We carried out this work in ultramafic soils with high Ni bioavailability (SAP), located in the municipality of Barro Alto, Goiás State, and in non-ultramafic typical Cerrado environments constituted by Cambisol (CAM) and Ferralsol (LV), with high Al bioavailability, located in Brasília, Federal District (FD) of Brazil.
Study area
The selected study areas are located in the municipality of Barro Alto, Goiás State and in Federal District (FD) of Brazil (Fig. 1). The first area is located in the Barro Alto mafic-ultramafic complex which is aligned with the intrusions of Niquelândia and Cana Brava for approximately 350 km extension along a NE-SW structural zone (CPRM 2010). The complex is subdivided into Lower Layered Series and Upper Layered Series. Our study area covers the Lower Layered Series which in turn is subdivided into Lower Mafic Zone and Ultramafic Zone. (Baeta Jr. 1986).
The ultramafic massif of Barro Alto occurs in shallow soils, with rocky outcrops, and strong undulating relief. The vegetation is adapted to the high levels of heavy metals and prolonged water deficit and is composed mostly of herbaceous plants, with sparsely distributed shrubs, and rare trees. According to the vegetation classification system proposed by Ribeiro and Walter (2008), there is a mosaic of phytophysiognomies composed of Campo Sujo (grassland), Campo Rupestre (rupestrian grassland) and Cerrado Rupestre (rupestrian Cerrado).
The second study area is located in the FD of Brazil, more specifically, in the ferruginous detritus-lateritic deposit of Tertiary-Quaternary age, which covers a large portion of the study area (CPRM, 2010). This unit is surrounded by different types of Neoproterozoic lithologies from Paranoá, Canastra, and Bambuí Groups. We can notice a large extension of urban areas covering this study site (Fi. 1).
Soil sampling for chemical and agronomic characterization of the study sites
In the ultramafic (SAP and LAT) and typical Cerrado soils (CAM and LV) sites, three composed soil samples, each one was constituted by 10–30 subsamples, were collected at the 0–20 cm soil depth using a “dutch” auger. The samples were air-dried, ground and passed through a 2 mm sieve in the Soil Laboratory of the Embrapa Cerrados. The chemical and agronomic characterization of the soils were carried out through the following analyzes, according to the procedures described by Embrapa (1999): organic matter (O.M.); pH in water (1:2.5); exchangeable Ca2+, Mg2+, and Al3+ extracted by KCl 1 N; P and K, extracted with Mehlich-1 solution (0.05 M HCl + 0.0125 M H2SO4); potential acidity (H + Al), extracted with 0.01 M calcium acetate at pH 7. Co, Cu, Fe, Mn, Ni and Zn were extracted with DTPA solution (bioavailable contents), according to methodologies described by Baker and Amacher (1982), and with aqua regia (extractable content) (ISO 1995), analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES, Thermo Fisher Scientific 7000).
Anglo American Mining Company provided information on the predominant mineralogy of these sites, such as the characteristics of the soil's parent material and total levels of Ni, MgO, Fe, SiO2 and Al2O3 were kindly provided by, with soil sampling density of 25 mx 25 m (data not shown).
Field plant sampling
In the region of ultramafic massif of Barro Alto, with soils with different levels of Ni availability (Andrade et al. 2015), above-ground tissues (leaves and stems) of Justicia lanstyakii Rizzini (Acanthaceae), Euploca salicoides (Cham.) J. I. M. Melo & Smir (Boraginaceae) and Oxalis hirsutissima Mart. Ex Zucc. (Oxalidaceae) plants species were sampled in areas with predominance of Campo Sujo (Table 1). In the botanic surveys mentioned above, at least one individual of those species collected in the area was found to hyperaccumulate Ni in above ground tissues. This also applies to specimens of E. salicoides sampled in ultramafic or typical Cerrado soils in relation to the hyperaccumulation of Al in the tissues.
Collection of plants for DNA, biochemical, and metal content analysis, and for localization of metals in leaf structures
In each site described in Table 1, at different times and according to the objective of the study, the shoot (leaves and stems) of various plants of each species, randomly distributed on the ground, was collected. The collected materials were packed in paper or plastic bags, identified and taken to the Laboratory of Chemical Analysis of Plants, for elemental characterization, and to the Laboratory of Plant Genetics and Molecular Biology for DNA analysis, both from Embrapa Cerrados. E. salicoides plants were taken to the Laboratory of Plant Physiology of the Department of Botany, University of Brasília (UnB), for analysis of non-structural carbohydrates and total proteins. With the authorization issued by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) (Special Authorization No. 02/2008), the plant tissue of E. salicoides (ES) species collected in the SAP5 site were prepared for identification, localization and semi-quantification of chemical elements at the tissue level, using scanning electron microscopy (SEM) and Energy-dispersion X-ray spectroscopy (EDXS), at the microscopy service (SCMEM) of the Faculty of Science and Technologies of Nancy (Université de Lorraine), Nancy, France.
a) Sample processing for DNA extraction
In the ultramafic (SAP5, SAP7, and SAP9) and the typical Cerrado (CAM) sites, shoots (leaves and stems) of two to three individuals were collected to form a composite sample of each species of E. salicoides (EU), O. hirsutissima (OH) and J. lanstyakii (JL), totaling 14 botanical accessions, as described in Table 2. The selected plants were in good vegetative condition and were growing more than two meters apart. The collected materials were packed in plastic bags, identified, placed in a polystyrene box with ice, until delivery, on the same day, to the Laboratory of Plant Genetics and Molecular Biology, where they were processed and aliquoted for DNA analysis and forwarding the rest of the plant material to the Laboratory of Plant Chemical Analysis.
Samples of genomic DNA from each access were extracted using the modified CTAB methodology (Faleiro et al. 2003) and with the use of washing in Sorbitol buffer (0.35 M). The amount of DNA was estimated by spectrophotometry at 260 nm (A260) and the A260/A280 ratio was used to evaluate the purity and quality of the samples (Sambroock et al. 1989). The DNA samples from each accession were diluted to 5 ng µL− 1. The amplification reactions to obtain Inter Simple Sequence Repeats (ISSR) markers were carried out with 4.9 µL of Milli Q water 1.3 µL of buffer; 0.39 µL of 50 mM MgCl2; 0.26 µL of deoxyribonucleotides (dATP, dTTP, dGTP and dCTP) 10 µM; 1.95 µL of a 2 µM primer (Operon Technologies Inc., Alameda, CA, USA); 0.2 µL of the Taq DNA polymerase enzyme (1 unit); and 3 µL of DNA (15 ng) (total volume of 13µL). Initially, 18 ISSR primers were tested (Table 3). From these tests, eight primers were selected and used to obtain ISSR markers that generated a greater number of polymorphic bands and presented better quality of amplifications.
The amplifications were performed in a thermocycler, in which the samples were initially denatured at 94°C for 2 min, followed by 37 cycles, starting with 15 seconds at 94°C; then 30 seconds at 47°C and 72°C per 1 min. At the end of all cycles, the process was completed in 7 min at 72°C and cooled to 4°C.
After amplification, 3 µl of a mixture of bromophenol blue (0.25 %) and glycerol (60 %) in water was added to each sample. These samples were applied on an agarose gel (1.2%), stained with ethidium bromide, submerged in a TBE buffer (90 mM Tris-Borate, 1 mM EDTA). The electrophoretic separation was approximately four hours, at 90 V. At the end of the race, the gels were photographed under ultraviolet light.
b) Preparation of samples for chemical analysis of leaf tissues
After removing the aliquot for DNA analysis, all remaining tissue material (leaves and stems) from each sample, together with samples of other specimens collected at the same sites, were sent to the Laboratory of Plant Chemistry Analysis for element characterization. The materials were slightly immersed in tap water and then in deionized water, to remove soil particles, and left on a bench until it dried at room temperature. The materials were then taken to a forced air oven at a temperature of 40°C, until reach constant weight. After drying, the leaf tissues were finely crushed in a knife mill and mineralized by moist digestion, in a mixture of perchloric acid and hydrogen peroxide, in the 2:1 proportion (v/v), respectively, to determine the levels of Ca, K, Mg, P, S, Co, Cu, Fe, Ni, Mn, and Zn by ICP-OES. The N was extracted by the micro-Khjeldahl method and determined by colorimetry (Embrapa 1999) or by flow injection analysis (FIA, Lachat Quikchem 6000 system), coupled with UV/VIS, method.
c) Morphological features and determination of non-structural carbohydrates and total proteins in E. salicoides plants originated from ultramafic (SAP) and Cerrado (LV) soils
The E. salicoides species were collected in two sites of ultramafic soils, previously characterized as contrasting in relation to the bioavailable levels of Ni (Andrade et al. 2015): ES-P6 access, in a site with ~ 100 mg Ni dm− 3 soil (LAT6), and ES-P5 access, in a site with ~ 600 mg Ni dm− 3 soil (SAP5). The ES-LV access is originated from plants growing in typical Cerrado soil (red Latosol), with low total and bioavailable levels of heavy metals (Freitas et al. 1978; Martins et. al. 2004). The site was located on the UnB campus, in Brasília, FD.
In each of the sites mentioned above, in the morning and, in the peak of dry season (June to August), three plants growing apart on the ground were collected to determine the non-structural carbohydrates (soluble sugars, glucose, fructose, sucrose, raffinose, and starch) and the total proteins. Before being sent for destructive analysis, the numbers of inflorescences/plant and branches/stem, as well as length of the stems and of the internodes for each plant were obtained using a pachymeter.
d) Determination of total soluble sugars (TSS)
Leaves samples of E. salicoides were lyophilized and ground (10 mg), for soluble sugars determination. The soluble sugars were extracted four times with 80 % ethanol (500 µL), at 80°C (40 min.). After centrifugation (10,000 g, 10 min), the supernatants were combined and depigmented by the modified Shannon method (Shanon 1968). In a separating funnel, the ethanolic fraction (2.0 mL), absolute ethanol (0.5 mL), chloroform (3.0 mL), and water (5.5 mL) were added in sequence. Separation occurred after a period of approximately 12 h. The measurement of TSS was performed according to the phenol-sulfuric method (Dubois et al. 1956). For comparison purposes, a standard glucose curve (SIGMA) was used at concentrations of 0, 5, 10, 20, 40, and 80 µg. Supernatants containing TSS were collected and reserved for further analysis. The precipitate was used to remove the other components.
For the analysis of TSS (sucrose, glucose, fructose, and raffinose), the alcoholic fractions were dried, resuspended in water (1 mL), passed through an anionic and cationic exchange column (Dowex), and analyzed by High Performance Ion Exchange Chromatography with Detector of Integrated Amperometric Pulse (HPAEC/IPAD), in CarboPac PA-10 column (Dionex Corporation, Sunnyvale, Ca, USA), using an elution gradient with 200 mM NaOH in water (30 min.). The detector responses were compared with the patterns of glucose, fructose, sucrose, and raffinose at 0.625, 1.25, 2.5, 5.0, 10.0, and 20.0 µM. The standard curve for each sugar was used to calculate the carbohydrate content in the leaves.
e) Starch determination
After removing the TSS, the starch in the precipitate was extracted twice with Bacillus licheniformis thermostable α-amylase (MEGAZYME − 120 U mL− 1) (0.5 mL) at 75°C, for 30 min, and twice with amyloglucosidase from the Aspergillus niger fungus (MEGAZYME − 15 U.mL− 1) (0.5 mL) at 50°C and for 30 min. At the end of the four incubations with the enzymatic extract (2.0 mL), 100 mL of 0.8 M perchloric acid was added to stop the reaction and to precipitate proteins. After centrifugation (13,000 g, 2 min), the glucose released (50 µL of extract) was dosed by the GODPOD enzymatic method, using 750 µL of the PAPLiquiform Glucose kit (CENTERLAB®), which contains glucose oxidase, peroxidase, 4-aminoantipyrine, and phenol. After incubation at 30°C and, for 15 min, the glucose content was determined in a spectrophotometer at 490 nm. The data obtained were compared with a standard glucose curve (SIGMA), at the concentrations of 0, 5, 10, 20, 30, and 40 µg. The released glucose was calculated and adjusted (-10 %) for the mass of bound glucose that is present in the starch.
f) Determination of total proteins
The proteins were extracted four times with sodium phosphate buffered saline (PBS, 100 mM pH 7.4) (0.5 mL) for 1 h, except the first, which remained 12 h at 4°C. After centrifugation (13,000 g, 10 min.), the total proteins were dosed using the Bradford method (Bradford 1976). The data obtained were compared with a standard glucose curve (SIGMA), at the concentrations of 0, 5, 10, 15, 20, and 25 µg. The released glucose was calculated and adjusted (-10 %) for the mass of bound glucose that is present in the starch.
g) Location of Ni in cell compartments of E. salicoides leaves, native to ultramafic soil, by SEM-EDXS
About three E. solicoides (ES-P5) plants were randomly collected, in the SAP5 ultramafic site for use in microscopic analysis for the location, distribution and relative concentration of Ni in the cells of the leaf tissue. Still in the field, the third pair of leaves that precedes the youngest leaflet were cut into small segments, with a stainless-steel blade. The cuttings were made in order to have leaf blade tissue and secondary ribs in the same segment and placed in a small test tube (10 mL) containing 5 mL of 70 % alcohol. In the laboratory, about 10 segments of each sample were cut into thin pieces (1 mm x 3 mm), fixed in adhesive tape, placed on carbon cassettes ("stub") and dried at room temperature. In these samples, the location of Ni accumulation in tissues was analyzed by using SEM-EDXS (Hitachi-S4800 SEM), with accelerated power ranging from 5 to 15 KV.
Statistical analysis
Due to the fact that the assumption of normality was not met, the data of metal content in soil and
in leaf tissues were processed using non-parametric statistical analysis. The comparison between the medians of treatment was made based on the Kruskal-Wallis test at the significance level of p ≤ 0.05. The principal component analysis (PCA) was used for the correlation of the sites according to the soil chemical characteristics and nutrient accumulation in leaf tissues. Understanding communality as the explanatory power of the variable by the factor, the value adopted was > 0.70. In order to evaluate how the soils are grouped according to all chemical attributes together, a cluster analysis was carried out by Ward's method, considering the Euclidean distance (Bussab et al. 1990). Optimum number of clusters was verified by the Average silhouette method (Rousseeuw 1987).
In the DNA analysis, the generated ISSR markers were converted into a matrix of binary data, from which the genetic dissimilarity between the different genotypes was estimated, based on the complement of the Nei and Li similarity coefficient (Nei and Li 1979), using the Genes Program (Cruz 2013). The genetic similarity (GS) was given by: Sgij = 2Nij / (Ni + Nj), where: Nij is the number of bands present in both i and j genotypes; and Ni and Nj are the number of bands present in i and j genotypes, respectively. By subtracting the unit's SG value by 1 (1 - SG), we obtained the genetic dissimilarity. The genetic dissimilarity matrix was used to perform cluster analysis, by the Unweighted pair group method arithmetic average (UPGMA) method (Sneath and Sokal 1973) as a clustering criterion, and the graphic dispersion based on multidimensional scales, using the main coordinate method.
To test the effects of treatments (type of soils) on the production of total sugars, proteins and starch by E. salicoides plants growing on different soils, the data were analyzed using a one-way ANOVA, followed by Tukey's test, when significance level was < 0.05.
Statistical analyzes were performed using the SAS (SAS Institute Inc. 2008), Statistica (StatSoft Inc. 2007), and R software, version 3.6.0 (R Core Team 2019).