	±	1.8	a
		dry	106.4	±	86.9	a	5.3	±	1.2	a	1.4	±	1.7	a	2.0	±	0.7	a	23.7	±	7.3	a
Pca	SF	wet	114.5	±	55.0	a	5.1	±	4.9	a	3.0	±	1.3	a	2.1	±	0.2	a	22.0	±	1.7	a
		dry	205.9	±	10.2	b	7.4	±	1.3	a	2.5	±	0.6	a	2.0	±	0.4	a	22.8	±	3.4	a
Cgu	SF	wet	26.7	±	6.4	aA	10.1	±	0.4	a	2.4	±	0.2	a	2.4	±	0.4	aA	19.4	±	2.9	aA
		dry	52.2	±	65.0	aA	10.2	±	0.1	a	3.3	±	1.2	a	2.0	±	0.02	aA	23.1	±	0.2	aA
Eex	SF	wet	110.6	±	93.9	aA	8.4	±	1.9	aA	2.6	±	0.4	aA	2.0	±	0.2	aA	23.2	±	2.3	aA
		dry	24.5	±	16.3	aA	9.9	±	6.1	aA	2.8	±	1.0	aA	2.1	±	0.4	aA	22.5	±	4.6	aA

). The data for Myrcia lingua (Mli), Campomanesia pubescens (Cpu), Eugenia cuprea (Ecu), and Campomanesia guaviroba (Cgu) revealed lower NRA in leaves relative to all other species, but no differences between seasons (Table 3), despite significant differences in NO₃⁻ availability (Table 2). Ecu and Marlierea suaveolens (Msu) had a low content in the xylem sap and equivalent values of NO₃⁻ in leaves in both seasons (Table 3 and

Table 4. Amino acid concentration (AA) and N transport in xylem sap of the Myrtaceae tree species during the wet and dry seasons. ASP = Aspartate, GLU = Glutamate, ASN = Asparagine, GLN = Glutamine, ARG = Arginine, CIT, Citruline, ALA = Alanine, PHE = Phenylalanine, MET = Methionine, VAL = Valine, LEU = Leucine, LYS = Lysine, Others = sum of remaining amino acids (each below 10%). Data are mean values. Acr—the species acronym, F—Forest type, S—Season. Types of tropical forest: OD—Ombrophylous Dense and SF—Semideciduous.

	F	S	Xylem sap
Acr			Nitrogen Transported (%)
			NO3−	ASP	GLU	ASN	GLN	ARG	CIT	ALA	PHE	MET	VAL	LEU	LYS	Others
Ecu	OD	wet	3.3	4.4	8.8	4.2	27.8	0.2	43.6							7.6
		dry	3.7	2.6	5.9	0.8	64.8	0.2	18.8							3.2
Msu	OD	wet	3.6	8.2	11.7	3.8	44.5	18.1								10.0
		dry	0.7	5.8	9.4	6.1	11.0	1.3	58.6							7.0
Mfl	SF	wet	0.6	2.7	7.7	0.5	50.1	11.0		25.5						2.0
		dry	0.6	4.6	6.5	6.2	41.3	2.4			20.1					18.4
Pca	SF	wet	0.6	2.5	7.9	1.1	54.0	10.9		16.8						6.2
		dry	0.8	6.1	18.2	0.0	18.3	3.4				15.8	25.2			12.2
Cgu	SF	wet	0.8	0.0	4.1	23.7	0.0	20.9			15.8			12.8		21.9
		dry	0.7	2.7	10.7	0.0	8.3	3.7			20.9				48.4	4.6
Eex	SF	wet	0.9	3.6	10.5	0.8	11.9	67.3								5.1
		dry	0.9	5.0	24.7	0.2	8.3	3.0				48.2				9.5

). In species in the genus Eugenia, the results for N_total were similar, with a low C:N ratio in both species. For the genus Campomanesia, the low N_total in Cpu and higher level found in Cgu were reflected in the significantly different values for the C:N ratio (Table 4).

Leaf δ¹⁵N values varied widely between Myrtaceae species (for example, from −0.11‰ to 3.0‰ in the wet season and −0.17‰ to 3.3‰ in the dry season). A notable amplitude variation occurred between forest types, with leaf δ¹⁵N decreasing in the following order: SF > OD > SW. Leaf δ¹⁵N values did not differ between the wet and dry seasons for any species (Table 3). In the wet season, plants showed mean leaf δ¹⁵N values of 1.5 ± 1.25‰, and for the dry season, 1.4 ± 1.36‰. However, when evaluated by type of forest formation, a positive value of foliar δ¹⁵N was observed in the forest species in OD and SF, whereas negative values were observed in SW species. When comparing species of Eugenia in OD and SF, significant differences were observed in δ¹⁵N in the dry period, with a value over five times higher attributed to Eex.

Overall, the apparent differences were related to forest type, with the species in SW standing out from those in OD and SF, having negative values for δ¹⁵N, higher C:N ratios, lower total N, and a tendency for lower NRA.

Analysis of the amino acid and NO₃⁻ contents representing the internal N transported by xylem sap, revealed low NO₃⁻ rates compared to amino acid rates. The NO₃⁻ content was lower than the amino acid content in all species (0.6 ≤ NO₃⁻ ≤ 3.7%) in both seasons (Table 4).

Significant differences between the wet and dry seasons were found in the NO₃⁻ concentration and free amino acids in the xylem sap of Mfl and in amino acids in Cgu. Among species of the same genera, Ecu and EEx showed differences in NO₃⁻ concentration in the dry period (Figure 1).

Our results show that eight amino acids predominated in the N transport profile of the xylem sap (GLN, ALA, VAL, LYS, MET, GLU, ARG, and CIT), depending on the species, season, and forest type (Table 4). In the wet season, OD, GLN, and CIT were most prevalent for Ecu and Msu, accounting for 44.5% (Msu) and 71.4% (Ecu), and in the dry season, 69.5% (Msu) and 83.6% (Ecu) of the total amino acid was transported in these forms. In SF, there was more variability in the form of N transported as amino acids: in the wet season, GLN, ALA, and ARG accounted for 81.7% (Pca) and 86.6% (Mfl), whereas in the dry season, GLN, VAL, LYS, and MET accounted for 59.3% (Pca) and 48.4% (Cgu) of the total xylem amino acids in Myrtaceae species. These percentages were low in all species relative to N transported as NO₃⁻, especially in Myrtaceae in SF. ASP and ASN were minor components in all species (Table 4).

Despite the low levels of ASP, amino acids synthesized from ASP stood out in N transport, with high amounts os MET (Eex—dry and Pca—dry). This finding suggests that the ASP family pathway in plants is essential for N transport and nutrition, possibly leading to the synthesis of other amino acids, such as LYS (Cgu—dry). However, although GLU was prominent in some cases, GLN was nearly always conspicuous and rarely less prominent than GLU. This observation is consistent with GLN being the first product of N assimilation, with GLU being the second. In Pca, Eex, and Mfl, we observed low foliar C:N, whereas GLN and ARG were the main transport compounds in the wet season. These species presented lower levels of foliar N_total and xylem sap NO₃⁻ than the other species. (Table 3 and Table 4).

4. Discussion

Overall, most soil chemical properties showed differences between areas, especially between the Atlantic Forest (SF and OD) and Cerrado (SW). The mesotrophic soil in the SF can be attributed to moderate acidity compared with SW and OD. Other characteristics aligned with the SF soil’s mesotrophic character were precipitation (<1500 mm) and moderate nutrient levels such as P and NO₃⁻, organic matter (OM), and organic carbon OC). In the OD and SW areas, the restricted availability of nutrients can be attributed to the dystrophy of the soils, which can be a limiting factor for nitrification. These differences can explain why soil characteristics influence the evolution of organic matter (OM), causing low reactivity and the accumulation of OM, notably in the following decreasing order: SF > OD > SW (Table 2). These conditions affect the availability of nutrients and biological activities, which could be a cause of the low concentration of inorganic N, for example, the lower concentration in SW compared to SF.

The variation in chemical properties is also influenced by the type of soil prevalent in each area (Table A1 and Table 2). The soils characteristically have low fertility (V% < 50%) and acidity and a high-water retention capacity, which would explain the predominance of NH₄⁺ over NO₃⁻ in the two seasons (Table 2). Clay, for example, influences the N mobility in the soil solution [43], and is more present in lower proportions in the Latosol (SW) than in the Red Argisol (SF). Furthermore, the precipitation regime causes variations in soil moisture, influencing the processes of N availability [44,45], which could explain the higher N concentration during the wet season.

In similar studies in tropical forests, refs. [13,25] showed that NRA was low in Myrtaceae species. This may reflect their adaptation to growth under low nutrient conditions in the soils in each area. Perhaps the low NRA can be explained by the better water availability in the soil, which could facilitate nitrification and the losses of NO₃⁻ by leaching or denitrification, which is common in tropical soils.

Although there is evidence that many species of tropical trees preferentially assimilate NO₃⁻ in the leaves [13,25], in our case, in Myrtaceae, NO₃⁻ reduction probably predominates in the roots. The ability of NO₃⁻ to induce NRA in higher plants is widely accepted, and different concentrations of NO₃⁻ are required for optimum induction, depending on the species [12]. The roots can be an important site of NO₃⁻ assimilation for some species at low external NO₃⁻ concentrations, whereas leaf NO₃⁻ assimilation becomes essential at high external NO₃⁻ levels when the capacity of the roots for NO₃⁻ assimilation is exceeded [35].

It is, therefore, possible that, for the species studied here, more reduction of NO₃⁻ occurs in the roots than in the leaves. The roots can act as sinks for NO₃⁻ from the soil, which can be converted to many forms of N for transport over long distances, mainly as amino acids [33]. This represents a mechanism for absorbing low levels of NO₃⁻ in the soil, thereby minimizing loss by leaching and the low nitrification caused by natural acidity [46]. Moreover, the concentration of NH₄⁺ in the soil (NH₄⁺ > NO₃⁻) suggests that the species studied absorb and assimilate this ion in the roots, which is supported by the fact that many amino acids found in the xylem sap have their origin in NH₄⁺ synthesis. This would explain why the NRA in the leaves was low.

The soil–plant relationship regarding N use observed in the studied species were consistent with those suggested by the literature. Meager availability of NO₃⁻ and low capability for its metabolism were observed among species, families, growth forms, and habitats for subalpine and alpine plants, where low concentrations of inorganic N in the soil are also found [7]. This situation was also observed in other species of Myrtaceae by [24,47] in savanna regions and in tropical forests [25]. Low NRA has been observed in plants that grow on acidic, ammonium-dominated soils [26,48], and is also prevalent among shrubs of the Ericaceae family [7,49,50].

The species studied here are slow-growing and commonly found in the understory of tropical forest types. Slow-growing plants have a limited ability to use NO₃⁻ as a significant form of inorganic N, as indicated by the very low in vivo NRA under field conditions [7]. A low level of leaf N was observed in some species, especially Cpu and Mli in SW. Low leaf N is also typical in Proteaceae [23] and some woody species [51], and these are also present in low leaf NRA.

In the case of the Myrtaceae species studied here, we verified N transport in the xylem sap in the form of amino acids generated in the roots with negligible transport of NO₃⁻, which would explain the low NRA in leaves. Amino acid transport is most likely a benefit in terms of carbon and nitrogen and was considerably higher in the Myrtaceae species than N transport via NO₃⁻, although the latter is considered an essential ion for N transport by xylem sap in many tropical tree species, e.g., the genera Cecropia sp. and Trema sp. [13].

Some studies have found that in tropical forest species, NO₃⁻ xylem concentrations may exceed those of organic compounds of N when assimilation occurs preferentially in the leaves [13,25,52]. In Cecropia sp., the high NO₃⁻ content in leaves was related to the levels of xylem transport [13]. However, in the Myrtaceae species studied here, NO₃⁻ uptake from the medium is unlikely to be limited to the assimilatory pathway. Slow-growing species, such as the studied Myrtaceae species, may have a more retentive strategy (e.g., low leaf N content) when compared with fast-growing species with an acquisitive resource strategy (e.g., high leaf N content) [53].

The variation in leaf δ¹⁵N may indicate that the Myrtaceae family uses specific strategies for N uptake according to each forest types. For example, in comparison with the SW species, Myrtaceae from SF showed higher N_total values and δ¹⁵N in their leaves. Various N uptake strategies, e.g., root-microorganism symbiosis, may be related to the variability in leaf δ¹⁵N, reflecting the varied ecological characteristics of tree species in different families [54]. However, further studies are needed on the capacity for N use and possible plant–soil interactions under different conditions in each forest type to better understand the N uptake strategy of the Myrtaceae studied here.

Our data provide information regarding internal N transport via the xylem sap of Myrtaceae species. The transport of N over a long distance is an essential process in plants, especially trees, where certain compounds often predominate in this role, transporting N from the roots to the leaves via the xylem. The amino acids GLN and ARG are the most common forms of N transported in the xylem. Mineral elements, such as NO₃⁻, are essentially used in the synthesis of organic compounds, principally amino acids. We observed that free NO₃⁻ was present in very low amounts in xylem sap (0.6 to 3.7% of the total N) (Figure 1 and Table 4). We, therefore, assume that the Myrtaceae studied had assimilated N in the roots, followed by translocation in the xylem to leaves as amino acids, mainly GLN, CIT, and ARG (Table 4), promoting low internal transport of NO₃⁻. This observation suggests that the relative amount of GLN, CIT, and ARG can serve as temporary N storage compounds when N assimilation is high. In plants, both NO₃⁻ and NH₄⁺ taken up by the roots are incorporated into GLN and GLU (primary nitrogen assimilation), which are then used to synthesize other amino acids and nitrogenous compounds by transamination. It is noteworthy that compared to most other amino acids, those that predominate in N transport have high C/N ratios, such as ARG (C/N ratio: 6:4), CIT (6:3), and GLN (5:2).

In the Myrtaceae species in the OD area, we observed a general tendency for GLN and CIT to predominate in xylem sap, but with fluctuations according to the wet and dry seasons. For example, Ecu transported N as CIT in the wet season and GLN in the dry season, whereas the inverse was observed in Msu (Table 4). The association with GLN indicates investment in a long-distance N transport strategy [13].

For species in SF, we observed a predisposition to transport N as GLN, more so in the wet season (Mfl and Pca). Other Myrtaceae species showed the predominance of different N transporters, for instance, ARG (wet) and MET (dry) in Eex and LYS in Cgu (dry). The significant contribution of MET (48.2%—dry) observed in Eex could be related to the metabolism of ASP and ASN [22], whereas the presence of LYS (48.4%-dry) in Cgu could be associated with the use of ASP as a precursor, an amino compound related to the formation of other amino acids such as THR, MET, and ILE [22]. Other amino acids observed (ALA in Mfl and Pca) suggest a stress response to root hypoxia generated by soil waterlogging [42,55] during the wet season.

Our data show that the Myrtaceae species, especially in SW, show little direct use of NO₃⁻, presenting a relatively low NO₃⁻ reduction in leaves (Table 3). The plants showed a better association with C:N, ARG, GLN, and CIT (Table 4), suggesting low responsiveness of NO₃⁻ in leaves and demonstrating that the root system of plants not only imports water and nutrients, such as NO₃⁻ from the soil solution, but is primarily responsible for N reduction in amino acids that are supplied to long-distance transport systems [56]. Due to differences in the biotic and abiotic factors in each forest type, nitrogen acquisition tradeoffs in plants likely occurs with compensation between NO₃⁻ and N reduced into amino acids in the root under edaphoclimatic conditions for each forest type.

Information on the ability of Myrtaceae species to reduce and assimilate NO₃⁻ in roots and leaves and understanding how different species can respond to N availability in neotropical forests are essential. In our study, we examined the uptake and use of nitrogen by comparing some Myrtaceae forest tree species. Leaf NRA was low in these Myrtaceae species, and the enzyme does not seem to have been influenced by NO₃⁻ levels in the leaves.

The poor relationships between NO₃⁻ availability in the soil and its consumption, accumulation, and transport by the plants, combined with low NRA values in the leaves, reinforce the idea that these species assimilate inorganic N preferentially in the roots. This enables the plants to use strategies for N utilization with an inadequate response to NO₃⁻ in leaves and a more significant role for organic nitrogen formation in the roots, e.g., the amino acids GLN, ALA, MET, GLU, ARG, and CIT.

We provide information on how the supply and demand for N by Myrtaceae species are modulated under three tropical forest formations. It is suggested that tropical Myrtaceae may be little or non-responsive to the internal transport and aboveground use of NO₃^−, whereas other N forms are used for internal transport during both seasonal periods.

5. Conclusions

Our findings show that in situ soil NO₃⁻⁻ availability in tropical forests has important implications for the forest types studied, not only in determining the composition of species or genera within the same family but also in understanding how some Myrtaceae utilizes the NO₃⁻ available, promoting its assimilation in the roots and transporting internal N via various nitrogenous forms, such as amino acids in the xylem sap. This is important because NO₃⁻ availability in tropical soils is crucial for predicting how plants respond to the seasonality of environmental factors to maintain their internal nitrogen status for survival.

Finally, studies of plant responses to NO₃⁻ have gained considerable interest, and our data concerning the nitrogen metabolism of different Myrtaceae species contribute to understanding strategies for nitrogen use in tropical and savanna forests. These data form the basis for future studies concerning the impacts of increases in N in such natural systems (e.g., the problem of increasing N deposition) and how this can influence tropical trees.